Frequently Asked Questions on iPSCs
Find answers to frequently asked questions (FAQs) about induced pluripotent stem cell (iPSC) lines from STEMCELL.
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When should I use iPSCs for my experiments?
Certain disorders are challenging to study in vitro due to an inaccessibility of donor tissue, e.g., neurodegenerative and psychiatric disorders of the brain and spinal cord and cardiomyopathies of the heart. Similar to embryonic stem cells, iPSCs have the capacity for unlimited self-renewal and the ability to differentiate into cell types from the three embryonic germ layers: ectoderm, mesoderm, and endoderm. Thus, human iPSCs make it possible to generate an unlimited capacity of previously inaccessible cell types, such as neurons and cardiomyocytes, while capturing the genetic diversity of patients with specific mutations or disease. iPSCs can be used to study the molecular mechanisms of disease, have the potential to be utilized for cell and gene therapies, and can be used in the drug discovery process for the evaluation of toxic compounds.
Why use human iPSC lines from STEMCELL in my research?
Start your research confidently with human iPSCs from STEMCELL that are ethically sourced, donor-consented, and Institutional Review Board (IRB)-approved for academic and commercial research. iPSC products purchased from STEMCELL are manufactured using stringent quality control parameters that either meet or exceed industry standards and are detailed in the lot-specific Certificate of Analysis (CoA). Additional transparency is provided by registration of iPSC lines in hPSCreg®—a global human pluripotent stem cell (hPSC) registry that improves visibility, confidence in ethical provenance, validation of characterization data, and comparability with other registered lines.
Use STEMCELL’s iPSC lines to establish a complete workflow for your cell culture system. Optimized for long-term maintenance and expansion in TeSR™ media, and validated for differentiation to a multitude of lineage-specific cell types and organoids using STEMdiff™ media systems, our iPSC lines provide flexibility for a wide range of research applications.
How are STEMCELL’s iPSC lines generated?
How are STEMCELL’s iPSC lines named?
STEMCELL recognizes that a lack of well-defined guidelines for naming human pluripotent stem cell (hPSC) lines has led to confusion and duplication of cell line names and identities in the research field. As a result, we use the standardized nomenclature established by hPSCreg® that (1) unambiguously identifies a registered cell line, (2) allows tracing of subclones, and (3) enables the assignment of different cell lines to a particular donor:
|Generator acronym||2 - 6||XXXXXX||Assigned by the hPSCreg® community|
|Cell line type||1||"e" or "i"||"e": embryonic stem cell
"i": induced pluripotent stem cell
|Donor ID||3||001||Alphanumeric; limited to 46,655 donors per generator|
|Clone number||2 - 3||-A||Alphabetic; preceded by hyphen; limited to 702 lines per donor|
|Identifier for subclone (only required if applicable)||0 - 3||-1||Alphanumeric; preceded by hyphen; limited to 1330 subclones per line|
|Total characters||8 - 16|
Explanation: STEMCELL Technologies iPSC Donor 3 Clone A
How are STEMCELL’s iPSC lines characterized?
Extensive quality control procedures are implemented at every stage of STEMCELL’s iPSC manufacturing process. Master cell banks are tested for identity, adventitious agents, genomic integrity and stability, survival, undifferentiated state, and pluripotency. For detailed information on the specific quality control tests performed on master cell banks, please refer to the Quality Control section of this FAQ.
Working cell banks and commercial vials are tested for a subset of these characterization criteria. For more information on the quality control (QC) of a particular iPSC line, please refer to the lot-specific Certificate of Analysis (CoA). For more information about STEMCELL’s commercial iPSC release criteria, see the Quality Control section of this FAQ.
Are additional clones available for a given iPSC line?
In some cases, additional clones may be available for an iPSC line. However, alternate clones will not have been characterized to the same extent as the commercial iPSC line and may only be available at an early passage number. To inquire about alternate clones for an iPSC line, please contact us.
Is a license required for use of STEMCELL’s iPSC lines?
Yes, customers are required to agree to the standard license agreement for iPSCs and pay an annual license fee for the use of STEMCELL’s iPSC lines. The annual license fee is priced at $5,000 USD per iPSC line and is capped at $20,000 USD per year (i.e. use of more than four iPSC lines will not incur higher annual license fees). Customers working for non-profit organizations (e.g. universities) may be eligible for a fee waiver. To inquire about the standard license agreement, annual license fees, or to discuss your eligibility for an annual license fee waiver, please contact us. Refer to the Licensing and Usage section of this FAQ for more information.
