Dual genetic reporters in human pluripotent cells for use as substitutes for mouse embryo assays

Abstract

Systems and methods for assessing cytotoxicity of cell culture media and devices. Such systems and methods include genetically modified stem cells that have been modified to express a reporter when a gene associated with pluripotent and / or trophoderm development is activated in the genetically modified stem cells.

Description

Background Art

[0001] The Mouse Embryo Assay (MEA) is a conventional bioassay used to evaluate the quality of reagents and laboratory equipment produced in the in vitro fertilization (IVF) industry. In this assay, single-cell or two-cell mouse embryos are cultured until the blastocyst stage in the presence of a test article (e.g., a test medium or a control medium exposed to a test device). The blastocyst morphology is evaluated, and if at least 80% of the embryos meet the morphological criteria for healthy blastocysts, normalized relative to the percentage of blastocysts under control conditions, the test article is scored as passing. In addition to being used by manufacturers of IVF media and devices, the MEA is also used by individual IVF clinics to meet certification requirements to demonstrate the proficiency of embryologists in embryo handling, and the MEA can be used to train entry-level embryologists.

[0002] One problem with the MEA is that the results are highly dependent on the exact procedure used. For example, studies have shown that starting from single-cell embryos is more sensitive to perturbations than starting from the two-cell stage. The commonly used CBA / B6 hybrid embryos in the MEA are less sensitive than other strains, resulting in variations in the results. Other factors, such as the presence or absence of proteins (such as albumin) in the medium that have a "chelating" effect on toxins, may also mask the detection of potential contaminants. In addition, MEA results are based on morphological scoring, which is subjective.

[0003] Another problem with the MEA is that it uses mouse embryos, which may not be a relevant model for the sensitivity and requirements of human embryos in culture. There are many differences between mouse and human embryonic development. For example, the transition from maternal transcription to zygotic transcription occurs at the 2-cell stage in mice, while in humans it occurs at approximately the 4-cell stage to the 16-cell stage. The practical result of the species difference is that mouse embryos seem to be very robust and are generally more tolerant of manipulation and perturbation than human embryos. In addition to the differences between mouse and human embryonic development, there are also ethical concerns regarding the large-scale need to produce and kill mouse embryos for the MEA.

[0004] There is still a need for materials and assays for evaluating reagent quality. The present disclosure provides such medical compositions and products, as well as methods for their preparation and use. Summary of the Invention

[0006] In some aspects, the present disclosure relates to unique systems and methods for evaluating the cytotoxicity of cell culture media. According to some forms, such systems and methods include one or more genetically modified stem cells. Thus, in one form the present disclosure provides a method for evaluating the cytotoxicity of cell culture media and devices, the method comprising: exposing a first target culture medium to a first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated in the genetically modified stem cells, and analyzing the culture medium for expression of the first reporter. In certain embodiments, the gene associated with trophectoderm development is CGA or CGB. In some forms, the method further comprises: exposing a second target culture medium to a second population of genetically modified stem cells, wherein the second population of genetically modified stem cells comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated in the genetically modified stem cells, and analyzing the second target culture medium for expression of the second reporter. In certain embodiments, the genes associated with pluripotency are selected from the group consisting of SOX2, NANOG, and POUF1. According to some forms, the first and / or second population of genetically modified stem cells comprises induced pluripotent stem cells.

[0007] In another embodiment, the present disclosure provides a system for detecting cell culture media, the system comprising: a first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated. In certain embodiments, the gene associated with trophectoderm development is CGA or CGB. In some forms, the system further comprises: a second population of genetically modified stem cells, wherein the second population of genetically modified stem cells comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated in the genetically modified stem cells. In certain embodiments, the genes associated with pluripotency are selected from the group consisting of SOX2, NANOG, and POUF1. According to some forms, the first and / or second population of genetically modified stem cells comprises induced pluripotent stem cells.

[0008] In another embodiment, the present disclosure provides a cell culture comprising a first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated. In certain embodiments, the gene associated with trophectoderm development is CGA or CGB. In some forms, the first reporter is a bioluminescent reporter. According to some forms, the first population of genetically modified stem cells comprises induced pluripotent stem cells.

[0009] In another embodiment, the present disclosure provides a laboratory test kit comprising: a first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated, wherein the first population of genetically modified stem cells is contained in a sterile package. In certain embodiments, the gene associated with trophectoderm development is CGA or CGB. In some forms, the laboratory test kit further comprises a second population of genetically modified stem cells, wherein the second population of genetically modified stem cells comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated in the genetically modified stem cells. In certain embodiments, the gene associated with pluripotency is selected from the group consisting of SOX2, NANOG, and POUF1. According to some forms, the first and / or second population of genetically modified stem cells comprises induced pluripotent stem cells.

