WO2025238202A1 - Production de cryonoïdes et de cellules sanguines issues de ceux-ci - Google Patents

Production de cryonoïdes et de cellules sanguines issues de ceux-ci

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Publication number
WO2025238202A1
WO2025238202A1 PCT/EP2025/063521 EP2025063521W WO2025238202A1 WO 2025238202 A1 WO2025238202 A1 WO 2025238202A1 EP 2025063521 W EP2025063521 W EP 2025063521W WO 2025238202 A1 WO2025238202 A1 WO 2025238202A1
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Prior art keywords
cells
cryonoids
hemanoids
hematopoietic
cryonoid
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Inventor
Nico LACHMANN
Marco António CARVALHO OLIVEIRA
Edwin Emilio VALDIVIA MALQUI
Charis-Patricia SEGERITZ
Marion Lydie Sophie CORNU
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Medizinische Hochschule Hannover
Novo Nordisk AS
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Medizinische Hochschule Hannover
Novo Nordisk AS
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Publication of WO2025238202A1 publication Critical patent/WO2025238202A1/fr
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/16—Physical preservation processes
    • A01N1/162—Temperature processes, e.g. following predefined temperature changes over time
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
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    • C12N2523/00—Culture process characterised by temperature

Definitions

  • the present invention relates to the production of particular cells, such as blood and immune cells, from cryopreserved organoids derived from pluripotent stem cells, and methods for preparing and recovering such cryopreserved organoids and compositions comprising same.
  • iPSC Human induced pluripotent stem cells
  • somatic cells derived thereof have been introduced for a variety of therapeutic and non-therapeutic applications.
  • Human immune cells have in the recent years proven to be especially promising for future therapies and drug applications.
  • iPSC-derived immune cells in particular platelets, T cells, NK cells and macrophages, have been shown to be of particular therapeutic interest and pave the way for next generation cell-based immunotherapies.
  • a general problem with the current cell-based immunotherapies is to obtain the suitable number of high-quality cells needed for treatment or diagnostic purposes.
  • iPSCs which are thawed when needed and subsequently cultured such as to produce the intended cell type, using for instance well established hematopoietic differentiation protocols using different cytokines and growth factors.
  • the currently available processes for producing iPSC derived cells face multiple limitations, such as complex culturing schemes, difficult distribution, and prohibition on work with iPSC in some countries, which hampers the more general use and availability of e.g., iPSC-based cell-based immunotherapy.
  • immune cells can also be produced from iPSC derived cell aggregates also described as hematopoietic organoids or hemanoids, which are not pluripotent, but have the ability to continuously produce different types of cells.
  • iPSC derived cell aggregates also described as hematopoietic organoids or hemanoids, which are not pluripotent, but have the ability to continuously produce different types of cells.
  • the ability of hemanoids to produce different cell types relies on the secretion of particular paracrine hormones within the hemanoid, which enables the aggregated cells to differentiate and produce particular cell types on-demand depending also on addition of particular factors in the culturing media.
  • hemanoids contain hematopoietic stem and progenitor cells as well as a unique structure, which support their functionality. Hematopoietic organoids may also be denoted for example but not limited to bone marrow organoids, blood organoids and hemanoids.
  • cryopreservation often destroys the intricate 3D architecture of the aggregates, such that the particular function of the cell aggregates and organoids is lost after cryopreservation.
  • methods that enable the long-term storage, transport and thawing of cellular aggregates, such as hemanoids are needed.
  • cryonoids frozen hemanoids
  • cryonoids may be long term stored and transported, thawed and used for production of various cells on demand, such as human immune cells.
  • This enables the continuous production of highly specialized immune cells directly from the thawed cryonoids for long periods of time, without the process of production of the desired cells needing to be restarted for every use, greatly simplifying the process for manufacturing e.g., immune cells needed for treatment.
  • a first aspect of the present disclosure relates to a method for cryopreserving a hematopoietic organoid comprising, a) obtaining a hematopoietic organoid in a suitable cryopreservation media, freezing said organoid to a temperature of from -40°C to -196°C to obtain a cryonoid, and optionally, storing said cryonoid in liquid nitrogen (LN2).
  • LN2 liquid nitrogen
  • less than 5% of the cells of the hematopoietic organoid are TRA-1-60 + , more preferably the hematopoietic organoid is TRA-1-60’.
  • TRA-1-60 + cells pluripotent cells
  • the process described herein overcomes these challenges, and enables that hematopoietic organoid in the form of cryonoids can be shipped around the world.
  • the absence of TRA-1-60 + cells simplifies downstream analysis/characterization of a product comprising said cryonoids devoid of TRA-1-60 + cells and makes for a simpler product release.
  • a second aspect of the present disclosure relates to a method for producing mammalian blood cells, wherein said method comprises; a) thawing a cryonoid as defined herein, culturing said cryonoid in a suitable medium, b) adding to said medium one or more stimulating agent(s) to promote production of mammalian blood cells, preferably immune cells, and c) optionally, harvesting said mammalian blood cells, preferably immune cells.
  • the mammalian is a human.
  • a third aspect of the disclosure relates to a composition
  • a composition comprising a cryonoid as defined herein, and a suitable medium.
  • the storage medium comprises DMSO, such as about 1-20% DMSO, or such as about 10% DMSO.
  • the medium preferably also comprises trehalose, such as in the range of 0.1-1 M trehalose, or such as about 0.5M trehalose.
  • the storage medium also comprises a ROCK inhibitor, such as e.g., Y-27632.
  • a fourth aspect of the disclosure relates to a cryonoid as defined herein, or a composition as defined herein, for use in the manufacturing of one or more mammalian blood cells, preferably one, or more human immune cells.
  • a fifth aspect of the disclosure relates to a mammalian blood cell obtainable from a method as disclosed herein.
  • FIGURE 1 A first figure.
  • Continuous iPSC-derived macrophage production from hemanoids A) Overview of the differentiation plan with the corresponding individual steps.
  • G FACS data showing the percentage of positive cells for CD34, CD144 and CD309 on day 10.
  • H FACS data showing induced pluripotent stem cells derived macrophages (iMAC) phenotype (CD45, CD11b, CD14 and CD163) at 1 st harvest.
  • I Cytospin picture demonstrating a mature iPSC-derived macrophage on the 1 st harvest.
  • J FACS data showing iMAC phenotype (CD45, CD11b, CD14 and CD163 expression markers) on the 2 nd harvest.
  • K Cytospin picture demonstrating a mature iPSC-derived macrophage on day 38 of differentiation (i.e. the 2 nd harvest).
  • Continuous iPSC-derived macrophage production from cryonoids cryopreserved on day 7 of Aggregation & Mesoderm priming step A) Representative microscopic picture from hemanoids on day 7 before cryopreservation. Arrows indicate primed hemanoids.
  • E FACS data showing the expression of macrophage's characteristic markers (CD45, CD11b, CD14 and CD163) from 1 st harvest.
  • F Representative microscopic picture of a Macrophage from 1 st harvest.
  • G FACS data showing the expression of macrophage's characteristic markers (CD45, CD11b, CD14 and CD163) from 2 nd harvest.
  • H Representative microscopic picture of a Macrophage from 2 nd harvest.
  • iPSC-derived macrophages Production of iPSC-derived macrophages from cryonoids cryopreserved on day 4 of Aggregation & Mesoderm priming step.
  • E Macrophage output per cryonoid over several harvests.
  • F FACS data showing the expression of macrophage's characteristic markers (CD45, CD11b, CD14 and CD163) from 1 st harvest.
  • G Representative microscopic picture of a Macrophage from 1 st harvest.
  • H FACS data showing the expression of macrophage's characteristic markers (CD45, CD11b, CD14 and CD163) from 2 nd harvest.
  • I Representative microscopic picture of a Macrophage from 2 nd harvest.
  • iPSC-derived macrophages Production of iPSC-derived macrophages from cryonoids cryopreserved on day 10 of Hematopoietic specification/cell production step.
  • F Representative microscopic picture of a Macrophage from 1 st harvest.
  • G FACS data showing the expression of macrophage's characteristic markers (CD45, CD11b, CD14 and CD163) from 2 nd harvest.
  • H Representative microscopic picture of a Macrophage from 2 nd harvest.
  • I FACS data showing the expression of macrophage's characteristic markers (CD45, CD11b, CD14 and CD163) from 3 rd harvest.
  • H Representative microscopic picture of a Macrophage from 3 rd harvest.
  • cryonoids capacity to produce different iPSC-derived immune cells A) Representative microscopic picture of a morphologically intact cryonoid. Arrows indicate intact cyronoids.
  • FIGURE 9 Graphical illustration of the different conditions used for cryopreservation of hematopoietic organoids at a fixed freezing rate. O/N means Over Night, which means 12 to 16 hours. FIGURE 9
  • A Size distribution of hematopoietic organoids and post-thawed cryonoids prepared by fixed freezing rate (1°C/min)
  • B Images of hematopoietic organoids or post-thawed cryonoids prepared by fixed freezing rate with different pre-cryopreservation incubation temperatures.
  • A Percentage of live cells present before or after controlled freezing cryopreservation
  • B Percentage of CD309 + TRA1-60 _ cells before or after cryopreservation following differential controlled freezing cryopreservation
  • C Percentage of CD309+TRA1-60' cells relative to CD34 + CD144 + cells
  • D Continuous harvest of macrophages from cryonoids following thawing from hematopoietic organoids or cryonoids.
  • A Size distribution of hematopoietic organoids and post-thawed cryonoids prepared by differential controlled freezing cryopreservation
  • B Images of hematopoietic organoids or post-thawed cryonoids prepared by differential controlled freezing cryopreservation with different initial freezing rates.
  • FIG. 1 Images depict CD34, CD144, and CD309 expression in Hemanoids and Cryonoids, detected by immunohistochemistry. Marker expression is observed with no significant differences between Hemanoids and Cryonoids. Bar plots show quantification of (B) CD34, (C) CD144, and (D) CD309 expression detected by immunohistochemistry.
  • DDO refers to the day on which Cryonoids were thawed. For Hemanoids, it represents the day the medium was changed from differentiation medium to production medium. Both Cryonoids and Hemanoids maintain their integrity during the production phase.
  • iMacs induced macrophages
  • hematopoietic organoids herein also denoted hemanoids
  • cryopreservation method that following cryopreservation maintain their function of producing hematopoietic derivable cells, in particular, myeloid cells and/or lymphoid cells in large quantities, preferably, allowing for continuous production over long periods of time, such as 1-4 months.