How can STEMCELL help me with my iPSC experiments?
STEMCELL is able to support your iPSC experiments with a suite of products designed to help you get the results you need. Attend one of our training programs to learn the latest iPSC techniques and protocols and perform your experiments with confidence. Our scientists and educators offer comprehensive virtual or hands-on technical training programs that are customizable for your specific needs.
How does STEMCELL collect donor demographic information?
STEMCELL collects donor demographic information ethically, using consent forms and protocols approved by either an Institutional Review Board (IRB), the Food and Drug Administration (FDA), the U.S. Department of Health and Human Services, and/or an equivalent regulatory authority. Donations are performed in the United States in compliance with applicable federal, state, and local laws, regulations, and guidance.
Our Quality Assurance, Quality Control, and Regulatory Affairs departments are ready to assist you with any necessary documentation to meet specific institutional requirements, including supplier approval for ease of procurement.
Contact us for further information on the collection of donor demographics.
What kind of donor information is available for STEMCELL’s iPSC lines and how are the data generated?
The following donor attributes are determined for each iPSC donor and are provided with each iPSC line. Full donor details are listed on the Product Information Sheet (PIS) and further in-depth analyses (including ClinVar and ancestry reports) are included in the Certificate of Analysis (CoA).
If additional donor attributes (not included in the table above) are required for your experiments, please contact us, and a representative will be in touch to work with you on your desired order.
What is STEMCELL’s definition of a “healthy” donor?
Healthy donors must be over the age of 18, weigh at least 120 lb, have a body mass index (BMI) between 18.5 - 24.9, demonstrate no use of tobacco products, and be in good general health.
Donors in our “healthy” pool are pre-screened using a health questionnaire aimed at excluding any donors with diseases, blood disorders, or other health concerns. Donors are tested for infectious diseases (HIV-1, HIV-2, hepatitis B, and hepatitis C), and other diseases or conditions are self-reported. An intake questionnaire containing questions about the donor’s health history and current condition is completed on the day of collection. Donors are restricted from donating if they are feeling unwell or have a fever. Additional blood parameter assessments are performed, including white blood cell counts, platelet levels, hematocrit, and blood pressure; any values outside of the acceptable normal range or minimum level would eliminate the sample from the healthy pool. Health questionnaires and donor inclusion/exclusion criteria are overseen by a medical director at each collection site.
Participants are not eligible for this program if they present with any of the following characteristics:
- High blood pressure
- Blood-borne or sexually transmitted disease(s) (hepatitis B or C, HIV-1 or HIV-2, syphilis)
- Diabetes (Type 1 or 2)
- Thyroid issues
- Heart, lung, kidney, or liver problems
- Currently pregnant or nursing
If specific health information is required or if your research project has any restrictions, please contact us to see if we can source an iPSC donor for you as a special request. Note: This may require donor recruitment or screening at an additional cost.
What is the difference between ancestry, race, and ethnicity?
Ancestry refers to the geographic origins of a person’s ancestors. For example, European, African, South Asian, or East Asian. Ancestry is a donor attribute that is genetically calculated.
Race refers to physical differences that groups and cultures consider socially significant. For example, people might identify their race as Aboriginal, African American or Black, Asian, European American or White, Native American, Native Hawaiian or Pacific Islander, Māori, or another race. Race is an attribute that is self-declared by STEMCELL’s iPSC donors.
Ethnicity refers to shared cultural characteristics such as language, ancestry, practices, and beliefs. For example, people might identify as Hispanic, Latino, or another ethnicity. Ethnicity is an attribute that is self-declared by STEMCELL’s iPSC donors.
Why does STEMCELL calculate donor ancestry?
Analysis of the coding regions of the human genome for germline and somatic mutations by whole exome sequencing (WES) is becoming commonplace in translational cancer genomics studies and in the field of precision medicine. Identifying a donor’s ancestry is essential for interpreting the impact of personal genetic variation on experimental results.
How does STEMCELL calculate donor ancestry?
