WO2008084214A1 - Assay for cell culture media and media supplements - Google Patents

Assay for cell culture media and media supplements Download PDF

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Publication number
WO2008084214A1
WO2008084214A1 PCT/GB2008/000048 GB2008000048W WO2008084214A1 WO 2008084214 A1 WO2008084214 A1 WO 2008084214A1 GB 2008000048 W GB2008000048 W GB 2008000048W WO 2008084214 A1 WO2008084214 A1 WO 2008084214A1
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Prior art keywords
stem cells
gene
test
cells
medium
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PCT/GB2008/000048
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French (fr)
Inventor
Paul Bello
Jacqui Johnson
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Stem Cell Sciences (Australia) Pty Ltd
Stem Cell Sciences (Uk) Ltd
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Priority to GB0810339A priority Critical patent/GB2449001A/en
Publication of WO2008084214A1 publication Critical patent/WO2008084214A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells

Definitions

  • the present invention relates to assays for assessing the suitability of cell culture media and medium supplements for the culture of particular cell types, particularly stem cells, including embryonic stem cells.
  • the invention relates to assays for assessing the suitability of a particular batch of an undefined medium or medium supplement, such as serum, for the culture of stem cells.
  • pluripotent stem cells such as embryonic stem (ES) cells
  • ES embryonic stem
  • LIF Leukaemia Inhibitory Factor
  • stem cell cultures can be further supported by culturing the stem cells in the presence of feeder cells or extracts thereof, e.g. mouse fibroblast cells. Under such conditions it is possible to maintain a number of stem cell types over many passages in culture whilst retaining the characteristic differentiation potential of the stem cells.
  • feeder cells or extracts thereof e.g. mouse fibroblast cells.
  • stem cells can only be maintained, or are best maintained, using medium that contains undefined components such as serum or serum extract.
  • fetal bovine serum variability in serum properties may be observed between serum originating from different geographical regions, different herds and different batches of serum. Such variability presents a particular impediment for the culture of stem cells, as serum from some sources or batches m may not support the maintenance and self-renewal of a particular type of stem cells.
  • some batches of serum may, for example, promote differentiation rather than self-renewal of stem cells.
  • FBS is subjected to a wide range of assays in an attempt to reduce the batch-to-batch variability.
  • assays typically include assays for the ability of FBS to support cloning and growth of murine myeloma cells and derived hybridomas; for the suitability of FBS for the attachment and proliferation of adherent cell lines, e.g. transformed human cells lines; and for the ability of FBS to support the growth of human diploid fibroblasts through multiple subcultures (such tests are described in the Invitrogen Corporation brochure "Fetal Bovine Serum").
  • further testing is required to determine whether serum from a particular batch or source is suitable for culturing ES cells.
  • further tests may include a plating efficiency assay using ES cells, a cytotoxicity assay testing the ability of media containing high proportions of serum to support the growth of low density ES and feeder cells, and assays of the morphology and differentiation characteristics of ES cells cultured in the presence of the FBS.
  • an object of the invention to provide an improved assay for determining the suitability of sera for the culture of stem cells.
  • a further object of the invention is to provide an assay suitable for testing the suitability of other cell culture media and medium supplements for stem cell cultures.
  • a still further object of the invention is to provide test cell lines suitable for use in the improved assay and kits for carrying out the assay.
  • a first aspect of the present invention provides a method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of desired stem cells in culture, the method comprising the steps of: a) culturing a first population of test stem cells in a reference medium so as to form ceil colonies; b) determining the proportion of cell colonies in step (a) retaining the differentiation potential of the desired stem cells; c) culturing a second population of test stem cells in a test medium which comprises the medium or medium supplement to be tested so as to form cell colonies; d) determining the proportion of cell colonies in step (c) retaining the differentiation potential of the desired stem cells; e) calculating the ability of the test medium, relative to the reference medium, to support the growth, maintenance and/or proliferation of the desired stem cells without loss of differentiation potential; wherein the test stem cells contain a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells.
  • a related aspect of the present invention provides a method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of desired stem cells in culture, the method comprising the steps of: a) culturing a first population of test stem cells in a reference medium; b) determining the proportion of cell colonies retaining the pluripotency of the desired stem cells in the culture of step (a); c) culturing a second population of test stem cells in a test medium; d) determining the proportion of cell colonies retaining the pluripotency of the desired stem cells in the culture of step (c); e) calculating the ability of the test medium, relative to the reference medium, to support the growth, maintenance and/or proliferation of the desired stem cells without loss of pluripotency; wherein the test stem cells contain at least one marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells.
  • the marker gene is preferentially expressed in the desired stem cells. This can be achieved using a promoter which regulates expression and which is preferentially active in the desired stem cells.
  • the promoter can be active in the desired stem cells and non-active in other cells.
  • stem cell embraces any cell having the capacity for self-renewal and the potential to differentiate into one or more other cell types.
  • stem cell includes pluripotential, multipotential or unipotential stem cells and progenitor cells from any tissue or stage of development.
  • the desired stem cells may be embryonic stem cells, gonadal stem cells, somatic stem cells, somatic progenitor cells, haematopoietic stem cells, epidermal stem cells or neuronal stem cells.
  • the stem cells are mammalian stem cells, for example murine stem cells, rat stem cells or human stem cells, preferably pluripotent cells, e.g. ES cells.
  • the methods of the invention can also be used to assess the suitability of culture media or medium supplements for the culture of other stem cells, including ES cells, derived from other organisms, such as American mink, hamster, pig, sheep, cow and primate stem cells.
  • test stem cells used in the assay of the invention may be any type of stem cell as described herein.
  • the test stem cells used in the assay will exhibit a similar phenotype to the desired stem cells, for example in respect of differentiation potential and source organism. It is preferred that the test stem cells used in the methods of the invention will exhibit the same differentiation potential as the desired stem cells, resulting in an assay that is specifically tailored to the type of stem cells that are to be cultured.
  • differentiation potential refers to the capacity of stem cells or progenitor cells to differentiate into one or more different cell types.
  • differentiation potential includes pluripotency, the capacity of stem cells to differentiate into cells derived from any of the three germ layers of endoderm, endoderm and mesoderm.
  • Stem cells may also have more limited differentiation potential.
  • multipotential stem cells can differentiate into cell types representative of a family of related lineages and unipotential stem cells can differentiate into cell types representative of only one lineage.
  • the test stem cells comprise a marker gene that encodes a gene product, the presence of which is visually detectable.
  • the marker gene product may be directly or indirectly detectable and many suitable marker genes are known.
  • the marker gene may encode a directly detectable protein, e.g. a fluorescent protein such as green fluorescent protein.
  • the presence of the marker gene product may be detected on exposure of the cells to an additional reagent or reagents.
  • the marker gene may encode an enzyme that can be detected on exposure of the cells to a chromogenic substrate.
  • ⁇ -galactosidase marker gene which will hydrolyse the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X- gal) to form a blue product.
  • Other indirect means of detecting marker gene expression include the use of a cell surface marker and detection using labelled antibodies.