[0010] Other embodiments of the invention and the features and advantages of the embodiments will be apparent from the description herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A Results of three independent tests of SOX2-snLuc cells for 48 hours in the presence of different concentrations of ethylene glycol are described. The amount of nanoluciferase normalized to tp 0h is shown as a fraction of control cells. "TE" refers to a population of control cells exposed to differentiation medium rather than pluripotency medium.

[0012] Figure 1B Results of three independent tests of CGA-snLuc cells for 65 hours in the presence of BAP differentiation medium with different concentrations of ethylene glycol are described. The amount of nanoluciferase is shown as a fraction of control cells. "Pluri" refers to a population of control cells exposed to pluripotency medium rather than differentiation medium.

[0013] Figure 1CDescribes the results of multiple independent tests of CGA-snLuc cells over 48 hours in the presence of DAF-BAP differentiation medium with different concentrations of ethylene glycol.

[0014] Figure 2A -C describes the immunostaining of side-by-side cultures of CGA-snLuc cells in the presence of DAF-BAP or BAP differentiation medium. Time points are expressed as days after exposure to each differentiation medium. Images were taken at 10X magnification. Figure 2A Shows the trophectoderm specification transcription factors GATA 2 / 3. Figure 2B Shows the pan-trophoblast cytoskeletal protein KRT7. Figure 2C Describes the differentiated syncytiotrophoblast marker CGA, which is undetectable at the earliest stages of differentiation. Note the presence of cyst-like syncytial structures in the DAF-BAP medium.

[0015] Description of the selected embodiments

[0016] To facilitate an understanding of the principles of the present invention, reference will now be made to the embodiments illustrated in the accompanying drawings, and specific language will be used to describe them. However, it is understood that no limitation of the scope of the claims is thereby intended, and changes and modifications of the shown drawings, and further applications of the principles of the disclosure shown therein, are covered herein as would normally be contemplated by a person skilled in the art related to the present disclosure.

[0017] As described above, aspects of the present disclosure relate to new methods and materials for using genetically modified stem cells in MEA replacement assays. Accordingly, the present disclosure provides assays, materials, and methods of performing assays for assessing the cytotoxicity of media, particularly media that has been exposed to devices and / or materials used in IVF procedures. The assays described herein are configured to quantitatively measure key features of early embryonic development. Development from the embryo to the blastocyst stage involves cell proliferation and the formation of the trophectoderm lineage (the future placenta), while maintaining a subset of cells, the inner cell mass, in a pluripotent state. According to certain embodiments, the present disclosure provides assays, materials, and methods for performing such assays that include at least one genetically modified stem cell line. In certain embodiments, the genetically modified stem cells have been modified to secrete a reporter or a detectable marker upon reaching a preselected developmental milestone. According to some forms, two or more genetically modified cell lines are used to indicate that the target cells have reached two or more developmental milestones. Accordingly, the present disclosure provides assays that are capable of validating the ability of a target media to support cell pluripotency and / or the formation of placental-like cells (trophectoderm). When used in parallel under their respective culture conditions, the modified cells provide a readout of perturbations that would result in cell death, reduced proliferation, failure to maintain pluripotency, and / or failure to differentiate into placental-like tissue.

[0018] In certain embodiments, the present disclosure provides a live cell reporter for pluripotency. According to certain preferred embodiments, the live cell reporter comprises a genetically modified stem cell that has been modified to have a cellular reporter for a gene associated with pluripotency. In certain embodiments, the genes associated with pluripotency include SOX2, NANOG, and / or POU5F1 (OCT4).

[0019] In certain embodiments, the present disclosure provides a live cell reporter for trophectoderm differentiation. According to certain preferred embodiments, the live cell reporter comprises a genetically modified stem cell that has been modified to have a cellular reporter for a gene associated with trophectoderm development. In certain embodiments, the genes associated with trophectoderm development include CGA, CBA, GATA2, GATA3, CDX2, KRT23, KRT7, HAND1, ISL1, TEAD1, TEAD4, TFAP2A, TFAP2C, and / or PGR.