Whole exome sequencing (WES) data is processed using EthSEQ, an analytical pipeline for identifying conserved ancestral groups. EthSEQ inspects WES data from each iPSC line, focusing on the differential single nucleotide polymorphism (SNP) genotype profile. This profile contains variants known to be particularly informative of ancestral heritage. Each donor profile is compared to a reference database containing thousands of genotypes of known ancestries collated from the 1000 Genomes Project. Subsequent principal component analysis (PCA) identifies donor ancestry by comparison to the reference database and calculates a percentage for each ancestry. STEMCELL’s internal ancestry determination workflow has been validated using WES data from the Simons Genome Diversity Project, a database that contains genotype data from hundreds of individuals with known ancestries, thus generating more comprehensive ancestral predictions.
- Romanel A et al. (2017) EthSEQ: ethnicity annotation from whole exome sequencing data. Bioinformatics. 33(15): 2402–4
- Mallick S et al. (2016) The Simons Diversity Project: 300 genomes from 142 diverse populations. Nature. 538(7624):201–6.
- The 1000 Genomes Project Consortium (2015) A global reference for human genetic variation. Nature. 526(7571): 68–74.
How does STEMCELL identify genetic variants?
Whole exome sequencing (WES) datasets are sequenced at a depth of 50x, indicating that each nucleotide of the exome (the collection of all coding sequences) is sequenced 50 times. From this redundant coverage, a highly accurate consensus sequence is derived, allowing for the identification of genomic single nucleotide polymorphisms (SNPs; representing genetic variants). The resulting profile of genetic variants is compared against ClinVar, a public archive of reports that detail relationships between human genetic variants and phenotypes, and any resulting pathogenic or likely pathogenic variants are determined. ClinVar analysis includes germline and somatic variants of any size, type, or genomic location.
Can I obtain whole exome sequencing (WES) and/or whole genome sequencing (WGS) data files for an iPSC line?
Yes, WES and WGS data files are available to customers of the corresponding iPSC line(s) for a fee. Please note that you must be purchasing or have previously purchased the iPSC line that you wish to acquire the WES and/or WGS data files for. Customers are required to acknowledge that no attempt will be made to identify the donor using sequencing data.
What iPSC quality standards does STEMCELL manufacture to?
STEMCELL is an advocate for standardizing human pluripotent stem cell data reporting and quality control measures to limit experimental variability and ensure that relevant, reproducible findings are shared. Our commercial iPSC quality assessments and release criteria have been developed based on recommendations and guidance from the International Stem Cell Banking Initiative (ISCBI) (2009), the Global Alliance for iPSC Therapy (GAiT) (2018), and consensus workshops hosted in 2022 by the International Society for Stem Cell Research (ISSCR) and International Stem Cell Initiative (ISCI).
- Ludwig, T & Andrews, PW. (Co-Chairs) ISSCR Standards Initiative Meeting. Presented June 19, 2022 at the ISSCR Annual Meeting in San Francisco, CA.
- Sullivan, S. et al. (2018) Quality control guidelines for clinical-grade human induced pluripotent stem cell lines. Regen Med. 13(7): 859–66.
- International Stem Cell Banking Initiative (2009) Consensus guidance for banking and supply of human embryonic stem cell lines for research purposes. Stem Cell Rev Rep. 5(4): 301–14.
What quality control procedures are performed on STEMCELL’s iPSC lines and at what stage(s) in the manufacturing process?
|Pre-Master||Master Cell Bank||Working Cell Bank||Commercial Cell Bank|
|Cell line identity||-||✓||✓||✓|
|T cell clonality assay*||✓||-||-||-|
|Copy number variants||-||✓||✓||✓|
|Whole exome sequencing||-||✓||-||-|
|Undifferentiated marker analysis||-||Yes, 5 passages after thaw||Yes, 3 passages after thaw||Yes, 3 passages after thaw|
* Blood-derived iPSC lines only
Note 1: Only iPSC vials from the Commercial Cell Bank are made available for commercial sale.
Note 2: Commercial Cell Bank iPSCs have been characterized for all assessment criteria at varying stages of the manufacturing process.
What are the acceptance criteria for release of a commercial iPSC line and what analytical methods are used?
Mesoderm: T+, NCAM+ ≥ 70%
Ectoderm: PAX6+, NESTIN+ ≥ 70%
STR = Short Tandem Repeat
TSB = Tryptic Soy Broth
FTB = Thioglycollate Broth
CLEAR = Cell Line Examination and Report
TCR = T cell receptor
How should SNP microarray data be interpreted?