  • the marker gene is operatively linked to a gene or gene fragment regulating expression, which gene or gene product is differentially active in stem and non-stem cells.
  • a gene or gene fragment regulating expression which gene or gene product is differentially active in stem and non-stem cells.
  • suitable regulatory elements e.g. promoters
  • the promoters of the Oct4 and Nanog genes would be suitable for directing ES cell-restricted expression of a marker gene.
  • the gene or gene fragment operatively linked to and regulating expression of the marker gene is associated with cells having the differentiation potential of the desired stem cells.
  • the gene or gene fragment will direct expression of the marker gene only in desired stem cells so that the assay can specifically target the effect of the test medium on the type of stem cell that is to be cultured.
  • the gene or gene fragment is associated with a pluripotential stage of development, that is the gene or gene fragment is active in pluripotential cells of the developing embryo, e.g. primitive ectoderm.
  • the gene or gene fragment may, for example be all or part of the Oct4 gene, such as the Oct4 promoter.
  • Octamer binding transcription factor 4 (Oct4) is a member of the POU family of transcription factors (reviewed by Scholer et al (1991) TIG, vol. 7, no. 10, pp323- 329). Oct4 transcription is activated between the 4- and 8-cell stages of the developing mouse embryo and is highly expressed in the expanding blastocyst and then in the pluripotent cells of the egg cylinder. Transcription is down-regulated as the primitive ectoderm differentiates to form mesoderm (Scholer et al (1990) Nature 344, pp435-439) and by 8.5 days post coitum is restricted to migrating primordial germ cells.
  • EP0695351 describes an embryonic stem cell line (OKO160) containing a ⁇ - galactosidase transgene under the control of the Oct4 promoter.
  • This cell line is particularly suitable in the methods of the invention, when used to assess the suitability of media or medium supplements for the culture of ES cells.
  • the methods of the invention can be used to test the suitability of any cell culture medium or medium supplement for culturing stem cells. Suitable cell culture media are well known in the art.
  • Cell culture medium supplements are also known, and include any component that is added to a cell culture medium to support or enhance the growth, maintenance or survival of cultured cells.
  • Medium supplements may be undefined or partially defined, e.g. sera, serum extracts, conditioned media and cell extracts.
  • the methods of the invention are particularly suitable for assaying the suitability of sera for the culture of stem cells.
  • the medium supplement can be any type of serum, including horse serum and fetal bovine serum.
  • Fully defined media for the culture of stem cells including ES cells
  • the methods of the present invention are suitable for assaying the suitability of such media for the culture of stem cells.
  • Fully defined media that may be assayed according to the methods of the invention include serum-free media and animal-free media. A number of such media are known in the art.
  • the assay method of the invention is a comparative assay in which the suitability for purpose of a test medium is evaluated with respect to a reference medium known to support the growth, maintenance and/or proliferation of the desired stem cells.
  • a reference medium known to support the growth, maintenance and/or proliferation of the desired stem cells.
  • the assay is carried out in respect of a cell culture medium
  • the reference medium and the test medium may differ in respect of one or more components.
  • the reference medium and test medium may be identically formulated, but result from different production batches.
  • the assay of the invention can be used to regulate and reduce inter-batch variability, resulting in a more reliable product, and to test the suitability of new medium formulations for stem cell culture.
  • the test medium when the assay is used to assess medium supplements, the test medium will differ from the reference medium in respect of the supplement to be tested.
  • the supplement may differ in nature from the supplement in the reference medium, for example if the assay is being used to evaluate the properties of an alternative medium component.
  • the supplement used in the test medium can be essentially the same as the supplement used in the reference medium, but differing in its source or production batch. Ideally, all other components of the reference medium and test medium will be the same.
  • the methods of the invention are particularly suitable for assessing the ability of different batches of serum, e.g. FBS, for the culture of stem cells.
  • serum e.g. FBS
  • Such assays have numerous applications including assessing and regulating the variability of serum properties between batches and identifying preferred sources of serum for a particular culture application.
  • the assay of the invention can be used to identify a geographical region, herd or abattoir that provides FBS with properties particularly suitable for the culture of desired stem cells.
  • the method includes further elements and steps, thereby providing a more detailed functional evaluation of the suitability of the medium of medium supplement for culturing stem cells.
  • the methods of the invention may further comprise assaying one or more of (i) the colony forming efficiency, (ii) the cloning efficiency, (iii) cytotoxicity, and (iv) the relative growth rate in the test medium, relative to the reference medium.
  • Such combined assays provide an advantage over known methods for assaying serum for ES cell culture in that all parts of the assay can be carried out using a single cell line.
  • a culture medium or medium supplement will be rejected as unsuitable for the culture of desired stem cells in the event that one or more of: the pluripotency efficiency or differentiation potential efficiency; the colony forming efficiency; the cloning efficiency; the cytotoxicity; and the relative growth rate is less than 100%. That is to say that the performance in one or more of the assay elements is decreased in the test medium in comparison to the reference medium. Conversely, if the performance in one or more of the assay elements is improved in the test medium, i.e. the relative performance in any one or more assay elements is greater or equal to 100%, then the medium or supplement will be accepted as suitable for the culture of desired stem cells.
  • the cut-off point for acceptance or rejection of a medium or medium supplement may be other than 100%. For example, in some circumstances a relative performance of about 1%, 5%, 10%, 20%, 30%, 40%, or 50% above or below 100% may be acceptable in one or more of the assay elements.
  • the method comprises the further steps of: f) determining the number of cell colonies in the cultures of steps (a) and (c); and g) calculating the relative colony forming efficiency.
  • test stem cells are seeded at low density in order to assess the ability of the test medium to support colony formation.
  • the cell colonies formed may or may not derive from a single originating cell.
  • the cells may be seeded at a density that delivers less than one cell per culture well in order to assay the cloning efficiency supported by the test medium. In this case, each resultant colony is assumed to be derived from a single originating cell.
  • the method further comprises the steps of: h) culturing a third population of test stem cells in a second reference medium comprising a higher concentration of reference medium supplement than the reference medium of step (a); i) culturing a fourth population of test stem cells in a second test medium comprising a higher concentration of the medium supplement than the test medium of step (c); j) determining the relative number of cell colonies in the cultures of steps (a) and (h) and of steps (c) and (i); and k) calculating the relative cytotoxicity of the medium supplement.
  • Th is embodiment of the invention allows any potential cytotoxic effect of the medium supplement being evaluated to be determined.
  • the reference and test media of cultures (a) and (c) may contain a low proportion of serum, typically 5% or 10%, and the reference and test media of cultures (h) and (i) may contain a high proportion of serum, typically about 30%.
  • a small reduction in colony formation at high serum concentration is acceptable, within defined limits.
  • the "high serum” test culture exhibits a significantly greater reduction in colony numbers relative to the "low serum” test culture than the reduction in colony numbers observed in the reference medium cultures, the serum being assayed will be rejected as unsuitable for the culture of stem cells.