[0020] As disclosed herein, the present disclosure provides systems comprising such stem cells that have been genetically modified to include one or more reporter genes. Any suitable reporter can be used. In certain embodiments, the reporter includes a fluorescent reporter, such as GFP, deGFP, mCherry, Venus, tdTomato, and / or mScarlet. In certain embodiments, the reporter includes a colorimetric reporter, such as lacZ. In certain embodiments, the reporter includes a bioluminescent reporter, such as luxCDABE, Renilla, and / or NanoLuc luciferase. In certain embodiments, the reporter is secreted into the test medium. In these embodiments, a portion of the test medium can be sampled to detect the secreted reporter. In certain embodiments, the reporter remains intracellular. In some forms, the present disclosure provides systems and methods that utilize two genetically modified cell lines, one cell line indicating pluripotency and the other indicating trophectoderm development. According to some forms, both of these modified stem cell lines can be configured to secrete the same or different reporters as described above. Additionally or alternatively, a single stem cell line that has been modified to contain two different reporter genes is within the scope of the present disclosure.

[0021] In certain embodiments, the present disclosure provides for the analysis of a target medium after it has been exposed to one or more of the genetically modified stem cells disclosed herein. Such an analysis is performed to detect the presence or absence of the reporter product. Those of ordinary skill in the art will readily understand the appropriate analysis required to determine the presence or absence of the reporter, depending on the selection of the reporter itself. For example, in certain embodiments, such an analysis includes observing the target medium or cells with ultraviolet light or other dedicated light sources.

[0022] According to some forms, the present disclosure provides materials and methods for assessing the cytotoxicity of IVF media. The materials and methods of the present disclosure can be used to assess any suitable IVF medium capable of supporting embryo development.

[0023] As described above, the assays disclosed herein utilize one or more genetically modified stem cells. The genetically modified stem cells can comprise any suitable stem cells, wherein the stem cells express one or more genes associated with pluripotency and / or trophectoderm development. For example, according to certain embodiments, the genetically modified stem cells comprise induced pluripotent stem cells. In some forms, the genetically modified stem cells may include mesenchymal stem cells, trophoblast stem cells, or placenta-derived stem cells. Providing assays that include different stem cell types is within the scope of the present invention. For example, stem cells that have been genetically modified to express a reporter when a gene associated with trophectoderm development is activated can comprise a different type of cell than stem cells that have been genetically modified to express a reporter when a gene associated with pluripotency is activated.

[0024] The present disclosure provides systems, kits, and methods for assaying the cytotoxicity of a target medium. According to some forms, the target medium is a cell culture medium for culturing embryonic cells. In certain embodiments, the target medium is a medium that has been exposed to one or more devices. In this manner, the present disclosure provides a method for assaying the cytotoxicity of a medium as a way to assay the cytotoxicity or contamination of a device or material (e.g., a pipette, glove, or culture dish) that has been in contact with the medium. In certain embodiments, the systems, kits, methods, and materials described in the present disclosure can be used to verify the performance of a technician or process in avoiding medium contamination.

[0025] The target medium can be exposed to the genetically modified stem cell population for any length of time sufficient to allow cell differentiation and reporter expression. According to certain embodiments, when validating a device or technique, for example, the target medium can be selected to support trophectoderm and / or placental differentiation. In some forms, the target medium comprises Essential8Flex medium. In certain embodiments, the genetically modified stem cells as described herein can be cultured in a medium containing BMP-4, A83-01, and / or PD173074. In some forms, the medium comprises a basal medium containing BMP-4, A83-01, and / or PD173074 (BAP). The BAP medium used herein refers to a basal medium configured to stimulate trophectoderm differentiation. In certain embodiments, the genetically modified stem cells as described herein can be cultured in a medium containing forskolin. For example, in certain embodiments, the medium containing BAP can comprise forskolin. The applicant has surprisingly found that, as described herein, using a medium containing forskolin accelerates the differentiation of the genetically modified stem cells. Figure 2 depicts the relative timing of the expression of the differentiation markers GATA2 / 3, KRT7, and CGA and shows that during differentiation in DAF-BAP medium, these markers are detectable approximately one day earlier than in BAP medium. An exemplary embodiment of such a medium containing forskolin, comprising DMEM, albumin, and forskolin (DAF-BAP), is shown in Table 1 below.

[0026] Table 1: DAF-BAP Medium Formulation

[0027]

[0028] Thus, according to certain embodiments, the genetically modified stem cells of the present disclosure can be exposed to the target medium for at least 24 hours, preferably at least 36 hours, and even more preferably at least 48 hours. In some forms, the target medium is exposed to the genetically modified stem cells modified to have a reporter indicative of pluripotency for at least 24 hours, preferably at least 36 hours, and even more preferably at least 48 hours. In certain embodiments, the target medium is exposed to the genetically modified stem cells for 12 to 120 hours, preferably 18 to 108 hours, and even more preferably 24 to 96 hours.

