WO2024252001A1

WO2024252001A1 - Methods for culturing cells

Info

Publication number: WO2024252001A1
Application number: PCT/EP2024/065825
Authority: WO
Inventors: Amy COCHRANE; Maricke Lauren ANGENENT; Erik VAN DER WAL; Kyle Bill QUINNEY; Ruud OUT; Sara PALACIOS ORTEGA
Original assignee: Meatable BV
Current assignee: Meatable BV
Priority date: 2023-06-08
Filing date: 2024-06-07
Publication date: 2024-12-12
Anticipated expiration: 2025-12-08

Abstract

The present invention relates to a method for culturing different types of cell in the same culture medium, to a method of culturing cells at high densities and to cell cultures comprising different types of cell.

Description

Methods for culturing cells

Field of the invention

The present invention relates to a method for culturing cells and to cell cultures. The invention also relates to a hollow fiber reactor, a food product and use comprising the methods and cell cultures.

Background of the invention

According to the most recent United Nations estimations, the current world population is 7.9 billion in July 2022 [https://www.worldometers.info/es/poblacion-mundial/#ref-1] and it is expected to reach 10 billion around the year 2056. This increase will be heterogeneously distributed around the globe, with nine countries covering half the projected growth of the global population in the next 30 years, including India, Nigeria, Pakistan, Egypt, and the United States of America. Population and economic growth are major drivers of increased meat consumption. According to the United Nations Food and Agricultural Organization (FAO, https://www.oecd- ilibrary.org/agriculture-and-food/oecd-fao-agricultural-outlook-2022-2031_f1 b0b29c-en), an estimated growth of 15% in global meat consumption is projected by 2031 . On the other hand, the correlation between income growth and higher meat consumption is clearly demonstrable at lower income rates but once consumers reach an adequate standard of living, they become more sensitive to environmental, ethical, and animal welfare and health concerns.

For this reason, there is a growing interest in finding alternative protein sources which ideally will be sustainable and will contain the nutrients normally provided by meat in the human diet. Cultured meat arises as another alternative to traditional animal agriculture that aims to produce the skeletal muscle and adipose tissues that normally comprise animal meats, except using in vitro tissue and biological engineering techniques. Despite efforts to develop robust protocols for scalable generation of animal cell types from easily accessible and renewable sources, the differentiation of animal (pluripotent) stem cells into specific cell types often remains cumbersome, lengthy, and difficult to reproduce and/or has not been established yet.

Additionally, to date, plant-based and cultured meat alternatives have focused mostly on mimicking the muscle component of meat. However, fat is also a crucial component of meat, contributing to sensory/ flavor, textural attributes and palatability (Zhang, Shu, et al. "DNA polymorphisms in bovine fatty acid synthase are associated with beef fatty acid composition 1." Animal genetics 39.1 (2008): 62-70.).

Accordingly, there remains a need for methods for culturing animal cells that are suitable for human consumption and that can be produced in a scalable and cost-effective manner.

Summary of the invention This invention is based on the development of methods for the co-culture of cells, for example the co-differentiation of cells, and the culture of cells at high densities, for example as carried out in hollow fiber reactors. Such co-culture may comprise co-differentiation of two or more cell types, for example differentiation of two or more cell types in the same culture medium and/or at the same time.

According to the invention, there is thus provided a method for culturing cells, which method comprises culturing in the same medium: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

The invention also provides a method for culturing cells, which method comprises culturing in the same medium or different media: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell, wherein differentiation of the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell are inducible or are induced by the same molecule.

In such a method, the same inducer may be used to induce the two or more cell types which are differentiated at the same time in the same culture medium.

In a method of the invention, the cells are cultured, for example differentiated, in one of the extra-capillary space or the intra-capillary space of a hollow fiber reactor. That is to say, more than one type of cell, for example (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell, is cultured, for example differentiated, in one of the extra-capillary space orthe intra-capillary space of a hollow fiber reactor. Where more than one type of cell is co-cultured, for example co-differentiated, in the extra-capillary space, there may be no or substantially no cells present in the intra-capillary space and vice versa.

The invention further provides a method for culturing cells, which method comprises culturing cells in a hollow fiber reactor, wherein: the density of the cell culture is at least 1 billion cells/ml; and/or the culturing of cells in the hollow fiber reactor is carried out substantially in the absence of an anchor point for the cells; and/or the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intra- capillary space of the hollow fiber reactor.

The invention in addition provides a method for culturing cells, which method comprises culturing in a hollow fiber reactor: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

The invention further provides: a cell culture obtainable by a method according to the invention; a cell culture which comprises: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell; a cell culture comprising cells in a hollow fiber reactor, wherein: the density of the cell culture is at least 1 billion cells/ml; and/or the cell culture comprises substantially no anchor point for the cells; and/or the cells are present in the extra-capillary space of the hollow fiber reactor and/or the intracapillary space of the hollow fiber reactor. a hollow fiber reactor comprising a cell culture of the invention; use of a cell culture according to the invention or use of the method according to the invention for tissue engineering, optionally for the production of cultured meat; a food product, for example cultured meat, comprising at least one cell obtained from the cell culture; and a structured product comprising the cell culture as described herein.

Description of the invention

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

As used herein, the term "and/or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... ,etc.

The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (i.e. of 10) more or less 0.1 % of the value.

The terms "expression vector" or “expression construct" refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame, inducible promoter As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. In the case of the present invention, the control is effected by the transcriptional regulator protein.

Any reference to nucleotide or amino acid sequences accessible in public sequence databases herein refers to the version of the sequence entry as available on the filing date of this document.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Cell culture

The inventors have surprisingly found that cells may be cultured in a hollow fiber bioreactor at high cell densities, for example cells densities of at least about 1 (one) billion cells/ml. In addition, the inventors have shown that it is possible to co-culture fat cells and muscle cells or cells which are capable of differentiating into fat cells or muscle cells. In such co-culture, the two types of cell may be capable of being cultured at the same time in the same culture medium and/or capable of being induced to undergo differentiation via addition of a single inducer (exogenous substance). The use of such approaches enables scalable production of cultivated meat in an economically feasible fashion. In addition, the inventors have surprisingly found that cells cultured in a hollow fiber bioreactor at high cell densities, for example cells densities of at least about 1 (one) billion cells/ml can be cultured at lower cell culture media volumes than previously described.

The invention thus concerns methods for culturing cells, including methods in which cells are cultured in a hollow fiber reactor and methods in which more than one type of cell, for example two types of cell or more are co-cultured. Cells suitable for use in such a method may be: fat cells or cells capable of being differentiated into fat cells; or muscle cells or cells capable of being differentiated into muscle cells. Where the method for culturing cells comprises the culture of more than one type of cell, the more than one type of cell may be cultured in the same or different media. The more than one type of cell are inducible or are induced to undergo differentiation by the same inducer (exogenous substance). Such co-culture may comprise culture of cells in a hollow fiber bioreactor.

A method of the invention may comprise the culture of cells in addition to fat cells (or cells capable of being differentiated into fat cells) or muscle cells (or cells capable of being differentiated into muscle cells), such as fibroblasts, fibroblast-like cells, engineered cells expressing one or more fibroblast-like characteristics or cells capable of differentiating into such cells.

The invention encompasses methods for co-culturing cells capable of being differentiated into fat cells and cells capable of being differentiated into muscle cells in the same cell culture medium which methods thus allow for the co-differentiation of said cells in the same cell culture medium (at the same time).

Cells

Method of culturing cells according to the invention may comprise culture of one or more types of cell, for example two types of cell, three types of cell or four types of cell. Preferably, a method of culturing cells comprises the culture of: a fat cell or a cell capable of differentiating into a fat cell; and/or a muscle cell or a cell capable of differentiation into a muscle cell.

Typically, a cell which is used in a method of the invention is one which is capable of differentiating to mature cells, for example mature fat cells or mature muscle cells quickly, for example within 14 days, such as within 10 days, for example within 8 days, preferably within 5 days or 4 days or more quickly. When two types of cell are used in a culture method of the invention it is preferably is those cells are capable of differentiating, for example to mature muscle and fat cells within about the same time period.

A fat cell, or “adipocyte” or “lipocyte” is a type of cell that primarily composes adipose tissue, specialized in storing energy as fat. Fat cells are derived from mesenchymal stem cells which give rise to fat cells through adipogenesis. In cell culture, fat cell progenitors can also form osteoblasts, myocytes and other cell types.

There are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT), which are also known as white and brown fat, respectively, and comprise two types of fat cells. Accordingly, a fat cell may be a white fat cell or a brown fat cell.

A muscle cell is any cell that makes up muscle tissue. There are 3 types of muscle cells in the human body; cardiac, skeletal, and smooth. Cardiac and skeletal myocytes are sometimes referred to as muscle fibers due to their long and fibrous shape. A muscle cell is also known as a myocyte when referring to either a cardiac muscle cell (cardiomyocyte), or a smooth muscle cell as these are both small cells. A skeletal muscle cell is long and threadlike with many nuclei and is called a muscle fiber. Muscle cells (including myocytes and muscle fibers) develop from embryonic precursor cells called myoblasts. Myoblasts fuse form multinucleated skeletal muscle cells known as syncytia in a process known as myogenesis. Skeletal muscle cells and cardiac muscle cells both contain myofibrils and sarcomeres and form a striated muscle tissue.