  • Hematopoietic organoids are three- dimensional cell aggregates derived from pluripotent stem cells (PSC), preferably induced pluripotent cells (iPSCs), which are typically of roundish morphology and a diameter size, which ranges from approx. 100 pm up to 500 pm, and which typically comprise immature hematopoietic cells and cells of the myeloid lineage. Hemanoids are also positive for the formation of hemogenic endothelium (HE).
  • PSC pluripotent stem cells
  • iPSCs induced pluripotent cells
  • HE hemogenic endothelium
  • HE hemogenic endothelium
  • CD34/CD144 hematopoietic progenitor markers
  • the hemanoids may also be positive for other cells, e.g. endothelial cells are present and express markers such as CD309.
  • cryopreservation of organoids is generally well known, however, when it comes to specific iPSC or ESC derived organoids, cryopreservation has shown to be difficult and often without success, due to damages to the organoids which disables the functionality of the organoid following thawing.
  • the inventors have herein shown the cryopreservation of particularly hematopoietic organoids is possible, without damaging the intricate 3D structure needed for the hematopoietic organoid to remain functional following cryopreservation.
  • the present invention relates to a method for cryopreserving a hematopoietic organoid comprising, a) obtaining a hematopoietic organoid in a suitable cryopreservation media, b) freezing said hematopoietic organoid to a temperature of from - 40°C to -196°C to obtain a cryonoid, and c) optionally, storing said cryonoid in liquid nitrogen (LN2).
  • LN2 liquid nitrogen
  • cryonoid intends to describe a hematopoetic organoid (also called hemanoid) which has been subjected to cryopreservation (see also cryopreservation method as described herein).
  • cryonoid term is intended to also encompass a cryonoid which has been thawn, in order to distinguish this type of cell aggregate from hemanoids which have never been subjected to cryopreservation.
  • Hemanoids can be formed from adult human stem cells, human multipotent stem cells, hematopoietic progenitor cells, iPSCs or ESCs.
  • the hemanoids are formed from induced pluripotent stem cells (iPSCs), obtained by e.g., somatic cell nuclear transfer or the reprogramming of somatic cells to yield iPSC.
  • pluripotent cell types from which hemanoids may be derived from include embryonic stem cells (ESCs) derived from the blastocyst stage of embryos, e.g., from mouse (mESC), primate, and human (hESC) sources. Methods for generating hemanoids are well known in the art.
  • hemanoids used for providing cryonoids are preferably not derived from stem cells, where destruction of human embryos was required.
  • the hemanoids are thus cryonoids derived from iPSC.
  • the hemanoid does not comprise any remnants of undifferentiated pluripotent stem cells before cryopreservation. Markers of undifferentiated pluripotent human stem cells commonly used is e.g., TRA-1-60 and/or TRA-1-81.
  • the amount of TRA-1-60 + cells declines during culturing of the hemanoids, thus it is optimal that during maturation of the hemanoid, the TRA-1-60 levels decrease to less than 5%, preferably the hemanoid is TRA-1-60; or substantially TRA-1-60’.
  • the term “substantially TRA-1-60’” or “essentially TRA-1-60’” is to be understood as having a TRA-1-60 + count of less than 5%, such as less than 4%, 3%, 2%, 1%, 0.1% or such as less than 0.01%.
  • the TRA-1-60 + count may be up to 10%, but is preferably less than 5%, more preferably less than 1%, such as less than 0.1% or less than 0.01% at the time of which the immune cells are produced by said cryonoids.
  • stem cells within hemanoids undergo differentiation and cell specification along the three germ lineages — endoderm, ectoderm, and mesoderm — which comprise all somatic cell types.
  • the stem cells are subjected to mesoderm priming for about 3- 15 days, preferably 4-10 days, in order to produce hemanoids.
  • mesoderm priming it is common that the number of cells positive for CD34/CD144 declines due to further maturation of HE and the further differentiation and growth of the hemanoid. Accordingly, it is preferred that the hematopoietic organoid comprises CD34/CD144 double positive cells.
  • the hemanoid comprises at least 3%, such as at least 4%, 5%, 8%, 10%, 15%, 17%, 20% 25%, 30%, 35% or such as at least 40% CD34/CD144 double positive cells, or between 5% and 40% CD34/CD144 double positive cells.
  • the hemanoid also comprises other cell types such as endothelial cells, which express markers such as CD309.
  • hemanoids useful for cryopreservation may comprise at least 20%, such as at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or such as at least 90% CD309 positive cells, or in a range from 20% to 95% CD309 positive cells.
  • the hemanoid comprises both HE i.e., CD34 + CD144 + cells, and endothelial cells i.e., CD309 + cells, while containing no, or essentially/substantially no TRA-1-60 + cells.
  • the ratio of CD309 + TRA1-60 _ to CD34 + CD144 + cells in the hematopoietic organoid and/or cryonoid is in the range of 1 :10-1 :3, such as about 1:10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4 or such as about 1:3.
  • hemanoids In contrast to monolayer differentiation cultures, however, the spheroid structures that are formed when PSCs aggregates enable the non-adherent culture of hemanoids in suspension. This makes hemanoids cultures inherently scalable, which is useful for bioprocessing approaches, whereby large yields of cells can be produced for potential clinical applications. Additionally, although hemanoids largely exhibit heterogeneous patterns of differentiated cell types, they are capable of responding to similar cues, such as e.g., cytokine stimulation, as those that direct embryonic development.
  • hemanoid formation often necessitates the use of inhibitors of the rho associated kinase (ROCK) pathway, e.g., Y-27632, HA-100, H-1152 and/or 2.4 disubstituted thiazole (Thiazovivin/Tzv), preferably, Y- 27632, e.g., about 10 pM.
  • ROCK rho associated kinase
  • Thiazovivin/Tzv disubstituted thiazole
  • hemanoids can be formed from PSCs such as iPSCs by manual separation of adherent colonies (or regions of colonies) or, preferably, by enzymatic treatment with collagenase IV, and subsequently cultured in suspension.
  • hemanoids in suspension allows for the formation of large quantities of hemanoids but provides little control over the size of the resulting aggregates, often leading to large, irregularly shaped hemanoids.
  • Mixed (or moved, e.g., stirred or shaken) culture platforms increase the homogeneity of hemanoid sizes when ESCs are inoculated within bulk suspensions.
  • the hemanoids used to produce the cryonoids of the invention are obtained by a method comprising cultivating pluripotent stem cells such as iPSC in suspension culture for a sufficient period of time, such as 3-15 days, preferably 4-10 days to produce hemanoids.
  • the hematopoietic organoid i.e. hemanoids
  • the iPSCs or ESCs are cultured for 7 to 15 days to obtain a TRA-1-60- hematopoietic organoid.
  • cryopreservation as described herein, which produces cryonoids, that upon thawing are still capable of producing several different cell types over long periods of time when cultured in absence of specific molecular cues, such as cytokines as disclosed herein.
  • the hemanoids are prepared for cryopreservation.
  • the cryopreservation may be done by harvesting of the hemanoids, by e.g., sedimentation, followed by one or more washing steps using e.g., DPBS (Gribco), whereafter a suitable cryoprotection media is added to the washed hemanoid and the cryopreservation is performed as described herein.
  • DPBS DPBS
  • the cryopreservation media may be selected from CryoStor® CS10 (Stemcell Technologies) or NutriFreez® (Sartorious), mFreSRTM (StemCell technologies), Vit Kit - Freeze NX (Fujifilm), and Sy nth-a- FreezeTM Cryopreservation Medium (ThermoFisher Scientific).
  • the cryopreservation media is CryoStor® CS10 (Stemcell Technologies).
  • the cryopreservation media may be supplemented with one or more sugars, preferably trehalose, such as e.g., about 0.1-1 M trehalose, preferably about 0.5M trehalose.
  • cryopreservation media may further be supplemented with one or more inhibitors of the rho associated kinase (ROCK) pathway inhibitors (Rl) as disclosed herein.
  • the cryopreservation media is CryoStor® CS10 (containing 10% DMSO) supplemented with about 0.5M trehalose and about 10nM Rl.
  • CryoStor® CS10 is a serum-free, animal-component free cryopreservation medium with 10% dimethyl sulfoxide (DMSO).
  • the hemanoids may be incubated for a period of time, such as 0-30 min, such as about 10-20 min, or such as about 15 min at 0°C- 20°C, such as 4°C, such as 10°C, such as 20°C, preferably at 4°C or on ice before initiating the cryopreservation.
  • the cryopreservation comprises one or more freezing steps.
  • the freezing is done in one step with a fixed freezing rate (°C/min), or freezing is done in more than one steps (differential controlled freezing cryopreservation), characterized in that each freezing step is done at a specific freezing rate (°C/min).
  • the freezing comprises a step of initial freezing from above 0°C, such as 0- 10°C to about -4°C, such as -3°C to -10°C, at a rate in the range of -0.01 to -10°C/min, such as -0.0rC/min, -0.05°C/min, -0.1°C/min, -0.3°C/min, -0.5°C/min, -1°C/min, -3°C/min, - 8°C/min or such as -10°C/min.
  • the freezing further comprises a step of lowering the temperature from about -4°C to about -40°C, at a rate in the range of - 10°C/min to -50°C/min, preferably -25°C/min.
  • the freezing further comprises a step of increasing the temperature followed by a step of lowering the temperature, wherein the heating step raises the temperature from about -40°C to about -12°C, at a rate in the range of 1°C/min to 30°C/min, preferably 10°C/min, and the freezing step comprises lowering the temperature from about - 12°C to about -40°C at a rate in the range of -0.1 to -10C/min, preferably a rate of -1°C/min.
  • the freezing further comprises a step of lowering the temperature from about -40°C to about -90°C at a rate in the range of -1°C/min to -30°C/min, preferably - 10°C/min.
  • the freezing comprises the following steps an initial freezing step to bring the sample to -4°C, performed at a rate of -O.rC/min, -1 °C/min, or -8°C/min; a second freezing step to bring the sample to -40°C, performed at a rate of - 25°C/min, a heating step to bring the sample to -12°C, performed at a rate of 10°C/min, a third freezing step to bring the sample to -40°C, performed at a rate of -1°C/min, and a fourth freezing step to bring the sample to -90°C, performed at a rate of -10°C/min.
  • the hemanoids are incubated for 0-30 min, such as about 10-20 min, or such as about 15 min, before initiating the cryopreservation protocol.
  • the storage of the hemanoids is done for a period of time that does not substantially influence the subsequent ability of the thawed cryonoid to continuously produce the desired cell types.
  • the cryopreservation protocol may e g., be conducted using a single controlled freezing rate or in using a series of multiple freezing rates and durations.
