EP0815206A1 - Procede de culture cellulaire - Google Patents

Procede de culture cellulaire

Info

Publication number
EP0815206A1
EP0815206A1 EP96907597A EP96907597A EP0815206A1 EP 0815206 A1 EP0815206 A1 EP 0815206A1 EP 96907597 A EP96907597 A EP 96907597A EP 96907597 A EP96907597 A EP 96907597A EP 0815206 A1 EP0815206 A1 EP 0815206A1
Authority
EP
European Patent Office
Prior art keywords
cells
selectable marker
population
selected sub
sub
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96907597A
Other languages
German (de)
English (en)
Inventor
Bradley Michael John Stringer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CellFactors PLC
Original Assignee
CellFactors PLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CellFactors PLC filed Critical CellFactors PLC
Publication of EP0815206A1 publication Critical patent/EP0815206A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12N5/0618—Cells of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to cell culture methods, and in particular to cell culture methods for the production of an essentially homogeneous population of cells (for example neuronal cells) in vitro.
  • the invention also relates to neural cells (e.g. human neural cells) having introduced therein a selectable marker (e.g. a positive and/or negative selectable marker) .
  • a selectable marker e.g. a positive and/or negative selectable marker
  • the central nervous sytem is presently the subject of intense research, but its enormous complexity at the cellular level has militated against a full understanding of its function. While increasingly selective methods of labelling specific sub-populations of neural cells ex vivo (such as immunostaining, in situ hybridization histochemistry etc.) have been developed, the separation and purification of such sub-populations as living cells presents severe difficulties.
  • FACS fluorescence-activated cell sorting
  • Another method is based upon the identification of extracellular membrane-bound markers specific for a given cell type (Urakami & Chiu (1990) J. Neuroscience 10:620).
  • separation in this example is achieved by panning the mixed cell population on an adherent antibody layer to produce cell-antibody complexes from which the cells of interest can later be dissociated for further study.
  • progenitor cells which give rise to specific cell populations are immortalized by oncogene transduction or by the sub-culturing of spontaneous cell outgrowths.
  • the degree of purification required to obtain homogeneity is often two or more orders of magnitude: such an enrichment is too great for FACS or panning, the result being a degree of contamination.
  • the present invention provides a method for selectively culturing a pre-selected sub-population of cells from a heterogenous population in vitro, comprising the steps of: (a) introducing a selectable marker (e.g. a positive and/or negative selectable marker) into the heterogenous cell population, which marker is subject to differential expression/activity in the pre-selected sub-population; and (b) selectively culturing the pre-selected sub-population on the basis of the differential expression/activity therein of the selectable marker.
  • a selectable marker e.g. a positive and/or negative selectable marker
  • the pre-selected sub-population of cells may be an essentially homogeneous population of cells of a particular cell type or cell class.
  • the pre-selected sub-population may be a particular class of neural cells.
  • the pre-selected population may be selected on the basis of transmitter characteristics, e.g. dopamine- or acetylcholine-containing neurones may be selectively cultured according to the method of the invention.
  • the selectable marker need not be introduced into every cell making up the heterogenous cell population: for most purposes it is sufficient if a significant proportion of the cells receive the selectable marker.
  • the selectable marker(s) are introduced into a large proportion (for example essentially all) of the heterogenous cell population.
  • the method of the invention finds particular application in the selective culture of particular classes of essentially normal neural cells.
  • the method of the present invention is of general application and may be used to selectively culture other sub-populations of cells.
  • mammalian neural cells can be transduced with heterologous genetic material. Many methods exist for transducing eukaryotic and other cells, but the characteristics of neural cells are such that natural methods of transfection are presently the most useful (Miller (1992) Nature 357:455). Thus, transduction with virally-packaged genetic material, for example, is not only more efficient, but also results in a lower neural cell mortality during the actual process than does, for example, calcium phosphate precipitation, electroporation, microprojectile bombardment or microinjection. Transduction of mammalian neural cells from the central nervous system both in vivo (Culver et al. (1992) Science 256:1550) and in vitro (Stringer & Foster (1994) Brain Res 79:267) has been described.
  • the genetic material that can be introduced into living cells may include both positive and negative selectable markers.
  • a positive selectable marker is one that permits survival of the transduced cell under conditions which would kill cells not expressing the selectable phenotype.