[0029] The genetically modified stem cells of the present disclosure can be provided as part of a kit or system. In some forms, the genetically modified stem cells are provided in a cryopreserved form and thawed prior to use. Thus, in some forms, the present disclosure provides a method of testing a cell medium, comprising thawing a cryopreserved cell composition comprising the genetically modified stem cells as described herein.

[0030] The determination can be performed in various forms. For example, the genetically modified stem cells can be cultured in a single-well plate or a multi-well plate in a conventional 2D adherent form. In another form, the genetically modified stem cells disclosed herein can be used to form embryoid bodies, and the determination can be performed when the cells are in an embryo-like 3D structure. In another form, a cryopreservation of single cells or embryoid bodies can be generated, and the determination can be performed on the thawed cells or embryoid bodies in a 3D structure.

[0031] Any or all of the components described herein can be provided in a sterile package for providing the necessary component or components to the user. For example, one or more vials containing genetically modified stem cells can be provided in a single sterile package or kit. For example, sterilization can be achieved by irradiation, ethylene oxide gas, or any other suitable sterilization technique, and the materials and other characteristics of the medical package will be selected accordingly. The package or kit of the components described herein can contain additional devices or tools that may be useful for the particular medical procedure being performed.

[0032] To facilitate further understanding of the embodiments disclosed herein and their features and advantages, the following specific examples are provided. It should be understood that these examples are illustrative and not restrictive.

[0033] Example 1: Generation of a Genetically Modified Cell Line

[0034] A live cell reporter for trophectoderm differentiation and pluripotency was created using the Crispr genome editing technique. To assess pluripotency, a transgene was inserted immediately before the stop codon of the SOX2 gene such that it was in-frame translationally with the SOX2 open reading frame. The transgene consisted of a T2A ribosomal skipping sequence, a furin cleavage site, and a NanoLuc luciferase coding sequence utilizing the IL6 signal peptide. Thus, activation of the genetically modified SOX2 gene was expected to produce two protein products: intracellular SOX2 and secreted NanoLuc luciferase. To minimize the possibility of re-cutting of the donor DNA or genomic DNA during the Crispr-mediated modification process, a silent mutation was introduced in the T2A linker region of the donor DNA. The homology arms were approximately 200 nucleotides in length.

[0035] To evaluate trophectoderm differentiation, a live cell reporter for human chorionic gonadotropin (hCG) was developed. hCG is a dimer of the protein products of the CGA and CGB genes. A transgene was inserted in-frame translationally immediately after the start codon of the CGA gene. The transgene consists of an IL6 signal peptide and a NanoLuc luciferase coding sequence, without a stop codon, followed by a T2A ribosome skipping sequence and a furin cleavage site. Thus, activation of the genetically modified CGA gene is expected to produce secreted NanoLuc luciferase and secreted CGA product. The homology arms are approximately 200 nucleotides in length.

[0036] The transgene insert was synthesized in the form of plasmid DNA. A double-stranded donor DNA fragment was generated by PCR amplification, and the products were pooled and purified using magnetic beads. The guide RNA sequences targeting the SOX2 gene are:

[0037] 5'-TGCCCCTCTCACACATGTGA-3'

[0038] And for the CGA gene are:

[0039] 5'-GTCTGTCTGCAGGAGCGCCA-3'.

[0040] Separate cell lines were prepared for each reporter using commercially available human induced pluripotent stem cells, which were cultured in E8-Flex medium on tissue culture plates coated with vitronectin (rhVTN-N). Ribonucleoprotein particles consisting of 20 pmol guide RNA (gRNA) and 2.5 μg of CAS9 protein were prepared by mixing with Celetrix electroporation buffer and, after a brief incubation, mixing with 0.75 to 1 μg of donor DNA and 500,000 cells. A 20 μl Celetrix tube was used and electroporation was performed according to the manufacturer's recommended settings. After the recovery and expansion period, the electroporated cells were dissociated with Accutase and plated at very low density using CloneR supplement to improve viability, and approximately 50 individual colonies were manually picked and cultured separately and screened using a combination of PCR and luciferase detection methods. A second round of low-density plating, colony isolation, and screening was performed for each cell line to increase the likelihood of generating clonal-derived homogeneous cell lines. Genomic DNA amplified at the 5' and 3' ligation regions between the transgene and native DNA was subjected to sequence analysis. Cell lines containing mutations predicted to result in amino acid sequence changes were removed for further analysis. Each cell line was amplified and cryopreserved to prepare a master cell bank.