Cardiac muscle cells form the cardiac muscle in the walls of the heart chambers, and have a single central nucleus. Cardiac muscle cells are joined to neighboring cells by intercalated discs, and when joined in a visible unit they are described as a cardiac muscle fiber.

Smooth muscle cells control involuntary movements such as the peristalsis contractions in the esophagus and stomach. Smooth muscle has no myofibrils or sarcomeres and is therefore non-striated. Smooth muscle cells have a single nucleus.

A method for culturing cells according to the invention may be any cell capable of differentiating into a desired cell type for example differentiating into a fat cell or a muscle cell.

Such cells include "pluripotent stem cells" which term as used herein includes embryonic stem cells, embryo-derived stem cells, induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSC). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of: OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 . Exemplary pluripotent stem cells can be generated using methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.

The pluripotent stem cells for use in the invention can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate embryonic stem cells and embryo- derived stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

Similarly, iPS cells can be from any species.

In certain embodiments, the pluripotent stem cell according to the invention, or for use in the invention is an animal cell. In certain embodiments the pluripotent stem cell according to the invention, or for use in the invention if from an edible animal species.

Preferably, the pluripotent stem cell according to the invention, or for use in the invention is from a livestock or poultry animal or a seafood animal. Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lamb, camels, water buffalo and rabbits.

Preferably, the pluripotent stem cell according to the invention, or for use in the invention is a porcine or a bovine pluripotent stem cell. Most preferably, a porcine pluripotent stem cell. In certain embodiments, the stem cell according to the invention is a porcine epiblast stem cell (pEpiSCs).

Poultry species include but are not limited to domestic chicken, turkeys, ducks, geese and pigeons. In certain embodiments, the cells originate from common game species such as wild deer, gallinaceous fowl, waterfowl and hare. Preferably a pluripotent stem cell according to the invention, or for use in the invention, is not a human cell.

Seafood species include, but are not limited to fish and shellfish.

A cell for use in a cell culture method of the invention, for example a pluripotent stem cell, may be one which can be induced to differentiate into a desired cell type, for example a fat cell or a muscle cell using, for example, the inducible expression platform as described in WO2018/096343 (the opti-ox™ approach).

Accordingly, a cell suitable for use in the invention may be a pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a protein involved in cell differentiation, wherein the coding sequence for the protein involved in cell differentiation is operably linked to an inducible promoter; and, optionally, iia) an expression construct for expression of a second protein involved in cell differentiation, wherein the coding sequence for the second protein involved in cell differentiation is operably linked to an inducible promoter; wherein the expression constructs of ii) and, optionally, iia) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.

A cell suitable for use in the invention may optionally comprise no expression constructs other than those set out in i) ii) and iia) above. Alternatively, a pluripotent stem cell of the invention may optionally comprise one or more expression constructs in addition to those set out in i), ii) and iia) above, for example into at least one further genetic safe harbour site that is not the first genetic safe harbour site. Such additional expression constructs may provide for the expression a further protein which regulates cell differentiation.

Proteins which regulate cell differentiation may be transcription factors. In the case of a cell which is intended for differentiation into a fat cell, the cell may express one or two or more transcription factors.

A cell suitable for use in a cell culture method of the invention may therefore be capable of expressing, for example, PPAR-y or CEPBa or a combination of both thereof.

In the case of a cell which is intended for differentiation into a muscle cell, the cell may express one or two or more transcription factors.

A cell suitable for use in a cell culture method of the invention may therefore be capable of expressing, for example, MYOD, MYOG or PAX7 or a combination of any two thereof, for example: a combination of MYOD and MYOG; or a combination of MYOD and PAX7. Such cells are described in W02024/084082 and patent application nos. PCT/EP2024/053897 and PCT/EP2024/053906. Any cell described in any one of those patent applications may be useful in a method of the invention for culturing cells.

Peroxisome proliferator- activated receptor gamma (PPAR-y) is a type II nuclear receptor functioning as a transcription factor that in humans is encoded by the PPARG gene. PPARG is mainly present in adipose tissue, colon and macrophages. Two isoforms of PPARG are detected in the human and in the mouse: PPAR-y1 (found in nearly all tissues except muscle) and PPAR-y2 (mostly found in adipose tissue and the intestine). In certain embodiments, the coding sequence of PPAR-y of the invention encodes PPAR-y2. PPARG regulates fatty acid storage and glucose metabolism. The genes activated by PPARG stimulate lipid uptake and adipogenesis by fat cells. PPARG knockout mice are devoid of adipose tissue, establishing PPARG as a master regulator of adipocyte differentiation.

In certain embodiments, PPAR-y is encoded by the sequence of SEQ ID NO: 1 and has the amino acid sequence of SEQ ID NO: 2.

CCAAT/enhancer-binding protein alpha (CEPBa) is a protein encoded by the CEBPA gene in humans. The protein encoded by this intronless gene is a bZIP transcription factor which can bind as a homodimer to certain promoters and gene enhancers. It can also form heterodimers with the related proteins CEBP-beta and CEBP-gamma, as well as distinct transcription factors such as c-Jun. The encoded protein is a key regulator of adipogenesis (the process of forming new fat cells) and the accumulation of lipids in those cells, as well as in the metabolism of glucose and lipids in the liver.

In certain embodiments, CEPBa is encoded by the sequence of SEQ ID NO: 3 and has the amino acid sequence of SEQ ID NO: 4.

MYOD, also known as myoblast determination protein 1 , is a protein in animals that plays a major role in regulating muscle differentiation. MYOD belongs to a family of proteins known as myogenic regulatory factors. MYOD is one of the earliest markers of myogenic commitment. MYOD is expressed at extremely low and essentially undetectable levels in quiescent satellite cells, but expression of MYOD is activated in response to exercise or muscle tissue damage. The effect of MYOD on satellite cells is dose-dependent: high MYOD expression represses cell renewal, promotes terminal differentiation and can induce apoptosis. Although MYOD marks myoblast commitment, muscle development is not dramatically ablated in mouse mutants lacking the MYOD gene. This is likely due to functional redundancy from Myf5 and/or Mrf4. Nevertheless, the combination of MYOD and Myf5 is vital to the success of myogenesis. The function of MYOD in development is to commit mesoderm cells to a skeletal myoblast lineage, and then to regulate that continued state. MYOD may also regulate muscle repair. MYOD mRNA levels are also reported to be elevated in aging skeletal muscle. One of the main actions of MYOD is to remove cells from the cell cycle (halt proliferation for terminal cell cycle arrest in differentiated myocytes) by enhancing the transcription of p21 and MYOD. MYOD is inhibited by cyclin dependent kinases (CDKs). CDKs are in turn inhibited by p21 . Thus MYOD enhances its own activity in the cell in a feedforward manner. Sustained MYOD expression is necessary for retaining the expression of muscle-related genes. MYOD is also an important effector for the fast-twitch muscle fiber (types HA, IIX, and IIB) phenotype.

In certain embodiments, MYOD is encoded by the sequence of SEQ ID NO: 5 and has the amino acid sequence of SEQ ID NO: 6.

PAX7, Paired box protein, is a protein that in humans is encoded by the PAX7 gene. Pax- 7 plays a role in neural crest development and gastrulation, and it is an important factor in the expression of neural crest markers such as Slug, Sox9, Sox10 and HNK-1. PAX7 is expressed in the palatal shelf of the maxilla, Meckel's cartilage, mesencephalon, nasal cavity, nasal epithelium, nasal capsule and pons. Pax7 is a transcription factor that plays a role in myogenesis through regulation of muscle precursor cells proliferation. It can bind to DNA as an heterodimer with PAX3. Also interacts with PAXBP1 ; the interaction links PAX7 to a WDR5-containing histone methyltransferase complex By similarity. Interacts with DAXX too. PAX7 functions as a marker for a rare subset of spermatogonial stem cells, specifically a sub set of Asingle spermatogonia. These PAX7+ spermatogonia are rare in adult testis but are much more prevalent in newborns, making up 28% of germ cells in neonate testis. Unlike PAX7+ muscle satellite cells, PAX7+ spermatogonia rapidly proliferate and are not quiescent. PAX7+ spermatogonia are able to give rise to all stages of spermatogenesis and produce motile sperm. However, PAX7 is not required for spermatogenesis, as mice without PAX7+ spermatogonia show no deficits in fertility. PAX7 may also function in the recovery in spermatogenesis. Unlike other spermatogonia, PAX7+ spermatogonia are resistant to radiation and chemotherapy. The surviving PAX7+ spermatogonia are able to increase in number following these therapies and differentiate into the other forms of spermatogonia that did not survive. Additionally, mice lacking PAX7 had delayed recovery of spermatogenesis following exposure to busulfan when compared to control mice.

In certain embodiments, PAX7 is encoded by the sequence of SEQ ID NO: 7 and has the amino acid sequence of SEQ ID NO: 8.

MYOG, or Myogenin, is a transcriptional activator encoded by the MYOG gene. Myogenin is a muscle-specific basic-helix-loop-helix (bHLH) transcription factor involved in the coordination of skeletal muscle development or myogenesis and repair. MYOG is a member of the MYOD family of transcription factors.