  • the cryopreservation protocol comprises a step of intermediate thawing.
  • the frozen hemanoids now denoted cryonoids
  • the cryonoids may be transferred to LN2.
  • the cryonoids may be readily transported to different manufacturing sites, that can upon demand thaw the cryonoid and initiate the production of the desired cell types.
  • the present invention also provides a composition comprising a cryonoid and a suitable storage medium.
  • the composition may be a cryopreserved composition, that upon thawing enables the on-demand production of immune cells. Accordingly, such a composition may comprise a cryonoid, a chemically defined medium and optionally additional supplements as described herein.
  • the composition comprises one or more TRA-1-60' or substantially TRA-1- 60’ cryonoids with a size of 100um-500um, and a chemically defined medium, preferably CryoStor® CS10 (Stemcell Technologies).
  • the composition comprises one or more saccharides, such as sugars, oligosaccharides or polysaccharides.
  • saccharide is a mono-, di- or trisaccharide, such as glucose, sucrose, trehalose or raffinose.
  • said composition comprises a suitable amount of ROCK inhibitors (Rl).
  • the storage medium may comprise DMSO, such as about 1-15% DMSO, or such as about 10% DMSO.
  • the storage medium may comprise one or more sugars, preferably trehalose, such as in the range of 0.1-1M trehalose, or such as about 0.5M trehalose.
  • the storage medium comprises one or more Rl(s), such as e.g., the Rl Y-27632, preferably about 10 nM Rl.
  • the composition may be provided as a part of a kit, which may comprise; a vial or another suitable container comprising the composition as disclosed herein stored in LN2, optionally, one or more vials/suitable containers comprising a suitable cultivation medium, optionally one or more vials/suitable containers comprising one or more cytokines, and instructions on o how to thaw the cryonoid, o optionally, how to prepare the cryonoid for culturing, o optionally, how to culture the cryonoid to produce one or more desired cell types, and o optionally, how to harvest the produced cells from the culturing medium.
  • a kit may comprise; a vial or another suitable container comprising the composition as disclosed herein stored in LN2, optionally, one or more vials/suitable containers comprising a suitable cultivation medium, optionally one or more vials/suitable containers comprising one or more cytokines, and instructions on o how to thaw the cryonoid, o optionally,
  • Suitable containers for storing the cryonoid are known in the art.
  • cryonoid may be stored and shipped in LN2, and the cryonoids may be thawed on demand, when needed.
  • Thawing as used herein means the process of bringing frozen cells/cryonoids from the solid frozen state to the defrosted state. Thawing may be performed according to any suitable method known to the person skilled in the art taking the cell type into account. Thus, thawing may be performed using an automatic thawing device such as e.g., ThawStar (BioLife Solutions), by collecting the LN2 stored cryonoids from the LN2 tanks and transferring them into the automatic thawing device. Following thawing the thawed cryonoids may be washed one or more times with a suitable wash medium that may e.g., comprise human albumin. Following wash of the thawed cryonoid, the washed cryonoid may be cultured with the desired culture and differentiation media, preferably supplemented with Rl, such as e.g., 10 nM.
  • Rl such as e.g. 10 nM.
  • the invention also relates to a cryonoid as disclosed herein, or a composition comprising a cryonoid as disclosed herein for use in the manufacturing of one or more mammalian blood cells, preferably one, or more human immune cells.
  • the cryonoid comprises CD34/CD144 double positive cells. Since HE is essential for the function of the cryonoid, it may be so that the cryonoid comprise at least 3%, such as at least 4%, 5%, 8%, 10%, 15%, 17%, 20% 25%, 30%, 35% or such as at least 40% CD34/CD144 double positive cells, or between 5% and 40% CD34/CD144 double positive cells. Besides HE, the cryonoid also comprises other cell types such as endothelial cells, which express markers such as CD309.
  • cryonoids may comprise at least 20%, such as at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or such as at least 90% CD309 positive cells, or in a range from 20% to 95% CD309 positive cells.
  • the cryonoid comprises both HE i.e., CD34 + CD144 + cells, and endothelial cells i.e., CD309 + cells, while containing no, or essentially/substantially no TRA-1-60 + cells.
  • the cryonoid should be capable of producing blood cells upon stimulation with one or more stimulating agents, as disclosed herein.
  • cryonoid intends to describe a hematopoetic organoid (also called hemanoid) with the above described characteristics, which has been subjected to cryopreservation (see also cryopreservation method as described herein).
  • cryonoid term is intended to also encompass a cryonoid which has been thawn, in order to distinguish this type of cell aggregate from hemanoids which have never been subjected to cryopreservation.
  • cryonoids as described herein may also produce IL-3 internally in an amount which may also lead to production of immune cells such as e.g., macrophages when e.g., M-CSF is added, however, the amount immune cells produced may be significantly improved upon addition of IL-3 to the cultivation media. Accordingly, cryonoids, once thawed are capable of producing different kinds of blood and/or immune cells (such as erythrocytes, macrophages, granulocytes, etc.) upon the addition of defined cytokines, such as but not limited to IL-3, optionally in combination with additional cytokines such as e.g., M-CSF or G-CSF.
  • defined cytokines such as but not limited to IL-3
  • additional cytokines such as e.g., M-CSF or G-CSF.
  • the one or more stimulating agents is IL-3 in combination with one additional stimulating agent. In embodiments, the one or more stimulating agents is IL-3 in combination with two additional stimulating agents. In embodiments, the one or more stimulating agents is IL-3 in combination with three additional stimulating agents. In embodiments, the one or more stimulating agent is IL-3 in combination with at least one additional stimulating agent(s), such as one, two, three, four, five, six, seven, eight, nine or ten additional stimulating agents. In embodiments, the one or more stimulating agent(s) does not comprise IL-3.
  • the hemanoids are unique as they release the target cell of choice for a time period of several months into the supernatant.
  • One major reason for the continuous production of cells relies on the formation of typical structures within the hemanoids (known as hemogenic endothelium; HE) and other cells which have the capacity to secrete hematopoietic promoting cytokines needed for the continuous production of immune cells from PSCs.
  • HE hemogenic endothelium
  • cryopreserved hemanoids i.e., cryonoids
  • the present disclosure therefore relates to a method for producing mammalian blood cells, wherein said method comprises culturing a cryonoid as defined herein in a suitable medium, adding to said medium one or more stimulating agent(s) to promote production of mammalian blood cells.
  • Such method may further comprise first thawing the cryonoid as described herein, and after culturing, the method may also comprise harvesting said mammalian blood cells.
  • suitable media refers to any media which may be used in the culturing of the cryonoids and is especially suitable for culturing the cryonoids in the production phase, where immune cells are produced. Examples of such media are e.g.
  • the present invention allows for work with the initial iPSCs and the thawed cryonoid to be separated, such that the production of the desired cells from the cryonoids can be done at different sites, where e.g., one site is responsible for all production and handling related to pluripotent cells, such as e.g., iPSC, the production of hemanoids and cryonoids, while a second manufacturing site focuses on the on-demand cell production from the cryonoids, wherein said production can be done in absence of reminiscent pluripotent cells.
  • iPSC pluripotent cells
  • FIG. 1 An example of this is illustrated in figure 1.
  • cryonoids provides a scalable solution for continuous on- demand production of different mammalian blood cells, which avoids the common legal issues related to handling and production from pluripotent cells such as e.g., iPSCs or ESCs.
  • pluripotent cells such as e.g., iPSCs or ESCs.
  • the presence of pluripotent cells may e.g., be revealed by the presence of the surface marker TRA-1-60.
  • the mammalian blood cells may e.g., be selected from the group consisting of platelets, erythrocytes, lymphocytes, such as NK cells, T cells or B cells, granulocytes such as neutrophils, eosinophils or basophils, macrophages and dendritic cells. It is preferred that the mammalian blood cells are immune cells, and as such the mammalian immune cells may e.g., be selected from the group consisting of NK cells, T cells, B cells, granulocytes, macrophages and dendritic cells. In case the cells are intended for treatment of a human, human immune cells, such as macrophages, NK cells, granulocytes, dendritic cells and/or progenitor cells are preferably produced.
  • human immune cells such as macrophages, NK cells, granulocytes, dendritic cells and/or progenitor cells are preferably produced.
  • cytokines e.g. SCF, BMP4, VEGF or small molecules including WNT pathway modulators such as CHIR99021 ((6-[[2-[[4-(2,4- Dichlorophenyl)-5-(5-methyl-1 H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3- pyridinecarbonitrile (TOCRIS)) and BIO ((2'Z,3'E)-6-Bromoindirubin-3'-oxime) (TOCRIS)).
  • the culture conditions include use of ROCK inhibitor, but addition of bFGF is not required.
  • the pluripotent stem cells from which the hemanoids are derived may be from any species, e.g., mouse, rat, rabbit, guinea pig, cat, horse, dog, cow, camel, pig, sheep, goat, monkey or primate, e.g., human. Human cells are preferred.
  • cytokines from the relevant species are used to produce the desired cells. These may be recombinant cytokines, optionally, comprising mutations, which do not diminish their functionality in the differentiation processes involved.
  • Culturing of cryonoids may be performed as adherent culture or as suspension culture. During culturing it is preferred that suspension cultures are used to reduce the adhesion of the cryonoids to surfaces of the culturing container.
  • the suspension culture used to culture the cryonoids of the invention prevents adhesion to surfaces, in particular to surfaces of containers.
  • the suspension culture also does not allow adhesion to carriers.
  • the suspension culture is moved to prevent adherence or sedimentation of cells, e.g., shaken, rotated or stirred.
  • the culture may take place in suspension plates, e.g., on an orbital shaker, or in roller bottles coated to prevent adhesion.
  • the suspension culture is carried out in a bioreactor allowing suspension culture such as an Erlenmeyer flask, spinner flask, stirred tank bioreactor, wave bioreactor and rotating wall bioreactor, preferentially in a stirred tank bioreactor, most preferably in an instrumented stirred tank bioreactor, i.e., a bioreactor equipped with technologies to monitor and control process parameters such as pH, pO2, temperature and stirring speed.
  • an instrumented stirred tank bioreactor i.e., a bioreactor equipped with technologies to monitor and control process parameters such as pH, pO2, temperature and stirring speed.
  • Such systems are scalable, i.e., the volume of the culture can be changed without significantly changing culture conditions.
  • the DASbox Mini bioreactor system, Eppendorf may be used, e.g., as described below. For compatibility with industrial processes, relative linear upscaling is probably the most important criteria.