Copy number variants (CNVs) determined by SNP microarray analysis are reported in the lot-specific Certificate of Analysis (CoA) for each iPSC line.
The SNP microarray can detect aneuploidy, deletions, and duplications of represented loci, and regions of loss/absence of heterozygosity (LOH), but cannot detect balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, and insertions), or point mutations.
Reportable copy number changes are gains or losses greater than 400 kb (highlighted in bold in the Copy Number Variant Report). Reportable regions of LOH are greater than 5 Mb. See Interpretation for copy number changes and regions of LOH that meet these criteria. See Call Table for all copy number changes identified by the analysis software. If mosaicism is detected, the approximate percentage of mosaicism is listed in the ‘Variant Type (% mosaic)’ column.
Where can I find lot-specific QC results for my iPSC line?
QC results are included in the lot-specific Certificate of Analysis (CoA), which can be retrieved or requested online. To retrieve a CoA, a catalog number and lot number are required. This information can be found on the product label or invoice and should be entered exactly as shown, including all numbers, letters, and dashes. In some cases, a CoA may not be available online. If your search does not retrieve a CoA, please complete the form in the Request Certificate tab. One of our representatives will contact you to provide the relevant information.
An example CoA for the SCTi003-A cell line can be found here.
Licensing and Usage Information
What are the Terms and Conditions of Sale?
The Terms and Conditions of Sale can be found here.
What is the Limited Use License for STEMCELL’s iPSCs?
STEMCELL provides iPSCs to researchers under the following Limited-Use License:
These iPSCs shall be used for customers’ internal research. These iPSCs and their modifications (including but not limited to derivatives or differentiated progeny) shall not be used or administered in (1) human subjects for human clinical use; (2) animals for veterinary use for therapeutic, diagnostic or prophylactic purposes; or (3) any subject in relation to, without limiting the generality of the foregoing, clinical applications, cell therapy, transplantation, and/or regenerative medicines.
Customers need a license to cover commercial uses of these iPSCs and/or their modifications (including but not limited to derivatives or differentiated progeny), including but not limited to the following: (a) for the manufacture of any product; (b) for screening compounds, antibodies, proteins, or peptides for commercial use, except for target discovery, target validation or assay development; (c) sale, lease, distribution, transfer or the use of such cells to provide a service, information or data to a third party; (d) use by a for-profit organization for its internal research and not for financial gain. All requests for a commercial license for these iPSCs should be directed to iPSCrequests@stemcell.com. Other than the rights granted herein, no other right, express or implied, is conveyed by the sale of these iPSCs.
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.
Where can I view STEMCELL’s standard license agreement for iPSCs?
STEMCELL’s standard license agreement for iPSCs can be found here.
What applications are/are not permitted under STEMCELL’s standard license agreement for iPSCs?
|Example Applications of iPSCs or Progeny||Permitted / Not Permitted||Notes|
|Academic research by a non-profit entity such as a university||Permitted|
|Educational purposes by a non-profit entity such as a university||Permitted|
|Internal research by a commercial entity||Permitted|
|Provide service(s), information, or data to a third party for no financial gain||Permitted|
|Transfer to a third party to perform service(s) on behalf of the customer, such as genome editing, QC, differentiation, or drug development assays||Permitted||After the service has been completed, any resulting cell product(s) may not be resold.|
|Screening compounds, antibodies, proteins, or peptides for commercial use||Partially permitted||Screening with the intent to discover a marketable compound is not permitted under STEMCELL’s standard license agreement and requires an additional license from iPS Academia Japan, Inc. Target discovery, target validation, disease modeling, and assay development are permitted.|
|Distribution or transfer to a third party for no financial gain||Partially permitted||An iPSC line from STEMCELL may be distributed or transferred free-of-charge to end users that (1) are members of the same institute and (2) work in the same physical location and/or site as the original laboratory that purchased the line.|
|Direct administration to (1) human subjects for human clinical use or (2) animals for veterinary use for therapeutic, diagnostic or prophylactic purposes, including clinical applications, cell therapy, transplantation, and regenerative medicines||Not permitted|
|Development of regulatory registered human diagnostic products (e.g. in vitro diagnostics)||Not permitted|
|Manufacture of related products for sale (e.g. cell culture medium or instruments)||Not permitted||Requires an additional license from iPS Academia Japan, Inc.|
|Sale, lease, distribution, transfer, or license for financial gain||Not permitted||Additional approval required. Please contact us for further information.|
|Provide service(s), information, or data to third parties for financial gain||Not permitted||Additional approval required. Please contact us for further information.|
How to inquire about the use of STEMCELL’s iPSCs (including derivatives and/or differentiated progeny) for:
- Commercial screening purposes* or for the manufacture of a related product for sale**:
An additional license is required from iPS Academia Japan, Inc. Inquiries should be directed to firstname.lastname@example.org.