In mice, MYOG is essential for the development of functional skeletal muscle. MYOG is required for the proper differentiation of most myogenic precursor cells during the process of myogenesis. When the DNA coding for myogenin was knocked out of the mouse genome, severe skeletal muscle defects were observed. Mice lacking both copies of myogenin (homozygous-null) suffer from perinatal lethality due to the lack of mature secondary skeletal muscle fibers throughout the body. In cell culture, myogenin can induce myogenesis in a variety of non-muscle cell types.

In certain embodiments, MYOG has the coding sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10.

In certain embodiments, the nucleic acid molecules encoding the proteins according to the invention are codon-optimized for expression in mammalian cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wildtype sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon. Preferably more than one nonpreferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon- optimized sequence. Replacement by preferred codons generally leads to higher expression.

A transcriptional regulator protein is a protein that bind to DNA, preferably sequence- specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor). Such entities are also known as transcription factors.

The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or nearthe promoter of the regulated DNA sequence.

Transcriptional activator proteins bind to a response element and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression.

A genetic safe harbour (GSH) site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighboring genes and expression of the inducible cassette minimizes interference with the endogenous transcription program. More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou et al, 201 1 , Nature Biotechnology, 29(1), 73-8. doi: 1 0. 1 038/nbt. 1 71 7.) These criteria include a site that is (i) 50 kb or more from the 5’ end of any gene, (ii) 300 kb or more from any gene related to cancer, (iii) 300 kb or more from any microRNA(miRNA), (iv) located outside a transcription unit and (v) located outside ultra-conserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH already identified do not fulfil all of the criteria. It is thought that a suitable GSH will satisfy at least 2, 3, 4 or all these criteria.

In certain embodiments of the invention, the first and further genomic safe harbour sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene or the CCR5 gene. In certain embodiments the first and further genomic safe harbour sites are located on chr1 : 152,360,840-152,360,859, chr1 : 175,942,362 -175,942,381 , chr1 :231 ,999,396-231 ,999,415, chr2: 45,708,354 - 45, 708, 373; chr8: 68,720,172 - 68,720,191 of the human genome.

In certain embodiments of the invention, the first and further genomic safe harbour sites are selected from any two of the safe harbour sites ROSA26, AAVS1 , the CLYBL gene or the CCR5 gene. Preferably, the genetic safe harbour sites are hROSA26 locus and the AAVS1 locus. In certain embodiments of the invention, the expression construct for expression of MYOD protein as described herein and the expression construct for expression of a MYOG protein as described herein are both inserted into a second genetic safe harbour site that is different from the first genetic safe harbour site. In certain embodiments, the expression construct that is inserted into the second genetic safe harbour site is capable of expressing both the MYOD protein and the MYOG protein simultaneously.

Transcriptional repressor proteins bind to a response element and prevent gene expression.

'activated or deactivated by a number of mechanisms including binding of a substance, interaction with other transcription factors (e.g., homo- or hetero-dimerization) or coregulatory proteins, phosphorylation, and/or methylation. The transcriptional regulator may be controlled by activation or deactivation.

If the transcriptional regulator protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation may be through any suitable means, but it is preferred that the transcriptional regulator protein is activated through the addition to the cell of an exogenous substance. The supply of an exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulator protein can be controlled. Alternatively, an exogenous substance can be supplied in order to deactivate a transcriptional regulator protein, and then supply withdrawn in orderto activate the transcriptional regulator protein.

If the transcriptional regulator protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires deactivation. Thus, a substance is supplied to prevent the transcriptional repressor protein repressing transcription, and thus transcription is permitted.

Any suitable transcriptional regulator protein may be used, preferably one that is activatable or deactivatable. It is preferred that an exogenous substance may be supplied to control the transcriptional regulator protein. Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins.

Accordingly, in certain embodiments, a pluripotent stem cell useful in the methods of the invention is controlled by an exogenously supplied substance. In a method of the invention where two types of pluripotent stem cell are used, preferably, they will be both be capable of being induced to undergo differentiation by the same exogenously supplied substance.

In certain embodiments, the exogenously supplied substance is selected from the group consisting of peptides (such as described by Klotzsche, et al; Journal of Biological Chemistry 280.26 (2005): 24591-24599 or Schlicht et al.; Applied and environmental microbiology 72.8 (2006): 5637- 5642) or the inducers described in Goeke, et al. Journal of molecular biology 416.1 (2012): 33-45; incorporated herein by reference), an aptamer (such as the RNA aptamer described in Hunsicker et al. “Chemistry & biology 16.2 (2009): 173-180; incorporated herein by reference), tetracycline, and anhydroteracyclin or a derivative thereof. Preferably, the exogenously supplied substance is doxycycline. In certain embodiments, the transcriptional regulator protein as described herein is selected from the group consisting of tetracycline responsive transcriptional activator protein (rtTa), Tetracycline repressor (TetR), VgEcR synthetic receptor or a hybrid transcriptional regulator protein comprising a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain from the human progesterone receptor or an activation domain from the human NF-kB.

Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression well known in the art where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline which is more stable). In this system, the transcriptional activator protein is tetracycline - responsive transcriptional activator protein (rtTa) ora derivative thereof. The rtTA protein is able to bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), which together form a tetracycline response element (TRE). There are two forms of this system, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or deactivates (Tet-Off) the rTA protein.

In a Tet-Off system, tetracycline or a derivative thereof binds rTA and deactivates the rTA, rendering it incapable of binding to TRE sequences, thereby preventing transcription of TRE- controlled genes. The Tet-On system is composed of two components; (1) the constitutively expressed tetracycline - responsive transcriptional activator protein (rtTa) and the rtTa sensitive inducible promoter (Tet Responsive Element, TRE). This may be bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, allowing it to bind to TRE sequences and inducing expression of TRE-controlled genes. In preferred embodiments of the invention the transcriptional regulator protein is rtTA.

If the transcriptional regulator protein is rtTA, then the inducible promoter inserted into the at least one further GSH hat is not the first GSH site includes the tetracycline response element (TRE). Thus, in certain embodiments the inducible promoter includes a Tet Responsive Element (TRE).

In some embodiments, where the transcriptional regulator protein is rtTA and includes TRE the exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

In certain embodiments of the invention, the expression construct that is inserted into the second genetic safe harbour site is a fusion protein that encodes two proteins, for example both the PPAR-y protein and the CEPBa protein as described herein or both the MYOD protein and the MYOG protein or both the MYOD protein and the PAX7 protein. In certain embodiments, the expression construct that is inserted into the second genetic safe harbour site encodes: a PPAR-y protein, a linker and a CEPBa protein; a MYOD protein; a MYOD protein, a linker and a MYOG protein; or a MYOD protein, a linker and a PAX7 protein.

Accordingly, in a method of the invention: at least one cell expressing a PPAR-y protein and a CEPBa protein and at least one cell expressing a MYOD protein may be co-cultured; at least one cell expressing a PPAR-y protein and a CEPBa protein and at least one cell expressing a MYOD and a MYOG protein may be co-cultured; or at least one cell expressing a PPAR-y protein and a CEPBa protein and at least one cell expressing a MYOD and a PAX7 protein may be co-cultured.

Preferred cells for use in the methods of the invention include:

A. A pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a PPAR-y protein, wherein the coding sequence for the PPAR-y protein is operably linked to an inducible promoter; and, iii) an expression construct for expression of a CEBPa protein, wherein the coding sequence for the CEBPa protein is operably linked to an inducible promoter; wherein the expression constructs of ii) and iii) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.

B. A pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a MYOD protein, wherein the coding sequence for the MYOD protein is operably linked to an inducible promoter; and, optionally, iii) an expression construct for expression of a MYOG protein, wherein the coding sequence for the MYOG protein is operably linked to an inducible promoter; wherein the expression constructs of ii) and, optionally, iii) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.

C. A pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a MYOD protein, wherein the coding sequence for the MYOD protein is operably linked to an inducible promoter; and, optionally, iii) an expression construct for expression of a PAX7 protein, wherein the coding sequence for the PAX7 protein is operably linked to an inducible promoter; wherein the expression constructs of ii) and, optionally, iii) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.

Preferred combinations of the aforementioned cells A, B and C for use in the invention are: A and B; and A and C. In certain embodiments, the linker sequence may be a cleavable linker. That is, the linker sequence may comprise a sequence of amino acids which are capable of being cleaved. For example, the linker sequence may comprise a sequence capable of acting as a substrate for an enzyme capable of cleaving peptide bonds--i.e. a cleavage site. Many such cleavage sites are known to and can be employed by the person skilled in the art of molecular biology. In some embodiments, the cleavable linker may comprise an autocleavage site. Autocleavage sites are automatically cleaved without the need for treatment with enzymes. For example, the family of 2A self-cleaving peptides, or 2A peptides have been described, which includes 2A peptides P2A, E2A, F2A, and T2A. F2A is derived from foot-and-mouth disease virus; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; T2A is derived from thosea asigna virus 2A. In certain embodiments, the cleavable linker is thus selected from the group consisting of P2A, E2A, F2A, and T2A.

In some preferred embodiments the expression construct comprises a Picornavirus 2A (P2A) linker.