  • the method further comprises the steps of:
  • the growth efficiency assay of steps (I) to (n) provides a measure of the ability of the test medium to sustain stem cell growth through multiple passages. Typically, the assay will require three sequential subcultures. At each passage, the cells in reference medium are harvested, counted, and seeded at the same cell density into fresh culture medium.
  • the invention provides a test stem cell line for use in a method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of the desired stem cells without loss of differentiation potential.
  • the test stem cell line is for use in the method of the invention, as described herein, and the test stem cells contain a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells.
  • the test stem cell line is obtainable by genetic modification of desired stem cells as described herein, for example via the introduction of a stably integrated or episomally maintained construct.
  • the genetic modification may comprise operatively inserting the marker gene into an endogenous gene of the desired stem cells and may involve any suitable method, for example transfection, lipofection, ballistic missile, viral vector, electroporation, or any other means.
  • Another aspect of the invention provides use of a stem cell comprising a marker gene, which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells in an assay for determining the ability of a cell culture medium or medium supplement to support the growth, maintenance and/or proliferation of desired stem cells.
  • the stem cell may be any type of stem cell as described herein, including an embryonic stem cell, a gonadal stem cell, a somatic stem/progenitor cell, a haematopoietic stem cell, an epidermal stem cell, or a neuronal stem cell.
  • the stem cell is an embryonic stem cell.
  • stem cells used according to this aspect of the invention are mammalian stem cells, such as murine stem cells and human stem cells.
  • the stem cells of this aspect of the invention will comprise a marker gene, as described herein in relation to other aspects of the invention.
  • the desired stem cells are ES cells
  • the OK0160 ES cell line is preferably used.
  • the stem cells may be used to determine the suitability of any medium or medium supplement as described herein to support the growth, maintenance and/or proliferation of desired stem cells.
  • a further embodiment of the invention provides a kit comprising, in one or more containers, (a) a population of test stem cells, wherein the test stem cells contain a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells, (b) a reference cell culture medium or medium supplement which supports the growth, maintenance and/or proliferation of the test stem cells, and (c) a test cell culture medium or medium supplement.
  • kits of the invention may also comprise further components that may assist in carrying out the assay of the invention. These may include cell culture flasks or plates, enzymatic or non-enzymatic cell dissociation buffers, and other components.
  • OKO160 Murine Oct4/ ⁇ -galactosidase transgenic ES cells, SCS Ltd.
  • OKO160 cells are normally cultured in a standard media DMEM supplemented with Na pyruvate, NEAA, 10% FBS, LIF, and 0.1mM ⁇ - mercaptoethanol on 0.1% gelatin-coated culture vessels OR, complete ESGROTM (Chemicon) serum-free media on 0.1% gelatin-coated culture vessels (Reference).
  • the OKO160 cells used cells are cultured in the 'Reference' complete, serum-free ESGROTM (Chemicon) media on both gelatin- and collagen IV-coated culture vessels.
  • ESGROTM serum-free ESGROTM
  • the plates are incubated for approximately 5-7 days at 37°C and 5% CO 2 in a humidified incubator with media changes every other day. This is to avoid build up of, primarily, ammonia from the break down of amino acids, glutamine in particular, which will affect the pH. - 14 -
  • % Colony Pluripotency Efficiency [Total 1/Total 1+Total 2] x 100
  • IV is optional. One can use one or the other, depending on experimental focus. For example, if comparing FBS then use gelatin and if comparing AF media, use collagen IV. If assay is comparing both, use gelatin and collagen IV if practical (dependent on number of Test media).
  • the three sequential subcultures ensure a true measurement of the test lot's growth-promoting ability by minimizing any residual effects of the previous growth medium.
  • the OKO160 cells will evaluate the serums ability to maintain mouse ES cell pluripotency using the Oct4/ ⁇ -galactosidase reporter.
  • the Certificate of Analysis provided with each lot of FBS, reports the average number of cells per duplicate flask from the third subculture for each cell type and assay.
  • Detroit551 Human Diploid Skin Fibroblast, ATCC #CCL-110. Detroit551 cells are normally cultured in a standard media DMEM supplemented with
  • OKO160 (Murine Oct4/ ⁇ -galactosidase transgenic ES cells, SCS Ltd). OKO160 cells are normally cultured in a standard media DMEM supplemented with 10% FBS, LIF, 0.1mM ⁇ -mercaptoethanol on 0.1 % gelatin- coated culture vessels.
  • the growth promotion assay is performed as follows:
  • OKO160 cells are inoculated in 9mL of the appropriate growth medium supplemented with 5% 'reference' and 'test' FBS in duplicate 25 cm 2 flasks. The flasks are incubated in a 4-6% CO 2 , 37°C ⁇ 2°C atmosphere for 7 days or 80-90% confluence, whichever comes first, with complete media replenishment every other day.
  • RGR relative growth rate
  • % RGR average cells/flask in test serum ⁇ average cells/flask in reference serum x 100 4b.
  • a measure of pluripotency maintenance capability of the reference versus test FBS-supplemented media can also be determined. This is done by performing a ⁇ -galactosidase assay at the end of passage 3 with one of the T25cm 2 flasks from each of the standard/10% FBS and low density/5% FBS conditions.
  • the relative pluripotency efficiency (RPE) in each test serum is expressed as a fraction of the ⁇ -galactosidase positive ( ⁇ -gal + ) colonies obtained in the reference serum:
  • % RPE ⁇ -gal + cells/flask in test serum ⁇ ⁇ -gal + cells/flask in reference serum x 100
  • Example 4 Combined colony forming efficiency, growth, cytotoxicity and pluripotencv assay
  • RPE % ⁇ -gal + Colonies Test ⁇ % ⁇ -gal + Colonies std x 100
  • RGR Cells Test /T25 + Cells std /T25 x 100
  • the Colony Forming Efficiency is essentially a measure cloning or plating efficiency by evaluating percentage of single cells seeded that give rise to single colonies.
  • the pluripotency efficiency is indicative of the percentage of these colonies formed that retain ⁇ -gal expression as driven by Oct4.
  • the former indicators are repeated for the high percentage serum concentration to assess cytotoxic effects.
  • the relative growth rate is evaluated by the stressful split ratio, number of population doublings and reduced serum as stringent indicators of the serum's ability to support growth of the cells.

Abstract

Assays are provided for assessing the suitability of cell culture media and medium supplements for the culture of particular cell types, particularly stem cells, including embryonic stem cells.

Description

ASSAY FORCELL CULTURE MEDIAAND MEDIUM SUPPLEMENTS
The present invention relates to assays for assessing the suitability of cell culture media and medium supplements for the culture of particular cell types, particularly stem cells, including embryonic stem cells. In particular, the invention relates to assays for assessing the suitability of a particular batch of an undefined medium or medium supplement, such as serum, for the culture of stem cells.
The establishment and maintenance of stem cell cultures in vitro, including cultures of pluripotent stem cells such as embryonic stem (ES) cells, is well known. For example, the culture of pluripotent stem cell cultures in the presence of medium containing serum and Leukaemia Inhibitory Factor (LIF) is described in Smith et al.