  • Preferred systems may already be utilized in the industry for other purposes, and may fulfil industry standards, e.g. they may be equipped with standard ports that allow incorporation of standard probes for process monitoring and control, and established sterilization and equipment validation procedures.
  • Bioreactors may be GMP- compatible.
  • Bioreactors used in the invention may be equipped with ports enabling continuous sampling, monitoring and harvest of cells, in particular immune cells. They may also be equipped with suitable cell retention systems allowing perfusion-based cell feeding and online monitoring of culture medium components such as metabolites e.g. glucose, lactate and ammonium.
  • the bioreactor may be a single-use (disposable) or reusable bioreactor, utilising glass, plastic or stainless-steel vessels.
  • the cultivation in a stirred tank bioreactor is carried out at about 37° C, with headspace gassing at about 3 L/h with about 21% O 2 , about 5% CO 2 , and stirring at about 50 revolutions per minute using a multiple blade (e.g., 4-12 blade)-pitched impeller.
  • “about” means +/- 10%, preferably, +/-5% or +1-2%.
  • the cultivation may be at 37° C, with headspace gassing at 3 L/h with 21% O 2 , 5% CO 2 , and stirring at 50 revolutions per minute using an 8 blade-pitched 60° C impeller and, optionally, with real-time monitoring of dissolved oxygen, pH, temperature, and impedance-based biomass assessment.
  • the method of the disclosure allows for continuous production of cells, preferably immune cells, which includes continuous production based on repeated batch methods, where a single cryonoid may be used for continuous production of the desired cells.
  • the continuous production of cells may enable production of different types of cells from the same cryonoid, such as either parallel production or different cell types or serial production, where the molecular cues, i.e., the culture media containing particular cytokines are changed between harvests to facilitate serial production of different cell types, such as e.g., first production of macrophages, followed by production of NK cells and vice versa.
  • the volume of the suspension culture is scalable, e.g., 50 mL-1000 L, 100 mL-500 L, 200 mL-100 L, 500 mL to 50 L or 1 L-20 L.
  • the medium used for cultivation is a chemically defined medium compatible with application in humans, e.g., X-VIVO 15 (Lonza) or APEL (Stem Cell Technologies), preferably, X-VIVO 15.
  • E8 or E6 media (Stem Cell Technologies) preferably, supplemented with ROCK-inhibitor, may also be used for the suspension cultures.
  • cryonoids results in the cellular aggregates being able to differentiate continuously to produce new cells depending on specific cues.
  • immature cells are delivered into the culture medium from the hemanoid in a continuous fashion.
  • additional cytokines are added to the culture medium.
  • Generation of immune cells from cryonoids typically takes about 4-30 days, depending on the specific cell type. Generation of first new cells from the hemanoid/cryonoid starts after formation of the hemanoid, and continuous generation is observed thereafter. Typically, the hemanoid is cryopreserved after 4-10 days of culturing, and following thawing of the cryonoid, harvest of immune cells is started about 3-30 days after thawing and culturing, depending on the desired cell type. In example for NK cells, a bi-weekly media change may be required, and the initial NK cell harvest be conducted after about 28 days, such as after about 20-40 days or such as about 25-30 days, of culturing in suitable medium, whereafter weekly harvests may be conducted.
  • the initial harvest may be conducted after about 8 days, such as after 6-20 days or such as 7-10 days of culturing in the suitable media, whereafter weekly harvests may be conducted.
  • the initial harvest may be conducted after about 7 days, such as 5-14 days, such as 6-9 days of culturing, whereafter weekly harvests can be performed.
  • the initial harvest may be conducted after about 15 days, such as after 12-25 days or such as 12-16 days of culturing in the suitable media, whereafter weekly harvests may be conducted.
  • a suitable time for the first harvest of immune cells from hemanoids is between 6-30 days, depending on the desired cell type, preferably about 7-8 days for macrophages and dendritic cell or about 28 days for NK cells.
  • at least weekly harvest of macrophages from a stirred bioreactor system is possible for several weeks.
  • stable production of about 2- 3x10 7 macrophages/week is possible as early as week three, which can be maintained over time for at least 5 weeks.
  • cultivation times and conditions may be varied depending on the phenotype of the desired cells. For example, harvesting continuously or in in small intervals may lead to production of less mature cells.
  • the harvested cells can than, optionally, as discussed later below, be subject to further maturation.
  • mammalian blood cells such as immune cells from the cryonoid requires the culturing in the presence of one or more stimulating agents, such as e.g., cytokines, antibodies, growth factors etc.
  • one or more stimulating agents such as e.g., cytokines, antibodies, growth factors etc.
  • one, two, three, four or five stimulating agents may be added to the differentiation medium. When more than one stimulating agent is added, these may be added simultaneously, or sequentially.
  • the one or more stimulating agent is selected from the group consisting of IL-3, M-SCF, G-CSF, IL-7, IL-15, FLT3, IL-4 and GM-CSF, and any combination thereof.
  • a cytokine such as IL-3 does not necessitate continuous presence of said cytokine. It is for example possible to culture the cells in the absence of IL-3 for time periods, but differentiation and, in particular, production of immune cells by the cryonoids requires presence of IL-3.
  • Suitable amounts or IL-3 are, e.g., 10-100 ng/mL, preferably, 20- 30 ng/mL, most preferably, about 25 ng/ml IL-3.
  • the differentiation media may comprise suitable concentrations of IL-3, or M-SCF and IL-3 (meaning a combination of M-SCF + IL-3), or G-CSF and IL-3 (meaning a combination of G-CSF + IL-3), or IL-7, IL-15, FLT3 and IL-3 (meaning a combination of IL-7 + IL-15 + FLT3 + IL-3), or IL-4, GM-CSF and IL-3 (meaning a combination of IL-4 +GM-CSF + IL-3).
  • M-SCF and IL-3 meaning a combination of M-SCF + IL-3
  • G-CSF and IL-3 meaning a combination of G-CSF + IL-3
  • IL-7, IL-15, FLT3 and IL-3 meaning a combination of IL-7 + IL-15 + FLT3 + IL-3
  • IL-4, GM-CSF and IL-3 meaning a combination of IL-4 +GM-CSF + IL-3
  • Production of immature cells may be obtained by culturing the cryonoid in the presence of the cytokine IL-3. Following production of the immature cells, said immature cells may be further cultured and matured to obtain cells of the desired phenotype, i.e., the immature cells may be matured into NK cells, macrophages, dendritic cells, granulocytes or further immune cells.
  • Culturing the cryonoid in the presence of IL-3 usually gives rise to production of immature cells positive for CD45, CD56 and negative for CD3, while further addition of M-CSF usually leads to production of macrophages with typical morphology and surface marker expression of CD45, CD11 b, CD14 and CD163, as is shown in figure 3 (hemanoids), 4, 5 and 6.
  • Culturing in the presence of IL-3 and G-CSF usually gives rise to production of CD45, CD11b, CD13, CD16, CD34, CD15, CD24, CD32, CD33, CD107a, CD164, CD69, MPO, CD612L and/or CD117 CD66b positive granulocytes.
  • Culturing in the presence of IL7, IL-15, FLT3 and IL-3 usually gives rise to production of NK cells with typical morphology and surface expression of CD45 and CD56 and which are negative for CD3, as shown in figure 7.
  • Culturing in the presence of IL-4, IL3, GM-CSF, FLT3 and/or SR1 usually gives rise to production of MHC-ll, CD141 , CD123, CD11c, CD80, CD86, CD11b l0W , CD123, CD207, CD205, CD317, CD172a and/or CD207; positive dendritic cells and which are negative for CD3, CD19, CD14, CD20, CD56 and/or CD235a as shown in figure 7.
  • a differentiation medium may be used e.g., a basic medium, preferably, a chemically defined medium such as X-VIVO 15 (Lonza) or APEL (Stem Cell Technologies), both suitable, e.g., for human cells, X-VIVO 15 is preferred further comprising suitable amounts, e.g., 1-100 ng/mL, preferably, 5-30 ng/mL, most preferably, about 25 ng/ml IL-3 (obtainable, e.g., from PeproTech).
  • the medium may further be supplemented with one or more, such as e.g., one, two, three, four, five or six, additional cytokines.
  • the basic medium may be supplemented with e.g., 40-60 ng/ml, preferably, about 50 ng/ml M-CSF (obtainable, e.g., from PeproTech), in addition to IL-3.
  • M-CSF obtainable, e.g., from PeproTech
  • the medium may further be supplemented with VEGF 40-60 ng/mL, preferably about 50 ng/mL, BMP4 40-60 ng/mL, preferably about 50 ng/mL, SCF 10- 30 ng/mL, preferably about 20 ng/mL, to further mature the cryonoid.
  • the IL-3 containing media may additionally be supplemented with e.g., about 1-30 ng/mL, preferably about 5 ng/mL IL-7, about 5-40 ng/mL, preferably about 20 ng/mL IL- 15, about 10-30 ng/ml, preferably about 20 ng/mL SCF and about 5-15 ng/ml, preferably about 10 ng/ml FLT3.
  • the IL-3 containing media may additionally be supplemented with about 40-120 ng/ml, preferably about 100 ng/ml IL-4 and about 40-120 ng/ml, preferably about 100 ng/ml GM-CSF.
  • the IL-3 containing media may additionally be supplemented with about 40-60 ng/ml, preferably about 50 ng/ml G-CSF.
  • the IL-3 containing media may additionally be supplemented with about 40-60 ng/ml, preferably about 50 ng/ml G-CSF.
  • NK cells the presence of about 3-8 ng/ml, preferably about 5 ng/ml of IL-3 in the culturing is preferred.
  • dendritic cells, macrophages, immature cells and granulocytes the presence of about 20-30 ng/ml IL-3 in the culturing medium is preferred.
  • the medium may optionally comprise suitable amounts of antibiotics, e.g., 1% penicillinstreptomycin. If the produced cells are not for use in humans, other basic media may be used, e.g., for mouse cells, RPMI1640 supplemented with 10% FCS, 2 mM L-glutamine, optionally, 1% penicillin-streptomycin and one or more cytokines, such as one, two, three, four, five, six, seven, eight, nine or ten cytokines.
  • antibiotics e.g., 1% penicillinstreptomycin.
  • other basic media may be used, e.g., for mouse cells, RPMI1640 supplemented with 10% FCS, 2 mM L-glutamine, optionally, 1% penicillin-streptomycin and one or more cytokines, such as one, two, three, four, five, six, seven, eight, nine or ten cytokines.
  • the culturing media contains IL-3 and an additional cytokine. In embodiments, the culturing media contains IL-3, and two or more additional cytokines.