*Other than target discovery, target validation, or assay development.
**For example, cell culture media or equipment.
- Therapeutic, diagnostic, or prophylactic purposes:
STEMCELL’s iPSCs, derivatives, or differentiated progeny may not be used for therapeutic, diagnostic, or prophylactic purposes. To register your interest in using STEMCELL’s iPSCs for these applications, please contact us.
- Use in a service performed for financial gain:
STEMCELL’s iPSCs, derivatives, or differentiated progeny may not be used in a for-profit service without approval from STEMCELL. If approval is granted, additional terms and conditions may apply. Please contact us with details about your inquiry, including your name, institute/company name, contact information, country/region, and proposed use.
- The resale of STEMCELL’s iPSCs:
STEMCELL’s iPSCs may not be resold without approval from STEMCELL. If approval is granted, additional terms and conditions may apply. Please contact us with details about your inquiry, including your name, institute/company name, contact information, country/region, and proposed use.
- The manufacture of derivatives or differentiated progeny for financial gain:
STEMCELL’s iPSCs may not be used to manufacture derivatives or differentiated progeny for financial gain without approval from STEMCELL. If approval is granted, additional terms and conditions may apply. Please contact us with details about your inquiry, including your name, institute/company name, contact information, country/region, and proposed use.
Where can I find the Product Information Sheet (PIS) that contains detailed instructions on how to thaw and maintain iPSCs?
As part of our continuous effort to reduce our environmental impact, we are eliminating paper-based Product Information Sheets (PISs). The PIS contains important information about your iPSC line, including recommended thawing instructions and culture conditions. A digital copy of the PIS can be accessed via the product web page for your iPSC line.
An example PIS for the SCTi003-A cell line can be found here.
How many iPSCs will I receive in one vial?
Upon manufacturing commercial vials of iPSCs, a clump culture expanded to optimal density is dissociated into smaller aggregates using ReLeSR™ and cryopreserved in CryoStor® CS10 at a density of >15,000 aggregates/mL. The resulting cell suspension is aliquoted into 2 mL cryovials at a volume of 1 mL per vial. One cryovial yields approximately 1 million viable iPSCs immediately after thawing.
How should I thaw cryopreserved iPSCs?
For product-specific directions on how to thaw and seed iPSCs, please refer to the Product Information Sheet (PIS) for your iPSC line.
The following are generalized thawing instructions for iPSCs cryopreserved as aggregates:
Wipe the outside of the vial with 70% ethanol or isopropanol. In a biosafety cabinet, twist the cap a quarter-turn to relieve internal pressure, then retighten. Quickly thaw cells in a 37°C water bath by gently shaking the vial. Remove the vial when a small frozen cell pellet remains. Do not vortex cells. Wipe the outside of the vial with 70% ethanol or isopropanol. Use a 2 mL serological pipette to transfer the contents of the cryovial to a 15 mL conical tube. Add 5 - 7 mL of warm mTeSR™ Plus dropwise to the 15 mL tube, gently mixing as the medium is added. Centrifuge cells at 300 x g for 5 minutes at room temperature (15 - 25°C). Aspirate the medium, leaving the cell pellet intact. Resuspend the cell pellet in mTeSR™ Plus by gently flicking the tube. Avoid pipetting up and down and take care to maintain the cells as aggregates.
For more information about thawing iPSCs, see our Tech Tip: Cryopreservation and Thawing of Pluripotent Stem Cells.
What is the first thing I should do upon receiving a new iPSC line from STEMCELL?
Cryopreserved iPSCs should be thawed and placed into routine maintenance by following the instructions in the cell line-specific Product Information Sheet (PIS). Cultures should be closely monitored to ensure the maintenance and expansion of healthy cells in the undifferentiated state over long periods of time. For guidance on this topic, refer to our Tech Tip: Assessing Morphology of hPSCs.
