AMSBIO announces its recent accreditation for ISO9001:2015

June 23, 2010

Culture Techniques for Expanding a Uniform Set of iPSC for Drug Discovery and Therapeutic Use

July 30, 2024

International Standards for publication of Stem Cell Research

10 minute read.

Post by Bethany James, Ph.D. (Stem cells and Neuroscience), Scientific Content Specialist at AMSBIO

1. Introduction: Why do the Standards Matter?

In June 2023, the International Society for Stem Cell Research (ISSCR) released their standards for human stem cell use in research. The overall purpose of these standards is to provide scientists with a set of recommendations in order to meet the minimum characterization and reporting criteria for research laboratories working with human stem cells. A checklist for reporting practices for publishing results with Human Pluripotent and Tissue Stem Cells has been put together from these standards, for use by reviewers and editors when assessing manuscripts.

It’s now essential that any publication includes detailed information on the parameters within the ISSCR Standards with the goal of increasing transparency of experimental details and promoting reproducibility. AMSBIO wants to help stem cell biologists with experimental design to ensure you meet publication standards. So, we’ve put together a list to highlight some of the key parameters to consider in the new ISSCR standards. We’ve also included a resource center which we will update regularly to keep it up to date with the latest guidelines for stem cell culture.

Introduction
Basic Characterization
Pluripotency and Genomic Characterization
1. Pluripotency
2. Assessing Genetic Stability
Conclusion
Resources

2. Basic Characterization of your Stem Cell Cultures

Section 1 of the ISSCR Standards provides advice on the basic principles of stem cell culture including information on acquisition/generation, preservation and early characterization which is essential to promote reproducibility and safety in the laboratory.

2.1. Cell Hygiene

One of the most important factors to consider for laboratory quality control and safety is Cell Hygiene. Cell cultures should undergo microbiological and viral testing including routine mycoplasma screening to promote sterility.

The recommended testing frequency from the ISSCR Standards is for initial screening of cultures upon entering the laboratory, to be followed by regular monitoring (quarterly at a minimum). A particularly important time-point is when sharing lines outside the laboratory, to prevent the spread of contamination between research laboratories.

Focus Area: Mycoplasma Contamination

Mycoplasma contamination is known to be a significant issue for cell culture with rates ranging from >80% depending on location and monitoring records (as reviewed in the ISSCR standards 2023).

Why is Mycoplasma contamination a widespread issue?

The contamination is undetectable with standard laboratory equipment, is resistant antibiotic treatment and evades filtration devices.

Why is this significant?

The infection affects both cell structure and function rendering any results obtained from infected cultures as unreliable.

How can I detect Mycoplasma?

Our MycoScope™ PCR Mycoplasma detection kit provides a sensitive and rapid detection of mycoplasma infection in cell cultures in less than 3 hours. The kit has been tested for use with a variety of commercially available DNA polymerases and is easy to use. To test your cell cultures simply remove cell supernatant and follow the kits instructions. The distinct banding at 500bp on an agarose gel allows easy detection using the same PCR primer set to allow you run multiple samples at once.

2.2. Acquisition of Materials

When acquiring new cell material (whether this be donor samples, primary tissues, or cell lines) appropriate transfer agreements are essential to provide background on all associated donor and supplier/provider restrictions. It is essential that researchers understand these restrictions and agreements as well as any additional local restrictions relating to the use of the materials.

Our extensive biorepository houses a wide variety of fresh tissues, which are ethically sourced and IRB-approved, and a range of cell lines. We have strict quality control measures in place and our experts are on hand to sort out all required transfer agreements to make sure you can trust your starting source.

2.3. Principles of Cell Line Biobanking

Once cell materials have been acquired, a Master Cell Bank should be generated from the earliest possible passage of an established cell line or development of a non-adherent culture before experimental use. This is a critical process for preserving quality-controlled cells. From this master bank it is strongly recommended to make a further working cell bank for experimental use. We provide a range of trusted and validated cryopreservation solutions for cells and tissues for long-term storage with high cell viability after freeze-thaw procedures. To find out more, you can browse our CELLBANKER® cell freezing media range. It’s important to establish the nomenclature/ unique identifier for each cell line to ensure it is persistent between laboratories and can be referenced in publications. More information is provided within the report for guidance on assigning cell line identifiers. After post-thaw, cells must be fully characterized and go through cell line authentication (Short Tandem Repeat analysis is recommended).

2.4. Reprogramming Transgene Elimination

An important factor to consider when forming new human induced pluripotent stem cell lines is the verification of the elimination of the transgene expression prior to biobanking and experimental use.

We offer a range of viral delivery systems that can be used to overexpress reprogramming factors. qPCR can be used to confirm vector clearance, ensuring clearance of transgene expression after reprogramming. Our PCR reagents and products are available to help you source materials for qPCR validation.

3. Pluripotency and Genomic Characterization of Human Pluripotent Stem Cells (hPSCs)

3.1. Pluripotency

The defining feature of hPSCs is their potential to self-renew as well as give rise to all somatic lineages – this is termed an undifferentiated developmental state. To characterize hPSCs, pluripotency should be rigorously demonstrated for not only newly derived cell lines, new culture systems and genetically modified lines, but also in established lines, to ensure their continued potency.

Pluripotency should be demonstrated experimentally by assays assessing differentiation capacity. Differentiation should be shown by quantitative measurements of the induction of marker combinations representative of ectoderm, endoderm and mesoderm lineages, alongside a loss of markers of the undifferentiated state.