(1988) Nature 336: 688-90. Maintenance and self-renewal of such stem cell cultures can be further supported by culturing the stem cells in the presence of feeder cells or extracts thereof, e.g. mouse fibroblast cells. Under such conditions it is possible to maintain a number of stem cell types over many passages in culture whilst retaining the characteristic differentiation potential of the stem cells.
In many cases stem cells can only be maintained, or are best maintained, using medium that contains undefined components such as serum or serum extract.
However, such undefined components frequently exhibit variable properties depending on the source or particular production batch of that component. In the case of serum, for example fetal bovine serum (FBS), variability in serum properties may be observed between serum originating from different geographical regions, different herds and different batches of serum. Such variability presents a particular impediment for the culture of stem cells, as serum from some sources or batches m may not support the maintenance and self-renewal of a particular type of stem cells.
Rather, some batches of serum may, for example, promote differentiation rather than self-renewal of stem cells.
Traditionally FBS is subjected to a wide range of assays in an attempt to reduce the batch-to-batch variability. In addition to standard physical, chemical, biochemical and microbiological tests, a number of functional assays are typically carried out in respect of the performance of the serum. Such assays typically include assays for the ability of FBS to support cloning and growth of murine myeloma cells and derived hybridomas; for the suitability of FBS for the attachment and proliferation of adherent cell lines, e.g. transformed human cells lines; and for the ability of FBS to support the growth of human diploid fibroblasts through multiple subcultures (such tests are described in the Invitrogen Corporation brochure "Fetal Bovine Serum"). Despite the use of several assays using different cell lines, further testing is required to determine whether serum from a particular batch or source is suitable for culturing ES cells. These further tests may include a plating efficiency assay using ES cells, a cytotoxicity assay testing the ability of media containing high proportions of serum to support the growth of low density ES and feeder cells, and assays of the morphology and differentiation characteristics of ES cells cultured in the presence of the FBS.
Such extensive testing does not guarantee the suitability of a given batch of FBS for the culture of a given ES cell line and provides little or no help in assessing the suitability of FBS for the culture of other types of stem cell. Moreover, the numerous assays used to test sera such as "ES cell qualified FBS" involve significant time and effort and require the maintenance and culture of many different cell lines. Despite this extensive testing, it may be necessary for the end user to carry out further testing of the suitability of a given batch of serum for culture of a particular cell line or cell type.
Thus, there is a need for a better method for assessing the suitability of sera for the culture of stem cells. Accordingly, it is an object of the invention to provide an improved assay for determining the suitability of sera for the culture of stem cells. A further object of the invention is to provide an assay suitable for testing the suitability of other cell culture media and medium supplements for stem cell cultures. A still further object of the invention is to provide test cell lines suitable for use in the improved assay and kits for carrying out the assay. Accordingly, a first aspect of the present invention provides a method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of desired stem cells in culture, the method comprising the steps of: a) culturing a first population of test stem cells in a reference medium so as to form ceil colonies; b) determining the proportion of cell colonies in step (a) retaining the differentiation potential of the desired stem cells; c) culturing a second population of test stem cells in a test medium which comprises the medium or medium supplement to be tested so as to form cell colonies; d) determining the proportion of cell colonies in step (c) retaining the differentiation potential of the desired stem cells; e) calculating the ability of the test medium, relative to the reference medium, to support the growth, maintenance and/or proliferation of the desired stem cells without loss of differentiation potential; wherein the test stem cells contain a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells.
A related aspect of the present invention provides a method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of desired stem cells in culture, the method comprising the steps of: a) culturing a first population of test stem cells in a reference medium; b) determining the proportion of cell colonies retaining the pluripotency of the desired stem cells in the culture of step (a); c) culturing a second population of test stem cells in a test medium; d) determining the proportion of cell colonies retaining the pluripotency of the desired stem cells in the culture of step (c); e) calculating the ability of the test medium, relative to the reference medium, to support the growth, maintenance and/or proliferation of the desired stem cells without loss of pluripotency; wherein the test stem cells contain at least one marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells.
In preferred embodiments of the invention the marker gene is preferentially expressed in the desired stem cells. This can be achieved using a promoter which regulates expression and which is preferentially active in the desired stem cells. For example the promoter can be active in the desired stem cells and non-active in other cells.
Such methods can be used to test the suitability of cell culture media or medium supplements for the culture of any type of stem cell that may be of interest, i.e. any desired stem cell. It is intended, for the purposes of the present invention, that the term stem cell embraces any cell having the capacity for self-renewal and the potential to differentiate into one or more other cell types. Thus, the term stem cell includes pluripotential, multipotential or unipotential stem cells and progenitor cells from any tissue or stage of development. For example, the desired stem cells may be embryonic stem cells, gonadal stem cells, somatic stem cells, somatic progenitor cells, haematopoietic stem cells, epidermal stem cells or neuronal stem cells. In one embodiment, the stem cells are mammalian stem cells, for example murine stem cells, rat stem cells or human stem cells, preferably pluripotent cells, e.g. ES cells.
The methods of the invention can also be used to assess the suitability of culture media or medium supplements for the culture of other stem cells, including ES cells, derived from other organisms, such as American mink, hamster, pig, sheep, cow and primate stem cells.
The test stem cells used in the assay of the invention may be any type of stem cell as described herein. Preferably the test stem cells used in the assay will exhibit a similar phenotype to the desired stem cells, for example in respect of differentiation potential and source organism. It is preferred that the test stem cells used in the methods of the invention will exhibit the same differentiation potential as the desired stem cells, resulting in an assay that is specifically tailored to the type of stem cells that are to be cultured.
The term differentiation potential refers to the capacity of stem cells or progenitor cells to differentiate into one or more different cell types. The term differentiation potential includes pluripotency, the capacity of stem cells to differentiate into cells derived from any of the three germ layers of endoderm, endoderm and mesoderm.
Stem cells may also have more limited differentiation potential. For example, multipotential stem cells can differentiate into cell types representative of a family of related lineages and unipotential stem cells can differentiate into cell types representative of only one lineage.
Typically the test stem cells comprise a marker gene that encodes a gene product, the presence of which is visually detectable. The marker gene product may be directly or indirectly detectable and many suitable marker genes are known. For example, the marker gene may encode a directly detectable protein, e.g. a fluorescent protein such as green fluorescent protein. Alternatively, the presence of the marker gene product may be detected on exposure of the cells to an additional reagent or reagents. For example, the marker gene may encode an enzyme that can be detected on exposure of the cells to a chromogenic substrate. One well- known system for achieving this is the use of a β-galactosidase marker gene, which will hydrolyse the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X- gal) to form a blue product. Other indirect means of detecting marker gene expression include the use of a cell surface marker and detection using labelled antibodies.