[0041] Example 2: Effect of doped ethylene glycol in the differentiation medium

[0042] For this assay, cells from the cell line described in Example 1 were thawed using standard procedures and plated in vitronectin-coated wells of a 96-well plate in Essential 8Flex medium with Revitacell supplement. The plating density was approximately 20,000 cells per well for SOX2-snLuc cells and approximately 15,000 cells per well for CGA-snLuc cells. Empty wells were filled with PBS to reduce variations in nanoluciferase concentration associated with evaporation. Cells were allowed to recover for 7 - 18 hours prior to exposure to the test medium.

[0043] For SOX2-snLuc cells, the test medium was Essential 8Flex. For CGA-snLuc cells, activation of the reporter required a medium that supported trophectoderm / placenta differentiation, such as BAP medium. DAF-BAP medium is an optimized formulation that accelerates differentiation and reporter activation compared to other BAP medium preparations lacking forskolin. DAF-BAP contains: DMEM with 25 mM HEPES and 4.5 g / L glucose and without L-glutamine, 100 ng / mL BMP-4, 1 μM A83-01, 0.1 μM PD173074, 5 mg / mL AlbumiNZ bovine albumin low free fatty acid, and 40 μM forskolin. The medium was pre-warmed prior to forskolin addition to prevent precipitation. Compounds to be tested for toxicity (such as ethylene glycol or triton-x-100) or devices and disposables to be tested were mixed with the test medium or exposed to the test medium prior to cell exposure.

[0044] After recovery from thawing, the medium was changed to the respective test medium for each cell line. In the case of CGA-snLuc cells, two partial exchanges were performed, equivalent to approximately 95% total exchange, to prevent loss of cell adhesion during medium exchange while still ensuring substantial removal of the pluripotent medium. In the case of SOX2-snLuc cells, optionally, a baseline read of luciferase expression was performed prior to medium change to assess variations in plating density or recovery after thawing. Cells were exposed to the test medium (24 - 96 hours for SOX2-snLuc cells and 48 - 96 hours for CGA-snLuc cells). Secreted nanoluciferase was detected by removing 10% of the medium and mixing with substrate and buffer in a white assay plate using the Promega Nano-Glo luciferase assay kit. Luminescence was measured using a BioTek Cytation 1 imaging reader.

[0045] Figure 1AThe results of three independent tests of SOX2-snLuc cells in the presence of different concentrations of ethylene glycol for up to 48 hours are described. The amount of nanoluciferase is normalized to tp 0h and is shown as a fraction of control cells. "TE" refers to a population of control cells exposed to differentiation medium rather than pluripotency medium.

[0046] Figure 1B The results of three independent tests of CGA-snLuc cells in the presence of BAP differentiation medium with different concentrations of ethylene glycol for up to 65 hours are described. The amount of nanoluciferase is shown as a fraction of control cells. "Pluri" refers to a population of control cells exposed to pluripotency medium rather than differentiation medium.

[0047] The assay was also tested in the context of detecting the contaminant ethylene glycol (EG), which is known to affect mouse embryo viability. For 1-cell MEA, 2-cell MEA, MEAlt (CGA-snLuc), and MEAlt (SOX2-SnLuc), the IC50 was calculated using the logistic4P Hill equation as shown in Table 2 below.

[0048] Table 2

[0049] IC50 1-cell MEA 2.14% EG 2-cell MEA 3.19% EG MEAlt(CGA-snLuc) 0.77% EG MEAlt(SOX2-snLuc) 1.25% EG

[0050] The results indicate that iPS cells are slightly less tolerant to ethylene glycol than mouse embryos, although overall the sensitivities between different systems are generally comparable. It was also found that the MEAlt assay can successfully detect low levels of additional contaminants such as triton-x-100 and formaldehyde.

[0051] Figure 1C The results of multiple independent tests of CGA-snLuc cells in the presence of DAF-BAP differentiation medium with different concentrations of ethylene glycol for up to 48 hours are described.

[0052] Embodiments

[0053] An enumerated list of some embodiments disclosed herein is provided below. It will be understood that this list is non-limiting and that individual features or combinations of features (e.g., 2, 3, or 4 features) as described in the detailed description above can be combined with the embodiments listed below to provide additional embodiments disclosed herein.

