Requirements for human-induced pluripotent stem cells.

'Requirements for Human-Induced Pluripotent Stem Cells' is the first set of guidelines on human-induced pluripotent stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, and instructions for use, labeling, packaging, storage, transportation, and waste handling for human-induced pluripotent stem cells, which apply to the production and quality control of human-induced pluripotent stem cells. It was released by the Chinese Society for Cell Biology on 9 January 2021 and came into effect on 9 April 2021. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human-induced pluripotent stem cells for applications.

SCOPE

This document specifies the technical requirements of human‐induced pluripotent stem cells and the requirements for the test methods, instructions for use, labeling, packaging, storage, transportation, and waste handling.

This standard applies to the production and testing of human‐induced pluripotent stem cells. All the citations in the text can be found in supplementary material.

NORMATIVE REFERENCES

The following documents constitute an indispensable part of this standard through normative reference. For dated references, only the edition cited applies. For undated references, only the latest edition (including all amendments) applies.

GB/T6682 Water for analytical laboratory use—specification and test methods

WS 213 Diagnosis for hepatitis C

WS 273 Diagnosis for syphilis

WS 293 Diagnosis for HIV/AIDS

WS 299 Diagnostic criteria for viral hepatitis B

T/CSCB 0001 General requirements for stem cells

T/CSCB 0002 Human embryonic stem cell

Pharmacopoeia of the People’s Republic of China

National Guide to Clinical Laboratory Procedures

TERMS AND DEFINITIONS

For the purposes of this document, the terms and definitions in T/CSCB 0001, T/CSCB 0002, and the following terms and definitions apply.

Human‐induced pluripotent stem cell

These stem cells are generated from human somatic cells by reprogramming and having the ability to self‐renew indefinitely and differentiate into all derivatives of three germ layers including ectoderm, mesoderm, and endoderm.

Reprogramming

It is the process to generate human‐induced pluripotent stem cells by transgene expression, chemical treatment, or epigenetic modifications, etc.

ABBREVIATIONS

DNA—deoxyribonucleic acid.

EBV—Epstein‐Barr virus.

HBV—hepatitis B virus.

HCMV—human cytomegalovirus.

HCV—hepatitis C virus.

hiPSC—human‐induced pluripotent stem cell.

HIV—human immunodeficiency virus.

HTLV—human T‐lymphotropic virus.

PCR—polymerase chain reaction.

STR—short tandem repeat.

The following abbreviations apply to this document.

TP—Treponema pallidum.

TECHNICAL REQUIREMENTS

Source materials and ancillary materials

5.1.1. The raw materials, reagents, consumables and other ancillary materials, and/or supplies (e.g., gases) shall meet the requirements of T/CSCB 0001.

5.1.2. To ensure the well‐being of the donor and the safety of the donated cells, the process for donor evaluation and screening, cell collection, transportation, and receipt shall be standardized.

5.1.3. The donors shall be screened for HIV, HBV, HCV, HTLV, EBV, HCMV, and TP, and the results shall be documented.

Critical quality attributes

Cell morphology

Cells grown under 2D conditions shall form compact colonies with clear edges, exhibit uniform morphology with a high nucleus‐to‐cytoplasm ratio, and have tight junctions between cells.

Chromosome karyotype

The normal karyotype shall be 46, XY, or 46, XX.

Cell viability

Cell viability shall be ≥90% before cryopreservation, and ≥60% post‐thaw.

Cell markers

The expression of at least two of the cell surface markers SSEA3, SSEA4, TRA‐1‐60, and TRA‐1‐81 shall be ≥70.0% of the cell population, and the expression of the intracellular marker OCT4 and NANOG shall be ≥70.0% of the cell population.

Teratoma formation

Teratoma formation shall be able to form teratomas with derivatives from all three germ layers in vivo.

Microorganisms

Microorganisms shall be negative for fungi, bacteria, mycoplasma, HIV, HBV, HCV, HTLV, EBV, HCMV, and TP.

Process control

Cell authentication

The identity of hiPSCs shall match that of donor cells by STR analysis.

Reprogramming method

The method used for hiPSC reprogramming shall be documented.

Exogenous reprogramming genes

The expression and genomic integration of exogenous reprogramming genes shall be tested and documented.

Note: if hiPSCs are used as the raw material for manufacturing cell products for clinical applications, the test results for the expression and genomic integration of the exogenous reprogramming genes shall be negative.