If you are establishing a new iPSC line in your laboratory, we highly recommend expanding the cells in culture and creating a well-characterized bank for safekeeping. For best practices on the banking and characterization of hPSCs for research purposes, please refer to the consensus guidance published by the International Stem Cell Initiative (2009).
STEMCELL has collaborated with the WiCell Research Institute to offer a characterization and banking service for iPSCs. To learn more about this service, explore our website or contact our Contract Assay Services Team for more information.
What is the best culture system to maintain iPSCs in?
- mTeSR™ Plus is used to produce STEMCELL’s commercial iPSC lines. Manufactured under relevant cGMPs, mTeSR™ Plus ensures the highest quality and consistency for fundamental iPSC research as well as for cell therapy and investigational new drug research applications. With stabilized critical medium components—including FGF2—and enhanced pH buffering, mTeSR™ Plus maintains cell quality attributes and increases cell expansion rates and seeding efficiencies with either daily or restricted feeding schedules. For full details about how to use mTeSR™ Plus, please refer to the mTeSR™ Plus Technical Manual.
- ReLeSR™ is used for enzyme-free dissociation and passaging of iPSCs as aggregates without manual selection or scraping. Passaging iPSCs with ReLeSR™ generates optimally sized aggregates, while eliminating the hassle and variability associated with manual manipulation. By eliminating the need for scraping, ReLeSR™ enables the use of culture flasks and other closed vessels, thus facilitating culture scale-up and automation. ReLeSR™, 500 mL, is manufactured following relevant cGMPs under a certified quality management system to ensure the highest quality and consistency for reproducible results. For full details on how to use ReLeSR™, please refer to the Product Information Sheet.
- Corning® Matrigel® hESC-Qualified Matrix (Corning Catalog #354277) is used as a cell culture substrate in the production of STEMCELL’s iPSCs. Matrigel® is a reconstituted basement membrane preparation that is extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. This material, once isolated, is approximately 60% laminin, 30% collagen IV, and 8% entactin.
For full details about the recommended culture conditions for your iPSC line, including medium, supplement, substrate, dissociation reagent, split ratio, and incubator atmosphere, please refer to the lot-specific Certificate of Analysis (CoA).
What is the percentage of viable iPSCs after thaw?
For iPSCs cryopreserved as aggregates, cell viability after thaw is >60%. One cryovial will yield approximately 1 million viable iPSCs. Lot-specific viability measurements are described in the Certificate of Analysis (CoA) for your iPSC line. For more details about STEMCELL’s iPSC quality control assessments and release criteria, please refer to Quality Control of this FAQ.
Can I thaw iPSCs into a different culture system from which they were cryopreserved?
We do not recommend thawing iPSCs into a different culture system from which they were cryopreserved. iPSCs transitioned into a different culture system will require time to adapt to their new environment. As such, we cannot guarantee the successful recovery of iPSCs when thawed into a different culture system than what is described in the cell line-specific Product Information Sheet (PIS) and Certificate of Analysis (CoA).
If your experiment absolutely requires thawing STEMCELL’s iPSCs into a different culture system from which they were cryopreserved, we highly recommend changing only one culture system constituent (i.e. medium or substrate) at a time. To increase the likelihood of success, a broad range of cell concentrations should be seeded. Maintenance media can also be supplemented with CloneR™2 to further enhance seeding efficiency.
For full details on the recommended culture conditions for your iPSC line, including medium, supplement, substrate, dissociation reagent, split ratio, and incubator atmosphere, please refer to the lot-specific CoA.
How do I transition iPSCs into a different culture system?
No adaptation step is required when transitioning iPSCs from mTeSR™ Plus to mTeSR™1, TeSR™-E8™, or TeSR™-AOF, as long as the cells are not being transitioned directly from thawing. Follow an enzyme-free passaging protocol and plate cell aggregates onto coated cultureware containing your new maintenance medium of choice.
It is recommended that a culture using mTeSR™ Plus is initially maintained in parallel to ensure that the chosen plating density in the new medium is appropriate. For iPSCs being transitioned into a low-protein medium (e.g. TeSR™-E8™), a reduced growth rate may be observed, resulting in reduced confluence during the usual passaging interval.