  • a negative selectable marker confers sensitivity on the cells which express it, such that they are destroyed under conditions which are relatively innocuous to other cells.
  • Genes that are widely applied as positive selectable markers include the bacterial neomycin phosphotransferase (neo; Colbere-Garapin et al. (1981) J. Mol. Biol. 150:1), hygromycin phosphotransferase (hph; Santerre et al. (1984) Gene 30:147) and xanthineguanine phosphoribosyl transferase (gpt; Mulligan & Berg (1981) Proc. Natl. Acad. Sci. USA 78:2072).
  • HSV-1 TK herpes simplex virus type 1 thymidine kinase
  • APRT adenine phosphoribosyl transferase
  • HPRT hypoxanthine phosphoribosyl transferase
  • Preferred negative selectable markers include genes encoding products involved in programmed cell death (apoptosis), for example the gene for p53. Such negative selectable markers may be activated by inducing the expression of the gene in question (for example by use of a tetracycline-responsive promoter, as described infra) .
  • the use of genes encoding products involved in apoptosis has the advantage that transient expression (in many cases 30 minutes or less) of the gene may be sufficient to commit the cell to death, permitting reliable and very stringent negative selection.
  • Some of the aforementioned genes also confer negative as well as positive selectable phenotypes. They include the HSV-1, APRT, HPRT and gpt genes. These genes encode enzymes which can catalyze the conversion of certain nucleoside or purine analogues to cytotoxic intermediates.
  • the nucleoside analogue ganciclovir GCV
  • GCV nucleoside analogue ganciclovir
  • Xanthineguanine phosphoribosyl transferase can be used for both positive and negative selection which expressed in wild type cells (Besnard et al. (1987) Mol. Cell Biol. 7:4139). Cytosine deaminase can also be used as a negative selection marker, converting the innocuous 5-fluorocytosine to the cytotoxic 5-fluorouracil (Polak & Scholer (1976) Chemotherapy (Basel) 21:113).
  • Selectable markers are usually used in both prokaryotic and eukaryotic genetic engineering to permit the recovery from a mixed population of cells those which have undergone a rare genetic change. For instance, they can be used in physical association with another gene which encodes a product of interest to select cells which have taken up that other gene along with the selectable marker. As an example, the neo gene has been used to monitor genetically modified cells taken from patient samples after gene therapy has taken place.
  • the method of the invention can include the prior induction of replication in mixed populations of embryonic neural cells, using supplements to the culture medium such as epidermal growth factor or fibroblast growth factor, or by prior transfection with immortalizing oncogenes, to elicit such replication.
  • non-expanding cell cultures can be used.
  • the cells are transduced with a positive selectable marker and a negative selectable marker, both linked operably to an expression element.
  • the expression element may be specific for a given central nervous system region, a given neural cell type, or a specific sub-population of neurones.
  • the cells may be allowed, at least partially, to differentiate.
  • the appropriate drug can then be applied, such that non- transduced cells and those transduced cells without the active specific expression element are eliminated, while transduced cells with the active element (which leads to the expression of the downstream selectable markers) will be resistant.
  • the expression elements for use in the invention may be selected from: promoters and/or enhancers which are specifically active in: (i) dopaminergic, serotoninergic, GABAergic, cholinergic or peptidergic neurones, or sub-populations thereof; (ii) Schwann cells, oligodendrocytes, astroycytes, microglia and sub-populations thereof; (iii) particular stages of embryogenesis and (iv) other specific non-neural tissues.
  • promoters and/or enhancers which are specifically active in: (i) dopaminergic, serotoninergic, GABAergic, cholinergic or peptidergic neurones, or sub-populations thereof; (ii) Schwann cells, oligodendrocytes, astroycytes, microglia and sub-populations thereof; (iii) particular stages of embryogenesis and (iv) other specific non-neural tissues.
  • Particularly preferred for use in the present invention
  • promoters and/or enhancers which direct the transcription of genes for: (i) neurotransmitter-specific receptors; (ii) ion channels; (iii) receptors involved in ion-channel gating, (iv) cytokines, growth factors and hormones and (v) any substance that is specifically produced and secreted in a paracrine, autocrine or endocrine fashion.
  • the invention provides a method of culturing human and other mammalian cells (e.g. neural cells) and, by selecting for a sub-population of cells on the basis of the genetic material contained within them, producing homogeneous cultures of a single cell type.
  • mammalian cells e.g. neural cells
  • Such cultures can be put to a variety of uses including basic electrophysiological, neurochemical and developmental experimentation.
  • the purified neural cell populations will be useful in more clinically applied studies, such as assessment of the feasibility of transplantation to alleviate the symptoms of central nervous sytem degenerative disease, and find application in various forms of therapy, prophylaxis and diagnosis.