Typically, the marker gene is operatively linked to a gene or gene fragment regulating expression, which gene or gene product is differentially active in stem and non-stem cells. Many suitable regulatory elements, e.g. promoters, are known and the skilled person would have no difficulty in selecting a promoter which directs expression in stem cells but not in non-stem cells. For example, the promoters of the Oct4 and Nanog genes would be suitable for directing ES cell-restricted expression of a marker gene.
In the context of the present invention, it is preferred that the gene or gene fragment operatively linked to and regulating expression of the marker gene is associated with cells having the differentiation potential of the desired stem cells. Ideally, the gene or gene fragment will direct expression of the marker gene only in desired stem cells so that the assay can specifically target the effect of the test medium on the type of stem cell that is to be cultured. Thus, in embodiments in which the suitability of media or supplements for the culture of ES cells is being assayed, it is preferred that the gene or gene fragment is associated with a pluripotential stage of development, that is the gene or gene fragment is active in pluripotential cells of the developing embryo, e.g. primitive ectoderm. The gene or gene fragment may, for example be all or part of the Oct4 gene, such as the Oct4 promoter.
Octamer binding transcription factor 4 (Oct4) is a member of the POU family of transcription factors (reviewed by Scholer et al (1991) TIG, vol. 7, no. 10, pp323- 329). Oct4 transcription is activated between the 4- and 8-cell stages of the developing mouse embryo and is highly expressed in the expanding blastocyst and then in the pluripotent cells of the egg cylinder. Transcription is down-regulated as the primitive ectoderm differentiates to form mesoderm (Scholer et al (1990) Nature 344, pp435-439) and by 8.5 days post coitum is restricted to migrating primordial germ cells. High level Oct4 expression is also observed in pluripotent embryo carcinoma and ES cell lines and is down regulated when these cells are induced to differentiate (Scholer et al (1989) EMBO J 8, pp2551-2557; Okamoto et al (1990) Cell 60, pp461-472).
EP0695351 describes an embryonic stem cell line (OKO160) containing a β- galactosidase transgene under the control of the Oct4 promoter. This cell line is particularly suitable in the methods of the invention, when used to assess the suitability of media or medium supplements for the culture of ES cells. The methods of the invention can be used to test the suitability of any cell culture medium or medium supplement for culturing stem cells. Suitable cell culture media are well known in the art. Cell culture medium supplements are also known, and include any component that is added to a cell culture medium to support or enhance the growth, maintenance or survival of cultured cells. Medium supplements may be undefined or partially defined, e.g. sera, serum extracts, conditioned media and cell extracts. The methods of the invention are particularly suitable for assaying the suitability of sera for the culture of stem cells. In such embodiments, the medium supplement can be any type of serum, including horse serum and fetal bovine serum.
There is also considerable interest in the development of fully defined media for the culture of stem cells, including ES cells, and the methods of the present invention are suitable for assaying the suitability of such media for the culture of stem cells. Fully defined media that may be assayed according to the methods of the invention include serum-free media and animal-free media. A number of such media are known in the art.
It is envisaged that the assay method of the invention is a comparative assay in which the suitability for purpose of a test medium is evaluated with respect to a reference medium known to support the growth, maintenance and/or proliferation of the desired stem cells. When the assay is carried out in respect of a cell culture medium, the reference medium and the test medium may differ in respect of one or more components. Alternatively, the reference medium and test medium may be identically formulated, but result from different production batches. Thus, the assay of the invention can be used to regulate and reduce inter-batch variability, resulting in a more reliable product, and to test the suitability of new medium formulations for stem cell culture.
Similarly, when the assay is used to assess medium supplements, the test medium will differ from the reference medium in respect of the supplement to be tested. The supplement may differ in nature from the supplement in the reference medium, for example if the assay is being used to evaluate the properties of an alternative medium component. Alternatively, the supplement used in the test medium can be essentially the same as the supplement used in the reference medium, but differing in its source or production batch. Ideally, all other components of the reference medium and test medium will be the same.
The methods of the invention are particularly suitable for assessing the ability of different batches of serum, e.g. FBS, for the culture of stem cells. Such assays have numerous applications including assessing and regulating the variability of serum properties between batches and identifying preferred sources of serum for a particular culture application. For example, the assay of the invention can be used to identify a geographical region, herd or abattoir that provides FBS with properties particularly suitable for the culture of desired stem cells.
In further embodiments of the invention, the method includes further elements and steps, thereby providing a more detailed functional evaluation of the suitability of the medium of medium supplement for culturing stem cells. The methods of the invention may further comprise assaying one or more of (i) the colony forming efficiency, (ii) the cloning efficiency, (iii) cytotoxicity, and (iv) the relative growth rate in the test medium, relative to the reference medium. Such combined assays provide an advantage over known methods for assaying serum for ES cell culture in that all parts of the assay can be carried out using a single cell line. Moreover, it is possible to combine one or more of the assay elements within a single experiment, thus providing a significant reduction in resources and time.
Typically a culture medium or medium supplement will be rejected as unsuitable for the culture of desired stem cells in the event that one or more of: the pluripotency efficiency or differentiation potential efficiency; the colony forming efficiency; the cloning efficiency; the cytotoxicity; and the relative growth rate is less than 100%. That is to say that the performance in one or more of the assay elements is decreased in the test medium in comparison to the reference medium. Conversely, if the performance in one or more of the assay elements is improved in the test medium, i.e. the relative performance in any one or more assay elements is greater or equal to 100%, then the medium or supplement will be accepted as suitable for the culture of desired stem cells. In some embodiments, the cut-off point for acceptance or rejection of a medium or medium supplement may be other than 100%. For example, in some circumstances a relative performance of about 1%, 5%, 10%, 20%, 30%, 40%, or 50% above or below 100% may be acceptable in one or more of the assay elements.
In one embodiment, the method comprises the further steps of: f) determining the number of cell colonies in the cultures of steps (a) and (c); and g) calculating the relative colony forming efficiency.
Typically, the test stem cells are seeded at low density in order to assess the ability of the test medium to support colony formation. The cell colonies formed may or may not derive from a single originating cell. Optionally, the cells may be seeded at a density that delivers less than one cell per culture well in order to assay the cloning efficiency supported by the test medium. In this case, each resultant colony is assumed to be derived from a single originating cell.
In a further embodiment, the method further comprises the steps of: h) culturing a third population of test stem cells in a second reference medium comprising a higher concentration of reference medium supplement than the reference medium of step (a); i) culturing a fourth population of test stem cells in a second test medium comprising a higher concentration of the medium supplement than the test medium of step (c); j) determining the relative number of cell colonies in the cultures of steps (a) and (h) and of steps (c) and (i); and k) calculating the relative cytotoxicity of the medium supplement. Th is embodiment of the invention allows any potential cytotoxic effect of the medium supplement being evaluated to be determined. For example, when the method is used to assay serum, the reference and test media of cultures (a) and (c) may contain a low proportion of serum, typically 5% or 10%, and the reference and test media of cultures (h) and (i) may contain a high proportion of serum, typically about 30%. Generally, a small reduction in colony formation at high serum concentration is acceptable, within defined limits. However, if the "high serum" test culture exhibits a significantly greater reduction in colony numbers relative to the "low serum" test culture than the reduction in colony numbers observed in the reference medium cultures, the serum being assayed will be rejected as unsuitable for the culture of stem cells.