Cultures should be monitored carefully after the transition to ensure selection of the optimal day for passaging, as it may be later than for cultures plated at the same density in stabilized media. Alternatively, the split ratio can be adjusted to compensate for reduced growth, for example, by seeding 20 - 25% more aggregates in TeSR™-E8™ if colonies are not reaching confluence at an acceptable rate.
How should STEMCELL’s iPSCs be passaged?
We recommend passaging iPSCs as small aggregates approximately 50 - 200 μm in diameter using ReLeSR™, an enzyme-free reagent for dissociating and passaging iPSCs without manual selection or scraping. This established method has been shown to allow the long-term expansion of many different cell lines while maintaining a normal karyotype. For full details on how to use ReLeSR™, please refer to the PIS. For a visual guide on how to generate cell aggregates and passage hPSCs, refer to this technical video.
Can I refreeze iPSCs after they have been expanded?
How many passages can STEMCELL’s iPSCs be expanded to?
Human iPSCs have a capacity for unlimited self-renewal and can be maintained in culture indefinitely. However, extensive passaging is not recommended. Cultures should be closely monitored to ensure the maintenance and expansion of healthy cells in the undifferentiated state over long periods of time.
If you are establishing a new iPSC line in your laboratory, we highly recommend creating a well-characterized master cell bank, and then generating a validated working cell bank that you can return back to every ~20 weeks. Unfortunately, somatic mutations that generate a selective advantage, such as a greater propensity for self-renewal, can become fixed in iPSC cultures over time. This selection leads to the development of nonrandom genetic changes found in human iPSCs maintained for long periods in culture. These changes, mostly detected by karyotypic analyses, commonly involve nonrandom gains of chromosomes 12, 17, 20, and X, or fragments of these chromosomes. As such, it is best practice to terminate cultures at approximately passage 50 and reinitiate cultures from a working cell bank.
At STEMCELL, we recommend routinely karyotyping your iPSCs by G-banding every 10 passages. For a rapid and cost-effective screening method for detecting the eight most commonly acquired abnormalities reported in iPSCs, consider implementing the hPSC Genetic Analysis Kit in your research. This qPCR-based kit enables the genetic screening of multiple hPSC lines and contains enough material to analyze 20 individual samples in triplicate.
What is the purity of OCT4+ and TRA-1-60+ cells in STEMCELL’s iPSCs?
STEMCELL analyzes markers of the undifferentiated state by quantifying OCT4 and TRA-1-60 expression levels by flow cytometry. Commercial iPSC lines express these markers at a level of >80%. Lot-specific undifferentiated marker expression levels are described in the Certificate of Analysis (CoA) for your iPSC line. For more details about STEMCELL’s iPSC quality control assessments and release criteria, please refer to the Quality Control section of this FAQ.
How long after thawing can iPSCs be differentiated into downstream cell types?
For iPSCs cryopreserved as aggregates, we recommend allowing the cells to stabilize in culture for a minimum of two passages prior to initiating downstream differentiation experiments, with the goal of establishing stable growth rates and high-quality morphological characteristics. Cryopreserved iPSCs should be thawed and placed into routine maintenance by following the instructions in the cell line-specific Product Information Sheet (PIS). Cultures should be closely monitored to ensure the maintenance and expansion of healthy cells in the undifferentiated state. It is essential that iPSCs are of high quality prior to initiating differentiation experiments.
What cell types can STEMCELL’s iPSCs be differentiated into?
All iPSC lines developed by STEMCELL are assessed for pluripotency using the STEMdiff™ Trilineage Differentiation Kit. Two lineage-specific markers are assessed by flow cytometry for each embryonic germ layer: PAX6 and NESTIN (ectoderm), BRACHYURY and NCAM (mesoderm), and CXCR4 and SOX17 (endoderm). Lot-specific trilineage marker expression levels can be found in the Certificate of Analysis (CoA) for your iPSC line.