Where a cell expresses two proteins, in certain embodiments, the inducible promotor that is operably linked to, for example the PPAR-y protein is different than the inducible promotor that is linked to the CEPBa protein. In certain embodiments, the inducible promotor that is operably linked to the PPAR-y protein is the same at the inducible promotor that is linked to the CEPBa protein. The same applies to cells which express a MYOD protein and a MYOG protein and a MYOD and a PAX7 protein. Typically, the inducible promoter will be the same, in order that expression of both proteins may be induced by the same molecule. Inducible promotors are well-known in the art, examples include but are not limited to CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.

In certain embodiments, the inducible promotor used in the present invention a tetOn promotor. Preferably a 3^rd generation TetOn promotor.

Culturing methods

In a culture method of the invention, cells may be proliferated and/or differentiated.

In certain embodiments, the method of the invention relates to a method for production of muscle cells and/or fat cells. In certain embodiments, the proliferation and/or differentiation medium does not comprise insulin and/or dexamethasone.

The proliferation medium and the differentiation medium may have the same composition. The same medium may be used for proliferation and differentiation. That is to say, the same culture system can be used for both proliferation and differentiation phases, for example via a medium change or by only adding an inducer of the opti-ox™ system within the bioreactor (the opti-ox™ approach is described herein and in WO2018/096343). The addition of an inducer for differentiation to the bioreactor is very attractive, as it minimizes capital investment in equipment, processing times and cell manipulation. Typically, the inducer is capable of inducing differentiation of all cell types used in a method of the invention such that they can induced simultaneously and/or using the same medium. That is to say, a single inducer of differentiation may be used in a method of the invention to simultaneously differentiate all of the cell types used in the method. In a method of the invention, one medium may be used to proliferate two or more cell types simultaneously and/or one medium may be used to differentiate those two or more cell types simultaneously.

The inventors have surprisingly found that use of the pluripotent cells as described herein allow for the co-culture of fat and muscle. Typically, such co-culture is a differentiation process in which differentiation of at least two types of cells is carried out simultaneously, ie. at the same time and wherein the different types of cell are in contact with the same medium. Such a method may be carried out so two or more types of cell are present in the same vessel. However, it is possible to carry out a method of the invention where in the different types of cell are present in different vessels, but wherein the same medium is circulated through those different vessels, i.e. the different vessels are connected in some way allowing a single medium to pass between them. The method as described herein optionally does not comprise an additional commitment phase induction step.

The invention accordingly concerns co-culture in which at least two different cell types can differentiate together in the same media (either muscle or fat differentiation defined recipes). Further, the cells may self-organize within the aggregates/tissues in the co-culture.

The method as described herein reduces the differentiation time of the pluripotent cells as described herein to mature muscle and fat cells dramatically. In certain embodiments, the time to produce mature fat cells and muscle cells as described herein, for example using a co-culture method, is at most 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days. Using the pluripotent cells as described in the method as described, it may be possible to achieve a conversion rate of at least 95% by day 4 of culture, meaning that at least 95% of the cells are mature after 4 days of culture. Accordingly, in certain embodiments the time to produce at least 95% mature fat cells and muscle cells is at most 4 days.

Thus is may be possible to simultaneously co-culture, for example differentiate, muscle and fat cells in, at most, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days or about 2 days. That is to say, muscle and fat cells may be co-differentiated in, at most, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days or about 2 days.

Equal amounts of at least two types of cell may be used in a method of the invention. However, more of one cell type may be used than the other(s). For example, in a method of the invention which comprises the use of fat and muscle cells or cells capable of being differentiated into such cells may be use at a ratio of about 90:10, about 80:20, about 70:30, about 60:40, about 50:50, about 40:60, about 30:70, about 20:80, about 10:90 based on muscle cells (or cells capable of being differentiated into muscle cells): fat cells (or cells capable of being differentiated into muscle cells).

In a further aspect, the invention provides for a cell culture comprising fat cells, preferably mature fat cells, and muscle cells, preferably mature muscle cells, as obtained by the methods as described herein.

Culturing the cells as described herein can be performed under so called 2D culturing conditions, which is considered the conventional approach to culturing cells. However, the method as described can also easily be adapted to allow culturing under 3D conditions as shown in the examples below.

3D cell culture is an artificially-created environment which enables cells to grow or interact with their surroundings in three dimensions. In such culture, cells typically form 3D colonies, which may be referred to as "spheroids". The 3D culture approach may more accurately model the cells' in vivo growth and behaviour. The skilled person is readily able to carry out 3D cell culture, for example by taking advantage of any of a number of commercially-available culturing tools. For example, the 3D culture may be carried out using scaffold or scaffold-free techniques. Scaffoldbased techniques make use of supports such as solid scaffolds and hydrogels to enable the cells to form a 3D culture. Such scaffolds may aim to mimic the natural extracellular matrix (ECM), which is present in vivo. Scaffold-free techniques dispense with the use of the scaffold on which to grow the cells. Instead, 3D spheroids may be established through the use of, for example, low-adhesion plates, hanging-drop plates, micro-patterned surfaces, rotating bioreactors, magnetic levitation and magnetic 3D bioprinting.

Cells that have been transduced with lentiviral vectors are not considered food safe or not safe for human and non-human dietary consumption. The pluripotent cells as described herein of the method as described herein obviate the need to use lentivirally transduced cells. Accordingly, in certain embodiments, the adipocytes and myocytes that are produced according to the method as disclosed herein are suitable for human and non-human dietary consumption. They may also be suitable in certain embodiments, the produced adipocytes and myocytes can be used in the production of cultured meat (or engineered meat) for human consumption.

Hollow fiber reactors

In a culture method of the invention, cells may be cultured using hollow fibers, for example edible hollow fibers. That is to say, the cells may be cultured in an apparatus comprising hollow fibers, such as a hollow fiber cartridge or hollow fiber bioreactor.

A method for culturing of cells according to the invention comprises using a hollow fiber bioreactor, wherein: the density of the cell culture is at least 1 billion cells/ml; and/or the culturing of cells in the hollow fiber reactor is carried out substantially in the absence of an anchor point for the cells; and/or the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intracapillary space of the hollow fiber reactor.

Culturing of cells in a hollow fiber reactor may be carried out substantially in the absence of an anchor point for the cells; and/or the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intracapillary space of the hollow fiber reactor.

Such a method of the invention may comprise culture of one cell type, but may comprise co-culture of two or more, for example three, four, five or more cell types. Suitable cell types are described herein. In one embodiment, the invention relates to a method of culturing muscle cells (or cells capable of differentiation into muscle cells such as herein described) using a hollow fiber bioreactor, wherein: the density of the cell culture is at least 1 billion cells/ml; and/or the culturing of cells in the hollow fiber reactor is carried out substantially in the absence or in the absence of an anchor point for the cells; and/or the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intracapillary space of the hollow fiber reactor and wherein the total external media supply is at most about 50mL - about 1500mL, about 100mL - about 1200mL, preferably about 200mL- about l OOOmL, more preferably about 200mL- about 400mL over a period of, for example, about 7 days.

In one embodiment, the invention relates to a method of culturing fat cells (or cells capable of differentiation into fat cells such as herein described) using a hollow fiber bioreactor, wherein: the density of the cell culture is at least 1 billion cells/ml; and/or the culturing of cells in the hollow fiber reactor is carried out substantially in the absence or in the absence of an anchor point for the cells; and/or the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intracapillary space of the hollow fiber reactor and wherein the total external media supply is at most about 50mL - about 1500ml, about 100mL - about 1200mL, preferably about 200mL- about 1000mL, more preferably about 200mL- to about 400mL over a period of, for example, 7 days.

In one embodiment, the invention relates to a method of culturing culturing in the same medium: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell using a hollow fiber bioreactor, wherein: the density of the cell culture is at least 1 billion cells/ml; and/or the culturing of cells in the hollow fiber reactor is carried out substantially in the absence or in the absence of an anchor point for the cells; and/or the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intracapillary space of the hollow fiber reactor and wherein the total external media supply is at most about 50mL - about 1500ml, about 100mL - about 1200mL, preferably about 200mL - about 1000mL, more preferably about 200mL - to about 400mL over a period of, for example, about 7 days.

Cells may be capable of forming macroscale tissue in a culture method of the invention. For example, where muscle cells are used (or cells capable of differentiating into such cells), for example at cell inoculation densities of from about 100 to about 300 million/ml, macroscale muscle tissue can be formed as aggregates fuse to forming a complete piece of tissue which is induced to differentiate into functional muscle. Such macroscale tissue may be at least about 2cm in length, for example at least about 3cm or longer in length.

Hollow fibers suitable for use in the invention may comprise one or more materials consisting of hydrocolloids and proteins. The hollow fibres may have an outer diameter of from about 0.2 mm to about 2.0 mm, and/or a porosity of from 0% to about 75% and/or a wall thickness of from about 0.05 mm to about 0.4 mm. The hollow fibers may have a wall thickness of from about 0.08 mm to 0.2 mm. The hollow fibers have a porosity of from about 40% to about 60%.