In another embodiment, the method further comprises the steps of:
I) propagating the stem cells of each of steps (a) and (c) through multiple sequential subcultures; m) determining the number of cell colonies in each of the final subcultures derived from steps (a) and (c); and n) calculating the relative growth efficiency.
The growth efficiency assay of steps (I) to (n) provides a measure of the ability of the test medium to sustain stem cell growth through multiple passages. Typically, the assay will require three sequential subcultures. At each passage, the cells in reference medium are harvested, counted, and seeded at the same cell density into fresh culture medium.
According to a further aspect, the invention provides a test stem cell line for use in a method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of the desired stem cells without loss of differentiation potential. Preferably, the test stem cell line is for use in the method of the invention, as described herein, and the test stem cells contain a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells. Preferably, the test stem cell line is obtainable by genetic modification of desired stem cells as described herein, for example via the introduction of a stably integrated or episomally maintained construct. The genetic modification may comprise operatively inserting the marker gene into an endogenous gene of the desired stem cells and may involve any suitable method, for example transfection, lipofection, ballistic missile, viral vector, electroporation, or any other means.
Another aspect of the invention provides use of a stem cell comprising a marker gene, which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells in an assay for determining the ability of a cell culture medium or medium supplement to support the growth, maintenance and/or proliferation of desired stem cells.
In this aspect of the invention the stem cell may be any type of stem cell as described herein, including an embryonic stem cell, a gonadal stem cell, a somatic stem/progenitor cell, a haematopoietic stem cell, an epidermal stem cell, or a neuronal stem cell. Preferably the stem cell is an embryonic stem cell. It is preferred that stem cells used according to this aspect of the invention are mammalian stem cells, such as murine stem cells and human stem cells.
It is envisaged that the stem cells of this aspect of the invention will comprise a marker gene, as described herein in relation to other aspects of the invention. In an embodiment in which the desired stem cells are ES cells, the OK0160 ES cell line is preferably used.
According to this aspect of the invention, the stem cells may be used to determine the suitability of any medium or medium supplement as described herein to support the growth, maintenance and/or proliferation of desired stem cells.
A further embodiment of the invention provides a kit comprising, in one or more containers, (a) a population of test stem cells, wherein the test stem cells contain a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells, (b) a reference cell culture medium or medium supplement which supports the growth, maintenance and/or proliferation of the test stem cells, and (c) a test cell culture medium or medium supplement.
It is envisaged that components (a) to (c) of the kit are as described herein in relation to other aspects of the invention. The kits of the invention may also comprise further components that may assist in carrying out the assay of the invention. These may include cell culture flasks or plates, enzymatic or non-enzymatic cell dissociation buffers, and other components.
The invention will now be described in more detail in the following examples.
Example 1 - Pluripotencv assay
Aim. To evaluate FBS and animal-free (AF) media formulations by a cell-based assay as a propagation and pluripotency maintenance media for HESC culture.
Methodology. The following cell lines are validated for use in the AF media comparison assays:
• OKO160 (Murine Oct4/β-galactosidase transgenic ES cells, SCS Ltd).
OKO160 cells are normally cultured in a standard media DMEM supplemented with Na pyruvate, NEAA, 10% FBS, LIF, and 0.1mM β- mercaptoethanol on 0.1% gelatin-coated culture vessels OR, complete ESGRO™ (Chemicon) serum-free media on 0.1% gelatin-coated culture vessels (Reference).
1. The OKO160 cells used cells are cultured in the 'Reference' complete, serum-free ESGRO™ (Chemicon) media on both gelatin- and collagen IV-coated culture vessels. 2. Pre-coat 2x 12-well plates separately with 0.1% (1mg/ml_) gelatin/PBS and 10ug/cm2 human collagen IV/0.1M acetic acid.
3. Note: Approximately 48mL of media for each reference and test sample will be required for the duration of the experiment with triplicate wells per 'Reference' (see above) and 'Test' samples.
4a. Dissociate OKO160 to single cells by enzymatic treatment (TrypLE or Trypsin/EDTA), quench by dilution into 1OmL DMEM-F12, and then determine cell viability and count by Trypan Blue/haemocytometer.
4b. Centrifuge down and resuspend cell pellet in DMEM-F12 to 1x106 cells/mL, then dilute 1 :10 (10OuL + 90OuL DMEM-F12) to finally give 1x104 cells/mL
4c. Dispense 12.5uL into each well of each plate (1250 cells/well or 330 cells/cm2). Note: Stagger cell addition if have multiple plates to avoid drying out.
4d. Dispense 2mL of appropriate media to wells.
5. The plates are incubated for approximately 5-7 days at 37°C and 5% CO2 in a humidified incubator with media changes every other day. This is to avoid build up of, primarily, ammonia from the break down of amino acids, glutamine in particular, which will affect the pH. - 14 -
Note: The number of days incubation is determined by the visibility/size of the colonies.
6. Following the incubation period, the plates are stained with X-gal, observed macroscopically, and the stained (blue, β-gal+) and unstained (white, β-gal") colonies, are counted. Data are recorded as indicated in Table 1.
Table 1
Figure imgf000015_0001
7. Pluripotency efficiencies are calculated as follows:
% Colony Pluripotency Efficiency (%CPE) = [Total 1/Total 1+Total 2] x 100
8. General Notes. (i) Reference media use (FBS-based versus ESGRO™) will depend if assay is geared towards comparing FBS or AF media.
(ii) Can be scaled up or down, depending on number of test media, by maintaining cell/cm2 concentration in culture vessel
(e.g., for 96-well plate, use 105 cells per well).
(iii) Culture vessel pre-coating by both gelatin and collagen
IV is optional. One can use one or the other, depending on experimental focus. For example, if comparing FBS then use gelatin and if comparing AF media, use collagen IV. If assay is comparing both, use gelatin and collagen IV if practical (dependent on number of Test media).
Example 2 - Combined colony-forming efficiency and pluripotencv assay
Aim. To evaluate FBS and animal-free (AF) media formulations by a cell-based clonal and growth assays as a prelude to their testing as a propagation and pluripotency maintenance media for HESC culture.
Methodology. The experimental protocol is as set out in Example 1.
Colony forming and pluripotency efficiencies are calculated as follows:
% Colony Forming Efficiency (%CFE) = [Total 1+ Total 2/1250 (cells inoculated per well)] x 100 % Colony Pluripotency Efficiency (%CPE) = [Total 1/Total 1+Total 2] x 100
Example 3 - Combined growth and pluripotency assay
Background. Normal diploid human fibroblasts (Detroit 551) and murine Oct4/βgalactosidase transgenic ES cells (OKO 160) are inoculated into duplicate 25 cm2 flasks at low density (2 x 105 cells/flask) with 5% test and reference FBS, and propagated through three sequential subcultures at 7-days or 80-90% confluence, whichever comes first. At each passage, the cells are subcultured to the original cell inoculation density. The stressful split ratio, number of population doublings, and reduced-serum concentration are stringent measurements of the serum lots ability to support growth of fastidious mammalian diploid cells. The three sequential subcultures ensure a true measurement of the test lot's growth-promoting ability by minimizing any residual effects of the previous growth medium. Moreover, the OKO160 cells will evaluate the serums ability to maintain mouse ES cell pluripotency using the Oct4/β-galactosidase reporter. The Certificate of Analysis, provided with each lot of FBS, reports the average number of cells per duplicate flask from the third subculture for each cell type and assay.