In vitro assays are recommended for showing differentiation to specific lineages/cell types. Evidence should be presented of differentiation into progenitors of definitive endoderm, mesoderm and neuroectoderm. At AMSBIO we offer a unique Quick-Trilineage™ Differentiation kit allowing researchers to quickly, easily, and efficiently differentiate PSCs into the cells of the three germ layers, using Sendai virus to induce differentiation in 6 days, without leaving a genetic footprint.

Evidence of differentiation should be based upon criteria such as: morphology, expression of cell type specific mRNAs or proteins, cell surface markers and functionality testing. Marker expression should be quantified where possible by flow cytometry or quantitative imaging. Highly specific antibodies for a range of protein detection are available through AMSBIO to validate your cell lineage of choice.

3.2. Assessing Genetic Stability

Culture-acquired genetic changes can have numerous irreversible effects on stem cells and their differentiation progeny including altered growth rate, tumorigenicity, differentiation potential, and functionality. Variant stem cells with a selective growth advantage can often rapidly out-compete wild-type cells and affect reproducibility of results. Therefore, cultures should be closely monitored for the presence of cultured-acquired genetic changes.

Currently, no single method can detect all types of genetic changes with high sensitivity and specificity. The specificity and detection limits of each individual assay should be considered when selecting a method for assessment. As a standard, cells should be monitored when forming a new master and working cell bank, through the time span of experiments and when a new cell line or derivative is generated.

Another important factor to consider is selecting cell culture conditions that support high rates of expansion while maintaining genetic stability. Chemically defined culture conditions provide a precisely controlled and reproducible environment for cell growth and maintenance. Animal-origin free media, such as StemFit, uses well-defined and purified components, eliminating variability and batch-to-batch inconsistencies. This standardization not only enhances the reproducibility of experiments but also minimizes the risk of introducing unwanted variables that could impact the genetic stability and differentiation potential of iPSCs. Additionally, single cell expansion is becoming a popular method for producing consistent iPSC cultures. iMatrix-511 laminin E8 fragments facilitates single-cell expansion by providing extracellular matrix (ECM) support, promoting cell survival through fast attachment of cells and appropriate migration.

At AMSBIO we offer stem cell culture media, matrix and cryopreservation solutions optimized to expand a uniform set of iPSCs and maintaining genetic stability. To find out more about what we can offer visit our expanded stem cell synergy webpage or read our expert blog Culture Techniques for Expanding a Uniform Set of iPSC for Drug Discovery and Therapeutic Use | AMSBIO.

4. Conclusion

In summary, we’ve explored the critical aspects in section 1-3 of the ISSCR standards for human stem cell use in research, covering essential topics such as basic characterization, pluripotency and the undifferentiated state, as well as genomic characterization. These foundational areas are crucial for ensuring the integrity and reliability of stem cell research.

In an upcoming blog, we’ll delve into the final sections of the ISSCR standards, focusing on stem cell-based model systems and the importance of accurate and transparent reporting in research. Stay tuned for insights into how these standards guide in the publication of ethical and reliable practices in stem cell research.

5. Resources

For further information, including information on standards for stem cell-based model systems and experimental reporting, see our resource centre below.

Standards Document — International Society for Stem Cell Research (isscr.org)

ISSCR_Standards_Dec23_Update.pdf (squarespace.com)

ISSCR_Standards_Checklist_2024Update.pdf (squarespace.com)

Affordable & consistent controls from 50 tumor cell lines, ideal for IHC, in situ hybridization & NGS applications.

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We are a global provider of human and animal biospecimens: including frozen & FFPE tissue, DNA, RNA, total proteins, blood products and primary cells.

We are a global provider of human and animal biospecimens: including frozen & FFPE tissue, DNA, RNA, total proteins, blood products and primary cells.

We are a global provider of human and animal biospecimens: including frozen & FFPE tissue, DNA, RNA, total proteins, blood products and primary cells.

We are a global provider of human and animal biospecimens: including frozen & FFPE tissue, DNA, RNA, total proteins, blood products and primary cells.

We are a global provider of human and animal biospecimens: including frozen & FFPE tissue, DNA, RNA, total proteins, blood products and primary cells.

We are a global provider of human and animal biospecimens: including frozen & FFPE tissue, DNA, RNA, total proteins, blood products and primary cells.

We are a global provider of human and animal biospecimens: including frozen & FFPE tissue, DNA, RNA, total proteins, blood products and primary cells.

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The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

The CELLBANKER® series of cryopreservation media allows for the stable long-term storage of any cell type, including sensitive cell lines.

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AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

AMSBIO offers one of the largest selection of active recombinant enzymes and enzyme activity assays for profiling, inhibitor screening assays, and drug discovery.

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Prepared from Arthrobacter luteus, Zymolyase has a highly effective lytic activity against yeast cell walls.

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AMSBIO announces its recent accreditation for ISO9001:2015

Culture Techniques for Expanding a Uniform Set of iPSC for Drug Discovery and Therapeutic Use

International Standards for publication of Stem Cell Research

1. Introduction: Why do the Standards Matter?

4. Conclusion

5. Resources

Related Product Info Pages

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MycoScope™ PCR Mycoplasma Detection Kit

STEM-CELLBANKER® Stem Cell Freezing Media

StemFit® Feeder-Free Stem Cell Culture Media

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