TEST METHODS

Cell morphology

Cell morphology observes the morphology of cells grown under 2D conditions in vitro using an inverted phase‐contrast microscope.

Chromosome karyotype

The method in the Pharmacopoeia of the People’s Republic of China (the provision “Preparation and quality control of animal cell substrates used in manufacturing and analysis of biological products”) shall be followed.

Cell viability

The method in Appendix A shall be followed.

Cell markers

The method in Appendix B shall be followed.

Exogenous reprogramming genes

The method in Appendix C shall be followed.

Teratoma formation

The method in Appendix D shall be followed.

Microorganisms

Fungi

The method in the Pharmacopoeia of People’s Republic of China (section 1101—Sterility tests) shall be followed.

Bacteria

The method in the Pharmacopoeia of People’s Republic of China (section 1101—Sterility tests) shall be followed.

Mycoplasma

The method in the Pharmacopoeia of People’s Republic of China (section 3301—Mycoplasma tests) shall be followed.

HIV

The nucleic acid method in WS 293 shall be followed.

HBV

The nucleic acid method in WS 299 shall be followed.

HCV

The nucleic acid method in WS 213 shall be followed.

HTLV

The nucleic acid method in the National Guide to Clinical Laboratory Procedures shall be followed.

EBV

The nucleic acid method in the National Guide to Clinical Laboratory Procedures shall be followed.

HCMV

The nucleic acid method in the National Guide to Clinical Laboratory Procedures shall be followed.

TP

The nucleic acid method in WS 273 shall be followed.

Adventitious viruses

The method in the People’s Republic of China Pharmacopoeia (section 3302—Adventitious virus tests) shall be followed.

INSPECTION RULES

Sampling method and quantity

7.1.1. Cells produced in the same production cycle with the same production line, the same source, the same passage number, and the same production process are considered to be the same batch.

7.1.2. The three smallest units of packaging shall be randomly sampled from the same batch .

Quality inspection and release

7.2.1. Each batch of products shall be subject to quality inspection before release, and inspection reports shall be attached.

7.2.2. The quality inspection shall include all items listed in Section 5.2.

Review inspection

Review inspection shall be performed by professional cytological testing organizations/laboratories as necessary.

Decision rules

7.4.1. Products that pass all requirements in Section 5.2 for the quality inspection for release are considered to be qualified. Products that fail to pass one or more requirements in Section 5.2 for the quality inspection for release are considered to be unqualified.

7.4.2. Products that pass all requirements in Section 5.2 for the quality review inspection are considered to be qualified. Products that fail to pass one or more requirements in Section 5.2 for the review inspection are considered to be unqualified.

INSTRUCTIONS FOR USE

The instructions for use shall include, but not limited to:

Product name;

Passage number;

Cell numbers;

Reprogramming method;

Test results of residual exogenous reprogramming genes;

Production date;

Batch number;

Production organization;

Storage conditions;

Shipping conditions;

Operation manual;

Executed standard number;

Note: according to what standards are the cells produced

Manufacturing address;

Note: alternatively refers to the laboratory where the cells were derived.

Contact information;

Postal code;

Matters that need attention.

Note: upon user’s requirement, endotoxin test results can be provided.

LABELS

The label shall include but not limited to:

Product name;

Passage number;

Cell numbers;

Batch number;

Production organization;

Production date.

PACKAGE, STORAGE, AND TRANSPORTATION

Package

The material and container selected shall not interfere with the critical quality attributes of hiPSCs.

Storage

10.2.1. T/CSCB 0001 and T/CSCB 0002 shall be followed.

10.2.2. Cryopreserved cell products shall be stored at temperatures below −130°C.

Transportation

10.3.1. T/CSCB 0001 and T/CSCB 0002 shall be followed.

10.3.2. Cryopreserved cell products shall be transported with dry ice or below −130°C. Non‐frozen cell products are recommended to transport at 2–8°C.

WASTE HANDLING

Waste generated during the production process and quality inspection tests shall be handled according to the requirements of T/CSCB 0001.

CONFLICT OF INTEREST

The authors declare no competing financial interests.

AUTHOR CONTRIBUTIONS

JY and TZ contributed to conception and design. YZ (Ying Zhang) drafted and revised the manuscript. AM, KZ, HD, JK, ZJ, GP, JH, YZ (Yong Zhang), BF, SH, JN, YL, JC, LW, and HZ critically read and revised the manuscript.

Supplementary Material

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