Aim. To evaluate 'reference' and 'test' FBS in a cell-based growth assay using diploid human and mouse cells.
Methodology. The following cell lines are validated for use in this growth assay:
• Detroit551 (Human Diploid Skin Fibroblast, ATCC #CCL-110). Detroit551 cells are normally cultured in a standard media DMEM supplemented with
10% FBS, 0.1mM β-mercaptoethanol. • OKO160 (Murine Oct4/β-galactosidase transgenic ES cells, SCS Ltd). OKO160 cells are normally cultured in a standard media DMEM supplemented with 10% FBS, LIF, 0.1mM β-mercaptoethanol on 0.1 % gelatin- coated culture vessels.
The growth promotion assay is performed as follows:
1. For each cell type, fresh 5% serum-supplemented growth medium is prepared for each media change and subculture during the assay, as required.
2. Cell culture flasks are seeded as indicated in Table 2; 2 x 105 Detroit551 and
OKO160 cells are inoculated in 9mL of the appropriate growth medium supplemented with 5% 'reference' and 'test' FBS in duplicate 25 cm2 flasks. The flasks are incubated in a 4-6% CO2, 37°C±2°C atmosphere for 7 days or 80-90% confluence, whichever comes first, with complete media replenishment every other day.
NOTE: Culture under standard media (see above), seeding (3-4 x 104 cells/cm2) and passaging conditions in a single T25cm2 is also included as a normal growth control for the OKO160 cells.
Table 2
Figure imgf000019_0001
* - For passage 3, seed 2 x 105 cells into 3x25cm2 flask as one flask will be used for a β-galactosidase assay (see 4b).
3. Morphology of each cell type is monitored and recorded and cells in each of the duplicate flasks per reference or test serum are quantitatively harvested by trypsinization (TrypLE) at day 7 or 80-90% confluence, whichever comes first, then pooled, counted and recorded. Cells are subcultured by inoculating 2 x 105 cells into new duplicate T25cm2 flasks containing fresh growth medium that has been supplemented with the same lot of reference or test FBS used for the previous passage. The flasks are then incubated as described in step 2.
4a. The assay is continued as described through three passages, and the average number of cells/flask for each reference or test serum is calculated and recorded at the end of each passage. The relative growth rate (RGR) in each test media during the final subculture is expressed as a fraction of the cell growth obtained in the reference serum:
% RGR = average cells/flask in test serum ÷ average cells/flask in reference serum x 100 4b. For the OKO160 mouse ES cells, a measure of pluripotency maintenance capability of the reference versus test FBS-supplemented media can also be determined. This is done by performing a β-galactosidase assay at the end of passage 3 with one of the T25cm2 flasks from each of the standard/10% FBS and low density/5% FBS conditions. The relative pluripotency efficiency (RPE) in each test serum is expressed as a fraction of the β-galactosidase positive (β-gal+) colonies obtained in the reference serum:
% RPE = β-gal+ cells/flask in test serum ÷ β-gal+ cells/flask in reference serum x 100
Example 4 - Combined colony forming efficiency, growth, cytotoxicity and pluripotencv assay
1. Overview.
1.1. Based on mouse Oct4/β-galactosidase transgenic embryonic stem cell line
(OKO160) where the pluripotent marker gene Oct4 regulatory elements drive expression of β-galactosidase (β-gal) which then hydrolyses the chromogenic substrate δ-bromo^-chloro-S-indolyl-beta-D-galactopyranoside (X-gal). 1.2. Adaptable "all-in-one" assay for cloning/plating efficiency, pluripotency, cytotoxicity and growth. 1.3. Scaleable (Multi-well format to T25cm2 flask).
2. Assay description.
2.1. Multi-well Format:
2.1.1. 330 cells/cm2 seeding in 10% and 30% FBS (Test and Std).
2.1.2. Colony Forming Efficiency (10% FBS) %CFE = Total Colonies/cm2 ÷ 330cm2 x 100 Relative CFE = %CFETest + %CFEstd
2.1.3. Relative Pluripotency Efficiency (10% FBS)
RPE = %β-gal+ ColoniesTest ÷ %β-gal+ Coloniesstd x 100
2.1.4. Cytotoxicity (30% FBS) %CFE = Total Colonies/cm2 ÷ 330cm2 x 100
Relative Cytotoxicity = %CFETest ÷ %CFEstd
2.2. T25cm2 Format:
2.2.1. Duplicates, 5% FBS (Test and Std), Low seeding density, minimum 3 passages. 2.2.2. Relative Growth Rate
RGR = CellsTest/T25 + Cellsstd/T25 x 100
The Colony Forming Efficiency is essentially a measure cloning or plating efficiency by evaluating percentage of single cells seeded that give rise to single colonies. The pluripotency efficiency is indicative of the percentage of these colonies formed that retain β-gal expression as driven by Oct4.
The former indicators are repeated for the high percentage serum concentration to assess cytotoxic effects.
The relative growth rate is evaluated by the stressful split ratio, number of population doublings and reduced serum as stringent indicators of the serum's ability to support growth of the cells.

Claims

1. A method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of desired stem cells in culture, the method comprising the steps of:
a) culturing a first population of test stem cells in a reference medium; b) determining the proportion of cell colonies retaining the differentiation potential of the desired stem cells in the culture of step (a); c) culturing a second population of test stem cells in a test medium which comprises the medium or medium supplement to be tested; d) determining the proportion of cell colonies retaining the differentiation potential of the desired stem cells in the culture of step (c); e) calculating the ability of the test medium, relative to the reference medium, to support the growth, maintenance and/or proliferation of the desired stem cells without loss of differentiation potential; wherein the test stem cells contain a marker gene which is differentially expressed in
(i) desired stem cells and (ii) cells other than desired stem cells.
2. A method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of desired stem cells in culture, the method comprising the steps of: a) culturing a first population of test stem cells in a reference medium; b) determining the proportion of cell colonies retaining the pluripotency of the desired stem cells in the culture of step (a); c) culturing a second population of test stem cells in a test medium; d) determining the proportion of cell colonies retaining the pluripotency of the desired stem cells in the culture of step (c); e) calculating the ability of the test medium, relative to the reference medium, to support the growth, maintenance and/or proliferation of the desired stem cells without loss of pluripotency; wherein the test stem cells contain at least one marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells.