The hollow fibers may comprise one or more of alginate, collagen, cellulose, chitosan, collagen, zein, agar, inulin, gluten, pectin, legume protein, methyl cellulose, pectin, gelatin, tapioca, xanthan gum, guar gum, tara gum, bean gum, plant protein, starch, plant isolates (e.g., soy/zein/casein/wheat protein), lipids, (e.g., free fatty acids, triglycerides, natural waxes, and phospholipids. The hollow fibers may comprise one of more of corn protein, potato protein, wheat protein, sorghum protein, animal protein, animal protein isolate, beef protein isolate, casein protein and whey protein. The plant isolates of the hollow fibers comprise one of more of soy, zein, casein, and wheat protein. In another aspect of the present invention, the lipids of the hollow fibers may comprise one or more of free fatty acids, triglycerides, natural waxes and phospholipids. The hollow fibers may comprise one or more legume proteins and one or more hydrocolloids.

Typically, each hollow fiber has a first end and a second end wherein the first end and the second end are positionally opposed to each other and, wherein a quantity of the hollow fibers are arranged in parallel or essentially in parallel and positioned such that the first ends of the hollow fibers are secured in a first holding device and the second ends of the hollow fibers are secured in a second holding device, the first and second holding devices being oriented perpendicular or essentially perpendicular to the longitudinal orientation of the hollow fibers and being orientated parallel or essentially parallel to each other, wherein at least one holding device allows for the flow of fluids to the interior of the hollow fibers, thereby creating a hollow fiber cartridge.

A hollow fiber cartridge typically comprises two compartments: the intracapillary space within the hollow fibers, and the extracapillary space surrounding the hollow fibers. Cells may be seeded and cultured within the extracapillary and/or intracapillary space in a method of the invention. The portion of the extracapillary compartment not filled with cells may be referred to as the “void” space. The hollow fibers of a hollow fiber cartridge may be at a density of from about 40 to about 120 per cm², for example at a density of from about 60 to about 100 per cm², such as at a density of from about 70 to about 90 per cm². A hollow fiber cartridge suitable for use in a method of the invention of the present invention may have an extracapillary space between the hollow fibers and the extracapillary space between the hollow fibers may be from about 25% to about 75% of the total volume of the hollow fiber cartridge, for example from about 40% to about 60% of the total volume of the hollow fiber cartridge.

The hollow fiber cartridge may be designed to be removably inserted into a housing, for example wherein the housing is part of a bioreactor or bioreactor system. Suitable for use in the invention is a hollow fiber cell culture reactor comprising a hollow fiber cell culture cartridge as described herein, a housing sized to hold the hollow fiber cartridge, a cell culture medium source fluidly connected to one or more inlets in the housing, one or more outlets in the housing and, one or more pumps to supply the medium to and/or remove waste medium and/or gases from the hollow fiber cartridge through the medium inlet(s) and/or outlet(s). The inlets may be fluidly connected to the interior of the hollow fibers. The inlets may be fluidly connected to the extracapillary space between the hollow fibers and the outlets may be fluidly connected to the interior of the hollow fibers, thereby creating a fluid flow from the outside to the inside of the hollow fibers. Optionally, the hollow fiber cell culture reactor may comprise an automated controller, for example with the automated controller may comprising a computer.

A method of the invention for culturing cells may comprise; seeding a hollow fiber reactor with one or more of a muscle cell, a fat cell, a cell capable of differentiating into a fat cell or a cell capable of differentiation into a muscle cell at a density of at least 10⁸ cells/ml. That is to say the hollow fiber reactor may be seeded with said cells at a density of at least about 100,000,000 cells/ml, for example at least about 110,000,000 cells/ml, at least about 120,000,000 cells/ml, at least about 130,000,000 cells/ml, at least about 140,000,000 cells/ml, at least about 150,000,000 cells/ml, at least about 160,000,000 cells/ml, at least about 170,000,000 cells/ml, at least about 180,000,000 cells/ml, at least about 190,000,000 cells/ml, such as at least about 200,000,000 cells/ml or a higher density.

Cells may be seeded into a hollow fiber reactor in the form of single cells and/or aggregates. Aggregates may fuse as the culture in the hollow fiber reactor takes place,

A method of the invention may be carried out, wherein the cells cultured in a hollow fiber reactor do not adhere or do not substantially adhere to the hollow fibers. For example, the material of the fibers may be one which has not been treated or formed in any way to promote cell adherence. Alternatively, the cells may be such that they do not adhere or attach or anchor to the hollow fibers. The hollow fiber reactor may be substantially free from anchor points. However, a hollow fiber reactor may comprise one or more fibres comprising one or more anchor points.

The cells may be cultured until achieving from about 80% to about 99% confluency, for example from about 85% to about 99% confluency, such as from about 90% to about 99% confluency. The void space may comprises less than about 90%, such as less than about 80%, such as less than about 70%, such as less than about 60%, such as less than about 50%, such as less than about 40%, such as less than about 30%, such as less than about 20%, such as less than about 10%, such as less than about 5%, confluency or less of the extracapillary space,

A method of the invention may additionally comprise removing the hollow fiber cartridge from the hollow fiber cell culture reactor after the cells have achieved desired confluency. A method of the present invention may comprise removing the first holding device and the second holding device from the first ends and second ends, respectively, of the hollow fibers.

Further, the method of the present invention may additionally comprise seeding (i.e., in addition to a fat cell, a muscle cell or a cell which may be differentiated into such a cell) the hollow fiber reactor with one or more other cells, for example fibroblasts, fibroblast-like cells, engineered cells expressing one or more fibroblast-like characteristics or cells capable of differentiating into such cells.

The method of the present invention may additionally comprise supplying media to the cells through one or both of the first end and second end of the hollow fibers into the interior of the hollow fibers, through the wall of the hollow fibers into the extracapillary space space between the hollow fibers where the cells may be seeded and through one or more of said outlets in said housing. The flow of media may be reversed for at least a portion of the culture method. The method of the present invention may comprise, for example after the cells have achieved confluency, infusing the interior of the hollow fibers and/or any remaining void space (i.e. the portion of the extracapillary space not filled with cells) between the cells with one or more of fats, flavors, colors, salts and preservatives. A substance which aids protein cross-linking, such as a transglutaminase may also be used. Alternatively, such substances may be added following harvesting of cells.

Optionally, a scaffold may be included in a hollow fiber reactor, for example in the extracapillary space.

Tissue engineering

In a further aspect, the invention provides for a use of a pluripotent stem cell as described herein or the fat cells and/or muscle cells obtained by a method as described herein for tissue engineering. Methods for tissue engineering may be in vitro or ex vivo methods. In certain aspect, the use is for the production of cultured meat. In certain aspect, the use is for the production of thick cuts of meat.

In yet a further aspect, the invention provides for a food product (also referred to as “foodstuff’) comprising the pluripotent stem cells as described herein and/or the fat cells and/or muscle cells produced and/or obtained by the method as described. In certain embodiments, the food product further comprises an edible composition for human or non-human consumption.

The edible composition for human or non-human consumption for example comprises at least one of mature fat cells and mature muscle cells. In addition, such an edible composition may comprise one or more further cell types.

In yet a further aspect, the invention provides for a method of producing a food product, the method comprising combining the pluripotent stem cells as described herein or the produced and/or obtained adipocytes with an edible composition for human consumption or non-human consumption as described herein. Such a food product may comprise edible hollow fibers. The food product may be cultured meat or a product comprising cultured meat.

The cultured meat product may be a structured product. Optionally, the product is structured with scaffolding. In one embodiment the structured product is free of scaffolding and the structure is provided by the cells as obtained by the methods as described herein. Differentiated fat cells distributed within co-cultured differentiated muscle cells produced according to a method of the invention may achieve a marbling effect.

One or more additives may be added to the food product, for example plant based proteins or proteins from microbial origin such as yeast proteins. Plant based proteins and yeast proteins suitable for the use in food products are known to the skilled person in the art.

A food product of the present invention may comprise one or more of minerals, synthetic substances, flavors, texture enhancers, nutritional additives, preservatives, and fats. In an aspect of the invention, the flavors are selected from one or more of essential oils, oleoresin (ESO), enzymes (ENZ), natural substances and extractives (NAT), non-nutritive sweetener (NNS), nutritive sweetener (NUTRS), herbs, spices, natural seasonings & flavorings (SP), and synthetic flavors (SY/FL), fumigant (FUM), artificial sweeteners and yeast extract. In another aspect of the present invention, the texture enhancers are selected from one or more of pureed plant material, guar gum, cellulose, hemicellulose, lignin, beta glucans, soy, wheat, maize and rice isolates and beet fiber, pea fiber, bamboo fiber, plant derived fiber, plant derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginate, natural polysaccharides, grain husk, calcium citrate, calcium phosphates, calcium sulfate, magnesium sulfate and salts. In another aspect of the present invention, the nutritional additives are selected from one or more of trace elements, bioactive compounds, endogenous antioxidants, A, B-complex, C, D, E vitamins, zinc, thiamin, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, protein, amino acids salt, creatine, taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoic acid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, Retinol, folic acid, dietary fiber and amino acids. In yet another aspect of the present invention, the fats are selected from one or more of saturated, monounsaturated, polyunsaturated fats, corn oil, canola oil, sunflower oil, safflower oil, olive oil, peanut oil, soybean, flax seed oil, sesame oil, canola oil, avocado oil, seed oils, nut oils, safflower and sunflower oils, palm oil, coconut oil, omega-3, fish oil, lard, butter, processed animal fat, adipose tissue, cellular agriculture derived fat essential oil and oleoresin. In another aspect of the present invention, the preservative and/or antioxidant is selected from one or more of: sodium salt, chloride salt, iodine salt. Nitrates, nitrosamines. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium benzoate, potassium benzoate and benzene ascorbic acid, citric acid, potassium, monosodium glutamate (MSG), sulphur dioxide, sulphites, antibiotics. It is noted here that any one additive, flavor, texture enhancer, nutrient additive, fat/oil and/or preservative/antioxidant may supply more than one attribute to food product of the present invention.