[0054] 1. A method for evaluating the cytotoxicity of a cell culture medium and a device, the method comprising:

[0055] Expose a first target medium to a first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated in the population of genetically modified stem cells; and

[0056] Detect the expression of the first reporter.

[0057] 2. The method according to embodiment 1, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

[0058] 3. The method according to any one of embodiments 1 or 2, wherein the first reporter comprises a bioluminescent reporter.

[0059] 4. The method according to any one of the above embodiments, wherein the first population of genetically modified stem cells comprises induced pluripotent stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated in the population of genetically modified stem cells.

[0060] 5. The method according to any one of the above embodiments, wherein the first target medium comprises a medium configured to support trophectoderm differentiation.

[0061] 6. The method according to any one of the above embodiments, wherein the first target medium comprises a control medium that has been exposed to a test device.

[0062] 7. The method according to any one of the above embodiments, wherein the first reporter is secreted into the first target medium.

[0063] 8. The method according to any one of the above embodiments, wherein the first target medium comprises a basal medium containing BMP-4, A83-01, and / or PD173074.

[0064] 9. The method according to any one of the above embodiments, wherein the first target medium comprises forskolin.

[0065] 10. The method according to embodiment 9, wherein the first target medium is exposed to the first population of genetically modified stem cells for about 48 hours.

[0066] 11. The method according to any one of embodiments 1 to 10, the method further comprising:

[0067] Expose a second target culture medium to a second population of genetically modified stem cells, wherein the second population of genetically modified stem cells comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated in the genetically modified stem cells; and

[0068] Analyze the second target culture medium for the expression of the second reporter.

[0069] 12. The method according to embodiment 11, wherein the gene associated with pluripotency is selected from the group consisting of SOX2, NANOG, and POUF1.

[0070] 13. The method according to any one of embodiments 11 or 12, wherein the second reporter comprises a bioluminescent reporter.

[0071] 14. The method according to any one of embodiments 11 to 13, wherein the second population of genetically modified stem cells comprises induced pluripotent stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated in the genetically modified stem cells.

[0072] 15. The method according to any one of embodiments 11 to 14, wherein the second target culture medium comprises a culture medium configured to support pluripotency.

[0073] 16. The method according to any one of embodiments 11 to 15, wherein the second target culture medium comprises a control culture medium that has been exposed to a test device.

[0074] 17. The method according to any one of the above embodiments, the method further comprising:

[0075] Thaw the first population of genetically modified stem cells.

[0076] 18. A system for detecting a cell culture medium, the system comprising:

[0077] A first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

[0078] 19. The system according to embodiment 18, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

[0079] 20. The system according to any one of embodiments 18 or 19, wherein the first reporter comprises a bioluminescent reporter.

[0080] 21. The system according to any one of embodiments 18 to 20, wherein the first genetically modified stem cell population comprises induced pluripotent stem cells.

[0081] 22. The system according to any one of embodiments 18 to 21, comprising:

[0082] A first target medium, the first target medium comprising a medium configured to support trophectoderm differentiation.

[0083] 23. The system according to embodiment 22, wherein the first target medium comprises a control medium that has been exposed to the test device.

[0084] 24. The system according to any one of embodiments 22 or 23, wherein the first reporter is secreted by the stem cells.

[0085] 25. The system according to any one of embodiments 22 to 24, wherein the first target medium comprises a basal medium containing BMP-4, A83-01, and PD173074.

[0086] 26. The system according to any one of embodiments 22 to 25, wherein the first target medium comprises forskolin.

[0087] 27. The system according to any one of embodiments 18 to 26, the system further comprising:

[0088] A second genetically modified stem cell population, wherein the second genetically modified stem cell population comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated.

[0089] 28. The system according to embodiment 27, wherein the gene associated with pluripotency is selected from the group consisting of SOX2, NANOG, and POUF1.

[0090] 29. The system according to any one of embodiments 27 or 28, wherein the second reporter comprises a bioluminescent reporter.

[0091] 30. The system according to any one of embodiments 27 to 29, wherein the second genetically modified stem cells comprise induced pluripotent stem cells.

[0092] 31. The system according to any one of embodiments 27 to 30, the system comprising:

[0093] A second target medium, the second target medium comprising a medium configured to support pluripotency.

[0094] 32. The system according to embodiment 31, wherein the second target culture medium comprises a control culture medium that has been exposed to the test device.