  • the additional cytokine is M-CSF (in a suitable concentration, e.g., 40-60 ng/mL, preferably, about 50 ng/mL M-CSF), and the produced immune cells are macrophages.
  • the additional cytokine is G-CSF (in a suitable concentration, e.g., 40-60 ng/mL, preferably, about 50 ng/mL G-CSF) and the produced immune cells are granulocytic precursor cells.
  • the granulocytic precursor cells may be further matured towards granulocytes upon culture in a media devoid of IL-3 but supplemented with G-CSF in a suitable concentration, e.g., 50-200 ng/mL, preferably, about 100 ng/mL G-CSF.
  • the additional cytokines are IL-4 (in a suitable concentration, e.g., 40-120 ng/ml) and GM-CSF (in a suitable concentration e.g., 40-120 ng/ml, preferably about 100 ng/ml), and the produced immune cells are dendritic cells.
  • the additional cytokines are IL-7 (in a suitable concentration e.g., 1-30 ng/mL, preferably about 5 ng/mL), IL-15 (in a suitable concentration e.g., 10-30 ng/mL, preferably about 25 ng/mL), SCF (in a suitable concentration e.g., 10-30 ng/ml, preferably about 20 ng/mL) and FLT3 (in a suitable concentration e.g., 5-15 ng/ml, preferably about 10 ng/ml) and the immune cells to be produced are NK cells.
  • IL-7 in a suitable concentration e.g., 1-30 ng/mL, preferably about 5 ng/mL
  • IL-15 in a suitable concentration e.g., 10-30 ng/mL, preferably about 25 ng/mL
  • SCF in a suitable concentration e.g., 10-30 ng/ml, preferably about 20 ng/mL
  • FLT3
  • additional cytokines are SCF and EPO (in suitable concentrations, e.g., 80-120 ng/mL, preferably, about 100 ng/mL SCF and 2-4 Units (U), preferably, about 3 U EPO), the produced myeloid cells comprise erythroid cells, which can be further differentiated upon culture in SCF and EPO only (in a suitable concentration, e.g., 50-200 ng/mL SCF, preferably, about 100 ng/mL SCF, and 2-4 Units (U) EPO, preferably, about 3 U EPO).
  • Other lineage-instructive cytokines may be applied to further broaden the spectrum of generated cell types towards dendritic cells or thrombocytes.
  • the cells may be harvested/separated from the cryonoid.
  • the mammalian blood cells are harvested after about 10 days of culturing, or after about 15, 20, 25, 30, 40, 50, 60, 70, 90, 100, 125, 150 or after about 200 days of culturing, or such after between 10-200 days of culturing, preferably after between 10-90 days of culturing.
  • the harvest is performed one or more times, such as 1, 2, 3, 4, 5, 6, 7, 10, 15 or such as 20 times, or such as between 1-20 times.
  • wherein the harvest is performed every 5-10 days for 0-4 months.
  • isolating/harvesting the produced cells may comprise a batch-continuous method comprising allowing for sedimentation of majority of the cryonoid in the suspension culture, e.g., by sedimentation for about 3-10 minutes, preferably, 6 minutes (e.g., by temporarily stopping movement (e.g., stirring) of the suspension culture, removing the supernatant,
  • the produced cells are isolated from the filtrate by sedimentation or centrifugation. They may then be formulated for administration to a patient, e.g., be taken up in a pharmaceutically acceptable carrier, e.g., a buffer such as PBS. Alternatively, they may be further cultivated, e.g., in suspension culture, e.g. in the presence of an activating and/or maturating agent selected form the group comprising one or more cytokines and/or active agent, e.g., IL-3, IL-4, IL-7, IL-15, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, SCF, FLT3, or LPS, and be formulated for administration later.
  • a pharmaceutically acceptable carrier e.g., a buffer such as PBS.
  • an activating and/or maturating agent selected form the group comprising one or more cytokines and/or active agent, e.g., IL-3, IL-4, IL-7,
  • the medium from the suspension culture may be removed through a filter having a mesh of about 70-100 pm, wherein the produced cells, e.g., macrophages, dendrites, granulocytes or NK cells, can be isolated, e.g., by centrifugation, from the filtrate, and, optionally, formulated for administration with or without further maturation.
  • This isolation procedure may be performed from time to time, allowing for a batch continuous production of produced cells, or continuously.
  • Further alternative methods for isolation of the produced myeloid cells comprise differential centrifugation, immunomagnetic cell separation, fluorescence-activated cell sorting, immunodensity cell isolation, microfluidic cell sorting or gradient centrifugation, e.g., with a sucrose gradient.
  • harvested myeloid cells may also be subjected to a terminal maturation, to further mature the harvested cells.
  • harvested macrophages are cultured for 3 to 10 days, preferable for 7 days in cultivation media supplemented with M-CSF.
  • harvested granulocytes are cultured for 3 to 10 days, preferable for 7 days on basic media supplemented with G-CSF.
  • harvested NK cells are cultured/expanded for 17 to 30 days, preferable for 21 days on basic expansion media supplemented with IL-2 and IL-15.
  • freshly harvested cells from cryonoids may be cultured in differentiation medium II (RPMI1640 medium supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine, 1% penicillin-streptomycin) containing 100 ng/ml SCF and 3U/ml EPO for erythroid differentiation for at least 7 days.
  • differentiation medium II RPMI1640 medium supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine, 1% penicillin-streptomycin
  • the herein disclosed method may comprise isolating the produced cells, preferably immune cells.
  • Such isolation may comprise purifying the produced cells, preferably, the macrophages, NK cells, dendritic cells, granulocytes or immature cells, to a purity of at least 50% or, preferably, at least 70%, at least 80%, at least 90% or at least 95%.
  • the produced cells may be identified from their expression of particular surface markers.
  • the purity of a population of cells, with regards to a particular cell type may be addressed by the identification of expression of particular makers on the surface of the cells.
  • macrophages may be identified as positive for CD45, CD11b, CD14, and CD163, and negative for CD34 and TRA-1-60 (CD45 + CD11b + CD14 + CD163 + CD34“TRA- 1-60“), which is indicative of macrophages.
  • the cells produced from the cryonoid following culturing in the presence of suitable concentrations of IL-3 and M-CSF for a suitable amount of time, is characterized in that preferably, at least 80%, at least 90% or at least 95% of the produced cells have an expression profile of CD45 + CD11 b + CD34’TRA-1- 60‘.
  • at least 50%, preferably, at least 60%, or 70-90% of the produced cells are CD45 + CD11b + CD34’TRA-1-60"CD14 + CD163 + macrophages.
  • At least 95% of the produced cells are CD45 + CD11b + CD34’TRA- 1-60’and 70-90% of the produced cells are CD45 + CD11b + CD34-TRA-1-60-CD14 + CD163 + .
  • produced cells may be identified from their expression of particular surface markers. As such, the purity (% cells) of a population of cells, with regards to a particular cell type may be addressed by the identification of expression of particular makers on the surface of the cells. In example, granulocytes may be identified as positive for CD45, CD11b, and CD66b Cd16 dim , and negative for CD34 and TRA-1-60 (CD45 + CD11b + CD66b + CD34"TRA-1-60"), which is indicative of granulocytes.
  • the cells produced from a cryonoid as described herein, following culturing in the presence of IL-3 and G-CSF, is characterized in that preferably, at least 80%, at least 90% or at least 95% of the produced cells have an expression profile of CD45 + CD11 b + CD34’TRA-1-60’.
  • at least 50%, preferably, at least 60%, or in the range of 70-90% of the produced cells are CD45 + CD11b + CD34’TRA-1-60"CD66b + granulocytes.
  • At least 95% of the produced cells are CD45 + CD11b + CD34’TRA- 1-60-and 70-90% of the produced cells are CD45 + CD11b + CD34-TRA-1-60- CD66b + .
  • NK cells may be identified as positive for CD45, CD56 and negative for CD3 and TRA-1-60' (CD45 + CD56 + CD3'TRA-1-60'), which is indicative of NK cells.
  • the cells produced from the cryonoid, following culturing in the presence of suitable concentrations of IL-3, IL-7, IL-15, SCF and FLT3 for a suitable amount of time is characterized in that preferably, at least 80%, at least 90% or at least 95% of the produced cells have an expression profile of CD45 + CD56 + CD3'TRA-1-60'.
  • at least 50%, preferably, at least 60%, or 70-90% of the produced cells are CD45 + CD56 + CD3 _ TRA-1-60' NK cells.
  • the invention also provides a cell population comprising at least 50%, preferably, at least 60% or more preferably at least 70% of erythroid cells, wherein, said cell population is obtainable by the method of the invention.
  • Cells which appear to be erythrocyte precursors may be identified as CD34'CD71 + CD235a + CD36 + erythroid cells.
  • CD34' CD71 + CD235a + CD36 + erythroid cells may be obtained by supplementing the cultivation media with IL-3, SCF and EPO.
  • the invention also provides a cell population comprising at least 10%, preferably, at least 20% or at least 30% of megakaryocytes, wherein, said cell population is obtainable by the method of the invention.
  • Analysis of expression of genes associated with pluripotency and activation of innate immune response confirmed efficient differentiation of the cryonoids into immune cells such as macrophages, NK cells, dendritic cells, granulocytes, and immature cells.
  • genes associated with macrophage function such as the toll-like receptors (TLR) 1 and 4, CD14, or components of the NF-KB signalling pathway (gene ontology (GO) Activation of innate immunity: 0002218) were significantly upregulated in induced Pluripotent Stem Cells derived Macrophages (iPSC-MACs) and Peripheral blood mononuclear cells derived macrophages (PBMC-MACs) versus induced Pluripotent Stem Cells (iPSCs).
  • iPSC-MACs induced Pluripotent Stem Cells derived Macrophages
  • PBMC-MACs Peripheral blood mononuclear cells derived macrophages
  • iPSCs induced Pluripotent Stem Cells
  • Functional evaluation of produced erythrocytes may be performed by the measurement of subject haemoglobin levels.
  • Produced dendritic cells have the capacity to uptake antigens (e.g. bacteria) and present them on their surface to activate other cells (e.g. NK, T cells).
  • antigens e.g. bacteria
  • NK, T cells e.g. NK, T cells
  • evaluation of the dendritic cells ability of take up and present antigens on the surface may be used to confirm that the generated cells are dendritic cells.
  • the present method makes it possible to produce a therapeutically relevant amount of cells comprising more than 1 xio 6 immune cells, at least 2xio 7 immune cells, at least 5x 10 7 immune cells, at least 1 x 1 o 8 immune cells, at least 1 x 10 9 immune cells or at least 1 X 10 1 ° immune cells from a cryopreserved hemanoid i.e., a cryonoid.