The inventors of present application have surprisingly found that, the cells as obtained by the methods as described herein and the tissue that can be engineered from the methods as described herein can be combined to obtain a large structured product such as a large cultivated meat product. This structured product does not require any scaffolding as the cells or tissue as obtained by the methods as described herein are capable of forming the structure. Even more surprisingly, the inventors were able to forming a thick slice (resembling a piece of meat, such as a steak) of the structured product (having a height of at least about 3 cm) without the use of an edible glue such as transglutaminase. The structured product is capable of being cooked. This cooking did not affect the structure of the product or the height of the product.

Accordingly, the invention provides for a structured product comprising the cell culture as described herein or the tissue that has been engineered by the methods as described herein. In one embodiment the structured product comprises scaffolding. In one embodiment the structured product is scaffold free. In one embodiment the structured product has a height of at least about 3 cm. In one embodiment, the structured product does not substantially shrink when exposed to heat.

Description of the sequences

Table 1 : Sequences

Description of the figures

Figure 1 : EpiSCs-MyoD1 cultured with EpiSCs-PPARy-CEBPa at a ratio of 90:10 after 7 days 3D differentiation in a shaker in suspension (A) and in a scaffold (B). Aggregates were stained for MHC (green) to identify differentiated muscle cells and LipidTOX (red) identifying neutral lipid droplets in differentiated adipocytes.

Figure 2: EpiSCs-MyoD1 cultured in the extra-capillary space of a hollow fiber bioreactor at a density of 10 million/ml. Muscle markers were observed 24-48 hours after differentiation was induced (data not shown). At 4 days of differentiation aggregates; expressing homogenously expressing muscle markers (MHC (green) and titin (red))(A), fused (B,C) forming a functional tissue, demonstrated by twitching muscle tissue (not shown). After 8 days of differentiation the harvested tissue homogenously expressed mature muscle markers (MHC (green) and titin (red)) with elongated striated muscle fibers (titin (red), arrows) (Fig. 2D, 2E), and long (300 urn) multinucleated fibers found throughout the tissue (Fig. 2F, 2G). At cell inoculation densities of 100-300 million/ml, macroscale muscle tissue was formed after aggregates fused forming a complete piece of tissue which was indued to differentiate into functional muscle (Fig. 2H).

Figure 3: EpiSCs-PPARy-CEBPa cultured in the extra-capillary space of a hollow fiber bioreactor at a density of 10 million/ml (A). Adipose differentiation was observed in the first 4-48 hours (not shown). The lipid accumulation, demonstrated by the presence of Lipid droplets; BODIPY (green), increased daily (B day 3), (C day 5), (D, day 10). Differentiated fat tissue (E) was also observed to float due to volume of lipid accumulation (F).

Figure 4: EpiSCs-MyoD1 cultured with EpiSCs-PPARy-CEBPa at a ratio of 90:10 in the extracapillary space of a hollow fiber bioreactor for 7 days at a density of 100-300 million/ml (A). Both cell lines were differentiated together using the same media recipe and inducer of differentiation. Aggregates fuse together to form a combination of tissue slurry and fibers (B,C). Cells self-organise during differentiation into muscle fibers (MHC, green) and lipid accumulating adipose cells (LipidTOX, red) (D).

Figure 5: EpiSCs-PPARy-CEBPa cultured at 1 billion/ml (A) in the extra-capillary space of a hollow fiber bioreactor all cells in culture efficiently differentiate (2B) and no necrosis observed (C, cross sections. Nuclei (blue), neutral lipids green (BODIPY) or red (LipidTOX)). Aggregates fuse to form thick slurry of fat tissue (D).

Figure 6: EpiSCs-MyoD1 cultured with EpiSCs-PPARy-CEBPa at a ratio of 90:10 cultured at 1 billion/ml. All extra-capillary space was filled with cells with no void space or necrotic core (Anuclei (blue)). Both cell lines were differentiated together using the same media recipe and inducer of differentiation. Longitudinal cross section demonstrates aggregates fusing into complete tissue with no void space left in the extra-capillary space outside hollow fibers (B). Moreover, intra-capillary spaces were filled at the end of culture with differentiated cultivated fat tissue (B, circle area). Tissue with hollow fibers at the end of culture dehydrated (using sucrose) and frozen then extracted from reactor cartridge housing as one solid tissue piece (C).

Figure 7: Fat tissue, muscle tissue or co-culture of muscle and fat tissue was harvested from hollow fiber bioreactor. 1 % Beetroot powder was added to muscle tissue and fat tissue was left uncoloured. Muscle and fat tissue were lightly blended into 3D printed molds. Due to the light mixing technique this allowed for marbling of fat between the coloured muscle tissue. (A-C) 5% Transglutaminase was added to some molds (A) otherwise molds were filled with harvested tissue only (Fig.7D ). Products were left in the fridge for 24-48 hours. Whole cuts were then removed from the molds (A-C) and on average cooked at 210°C for 5-10 minutes (fig.7D).

Figure 8: Harvested tissue from 3mL working volume (EpiSCs-PPARy-CEBPa derived adiposetissue, or co-culture; EpiSCs-MyoD1 + EpiSCs-PPARy-CEBPa at a ratio of 90:10 at several different media volumes (l OOOmL (co-culture), 400mL and 200mL (EpiSCs-PPARy-CEBPa)).

Examples

The present invention is further illustrated by the following Examples which should not be construed as limiting the scope of the invention.

Materials and Methods

Porcine Epiblast-derived Stem Cell (pEpiSCs) differentiated towards muscle, adipose or co-culture inside hollow fiber bioreactors

Undifferentiated pEpiSCs with EpiSC-MyoD I or co-culture of EpiSCs-MyoD with EpiSCs- PPARG-CEPBA were inoculated at a density of 10-1000 million/ml in the extra-capillary (EC) space of the hollow fiber bioreactor cartridge. For co-culture experiments, cells were inoculated at a ratio 90:10 muscle : fat. For cell densities less than 500 million/ml, cells were first cultured for 2-5 days further in proliferation media inside the hollow fiber bioreactor. Proliferation media is described in patent application nos. PCT/EP2024/053897 and PCT/EP2024/053906 both of which are incorporated herein by reference. Next, media was switched to differentiation media. Differentiation media is described in patent application nos. PCT/EP2024/053897 and PCT/EP2024/053906 both of which are incorporated herein by reference. Next, media was switched to differentiation media. Differentiation medium was additionally supplemented with 1 ug doxycycline to activate Opti-ox system.

EpiSCs-PPARG-CEPBA cell densities less than 500 million/ml were cultured for 2 days in proliferation media (proliferation media is described in W02024/084082 which is incorporated herein by reference). Media was switched to differentiation media for 7-10 days to induce adipogenesis (differentiation media is described in W02024/084082 which is incorporated herein by reference). Differentiation medium was additionally supplemented with 1 ug doxycycline to activate Opti-ox system. Hollow fiber bioreactor cartridges (FiberCell/KD Bio C 7025, C 7011 ).

Porcine Epiblast-derived Stem Cell (pEpiSCs) co-culture differentiated towards muscle and adipose in suspension.

Undifferentiated EpiSC-MyoD I or co-culture of EpiSCs-MyoD with EpiSCs-PPARG-CEPBA were inoculated at a density of 8-1000 million cells/ml at a ratio 90:10 muscle : fat. Cells were cultured in differentiation medium for 7-8 days (differentiation media is described in W02024/084082 and patent application nos. PCT/EP2024/053897 and PCT/EP2024/053906 both of which are incorporated herein by reference). Differentiation medium was additionally supplemented with 1 ug doxycycline to activate Opti-ox system.

IF staining

Cells were fixed for 30 minutes using 4% PFA then washed 3 times with PBS. Blocking with 3% BSA, 0,1 % Tween and 0.1 % Triton-X100 in PBS for 1 hour with gentle shacking. Primary antibodies for MHC and Titin were incubated in 1 % BSA, 0,1 % Tween and 0.1 % Triton-X100 1 :50 for 2 hours at room temperature with gentle shaking. Samples washed 3 times with PBS then secondary antibodies or LipidTOX stain for 1 hour at room temperature with gentle shaking. Nuclei were stained with DAPI (10 mins in PBS). For lipid staining BODIPY was added to live cells or post fixation using PFA, for 10 mins and washed 3 times with PBS. Images were taken using the Andor BC43 confocal or EVOS M7000 microscope.

Cryosectioning

Increasing concentrations of sucrose solutions (10%, 20%, 30%) were perfused through the fibers to dehydrate the tissue over 24 hours. Samples were frozen using liquid nitrogen, pushed out of cartridge housing, rolled in mounting media and to cut cryosection of 20 urn.