3. A method according to Claim 1 or Claim 2, wherein the desired stem cells are selected from embryonic stem cells, gonadal stem cells, somatic stem cells, somatic progenitor cells, haematopoietic stem cells, epidermal stem cells, and neuronal stem cells.
4. A method according to Claim 3, wherein the desired stem cells are pluripotent stem cells, e.g. embryonic stem cells.
5. A method according to Claim 3 or Claim 4, wherein the desired stem cells are mammalian stem cells.
6. A method according to Claim 5, wherein the desired stem cells are murine stem cells.
7. A method according to Claim 5, wherein the desired stem cells are human stem cells.
8. A method according to any preceding claim, wherein the test stem cells and the desired stem cells possess the same differentiation potential.
9. A method according to any preceding claim, wherein the medium supplement is serum.
10. A method according to Claim 9, wherein the serum is fetal bovine serum (FBS).
11. A method according to any one of Claims 1 to 8, wherein the cell culture medium is a serum-free medium or an animal-free medium.
12. A method according to any preceding claim further comprising assaying one or more of (i) the colony forming efficiency, (ii) the cloning efficiency, (iii) cytotoxicity, and (iv) the relative growth rate in the test medium, relative to the reference medium.
13. A method according to any preceding claim further comprising the steps of: i) determining the number of cell colonies in the cultures of steps (a) and (c); and ii) calculating the relative colony forming efficiency.
14. A method according to any one of Claims 1 to 10 or 12 to 13 further comprising the steps of: i) culturing a third population of test stem cells in a second reference medium comprising a higher concentration of reference medium supplement than the reference medium of step (a); ii) culturing a fourth population of test stem cells in a second test medium comprising a higher concentration of the medium supplement than the test medium of step (c); iii) determining the relative number of cell colonies in the cultures of steps (a) and (h) and of steps (c) and (i); and iv) calculating the relative cytotoxicity of the medium supplement.
15. A method according to any preceding claim further comprising the steps of: i) propagating the stem cells of each of steps (a) and (c) through multiple sequential subcultures; ii) determining the number of cell colonies in each of the final subcultures derived from steps (a) and (c); and iii) calculating the relative growth efficiency.
16. A method according to any preceding claim, wherein the marker gene encodes a gene product, the presence of which is visually detectable.
17. A method according to Claim 16, wherein the presence of the marker gene product is detected on exposure of the cells to a chromogenic substrate.
18. A method according to Claim 17, wherein the marker gene product is β- galactosidase.
19. A method according to any preceding claim, wherein the marker gene is operativeiy linked to a gene or gene fragment regulating expression, which gene or gene fragment is differentially active in stem and non-stem cells.
20. A method according to any preceding claim, wherein a gene or gene fragment operatively linked to and regulating expression of the marker gene is associated with cells having the differentiation potential of the desired stem cells.
21. A method according to Claim 20, wherein the gene or gene fragment is associated with a pluripotential stage of cellular development.
22. A method according to Claim 21 , wherein the gene or gene fragment is active in pluripotential cells of the developing embryo.
23. A method according to Claim 21 or Claim 22, wherein the gene or gene fragment is active in primitive ectoderm.
24. A method according to any of Claims 20 to 23, wherein the gene or gene fragment is all or part of the Oct4 gene.
25. A method according to any of claims 20 to 24, wherein the gene or gene fragment is the Oct4 promoter.
26. A method according to any preceding claim, wherein the test stem cells are OKO160 embryonic stem cells.
27. A test stem cell line for use in a method for assaying the ability of a cell culture medium or a cell culture medium supplement to support the growth, maintenance and/or proliferation of the desired stem cells without loss of differentiation potential.
28. A test stem cell line for use in the method of any one of Claims 1 to 26, wherein the test stem cells contain a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells.
29. A test stem cell line according to Claim 27 or Claim 28, wherein the test stem cell line is obtainable by genetic modification of desired stem cells.
30. A test stem cell line according to Claim 29, wherein the genetic modification comprises the introduction of a stably integrated or episomally maintained construct.
31. A test stem cell line according to Claim 29 or Claim 30, wherein the genetic modification comprises operatively inserting the marker gene into an endogenous gene of the desired stem cells.
32. A test stem cell line according to any one of Claims 29 to 31 , wherein the genetic modification is achieved by transfection, lipofection, ballistic missile, viral vector, electroporation, or any other means.
33. Use of a stem cell comprising a marker gene which is differentially expressed in (i) desired stem cells and (ii) cells other than desired stem cells in an assay for determining the ability of a cell culture medium or medium supplement to support the growth, maintenance and/or proliferation of desired stem cells.
34. Use according to Claim 33, wherein the stem cell is an embryonic stem cell, a gonadal stem cell, a somatic stem/progenitor cell, a haematopoietic stem cell, an epidermal stem cell, or a neuronal stem cell.
35. Use according to Claim 33, wherein the stem cell is an embryonic stem cell.
36. Use according to Claim 33 or Claim 34, wherein the stem cell is a mammalian stem cell.
37. Use according to Claim 36, wherein the stem cell is a murine stem cell.
38. Use according to Claim 36, wherein the stem cell is a human stem cell.
39. Use according to any one of Claims 33 to 38, wherein the medium supplement is serum.
40. Use according to Claim 39, wherein the serum is fetal bovine serum (FBS).
41. Use according to any one of Claims 33 to 38, wherein the cell culture medium is a serum-free medium or an animal-free medium.
42. Use according to any one of Claims 33 to 41 , wherein the marker gene encodes a gene product, the presence of which is visually detectable.
43. Use according to Claim 42, wherein the presence of the marker gene product is detected on exposure of the cells to a chromogenic substrate.
44. Use according to Claim 43, wherein the marker gene product is β-galactosidase.
45. Use according to any one of Claims 33 to 44, wherein the marker gene is operatively linked to a gene or gene fragment regulating expression, which gene or gene fragment is differentially active in stem and non-stem cells.
46. Use according to any one of Claims 33 to 45, wherein a gene or gene fragment operatively linked to and regulating expression of the marker gene is associated with cells having the differentiation potential of the desired stem cells.
47. Use according to Claim 46, wherein the gene or gene fragment is associated with a pluripotential stage of cellular development.
48. Use according to Claim 47, wherein the gene or gene fragment is active in pluripotential cells of the developing embryo.
49. Use according to Claim 47 or Claim 48, wherein the gene or gene fragment is active in primitive ectoderm.
50. Use according to any of Claims 47 to 49, wherein the gene or gene fragment is all or part of the Oct4 gene.
51. Use according to any of Claims 47 to 50, wherein the gene or gene fragment is the Oct4 promoter.
52. Use according to any preceding claim, wherein the stem cell is an OKO160 embryonic stem cell.
53. A kit comprising, in one or more containers, (a) a population of test stem cells, wherein the test stem cells contain a marker gene which is differentially expressed in
(i) desired stem cells and (ii) cells other than desired stem cells, (b) a reference cell culture medium or medium supplement which supports the growth, maintenance and/or proliferation of the test stem cells, and (c) a test cell culture medium or medium supplement.
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