  • the cells obtainable from the cryonoid of the invention typically have a significantly higher surface expression (as determined by FACS) of CD14 and C163 and a significantly lower surface expression of HLA than macrophages obtained from PBMC.
  • the macrophages produced in the examples were shown to produce pro-inflammatory cytokines such as IL-6, IL-8, or TNF-alpha. Accordingly, they can be considered be more pro-inflammatory macrophages than anti-inflammatory macrophages.
  • suitable cytokines e.g. IL- 13, IL-10, IL-4
  • the phenotype can be redirected to anti-inflammatory macrophages.
  • other substances e.g. corticosteroids, may be used to induce an anti-inflammatory phenotype.
  • compositions for use in therapy may be prepared, such composition are referred to herein as pharmaceutical compositions.
  • compositions as described herein may be freshly prepared from the culture or may be stored as long as the majority of the produced cells are viable, preferably at least 80% of the produced cells are viable after storage.
  • Storage of produced cells in contact with a hypertonic solution may be, e.g., for up to 48 h, up to 24 h or over-night.
  • the produced cells may be stored in an isotonic solution at 4° C for up to 12 h, up to 24, or up to 48 h, and incubated with the hypertonic solution before application.
  • Kits for storage of the produced cells e.g., macrophages, dendritic cells, immature cells, granulocytes or NK cells of the present invention, and reducing the size thereof are also provided herewith, e.g., two chamber systems, one chamber comprising the cells in isotonic solution, and one chamber comprising a hypertonic solution, wherein mixing of the two solutions leads to the cells being contacted with a hypertonic solution as defined herein, wherein, consequently, the size of the cells is reduced. Sterility of the cells in the kit may be easily preserved.
  • this kit can be linked to a device suitable for spraying the cells, or it is a part of a device for spraying the cells.
  • iPSC cultivation iPSC were cultured on Geltrex-coated tissue culture plates (ideally 6-well size plates) or T25/T75 flasks (depending on the amount of iPSCs needed) using Essential 8 (E8) medium (containing DMEM/F-12 (GIBCO, Life Technologies), 64 mg/L Ascorbic acid 2-phosphate, 14 ug/L Sodium Selenite, 543 mg/L, NaHCO3 and 20 mg/L Insulin (all from Sigma-Aldrich)) supplemented with 100 ng/mL hbFGF and 2 ng/mL hTGFIS (both from Peprotech) in an incubator at 37 °C and 5% CO2.
  • the cells were split/expanded every three days using Accutase (Stemcell Technologies) and supplemented with 10 nM ROCK inhibitor (RI;Tocris, Bristol, UK). Complete medium changes using ROCK inhibitor-free medium were performed every day until the next splitting.
  • hemanoids were performed together with the mesoderm priming step.
  • mesoderm priming la medium Essential 8 medium supplemented with 10 nM Rl, 50 ng/mL hVEGF and hBMP4 and 20 ng/mL hSCF (all from Peprotech)
  • the cells were either seeded on CELLSTAR 6-well plates placed on an orbital shaker with RPM70, or seeded on a 50 mL CERO tube and placed on a 3D CERO device at 80RPM.
  • the medium is changed to Essential 6 medium (supplemented with hVEGF, hBMP4, hSCF and Rl) and changed to 85 revolutions per minute or 65RPM, 6-well plate or CERO device, respectively.
  • Essential 6 medium supplied with hVEGF, hBMP4, hSCF and Rl
  • 65RPM revolutions per minute
  • 6 well plate 6-well plate or CERO device, respectively.
  • mesoderm priming II medium Essential 6 medium supplemented with 50 ng/mL VEGF and BMP4, 20 ng/mL hSCF and 25 ng/mL hlL-3) were added.
  • the mesoderm priming medium II were refreshed before hematopoietic differentiation were started at day 10 or, alternatively, the hematopoietic differentiation may be started on day 7 of mesoderm priming step.
  • hemanoids were harvested and washed with DPBS (GIBCO). After the addition of DPBS, the hemanoids were let settling down through gravity, or alternatively, centrifuged for 1 min at 40-80G using a tabletop centrifuge. After washing, the supernatant was removed and 1 ml of CryoStor® CS10 (Stemcell Technologies) supplemented with 0.5 M of Trehalose and 10 nM Rl was added. The aggregates were then incubated at 4°C for 15min before cryopreservation process was initiated.
  • DPBS Gibcell Technologies
  • the cryopreservation program was initiated using a controlled-rate freezer (CryoMed, ThermoFisher Scientific).
  • the program starts with a reduction of temperature from 4°C to -4°C by one degree Celsius per minute.
  • the temperature drops to -40°C by 25°C per minute.
  • the program changes and the temperature rise to -12°C by 10°C/min.
  • the second last step involves the reduction of temperature to -40°C by 1°C/min.
  • the last step decreases the temperature to -90 at a pace of 10°C/min.
  • the hemanoids were then transferred to liquid nitrogen (LN2).
  • the thawing of cryonoids was performed using an automatic thawing device (ThawStar, BioLife solutions). Briefly, the cryovials were collected from LN2 tanks and transferred into the automatic thawing device. After thawing, the cryonoids were washed with basic medium supplemented with human albumin and centrifuged for 30 seconds at 40-80G. After removing the supernatant, new differentiation medium supplemented with 10 nM Rl was added.
  • the aim of the present example was to investigate whether cryonoids can be prepared from hemanoids without disrupting the 3D architecture of the hemanoids, which is essential for formation of new cells from the hemanoids.
  • hemanoids are formed during the period of mesoderm priming which is from day 4 to day 7 ( Figure 3A).
  • hemanoids from different days of mesoderm priming and specification e.g. day 4, day 7 and day 10
  • Figure 3B, D, F hemanoids from different days of mesoderm priming and specification
  • Figure 3C, E and G hemanoids from different days of mesoderm priming and specification
  • Figure 3C, E and G show typical roundish morphology and a diameter size, which ranges from about 100pm up to 500pm
  • Analysis of cells revealed the presence of CD34/CD144 double positive cells, indicating the presence of hemogenic endothelium (HE), which is a prerequisite for the successful generation of hematopoietic cells.
  • HE hemogenic endothelium
  • FIG 3H a typical flow cytometry profile where macrophages are characterized as CD45 + , CD11 b + , CD14 + and CD163 + from the first harvest (harvest 1) performed between day 5 to 12 preferably at day 7.
  • FIG 3I a representative image of a macrophage harvest at day 7 are shown. Similar macrophages with correspondingly characteristics were observed at the second and third harvest (harvest 2 and 3; figure 3J-3M).
  • cryopreservation strategies as described in the Methods section were used to cryopreserve the entire hemanoid aiming to maintain the entire cell aggregate and the 3D organization of cells within the cell cluster (Figure 4).
  • hemanoids from day 7 of mesoderm priming were used since day 7 hemanoids are of the right size and morphology and show stable expression of CD34/CD144 double positive HE cells, while pluripotent TRA-1-60 positive cells are almost absent (Figure 4B).
  • day 4 day 4, 7 and 10 iPSC-hemanoids were frozen as described in the Methods section, and stored in LN2, resulting in a cryopreserved preparation of the hemanoids, i.e., cryonoids, which can easily be stored and transported.
  • cryonoids were thawed as described in the Methods section and the cellular integrity of the thawed cryonoids was verified by microscopic evaluation as well as functional evaluation (production of immune cells) as described in the methods section, and as is shown in figure 4A, 5A and 6A, the 3D architecture of the original hemanoids were maintained sufficiently well for the cells to be further cultured and differentiated.
  • the present example shows that the process as described is a viable solution that enables cryopreservation of hemanoids, into functional cryonoids, that upon thawing maintains their overall 3D architecture.
  • the present example also shows that day 4, 7 and 10 iPSC-hemanoids are equally promising as no difference in the 3D architecture could be demonstrated.
  • the aim of the present example was to investigate whether macrophages can be generated in a continuous manner from cryonoids.
  • cryonoids Following thawing of the cryonoids as described above the cryonoids were washed with basic media and new differentiation media was added.
  • cryopreserved hemanoids new Essential 6 (E6) media supplemented with VEGF (50 ng/mL), BMP4 (50 ng/mL), SCF (20 ng/mL) and IL-3 (25 ng/mL) was added.
  • E6 new Essential 6
  • BMP4 50 ng/mL
  • SCF 20 ng/mL
  • IL-3 25 ng/mL
  • new X-VIVO 15 media supplemented with M-CSF (50 ng/mL) and IL-3 (25 ng/mL) was added.
  • Cryonoid-derived cells showed macrophage morphology and positive staining for the macrophage markers CD45, CD11b, CD14 and CD163 (Figure 4E, F). Overall, continuous production of cells resulted in a cell quantity of about 30.000 cells per cryonoids per week ( Figure 4D).
  • day 4 and day 10 cryonoids i.e., hemanoids cultivated for 4 or 10 days
  • day 4 and day 10 hemanoids were also tested for their ability to continuously produce macrophages. Similar to the day 7 Cryonoids, the day 4 and day 10 hemanoids could be cryopreserved and successfully thawed ( Figure 5A and 6A).
  • thawed Cryonoids show intact cell aggregate morphology (i.e., intact 3D cellular architecture) with typical roundish morphology and size ( Figure 5A (day 4) and Figure 6A (day 10)).
  • Cryonoids can be obtained from different stages of hematopoietic differentiation, resulting in Cryonoids with a different composition of CD34/CD144 double positive committed HE cells as well as TRA-1-60 positive pluripotent cells.
  • the aim of the present example was to investigate whether immune cells can be generated from cryonoids in a continuous fashion.
  • cryonoids After thawing the cryonoids (frozen at day 7 or day 10 of mesoderm priming step), different cells can be obtained if proper differentiation media is used. Depending on the chosen platform, media quantities need to be adjusted.
  • cryonoids Following thawing of the cryonoids as described above the cryonoids were washed with basic media and new differentiation media was added as follows:
  • NK cells Natural killer (NK) cells new X-VIVO 15 media supplemented with IL-3 (5 ng/mL), IL-7 (20 ng/mL), IL-15 (10 ng/mL), SCF (20 ng/mL) and FLT3 (10 ng/mL) was added and for NK cells, bi-weekly media change was required. After about 28 days of differentiation the first NK cell harvest occurred.