Select iPSC lines undergo more extensive differentiation testing than others. For example, we have confirmed compatibility of SCTi003-A with the following STEMdiff™ kits:
- STEMdiff™ SMADi Neural Induction Kit
- STEMdiff™ Forebrain Neuron Differentiation Kit
- STEMdiff™ Midbrain Neuron Differentiation Kit
- STEMdiff™ Astrocyte Differentiation Kit
- STEMdiff™ Choroid Plexus Organoid Differentiation Kit
- STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit
- STEMdiff™ Cerebral Organoid Kit
- STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit
- STEMdiff™ Atrial Cardiomyocyte Differentiation Kit
- STEMdiff™ Hematopoietic Kit
- STEMdiff™ Microglia Differentiation Kit
- STEMdiff™ Intestinal Organoid Kit
The majority of differentiation data can be found in the “Data and Publications” section on the iPSC line-specific product web page. If you require further information about differentiation towards a particular lineage or cell type, please contact us.
Can cryopreserved iPSCs be seeded directly into a 3D culture system?
We do not recommend thawing cryopreserved iPSC aggregates directly into a 3D suspension culture system for scale-up. Once thawed iPSCs have stabilized as a 2D culture in mTeSR™ Plus on Corning® Matrigel® hESC-Qualified Matrix (Corning Catalog #354277), which takes approximately 2 passages, cells can be transitioned directly into a dynamic suspension culture with no separate adaptation step.
To successfully expand iPSCs and maintain pluripotency in a suspension culture system, it is crucial to begin with high-quality iPSC cultures. Typically, iPSCs are maintained in 2D in mTeSR™ Plus on Corning® Matrigel® hESC-Qualified Matrix and are passaged with ReLeSR™ as clumps that are 50 - 200 µm in diameter. Prior to transitioning into a 3D suspension culture system, high-quality iPSCs should display the following characteristics:
- Express high levels of undifferentiated markers (>90%), including OCT4, TRA-1-60, and SSEA-3 as assessed by flow cytometry
- Display high-quality colony morphology and a low percentage of differentiated cells (<10%)
- Have normal growth rates
- Retain karyotype as assessed by G-banding analysis or qPCR
Is it necessary to add antibiotics to iPSC maintenance media?
No, it is not necessary to add antibiotics to the media as aseptic technique should be sufficient to maintain sterile iPSC cultures. We recommend abstaining from the use of antibiotics in cell culture where possible, as their presence can mask any underlying contaminations.
Are STEMCELL’s iPSC lines manufactured under cGMP?
STEMCELL’s iPSC lines are not manufactured under cGMP. However, cGMP reagents are used for key stages of the iPSC manufacturing process, such as mTeSR™ Plus for maintenance and expansion, ReLeSR™ for dissociation and passaging, and CryoStor® CS10 for cryopreservation.
It is important to note that “GMP” is not a grade, but instead refers to the series of processes, policies, and controls within a quality management system (QMS) that ensures the resulting manufactured product is safe and efficacious for its intended use. A “GMP-ready” iPSC manufacturing process includes additional controls that are implemented at every stage of production, including GMP production team training, GMP facility preparation, and qualification of USP-grade characterization assays, among numerous other controls in the core areas of donor recruitment, tissue procurement and processing, supply chain, reprogramming, cell banking, and characterization.
To register your interest in the use of iPSC lines manufactured under cGMP, please contact us.
For iPSC lines derived from T cells, what information is provided about the T cell of origin?
For peripheral blood mononuclear cell (PBMC)-derived iPSC lines, we perform the T cell clonality assay to determine if the reprogrammed cell of origin was a T cell or not. For T cell-derived iPSC lines, the T cell receptor (TCR) is characterized as follows:
- TCR-αβ positive
- TCR-λβ positive
- TCR-αβ/λβ double positive
As we do not sort PBMCs prior to reprogramming, we are unable to determine if a T cell-derived iPSC line originated from a naïve or memory CD4 or CD8 T cell. For more information about the derivation cell of origin for your iPSC line, please refer to the Product Information Sheet (PIS) or Certificate of Analysis (CoA).
Shipping and Delivery
How are cryopreserved (frozen) iPSCs shipped?
STEMCELL’s iPSCs are shipped in CryoStor® CS10 cryopreservation medium, in a box within a container rated for 36 - 48 hours of dry ice preservation. Liquid nitrogen options may be available but are subject to higher shipping fees. For further information about shipping methods, please contact your STEMCELL sales representative or use our contact us form.
Are cryopreserved iPSCs available in my region?
STEMCELL provides iPSCs to most global regions. Some jurisdictions require additional permits. Please contact your STEMCELL sales representative or use our contact us form for the most up-to-date information on the availability of iPSCs in your region.
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Frequently Asked Questions on iPSCs