  • Such diseases include: (i) Parkinson's disease or parkinsonism, the pre-selected sub-population of cells being dopaminergic neurones of the substantia nigra; (ii) Huntington's disease, the pre-selected sub-population of cells being neural cells of the striatum; (iii) Alzheimer's disease, the pre-selected sub-populations of cells being acetylcholine-, serotonin-, and/or noradrenaline-containing neurones associated with the neo- and palaecortex; or (iv) multiple sclerosis, the pre-selected sub-population of cells being brain oligodendrocytes.
  • Thyrogiobuiin Thyroid cells Hypothyroidism Pichon et al. (1994) Biochem J 298.537-41
  • HSV Herpes simplex virus
  • tk thymidine kinase gene
  • neo gene operably linked to a promoter which is active only in dopamine-containing neurones, e.g. that controlling expression of tyrosine hydroxylase (see e.g. that described by Harrington et al. (1987) Nucl. Acids Res. 15:2363).
  • the construct is then cloned into the appropriate cloning site of a retroviral vector, and used to transfect an amphotropic retroviral packaging cell line (e.g. f-crip (for review see Molecular Virology: A Practical Approach (Eds. AJ Davison & RM Elliott) IRL Press, 1993).
  • Tissue is dissected from embryonic (approximately 5-8 weeks of gestation) human ventral mesencephalon and grown in dissociated culture.
  • the dopaminergic precursor cells are induced to replicate, by application of fibroblast growth factor (Mayer et al. (1993) Neuroscience 56:389), epidermal growth factor (Reynolds & Weiss (1992) Science 255:1707) or by oncogene transduction (Stringer et al. (1994) Brain Res. 79:267).
  • the cultured cells are transduced with the retrovirally-packaged selectable markers, and the cultures allowed to expand. When sufficient numbers of cells are produced, the cultures are incubated under conditions leading to cessation of neuronal division.
  • the cultures are then treated with geneticin to eliminate non-transduced cells as well as transduced cells not expressing tyrosine hydroxylase, but leaving transduced, tyrosine hydroxylase-containing neurones.
  • Example 2 Preparation of a homogeneous culture of human oligodendrocvtes
  • HSV Herpes simplex virus
  • tk thymidine kinase gene
  • neo gene operably linked to a promoter which is active only in oligodendrocytes, that controlling expression of the oligodendrocyte-specific enzyme galactocerebrosidase.
  • the construct is virally packaged as in Example 1.
  • a virus such as adenovirus could alternatively be used.
  • Tiossue from embryonic or adult brain is dissected and grown in dissociated culture. If necessary, cell replication is induced (for example by using cells from HS2ts6 mice (Noble et al.
  • the cells are transduced with the genes coding for the positive selectable marker linked to, for example, the galactocerebrosidase promoter, and for the negative selectable marker linked to a constitutively active promoter, such as cytomegalovirus.
  • Cell selection is obtained as in Example 1, yielding pure populations of oligodendrocytes.
  • Example 3 Preparation of a homogeneous culture of essentially normal human dorsal root ganglion cells expressing calcitonin gene related peptide
  • Neurones from dorsal root ganglia can be grown in vivo using either embryonic, neonatal or adult tissue as a source material.
  • the mixed cell population will be grown on, for example, a background layer of e.g. previously prepared neomycin resistant non-neuronal cells to provide trophic support (Brenneman et al. (1987) J. Cell Biol. 104:1603).
  • the DRG cells are transfected using adenoviral technology with a neo gene linked operably to the promoter for calcitonin gene related peptide (CGRP) expression.
  • CGRP calcitonin gene related peptide
  • retroviral particles comprising a construct (tsA58) incorporating a resistance marker to geneticin (G418 r ) linked to an SV40T promoter were added to the medium together wtih 0.8 ⁇ g/ml polybrene. After lh the culture medium was replaced with fresh medium. After 5 days, geneticin was added to the culture medium (0.4 mg/ml) for a further 10 days to eradicate cells which had not incorporated the retroviral vector.
  • clusters of human neural precursor cells could be found from all of the areas listed above which were able to replicate in the FGF-containing medium and which were also geneticin resistant. All exhibited a neuronal phenotype (they were for example neurone specific enolase positive) .
  • the construct can include a selection marker such as geneticin resistance.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne un procédé permettant la culture sélective in vitro d'une sous-population présélectionnée de cellules provenant d'une population hétérogène de cellules. Ce procédé consiste (a) à introduire un marqueur sélectionnable (par exemple positif et/ou négatif) au sein de la population hétérogène de cellules, ledit marqueur étant soumis à une activité ou à une expression différentielle en présence de la sous-population, et (b) à cultiver de manière sélective la sous-population en fonction de l'activité ou de l'expression différentielle du marqueur sélectionnable au sein de ladite population.
EP96907597A 1995-03-21 1996-03-20 Procede de culture cellulaire Withdrawn EP0815206A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9505663 1995-03-21
GBGB9505663.6A GB9505663D0 (en) 1995-03-21 1995-03-21 Genetically modified neural cells
PCT/GB1996/000671 WO1996029395A1 (fr) 1995-03-21 1996-03-20 Procede de culture cellulaire