Porcine Epiblast-derived Stem Cell (pEpiSCs) differentiated towards muscle, adipose or co-culture inside hollow fiber bioreactors using reduced media volumes

Undifferentiated pEpiSCs, EpiSCs-PPARG-CEPBA I or co-culture of EpiSCs-MyoD with EpiSCs- PPARG-CEPBA were inoculated at a density of 1000 million/ml in the extra-capillary (EC) space of the hollow fiber bioreactor cartridge (FiberCell/KD Bio C7025). For co-culture experiments, cells were inoculated at a ratio 90:10 muscle : fat. Cells were cultured with circulating differentiation media for 7 consecutive days. Differentiation medium was additionally supplemented with 0.5 pg/mL doxycycline on the inoculation day to activate the Opti-Ox system. Each HFB working volume was 3 ml and attached to an external media supply for continuous circulation through the fibers. This external media supply was 100 ml, 400ml or 200 ml. No media refreshments nor additional supplementations were added over the course of the differentiation period. After 7 days, the tissue was harvested by breaking open the hollow fiber cartridge and collecting the cellular material in a pre-weighed tube. The obtained cell pellet was washed with PBS. The harvest was centrifuged for 5 minutes at 500 g and excess supernatant was removed completely. The pellet was weighed with a precision scale, resulting in the wet weight harvest results expressed in grams.

Results

Example 1: Development of an Inducible Transgene Overexpression Myogenic Reprogramming Method by Dual Genomic Safe Harbor (GSH) Targeting in Animal Cells

The development generation of EpiSCs-MYOD cell lines and EpiSCs-MYOD-MYOG and EpiSCs- MYOD-PAX7 (pluripotent stem cell lines expressing MYOD, MYOD + MYOG and MYOD + PAX7 using the opti-ox™ approach) has been described in patent application nos. PCT/EP2024/053897 and PCT/EP2024/053906 both of which are incorporated herein by reference.

Example 2: Development of an Inducible Transgene Overexpression Adipogenic Reprogramming Method by Dual Genomic Safe Harbor (GSH) Targeting in Animal Cells

The generation of EpiSCs-CEBPa-PPARy cell lines (pluripotent stem cell lines expressing CEBPa- PPARy using the opti-ox™ approach) has been described in W02024/084082.

Example 3: 3D Co-Culture of muscle (EpiSCs-MyoD) and fat cells (EpiSCs-PPARG-CEPBA))

To examine if both fat and muscle cells could be grown as a co-culture, EpiSCs-MyoD and EpiSCs- PPARG-CEPBA single cells were added to a shaker flasks at a ratio of 90:10 muscle:fat. Simultaneous differentiation of both cell lines was induced using one inducer (doxycycline) and differentiation of both cell lines to muscle and fat cells occurred at the same time with the same media condition. The cells self-organized within the aggregates and after 7 days aggregates were positive for muscle and fat cells demonstrated by elongated MHC positive muscle fibers (green) and lipid droplets accumulating inside adipocytes (LipidTOX; red) (Fig. 1A). Samples from suspension culture (shaker flask, not shown) and hollow fiber reactor (Fig. 2A) start expressing muscle markers 4 days after differentiation is induced.

To examine if cells could also differentiate simultaneously in a scaffold, EpiSCs-MyoD and EpiSCs- PPARG-CEPBA single cells were mixed with fibrinogen at a ratio of 90:10 muscle:fat and cultured for 5-8 days. Muscle and fat were also observed here to self-organize into muscle fibers and lipid rich adipocytes, with adipose cells self-organising into intra-muscular localisation (Fig. 1 B).

Example 4: 3D culture of EpiSCs-MyoD in hollow fiber reactors

To examine if EpiSCs-MyoD cells could be cultured in hollow fibers, 10-1000 million cells/ml were inoculated into the extra-capillary space. For inoculation densities less than 1000 million/ml cells were cultured for 2 further days in proliferation medium. This process is successful with a starting cell culture of single cells or aggregates. After 2 days of proliferation an increase in total amount of cell mass was observed (data not shown), indicating capability of cells to continue to proliferate in the hollow fiber reactor. Subsequently, the media was switched to differentiation media for 7-8 days. During culture, it was observed that aggregates fused (Fig. 2B, 2C). Twitching of single and fused aggregates was observed indicating muscle functionality (not shown). Cells within the aggregates started to differentiate and expressed muscle cell markers like MHC (green) and Titin (red) in early days of differentiation (Fig. 2A, day 4). After 7-8 days of differentiation, the cells matured further (Fig. 2D), forming aligned myotubes with clear z-striation pattern (Fig. 2E, white arrows). Fibers were around 200-300 urn in length and multinucleation was observed (Fig. 2F).

It was observed that cells only loosely attached to the fibers and could detach without the need for enzymatic reactions. Instead, the aggregates fuse with each other (Fig. 2B, 2C, 2G, 2H) forming a structured piece of tissue. The constant perfusion of media ensured no necrosis occurred at the core of the tissue and homogeneous differentiation observed throughout the tissue.

Example 5: 3D culture of EpiSCs-PPARG-CEPBA in hollow fiber reactors

To examine if EpiSCs-PPARG-CEPBA cells could be cultured in hollow fibers, 10-1000 million cells/ml were inoculated into the extra-capillary space (Fig.3A). For inoculation densities less than 1000 million/ml cells were cultured for 2 further days in proliferation medium. This process is successful with a starting cell culture of single cells or aggregates. After 2 days of proliferation aggregates formed and an increase in total amount of cell mass was observed (data not shown), indicating proliferation of EpiSCs-PPARG-CEPBA cells in hollow fiber reactor with uniform aggregate size observed along hollow fibers (Fig. 3A). Subsequently, the media was switched to differentiation media for 5-10 days. The constant perfusion of media ensured no necrosis occurred at the core of the tissue and homogeneous differentiation observed throughout the tissue. Cells only loosely attached to the fibers and could detach without the need for enzymatic reactions to easily harvest tissue (Fig. 3E). During the differentiation, cells within the aggregates accumulated lipids demonstrated with BODPIY positive lipid droplets (Fig. 3.B-D), expression of mature adipocyte markers (not shown) and many of the cells floated (Fig. 3F).

Example 6: 3D co-culture of EpiSCs-PPARG-CEPBA and EpiSCs-MyoD in hollow fiber reactors To examine if EpiSCs-PPARG-CEPBA and EpiSCs-MyoD cells could be co-cultured in hollow fiber reactors, 10-1000 million cells/ml were inoculated into the extra-capillary space at a ratio of 90:10. For inoculation densities less than 1000 million/ml cells were cultured for 2 further days in proliferation medium. Media was changed to differentiation media for 6-8 days. Both EpiSCs-MyoD and EpiSCs-PPARG-CEPBA cells differentiated simultaneously using one media recipe and one inducer of differentiation (doxycycline). (Fig. 4A). When harvested, tissue formed a complex slurry (Fig. 4B) comprised of many tissue fibers (Fig. 4C). Cells self-organised within the tissue with LipidTOX (red) staining demonstrates presence of intramuscular adipose lipid rich adipose cells and MHC (green) identifying elongated fibers (Fig. 4D, white arrow showing striation). Exam pie 7 : High cell density culture of cells in hollow fiber reactors for structured meat product To examine if cells could also be cultured at high densities required for thick cut products EpiSCs- PPARG-CEPBA were inoculated at a density of 1 billion cells/ml. Fig.5A demonstrates that all extracapillary space is utilised by cell mass leaving little to no void space. The hollow fibers supplied sufficient perfusion of media/extraction of waste to ensure no necrosis was observed and homogenous differentiation of fat tissue (Fig. 5B). Cryosections of the sample demonstrated confluent cell mass in extra-capillary space with differentiation of adipose tissue activated throughout the whole cell mass (Fig.5C) with unilocular lipid droplets observed (white arrows). Harvested tissue of thick slurry of fat (Fig.5D).

Upon culture of EpiSCs-PPARG-CEPBA and EpiSCs-MyoD at a density of 1 billion cells/ml and ratio of 90:10. Fig. 6A demonstrates all extra-capillary space is filled with viable cell mass. At the end of culture, the intra-capillary space of the hollow fiber reactor was filled with differentiated tissue (Fig. 6B, red circle) which gives the complexity of marbling required for thick meat cuts.

Dehydrating the sample then freezing resulted in the tissue easily removed from housing holding together as one structure (Fig. 6C).

Example 8: Production of a structured meat product

Harvested tissue was combined into a 3D printed mold without any scaffold or post processing components . Optionally, 5% transglutaminase was added to the product to increase mass and give a different texture. Muscle tissue was dyed using food grade colours such as beetroot powder, fat tissue left un-coloured (Fig.7A). When fat tissue was added using a “marble cake” technique this resulted in fat marbling throughout the tissue. Furthermore, when using muscle tissue that was cocultured with fat the cooked product visibly demonstrated intramuscular fat (Fig.7B). After 24-48 hours in the fridge the product was free standing without the mold with ( Fig.7A) or without ( Fig. 7C) the addition of transglutaminase. This was replicated using larger molds with a height of at least 3cm and the tissue could here also be free standing without any scaffold or transglutaminase with similar results observed with the addition of transglutaminase . This structure was maintained after cooking with minimum shrinkage observed (Fig. 7D).

Example 9: Cell media requirements can be reduced up to 25% independent of cell source and type without affecting the final yield .