  • IL-3 25 ng/mL
  • IL-4 50 ng/mL
  • GM-CSF 50 ng/mL
  • day 7 Cryonoids were thawed, further matured (as described in the method section) and differentiated in the presence of:
  • I L-3/GM-CSF/I L-4 to obtain Dendritic cells
  • CFU-GM colony forming units-granulocyte-Monocytes
  • CFU-M colony Megakaryocytic colony forming units
  • NK cells natural killer cells
  • NK cells appear much later than macrophages or immature cells, small round shaped suspension cells which are positive for CD45, CD56 and negative for CD3 appeared continuously in the culture ( Figure 7G and could be continuously harvested following about 28 days of culturing.
  • the aim of the present example was to investigate the effect of the temperature of the pre- cryopreservation incubation step when using a fixed freezing rate.
  • hemanoids was performed together with the mesoderm priming step.
  • mesoderm priming la medium Essential 8 medium supplemented with 10 nM Rl, 50 ng/mL hVEGF and hBMP4 and 20 ng/mL hSCF (all from Peprotech)
  • the cells were either seeded on 6-well plates placed on an orbital shaker with 70 RPM, or seeded in a 50mL CERO tube and placed in a 3D CERO device at 80 RPM.
  • the medium was changed to Essential 6 medium (supplemented with hVEGF, hBMP4, hSCF and Rl) and changed to 85 revolutions per minute or 65 RPM, 6-well plate or CERO device, respectively.
  • Essential 6 medium supplied with hVEGF, hBMP4, hSCF and Rl
  • mesoderm priming II medium Essential 6 medium supplemented with 50 ng/mL VEGF and BMP4, 20 ng/mL hSCF and 25 ng/mL hlL-3) was added.
  • mesoderm priming medium II is refreshed before hematopoietic differentiation is started at day 10.
  • hemanoids were harvested and washed with DPBS (GIBCO). After the addition of DPBS, the hemanoids are let settling down through gravity, or alternatively, centrifuged for 1 min at 40-80xg using a tabletop centrifuge. After washing, the supernatant was removed and 1 ml of CryoStor CS10 (Stemcell Technologies) supplemented with 0.5M of Trehalose and 10 nM Rl was added.
  • DPBS Gibcell Technologies
  • the evaluation of size was performed by obtaining images from aggregates immediately before cryopreservation and from Cryonoids immediately after thawing. Images were taking using a Vert.AI Zeiss microscope and ZEN microscopy software. For the calculation of the size, images were processed using ImageJ software. A macro was generated to optimized and ensure the identical processing of the images. Analysis of the data was performed using Graph Pad software. In the case of the Cryonoids' analysis, it was performed immediately after thawing.
  • hemanoids and cryonoids respectively to produce immune cells were culture on X-VIVOTM 15 medium supplemented with MSCF 50 ng/mL and IL-325 ng/mL (differentiation medium).
  • X-VIVOTM 15 medium supplemented with MSCF 50 ng/mL and IL-325 ng/mL (differentiation medium).
  • hemanoids after mesoderm priming the media was changed to supplemented X-VIVOTM 15 media and produced cells (macrophages) were harvested every 7 days for at least 4 weeks.
  • Cryonoids after being thawed, they were immediately cultured in supplemented X-VIVOTM 15 medium, and cells produced were harvested every 7 days for at least 4 weeks.
  • the aim of the present example was to investigate the effect of the pre-cryopreservation incubation temperature on the viability, cell composition, cell productivity and cryonoid recovery.
  • cryonoids were counted before and after cryopreservation (immediately, 1 day, 7 days and 14 days after thawing, respectively).
  • the aim of the present example is to investigate the effect of the temperature gradient of the initial freezing step in a controlled freezing program.
  • Step 1 wait at 4°C for 15 min
  • Step 2 0.1 °C/min to -4°C or 1 °C/min to -4°C or 8°C/min to -4°C ;
  • Step 3 25°C/min to -40°C;
  • Step 5 1 o C/min to -40°C;
  • Step 6 10°C/min to -90°C;
  • the aim of the present example is to investigate the effect of modifying the first freezing rate (0.1°C/min, 1 °C/min or 8°C/min) of a controlled cryopreservation protocol on both the recovery, viability and ability to produce macrophages.
  • the controlled freezing using the CryoMed program affected the size of the cryonoids, where under all conditions, the cryonoids produced using the CryoMed program were overall larger than the non-cryopreserved hemanoids ( Figure 15(A) and 15(B).
  • cryonoid which is comparable to the number of macrophages harvested when using a fixed rate freezing like MrFrostyTM and a preincubation at 4°C (See example 4).
  • the recovery and stability of the cryonoids was also addressed by counting the cryonoids before and after cryopreservation (immediately, 1 day, 7 days and 14 days after thawing, respectively). While no statistical difference was observed between the two conditions, and the overall tendency observed was that less cryonoids were present after 14 days of culturing when the initial freezing rate was 1°C/min or 8°C/min, compared to the cryonoids prepared by a lower initial freezing rate of 0.1°C/min ( Figure 14 (A) and 14(B), where on average about 90% of the cryonoids was still observed after 14 days of post-thaw culturing, while on average about 60% or 75% of the cryonoids were still present after 14 days of post-thaw culturing for 1°C/min or 8°C/min, respectively.
  • the present example shows that the optimal recovery of cryonoids was obtained when using an initial freezing rate of 0.1°C/min.
  • Hematopoietic organoids are three-dimensional cell aggregates derived from pluripotent stem cells (PSC), preferably induced pluripotent cells (iPSCs).
  • hemogenic endothelium is essential for the function of a humanoid and may e.g., be verified by the presence of the progenitor markers CD34/CD144, which may be revealed e.g., by staining of the hemanoids with anti-CD34 and anti-CD144 markers.
  • the hemanoids may also be positive for other cells, e.g. endothelial cells are present and express markers such as CD309.
  • cryopreserved hemanoids i.e., cryonoids
  • the present example 6 and the following examples 7-11 serve to compare the performance of hemanoids and cryonoids and evaluate whether the cryopreservation of the hemanoids has an influence of the humanoid performance post-thawing.
  • Hemanoids or Cryonoids were fixed in 4% paraformaldehyde (PFA) overnight at 4°C and then washed with PBS at least twice. After dehydration with a graded ethanol series, the samples were embedded in paraffin. For staining, 3 pm sections were cut using an RM 2265 microtome (Leica), and antigen retrieval was performed using antigen retrieval buffer (Dako). Endogenous peroxidase activity was blocked by treating the samples with 3% hydrogen peroxide for 10 minutes. After washing the samples with PBS, nonspecific antigen binding was blocked by incubating the slides with 1% BSA in PBS at room temperature.
  • PFA paraformaldehyde
  • CD309 Cell Signaling #9698
  • CD144 Invitrogen #PA5-19612
  • CD34 Abeam #ab81289
  • ImmPRESS anti-Rabbit IgG Reagent Biozol #Vec-MP-7451-15
  • DAB 3,3'-diaminobenzidine
  • slices were counterstained with hematoxylin to allow analysis of the images. Images were obtained with Zeiss Observer.ZI microscope equipped with an AxioCam MRm digital camera. Image analysis was performed using QuPath software.
  • CD34 + , CD144 + and CD309 + cells were similar in Hemanoids and Cryonoids (Fig. 16A).
  • the percentage of cells expressing CD34, CD144 and CD309 in Hemanoid slices was 10.7 ⁇ 9.7%, 15.1 ⁇ 9.9% and 30.2 ⁇ 12.3%, respectively, while in Cryonoids, it was 8.4 ⁇ 6.3%, 17.1 ⁇ 10.8% and 24.3 ⁇ 15.1%, respectively (Fig.16B-D).
  • Hemanoids or cryonoids were washed with dPBS and centrifuged at 100 xg for 1 min. the supernatant was discarded, and 5 mL TrypLETM Express (Invitrogen #12605-028) was added. To dissociate the Hemanoids or Cryonoids, they were incubated for 5-10 min in TrypLETM Express at 37°C in water bath, with active pipetting to facilitate aggregate dissociation. Once dissociated, the cell suspension was diluted with MACS buffer and centrifuged at 300 xg for 5 min. Afterwards, the supernatant was discarded, and the cell pellet resuspended in MACS buffer and dispensed into FACS tubes.
  • TrypLETM Express Invitrogen #12605-028
  • cells were labeled with Live/dead staining (Biolegend #423102), CD34 (eBioscience #11- 0349-41), CD144 (Invitrogen #17-1449-42) and CD309 (Biolegend #359911) antibodies. Following a 30-min incubation at 4°C, dPBS was added to remove unbound antibodies, and the tubes centrifuged at 300xg for 5 min. After centrifugation, the supernatant was discarded and 100 pl of dPBS was added. Samples were analyzed using CytoFlex cytometer.
  • CD34, CD144, and CD309 were observed to remain similar between Hemanoids and Cryonoids (Fig. 17A-C).
  • the mean expression levels of CD34, CD144 and CD309 were 14.8 ⁇ 7.2%, 11.1 ⁇ 6.0% and 93.5 ⁇ 4.9 for Hemanoids, and 14.9 ⁇ 15.53%, 21.7 ⁇ 17.8 and 93.4 ⁇ 4.7% for Cryonoids.
  • Statistical analysis showed no significant differences between Hemanoids and Cryonoids for any of the three markers.
  • the expression of CD34, CD144 and CD309 in cells within Cryonoids remains similar to those in Hemanoids, which corroborates the results observed in example 6 and validate the robustness of cryopreservation of Hemanoids.
  • Example 8 Recovery of cryonoids post-thawing
  • Hemanoids After mesoderm priming, a known number of Hemanoids were cultured. For Cryonoids, they were thawed, counted and cultured. Hemanoids and Cryonoids were manually counted on DDO, DD1 , DD7, DD14, DD21 and DD28.
  • the culture medium used for Hemanoids and Cryonoids was Xvivo15 supplemented with M-SCF (Prepotech, #300-25) and IL-3 (Prepotech, #200-03).
  • the survival/recovery percentage was evaluated by dividing the number of aggregates (Hemanoids or Cryonoids) observed at each counting day by the number of aggregates counted the first day of culture DDO and multiply by 100.
  • the survival/recovery rate of Cryonoids post-thaw and production phase was observed to be similar to Hemanoids (Fig.18).
  • the statistical analysis showed no significant difference between the groups at any of the evaluated time points (DDO, DD1 , DD7, DD14, DD21 and DD28).