[0095] 33. A cell culture, comprising:

[0096] A first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

[0097] 34. The cell culture according to embodiment 33, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

[0098] 35. The cell culture according to any one of embodiments 33 or 34, wherein the first reporter comprises a bioluminescent reporter.

[0099] 36. The cell culture according to any one of embodiments 33 to 35, wherein the first population of genetically modified stem cells comprises induced pluripotent stem cells.

[0100] 37. The cell culture according to any one of embodiments 33 to 36, wherein the first population of genetically modified stem cells is cryopreserved.

[0101] 38. A laboratory test kit, comprising:

[0102] A first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated, wherein the first population of genetically modified stem cells is contained in a sterile package.

[0103] 39. The laboratory test kit according to embodiment 38, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

[0104] 40. The laboratory test kit according to any one of embodiments 38 or 39, wherein the first reporter comprises a bioluminescent reporter.

[0105] 41. The laboratory test kit according to any one of embodiments 38 to 40, wherein the first population of genetically modified stem cells comprises induced pluripotent stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

[0106] 42. The laboratory test kit according to any one of embodiments 38 to 41, the laboratory test kit comprising:

[0107] A first target culture medium, the first target culture medium comprising a culture medium configured to support trophectoderm differentiation.

[0108] 43. The laboratory test kit according to embodiment 42, wherein the first target culture medium comprises forskolin.

[0109] 44. The laboratory test kit according to any one of embodiments 38 to 43, the laboratory test kit further comprising:

[0110] A second genetically modified stem cell population, wherein the second genetically modified stem cell population comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated, wherein the second genetically modified stem cell population is contained in a sterile package.

[0111] 45. The laboratory test kit according to embodiment 44, wherein the gene associated with pluripotency is selected from the group consisting of SOX2, NANOG, and POUF1.

[0112] 46. The laboratory test kit according to any one of embodiments 44 or 45, wherein the second reporter comprises a bioluminescence reporter.

[0113] 47. The laboratory test kit according to any one of embodiments 44 to 46, wherein the second genetically modified stem cells comprise induced pluripotent stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated.

[0114] 48. The laboratory test kit according to any one of embodiments 44 to 47, comprising:

[0115] A second target culture medium, the second target culture medium comprising a culture medium configured to support pluripotency.

[0116] In the context of describing the invention, particularly in the context of the following claims, the use of the terms "a", "an", "the", and similar terms referring to an object shall be understood to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context. Unless otherwise specified herein, the recitation of a range of values herein is merely a shorthand method for referring individually to each value within the range, and each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "such as", "etc.") provided herein is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention, unless otherwise claimed. Any language in this specification should not be construed as indicating any non-claimed element as essential to the practice of the invention.

Claims

1. A method for evaluating the cytotoxicity of a cell culture medium and a device, the method comprising: Exposing a first target culture medium to a first population of genetically modified stem cells, wherein the first population of genetically modified stem cells comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated in the population of genetically modified stem cells; and Detecting the expression of the first reporter.

2. The method according to claim 1, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

3. The method according to any one of claims 1 or 2, wherein the first reporter comprises a bioluminescent reporter.

4. The method according to any one of the preceding claims, wherein the first population of genetically modified stem cells comprises induced pluripotent stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated in the population of genetically modified stem cells.

5. The method according to any one of the preceding claims, wherein the first target culture medium comprises a culture medium configured to support trophectoderm differentiation.

6. The method according to any one of the preceding claims, wherein the first target culture medium comprises a control culture medium that has been exposed to a test device.

7. The method according to any one of the preceding claims, wherein the first reporter is secreted into the first target culture medium.

8. The method according to any one of the preceding claims, wherein the first target culture medium comprises a basal medium containing BMP-4, A83-01, and / or PD173074.

9. The method according to any one of the preceding claims, wherein the first target culture medium comprises forskolin.

10. The method according to claim 9, wherein the first target culture medium is exposed to the first population of genetically modified stem cells for about 48 hours.

11. The method according to any one of claims 1 to 10, the method further comprising: Exposing a second target culture medium to a second population of genetically modified stem cells, wherein the second population of genetically modified stem cells comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated in the second population of genetically modified stem cells; and Detecting the expression of the second reporter.

12. The method according to claim 11, wherein the gene associated with pluripotency is selected from the group consisting of SOX2, NANOG, and POUF1.

13. The method according to any one of claims 11 or 12, wherein the second reporter comprises a bioluminescent reporter.

14. The method according to any one of claims 11 to 13, wherein the second population of genetically modified stem cells comprises induced pluripotent stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated in the second population of genetically modified stem cells.

