Critical Quality Attributes and Assays for Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are a revolutionary type of stem cell that can differentiate into various cell types, making them highly valuable for regenerative medicine and disease modeling. However, ensuring the critical quality attributes (CQAs) of iPSCs is of utmost importance to ensure their safety and efficacy in clinical applications. The achievement of this goal depends on demonstrating comparability between cell lines and between products made from them, from different donors (whether autologous or allogeneic), between products from different manufacturers, and between different passages of the same cell line.

Identity

The risk of accidental line switching and cross-contamination of lines with other cell lines necessitates a stringent assay for cell line identification. Single tandem repeat (STR) genotyping of source tissue or cells and iPSC seed and master cell banks is recommended using a commercially available kit performed by an accredited laboratory.

Microbiological Sterility

• Maintaining microbiological sterility is crucial for the quality assurance of iPSCs to avoid contamination and subsequent adverse effects. In general, testing for mycoplasma, bacteriology, viral detection, and endotoxin is required.
• The mycoplasma and bacteriology tests could be PCR-, broth-, culture- or VERO incubation-based. All mandatory tests for human adventitious agents should be performed, such as hepatitis B virus, hepatitis C tests (HCV), human immunodeficiency virus, and nucleic acid testing. Amoebocyte Lysate (LAL) assay, is a widely accepted method, to quantify endotoxin levels in iPSC products.

Genetic Fidelity & Stability

• Genetic stability is a fundamental aspect of iPSCs to ensure that the desirable characteristics are preserved throughout long-term culture. Residual vector testing and karyotype are mandatory, while single nucleotide polymorphism arrays are for reference.
• Testing for reprogramming vectors is mandatory as there are potential safety issues if vectors have integrated into the host genome. Initial recommendations cover reprogramming by episomal vectors, as currently, this is the reprogramming technique most commonly considered for generating clinical-grade iPSC lines. Procedures for showing transgene clearance by Sendai virus or mRNA methods are currently being developed and may be included in the second iteration of these guidelines.
• To test for karyotype, it is recommended that a representative aliquot be resuscitated according to best practice and cultured for 48-72 h before cells are harvested for karyotypic analysis. A 20-metaphase karyotypic analysis is recommended since it is a universal clinical standard and is widely accepted by regulators worldwide, giving 95% certainty of diploidy.

Viability

Viability assessment is crucial to determine the functional state of iPSCs and their potential for successful engraftment or in vitro applications. It is recommended that viability should be quantified using a validated method and that a cell viability test be carried out on iPSC cultures 48 hours after resuscitation.

Characterization

In-depth characterization of iPSCs is essential to assess their biological properties and developmental potential. Immunophenotyping with a minimum of two markers from the standard hPSC panel (positive for OCT4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, Sox2, and Nanog) is mandatory. A combination of one intracellular (e.g., OCT4, SOX2, or Nanog) and one extracellular (e.g., SSEA-4 or TRA-1-60) should be used.

Potency

• Potency is the qualitative measure of the biological activity of the cells, which is linked to the relevant biological properties of the product. Risks associated with inadequate control of iPSC potency include lack of product efficacy, tissue chimerism, and inappropriate cell function.
• Self-renewal and undifferentiated cell markers are often used as surrogates to assess pluripotency but there is a need to functionally test for pluripotency. Embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers should be performed.

Creative Bioarray Relevant Recommendations

Our extensive expertise, combined with cutting-edge technologies and assays, ensures the reliability, efficacy, and safety of iPSCs for various applications. Creative Bioarray is your trusted partner in acquiring top-notch iPSCs with critical quality attributes.

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