To examine if differentiated pEpiSCs can be efficiently cultured inside hollow fiber bioreactors at high densities (1 billion/mL) in reduced media volumes without compromising yield, EpiSCs-PPARy- CEBPa derived adipose- tissue, or co-culture; EpiSCs-MyoD1 + EpiSCs-PPARy-CEBPa at a ratio of 90:10 were cultured in 100 ml, 400ml or 200 ml in the extra-capillary space of a hollow fibre reactor. No media refreshments nor additional supplementations were added over the course of the differentiation period. After 7 days, the tissue was harvested by breaking open the hollow fiber cartridge and collecting the cellular material in a pre-weighed tube. As can be seen in Figure 8, the so cultured pEpiSCs have reproducible harvest weights independent of the supplemented media volume. Example 10: Culture of EpiSCs-PPARG-CEPBA and EpiSCs-MYOD-MYG

The above Examples were repeated with EpiSCs expressing a combination of MYOD and MYOG. It was shown that such cells could be co-cultured and co-differentiated in 3D together with EpiSCs- PPARG-CEPBA in hollow fiber reactors at comparably high densities and conditions as those obtained with EpiSCs expressing only MYOD.

Statements (features) and embodiments of the methods and compositions as disclosed herein are set out below. Each of the statements and embodiments as disclosed by the invention so defined may be combined with any other statement and/or embodiment unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Embodiments

The present invention provides at least the following numbered statements/embodiments:

1 . A method for culturing cells, which method comprises culturing in the same medium: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

2. A method for culturing cells, which method comprises culturing in the same medium or different media: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell, wherein differentiation of the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell is inducible by the same molecule.

3. A method according to embodiment 1 or 2, wherein the culturing is carried out in a hollow fiber reactor.

4. A method according to any one of the preceding embodiments, wherein the method comprises differentiating the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and differentiating the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell. 5. A method according to any one of the preceding embodiments, wherein the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell are each a pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a protein involved in cell differentiation, wherein the coding sequence for the protein involved in cell differentiation is operably linked to an inducible promoter; and, optionally, iia) an expression construct for expression of a second protein involved in cell differentiation, wherein the coding sequence for the second protein involved in cell differentiation is operably linked to an inducible promoter; wherein the expression constructs of ii) and, optionally, iia) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.

6. A method according to embodiment 5, wherein the at one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell is capable of expressing one or more of a MYOD protein, a MYOG protein and a PAX7 protein and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell is capable of expressing one or both of a PPAR-y protein and a CEPBa protein.

7. A method for culturing cells, which method comprises culturing cells in a hollow fiber reactor, wherein: the density of the cell culture is at least about 1 billion cells/ml; and/or the culturing of cells in the hollow fiber reactor is carried out substantially in the absence of an anchor point for the cells; and/or the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intra-capillary space of the hollow fiber reactor.

8. A method according to embodiment 7, wherein the method of is for culturing: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and/or (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

9. A method for culturing cells, which method comprises culturing in a hollow fiber reactor: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and/or (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell. A method according to any one of the preceding embodiments, wherein differentiation of the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell is inducible by the same molecule. A method according to embodiment 10, wherein the density of the cell culture is at least about 1 billion cells/ml. A method according to any one of embodiments 7 to 11 , wherein the method comprises differentiating the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and differentiating the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell. A method according to any one of the preceding embodiments, wherein the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell are each a pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a protein involved in cell differentiation, wherein the coding sequence for the protein involved in cell differentiation is operably linked to an inducible promoter; and, optionally, iia) an expression construct for expression of a second protein involved in cell differentiation, wherein the coding sequence for the second protein involved in cell differentiation is operably linked to an inducible promoter; wherein the expression constructs of ii) and, optionally, iia) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein. A method according to embodiment 13, wherein the at one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell is capable of expressing one or more of a MYOD protein, a MYOG protein and a PAX7 protein and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell is capable of expressing one or both of a PPAR-y protein and a CEPBa protein A method according to any one of embodiments 3 to 14, wherein the culturing of cells in the hollow fiber reactor is carried out substantially in the absence of an anchor point for the cells. 16. A method according to any one of embodiments 3 to 15, wherein the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intra-capillary space of the hollow fiber reactor.

17. A method according to any one of embodiments 3 to 16, wherein the cells are cultured in the extra-capillary space of the hollow fiber reactor or the intra-capillary space of the hollow fiber reactor.

18. A method according to any one of the preceding embodiments, wherein the cells produced by the method are in the form of a tissue.

19. A method according to any one of the preceding embodiments, wherein the cells produced by the method are suitable for human and non-human dietary consumption.

20. A cell culture or a tissue obtainable by a method according to any one of the preceding embodiments.

21 . A cell culture which comprises: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

22. A cell culture according to embodiment 21 , wherein differentiation of the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell are inducible by the same molecule.

23. A cell culture comprising cells in a hollow fiber reactor, wherein: the density of the cell culture is at least 1 billion cells/ml; and/or the cell culture comprises substantially no anchor point for the cells; and/or the cells are present in the extra-capillary space of the hollow fiber reactor and/or the intra-capillary space of the hollow fiber reactor.

24. A hollow fiber reactor comprising a cell culture according to any one of embodiments 20 to 23.

25. Use of a cell culture according to any one of embodiments 20 to 23 or use of the method according to any one of embodiments 1 to 19 fortissue engineering, optionally for the production of cultured meat. 26. A food product comprising a least two cell types obtained from the cell culture according to any of embodiments 20 to 23 or use of the method according to any one of embodiments 1 to 19. 27. A food product according to embodiment 26, wherein the food product is cultured meat.

28. A structured product, optionally a scaffold free structured product, comprising the cell culture according to any of embodiments 20 to 23.

Claims

3. A method according to claim 1 or 2, wherein the culturing is carried out in a hollow fiber reactor.

4. A method according to any one of the preceding claims, wherein the method comprises differentiating the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and differentiating the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

5. A method according to any one of the preceding claims, wherein the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell are each a pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a protein involved in cell differentiation, wherein the coding sequence for the protein involved in cell differentiation is operably linked to an inducible promoter; and, optionally, iia) an expression construct for expression of a second protein involved in cell differentiation, wherein the coding sequence for the second protein involved in cell differentiation is operably linked to an inducible promoter; wherein the expression constructs of ii) and, optionally, iia) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.

6. A method according to claim 5, wherein the at one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell is capable of expressing one or more of a MYOD protein, a MYOG protein and a PAX7 protein and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell is capable of expressing one or both of a PPAR-y protein and a CEPBa protein.

8. A method according to claim 7, wherein the method of is for culturing: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and/or (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

9. A method for culturing cells, which method comprises culturing in a hollow fiber reactor: (i) at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell; and/or (ii) at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

10. A method according to any one of the preceding claims, wherein differentiation of the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell is inducible by the same molecule.

11. A method according to claim 10, wherein the density of the cell culture is at least about 1 billion cells/ml.

12. A method according to any one of claims 7 to 11 , wherein the method comprises differentiating the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and differentiating the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell.

13. A method according to any one of the preceding claims, wherein the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell are each a pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; ii) an expression construct for expression of a protein involved in cell differentiation, wherein the coding sequence for the protein involved in cell differentiation is operably linked to an inducible promoter; and, optionally, iia) an expression construct for expression of a second protein involved in cell differentiation, wherein the coding sequence for the second protein involved in cell differentiation is operably linked to an inducible promoter; wherein the expression constructs of ii) and, optionally, iia) are inserted into at least one further genetic safe harbour site that is not the first genetic safe harbour site, and wherein the inducible promotor is regulated by the transcriptional regulator protein.

14. A method according to claim 13, wherein the at one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell is capable of expressing one or more of a MYOD protein, a MYOG protein and a PAX7 protein and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell is capable of expressing one or both of a PPAR-y protein and a CEPBa protein

15. A method according to any one of claims 3 to 14, wherein the culturing of cells in the hollow fiber reactor is carried out substantially in the absence of an anchor point for the cells.

16. A method according to any one of claims 3 to 15, wherein the cells are cultured in the extra-capillary space of the hollow fiber reactor and/or the intra-capillary space of the hollow fiber reactor.

17. A method according to any one of claims 3 to 16, wherein the cells are cultured in the extra-capillary space of the hollow fiber reactor or the intra-capillary space of the hollow fiber reactor.

18. A method according to any one of the preceding claims, wherein the cells produced by the method are in the form of a tissue.

19. A method according to any one of the preceding claims, wherein the cells produced by the method are suitable for human and non-human dietary consumption.

20. A cell culture or a tissue obtainable by a method according to any one of the preceding claims.

22. A cell culture according to claim 21 , wherein differentiation of the at least one cell which is a muscle cell or a cell capable of being differentiated into a muscle cell and the at least one cell which is a fat cell or a cell capable of being differentiated into a fat cell are inducible by the same molecule.

24. A hollow fiber reactor comprising a cell culture according to any one of claims 20 to 23.

25. Use of a cell culture according to any one of claims 20 to 23 or use of the method according to any one of claims 1 to 19 fortissue engineering, optionally for the production of cultured meat.

26. A food product comprising a least two cell types obtained from the cell culture according to any of claims 20 to 23 or use of the method according to any one of claims 1 to 19.

27. A food product according to claim 26, wherein the food product is cultured meat.

28. A structured product, optionally a scaffold free structured product, comprising the cell culture according to any of claims 20 to 23.