15. The method according to any one of claims 11 to 14, wherein the second target culture medium comprises a culture medium configured to support pluripotency.

16. The method according to any one of claims 11 to 15, wherein the second target culture medium comprises a control culture medium that has been exposed to a test device.

17. The method according to any one of the preceding claims, the method further comprising: Thawing the first genetically modified stem cell population.

18. A system for testing a cell culture medium, the system comprising: A first genetically modified stem cell population, wherein the first genetically modified stem cell population comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

19. The system according to claim 18, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

20. The system according to any one of claims 18 or 19, wherein the first reporter comprises a bioluminescent reporter.

21. The system according to any one of claims 18 to 20, wherein the first genetically modified stem cell population comprises induced pluripotent stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

22. The system according to any one of claims 18 to 21, comprising: A first target culture medium, the first target culture medium comprising a culture medium configured to support trophectoderm differentiation.

23. The system according to claim 22, wherein the first target culture medium comprises a control culture medium that has been exposed to a test device.

24. The system according to any one of claims 22 or 23, wherein the first reporter is secreted by the stem cells.

25. The system according to any one of claims 22 to 24, wherein the first target culture medium comprises a basal medium containing BMP-4, A83-01, and PD173074.

26. The system according to any one of claims 22 to 25, wherein the first target culture medium comprises forskolin.

27. The system according to any one of claims 18 to 26, the system further comprising: A second genetically modified stem cell population, wherein the second genetically modified stem cell population comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated.

28. The system according to claim 27, wherein the gene associated with pluripotency is selected from the group consisting of SOX2, NANOG, and POUF1.

29. The system according to any one of claims 27 or 28, wherein the second reporter comprises a bioluminescent reporter.

30. The system according to any one of claims 27 to 29, wherein the second genetically modified stem cell population comprises induced pluripotent stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated.

31. The system according to any one of claims 27 to 30, the system comprising: A second target culture medium, the second target culture medium comprising a culture medium configured to support pluripotency.

32. The system according to claim 31, wherein the second target culture medium comprises a control culture medium that has been exposed to the test device.

33. A cell culture comprising: A first genetically modified stem cell population, wherein the first genetically modified stem cell population comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

34. The cell culture according to claim 33, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

35. The cell culture according to any one of claims 33 or 34, wherein the first reporter comprises a bioluminescent reporter.

36. The cell culture according to any one of claims 33 to 35, wherein the first genetically modified stem cell population comprises induced pluripotent stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

37. The cell culture according to any one of claims 33 to 36, wherein the cell culture is frozen.

38. A laboratory test kit comprising: A first genetically modified stem cell population, wherein the first genetically modified stem cell population comprises stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated, wherein the first genetically modified stem cell population is contained in a sterile package.

39. The laboratory test kit according to claim 38, wherein the gene associated with trophectoderm development is selected from the group consisting of CGA and CGB.

40. The laboratory test kit according to any one of claims 38 or 39, wherein the first reporter comprises a bioluminescent reporter.

41. The laboratory test kit according to any one of claims 38 to 40, wherein the first genetically modified stem cell population comprises induced pluripotent stem cells that have been modified to express a first reporter when a gene associated with trophectoderm development is activated.

42. The laboratory test kit according to any one of claims 38 to 41, comprising: A first target culture medium, the first target culture medium comprising a culture medium configured to support trophectoderm differentiation.

43. The laboratory test kit according to claim 42, wherein the first target culture medium comprises forskolin.

44. The laboratory test kit according to any one of claims 38 to 43, further comprising: A second genetically modified stem cell population, wherein the second genetically modified stem cell population comprises stem cells that have been modified to express a second reporter when a gene associated with pluripotency is activated, wherein the second genetically modified stem cell population is contained in a sterile package.

45. The laboratory test kit according to claim 44, wherein the gene associated with pluripotency is selected from the group consisting of SOX2, NANOG, and POUF1.

46. The laboratory test kit according to any one of claims 44 or 45, wherein the second reporter comprises a bioluminescent reporter.

47. The laboratory test kit according to any one of claims 44 to 46, wherein the second genetically modified stem cell comprises an induced pluripotent stem cell that has been modified to express the second reporter when a gene associated with pluripotency is activated.

48. The laboratory test kit according to any one of claims 44 to 47, comprising: a second target medium, the second target medium comprising a medium configured to support pluripotency.

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