  • the mean survival/recovery rate at DD1, DD7, DD14, DD21 , DD28 for Hemanoids was 98.7 ⁇ 3.4%, 96.7 ⁇ 5.2%, 94.6 ⁇ 6.4%, 93.8 ⁇ 6.6% and 94.0 ⁇ 6.6% while the mean survival/recovery rate for Cryonoids was 99.9 ⁇ 0.3%, 92.7 ⁇ 9.4%, 89.9 ⁇ 9.9%, 88.8 ⁇ 9.8% and 86.6 ⁇ 10.7%, respectively.
  • the integrity of the Cryonoids post-thawing remains similar to the integrity of the Hemanoids never subjected to cryopreservation, suggesting that the cryopreservation process, thawing and culture to which Cryonoids were subjected does not affect their survival and indicate that the Cryonoids maintain their ability to continuously produce immune cells after thawing.
  • Hemanoids and Cryonoids were stained with Live/dead Viability/cytotoxicity Kit (ThermoFisher, #L3224) to discriminate between live and dead cells within each aggregate. Cryonoids and Hemanoids were washed twice with dPBS and stained using 1mL PBS containing 2 M calcein-AM and 4pM EthD-1. After 30 min of incubation at room temperature, fluorescent images (Live: ex/em ⁇ 495nm/515nm and Dead: 495nm/635nm) were taken using Olympus microscope. The total area of the aggregate was estimated as the sum of the live and dead signal areas. The percentage of live cells was calculated as the ratio of the live signal area to the total aggregate area, multiplied by 100. Similarly, the percentage of dead cells was calculated as the ratio of the dead signal area to the total aggregate area, multiplied by 100.
  • the mean percentage of live cells for Hemanoids and Cryonoids was 99.9 ⁇ 0.1% and 99.7 ⁇ 0.3%, respectively.
  • the mean percentage of dead cells in Hemanoids and Cryonoids was 0.1 ⁇ 0.1 % and 0.3 ⁇ 0.2%, respectively (Fig.19A).
  • Statistical analysis showed no significant differences in the proportion of live and dead cells between Hemanoids and Cryonoids. Additionally, the evaluated images showed that cryopreservation does not compromise morphology or viability of aggregates (Fig.19B). Cryonoids maintained similar structural appearance compared to Hemanoids.
  • cryopreservation of Hemanoids using the method of the present invention and the following thawing process does not cause massive cell death; on the contrary, it supports cell integrity and survival compared to non-cryopreserved aggregates (Hemanoids).
  • Phase-contrast images from Hemanoids and Cryonoids were captured using Olympus microscope. The total area and circularity of aggregates were calculated automatically using Imaged software. The percentage of cystic Area for each aggregate (Hemanoids or Cryonoids) was determined by measuring the cystic area, and dividing it by total aggregate area, and then multiplying by 100.
  • the mean area of Hemanoids and Cryonoids was 240211 ⁇ 113231 pm 2 and 248338 ⁇ 129202pm 2 , respectively (Fig. 20A).
  • the mean circularity of Hemanoids and Cryonoids was 0.5 ⁇ 0.2 and 0.6 ⁇ 0.2, respectively (Fig. 20B).
  • the mean percentage of cystic Area in Hemanoids and Cryonoids was 11.4 ⁇ 12.9% and 9.7 ⁇ 9.6%, respectively (Fig. 20C).
  • Statistical analysis showed no significant differences between Hemanoids and Cryonoids for Area, circularity or percentage of Cystic area.
  • cryopreservation of Hemanoids using the method of the present invention and the following thawing process does not impact the structural characteristics of the aggregate.
  • the comparable area of Hemanoids and Cryonoids indicates that cryopreservation does not cause substantial shrinkage or expansion.
  • the mean circularity values suggest that overall shape and compactness of aggregates are maintained after cryopreservation.
  • the mean percentage of cystic area were similar between Hemanoids and Cryonoids, which indicates that cryopreservation does not disrupt cyst formed within aggregate.
  • a determined amount of Hemanoids and Cryonoids were cultured using Xvivo15 medium supplemented with M-SCF (Prepotech, #300-25) and IL-3 (Prepotech, #200-03). Periodic collections of produced cells were performed every 7 days. On the day of harvest, the medium was refreshed until the next harvest. Harvested cells were collected and counted. The amount of cells was calculated based on the number of Hemanoids or Cryonoids in culture.
  • Hemanoids was 35741 ⁇ 22506, 1174 ⁇ 6150, 8258 ⁇ 7525 and 6873 ⁇ 5983, respectively.
  • the corresponding values were 24644 ⁇ 15891 , 1044418699, 758815483 and 883916504.
  • the number of cells produced per aggregate remained stable across all four harvests for both Hemanoids and Cryonoids (Fig.21). Additionally, there was no statistically significant difference between groups in any of the four harvests.
  • a method for cryopreserving a hematopoietic organoid comprising: a) Obtaining a hematopoietic organoid in a suitable cryopreservation media, b) Freezing said hematopoietic organoid to a temperature of from -40°C to - 196°C to obtain a cryonoid, and c) Optionally, storing said cryonoid in liquid nitrogen (LN2).
  • LN2 liquid nitrogen
  • the hematopoietic organoid comprises at least 3%, such as at least 4%, 5%, 8%, 10%, 15%, 17%, 20% 25%, 30%, 35% or such as at least 40% CD34/CD144 double positive cells, or in a range from 5% to 40% CD34/CD144 double positive cells.
  • the hematopoietic organoid comprises at least 15%, such as at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or such as at least 90% CD309 positive cells, or in a range from 15% to 95% CD309 positive cells.
  • the ratio of CD309 + TRA1-60' to CD34 + CD144 + cells in the hematopoietic organoid is in the range of 1 :10-1 :3, such as about 1:10, 1 :9, 1 :8, 1:7, 1 :6, 1:5, 1 :4 or such as about 1:3.
  • the cryonoid comprises CD34/CD144 double positive cells.
  • cryonoid comprises at least 3%, such as at least 4%, 5%, 8%, 10%, 15%, 17%, 20% 25%, 30%, 35% or such as at least 40% CD34/CD144 double positive cells, or in a range from 5% to 40% CD34/CD144 double positive cells.
  • cryonoid comprises at least 20%, such as at least 25%, 30%, 40%, 50%, 60%, 70%, 80% or such as at least 90% CD309 positive cells, or in a range from 20% to 95% CD309 positive cells.
  • the ratio of CD309 + TRA1-60 _ to CD34 + CD144 + cells in the cryonoid is in the range of 1:10-1 :3, such as about 1 :10, 1 :9, 1:8, 1 :7, 1 :6, 1 :5, 1:4 or such as about 1 :3.
  • the hematopoietic organoid is derived from adult human stem cells, human multipotent stem cells, hematopoietic progenitor cells, iPSCs or ESCs.
  • the hematopoietic organoid is obtained by culturing iPSCs or ESCs in a suitable media, to obtain a TRA-1-6CT hematopoietic organoid.
  • the iPSCs or ESCs are cultured for 7 to 15 days to obtain a hematopoietic organoid, wherein less than 5% of the cells of the are TRA-1-60T
  • a method for producing mammalian blood cells comprising; a) thawing a cryonoid as defined in any of embodiments 1-16, b) culturing a cryonoid as defined in any of embodiments 1-16 in a suitable media, c) adding to said media one or more stimulating agent(s) to promote production of mammalian blood cells, and d) optionally, harvesting said mammalian blood cells.
  • the mammalian blood cells are selected from the group consisting of platelets, erythrocytes, lymphocytes, such as NK cells, T cells or B cells, granulocytes such as neutrophils, eosinophils or basophils, macrophages and dendritic cells.
  • the mammalian immune cells are selected from the group consisting of NK cells, T cells, granulocytes, macrophages and dendritic cells.
  • the mammalian cells are human cells.
  • the one or more stimulating agent is selected from the group consisting of IL-3, M-SCF, G-CSF, IL7, IL-15, FLT3, IL-4 and GM-CSF, and any combination thereof.
  • composition comprising a cryonoid as defined in any of embodiments 1 to 16, and a suitable medium.
  • the medium comprises DMSO, such as about 1-15% DMSO, or such as about 10% DMSO.
  • a cryonoid as defined in any of embodiments 1 to 16, or a composition according to any of embodiments 28 to 31 for use in the manufacturing of one or more mammalian blood cells, preferably one, or more human immune cells.
  • a mammalian blood cell obtainable from a method according to any of embodiments 17 to 27.

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Abstract

La présente invention concerne la production de cellules particulières, telles que des cellules sanguines et immunitaires, à partir d'organoïdes cryoconservés issus de cellules souches pluripotentes, ainsi que des procédés de préparation de ces organoïdes cryoconservés et des compositions les comportant.
PCT/EP2025/063521 2024-05-16 2025-05-16 Production de cryonoïdes et de cellules sanguines issues de ceux-ci Pending WO2025238202A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017078807A1 (fr) * 2015-11-04 2017-05-11 Fate Therapeutics, Inc. Procédés et compositions pour induire la différenciation de cellules hématopoïétiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017078807A1 (fr) * 2015-11-04 2017-05-11 Fate Therapeutics, Inc. Procédés et compositions pour induire la différenciation de cellules hématopoïétiques

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHRISTIE MUNN: "Generation of cryopreserved macrophages from normal and genetically engineered human pluripotent stem cells for disease modelling | PLOS ONE", 22 April 2021 (2021-04-22), XP093129166, Retrieved from the Internet <URL:https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250107#sec017> [retrieved on 20240208] *
HENGXIN HAN ET AL: "Cryopreservation of organoids: Strategies, innovation, and future prospects", BIOTECHNOLOGY JOURNAL, WILEY-VCH VERLAG, WEINHEIM, DE, vol. 19, no. 2, 25 February 2024 (2024-02-25), pages n/a, XP072593149, ISSN: 1860-6768, DOI: 10.1002/BIOT.202300543 *
KIM JIHOON: "Human organoids: model systems for human biology and medicine", 1 October 2020 (2020-10-01), XP093216329, Retrieved from the Internet <URL:https://www.nature.com/articles/s41580-020-0259-3> *
MANIA ACKERMANN: "A 3D iPSC-differentiation model identifies interleukin-3 as a regulator of early human hematopoietic specification", vol. 106, no. 5, 23 April 2020 (2020-04-23), pages 1354 - 1367, XP093159504, ISSN: 0390-6078, Retrieved from the Internet <URL:https://haematologica.org/article/download/9728/70805> DOI: 10.3324/haematol.2019.228064 *
ROGULSKA OLENA ET AL: "Cryopreservation of Organoids", CRYO LETTERS, vol. 44, no. 2, 1 March 2023 (2023-03-01), UK, pages 65 - 75, XP093215985, ISSN: 0143-2044, DOI: 10.54680/fr23210110112 *

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