Publications (1)

Publication Number Publication Date
EP0815206A1 true EP0815206A1 (fr) 1998-01-07

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EP96907597A Withdrawn EP0815206A1 (fr) 1995-03-21 1996-03-20 Procede de culture cellulaire

Country Status (10)

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EP (1) EP0815206A1 (fr)
JP (1) JPH11502702A (fr)
KR (1) KR19980703205A (fr)
AU (1) AU5116596A (fr)
CA (1) CA2214385A1 (fr)
CZ (1) CZ295997A3 (fr)
GB (1) GB9505663D0 (fr)
HU (1) HUP9802640A3 (fr)
NZ (1) NZ304076A (fr)
WO (1) WO1996029395A1 (fr)

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GB9807935D0 (en) * 1998-04-14 1998-06-10 Univ Edinburgh Lineage specific cells and progenitor cells
US6482937B1 (en) * 1997-10-09 2002-11-19 Biotransplant, Inc. Porcine Oct-4 promoter
US6576464B2 (en) 2000-11-27 2003-06-10 Geron Corporation Methods for providing differentiated stem cells
EP1740945B1 (fr) 2004-04-07 2018-09-19 Ncardia AG Systemes de dosage tissulaire fonctionnel in vitro, non invasifs
US8318488B1 (en) 2004-05-11 2012-11-27 Axiogenesis Ag Assay for drug discovery based on in vitro differentiated cells
CN105671027A (zh) * 2008-09-30 2016-06-15 诺维信股份有限公司 在丝状真菌细胞中使用阳性和阴性选择性基因的方法
WO2012048276A2 (fr) 2010-10-08 2012-04-12 Caridianbct, Inc. Procédés et systèmes configurables pour la culture et la récolte de cellules dans un système de bioréacteur à fibres creuses
WO2015073913A1 (fr) 2013-11-16 2015-05-21 Terumo Bct, Inc. Expansion de cellules dans un bioréacteur
WO2015148704A1 (fr) 2014-03-25 2015-10-01 Terumo Bct, Inc. Remplacement passif de milieu
EP3198006B1 (fr) 2014-09-26 2021-03-24 Terumo BCT, Inc. Alimentation programmée
WO2017004592A1 (fr) 2015-07-02 2017-01-05 Terumo Bct, Inc. Croissance cellulaire à l'aide de stimuli mécaniques
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
CN117247899A (zh) 2017-03-31 2023-12-19 泰尔茂比司特公司 细胞扩增
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US12234441B2 (en) 2017-03-31 2025-02-25 Terumo Bct, Inc. Cell expansion
EP4314244B1 (fr) 2021-03-23 2025-07-23 Terumo BCT, Inc. Capture et multiplication cellulaire
US12209689B2 (en) 2022-02-28 2025-01-28 Terumo Kabushiki Kaisha Multiple-tube pinch valve assembly
USD1099116S1 (en) 2022-09-01 2025-10-21 Terumo Bct, Inc. Display screen or portion thereof with a graphical user interface for displaying cell culture process steps and measurements of an associated bioreactor device

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WO1994024274A1 (fr) * 1993-04-21 1994-10-27 The University Of Edinburgh Isolation, selection et propagation de cellules souches d'animaux transgeniques

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CA1228025A (fr) * 1983-05-20 1987-10-13 Paul I. Terasaki Methode et composes pour isoler les leucocytes
AU4995193A (en) * 1992-08-04 1994-03-03 Regeneron Pharmaceuticals, Inc. Method of enhancing differentiation and survival of neuronal precursor cells
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Patent Citations (1)

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WO1994024274A1 (fr) * 1993-04-21 1994-10-27 The University Of Edinburgh Isolation, selection et propagation de cellules souches d'animaux transgeniques

Also Published As

Publication number Publication date
HUP9802640A2 (hu) 1999-03-29
GB9505663D0 (en) 1995-05-10
MX9707195A (es) 1997-11-29
JPH11502702A (ja) 1999-03-09
KR19980703205A (ko) 1998-10-15
WO1996029395A1 (fr) 1996-09-26
NZ304076A (en) 2001-01-26
HUP9802640A3 (en) 1999-04-28
CA2214385A1 (fr) 1996-09-26
CZ295997A3 (cs) 1998-01-14
AU5116596A (en) 1996-10-08

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