Requirements for human embryonic stem cells.

'Requirements for Human Embryonic Stem Cells' is the first set of guidelines on human embryonic 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, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for human embryonic stem cells, which is applicable to the quality control for human embryonic stem cells. It was originally released by the China Society for Cell Biology on 26 February 2019 and was further revised on 30 April 2020. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human embryonic stem cells for applications.

SCOPE

This document specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for human embryonic stem cells.

This standard is applicable for the quality control of human embryonic stem cells.

NORMATIVE REFERENCES

The following content constitutes indispensable articles 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.

WS213 Diagnosis for hepatitis C

WS273 Diagnosis for syphilis

WS293 Diagnosis for HIV/AIDS

T/CSCB 0001‐2020 General requirements for stem cells

Pharmacopoeia of the People's Republic of China, Volume Ш

National Guide to Clinical Laboratory Procedures

Ethical guidelines for research of human embryonic stem cells

TERMS, DEFINITIONS AND ABBREVIATIONS

Terms and definitions

For the purposes of this document, the terms and definitions in T/CSCB 0001‐2020 and the following terms and definitions apply.

Human embryonic stem cell

The undifferentiated cells derived from pre‐implantation human embryo, which are able to self‐renew indefinitely in vitro and can differentiate into all cells of the three embryonic germ layers.

Karyotype

Chromosomal characteristics of a cell in the mitotic metaphase, including chromosome number, length, centromere position, satellites, primary constriction and secondary constriction.

Teratoma

A type of benign tumour containing representative differentiated tissues and cells from all three embryonic germ layers.

Mycoplasma

A class of small and facultatively anaerobic prokaryotic microbes which lack cell walls and can survive independently.

Abbreviations

The following abbreviations are applicable for this document.

DNA: deoxyribonucleic acid.

EBV: Epstein‐Barr virus.

HBV: hepatitis B virus.

HCMV: human cytomegalovirus.

HCV: hepatitis C virus.

HIV: human immunodeficiency virus.

HTLV: human T‐lymphotropic virus.

PCR: polymerase chain reaction.

STR: short tandem repeat.

TP: Treponema pallidum

TECHNICAL REQUIREMENTS

Source materials and auxiliary materials

For the harvesting of human biological source material (source materials), the Ethical guidelines for research on human embryonic stem cells shall be followed.

The source materials, reagents, consumables and other auxiliary materials and/or supplies (eg gases) shall meet the requirements of T/CSCB 0001‐2020.

The donor shall be negative for HIV, HBV, HCV, HTLV and TP.

Primary quality attributes

Primary quality attributes shall meet the requirements in Table 1.

Table 1

Primary quality attributes

Attributes	Requirements
Cell morphology	Cells grown in 2D conditions shall exhibit growth as colonies with clear boundaries, high nuclear‐cytoplasmic ratios and uniform morphology. Within each colony, cell‐cell contact should be tight.
Cell authentication	Cell lines shall have a known unique genetic profile to facilitate exclusion of cross‐contamination with other cells and confirm donor origin. Human short tandem repeat (STR) analysis shall include the following 16 human‐specific alleles: D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D16S539, TH01, TPOX, CSF1PO, D7S820, AMEL, PentaE and PentaD.
Chromosome karyotype	46, XX or 46, XY
Cell viability	≥90% before cryopreservation and ≥60% after resuscitation
Cell markers	Cell surface markers: ≥70.0% of the cell population express any two of the following genes: SSEA3, SSEA4, TRA‐1‐60, TRA‐1‐81, for example, TRA‐1‐81–positive rate ≥70.0% and SSEA4–positive rate ≥70.0%; intracellular markers: OCT4‐positive rate ≥70.0% and NANOG‐positive rate ≥70.0%
Teratoma formation	Shall generate teratomas with cells from all three germ layers
Microorganisms	Shall be negative for fungi, bacteria, mycoplasma, HIV, HBV, HCV, HTLV, EBV, HCMV and TP.

Process control

The process of cell expansion, cryopreservation and resuscitation shall follow the requirements of T/CSCB 0001‐2020.

INSPECTION METHODS

Cell morphology

Observe the morphology of cells grown in 2D condition using a microscope.

Cell authentication

The method in Appendix A shall be followed.

Chromosome karyotype

The method in the Pharmacopoeia of the People's Republic of China (Volume III) shall be followed.

Cell viability

The method in Appendix B shall be followed.

Cell markers

The method in Appendix C shall be followed.

Teratoma formation

The method in Appendix D shall be followed.

Microorganisms

Fungi

The method in Pharmacopoeia of the People's Republic of China (Volume III) shall be followed.

Bacteria

The method in Pharmacopoeia of the People's Republic of China (Volume III) shall be followed.

The method in Pharmacopoeia of the People's Republic of China (Volume III) shall be followed.

Human immunodeficiency virus

The method in WS 293 shall be followed.

Hepatitis B virus

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

Epstein‐Barr virus

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

Treponema pallidum

The method in WS 293 shall be followed.

INSPECTION RULES

Sampling method

Cells produced from the same production cycle, same production line, same source, same passage and same method are considered to be the same batch.

Three smallest units of packaging shall be randomly sampled from the same batch.

Quality inspection and release

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

The quality inspection items shall include all the attributes specified in 4.2.

Review inspection

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

Decision rules

Products that pass all requirements in 4.2 for the quality inspection for release are considered to be qualified. Products that fail to pass one or more requirements in 4.2 for the quality inspection for release are considered to be unqualified.

Products that pass all requirements in 4.2 for the quality review inspection are considered to be qualified. Products that fail to pass one or more requirements in 4.2 for the review inspection are considered to be unqualified.

INSTRUCTIONS FOR USAGE

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

Product name;

Passage number;

Cell numbers;

Production date;

Lot number;

Production organization;

Storage conditions;

Shipping conditions;

Contact information;

Operation manual ;

Execution standard number;

Note: according to what standards are the cells produced

Manufacturing address;

Note: alternatively refers to the derivation laboratory

Postal code;

Matters that need attention.

LABELS

The label shall include but not limited to:

Product name;

Passage number;

Cell number;

Lot number;

Production organization;

Production date.

PACKAGE, STORAGE AND TRANSPORTATION

Package

The appropriate materials and containers shall be selected to ensure maintenance of the primary quality attributes of human embryonic stem cells.

Storage

T/CSCB 0001‐2020 shall be followed.

Productions should be stored in liquid nitrogen.

Transportation

T/CSCB 0001‐2020 shall be followed.

Cryopreserved cell products shall be transported in dry ice or liquid nitrogen.

CONFLICT OF INTEREST

No potential conflicts of interest are disclosed.

AUTHOR CONTRIBUTIONS

QZ and TZ contributed to conception and design. JH, JC, LW, AM and SC drafted and revised the manuscript. JD, LW, BF, YZ, XP, PX, QL, YZ, JZ, SH, JY, TW, HZ and GS critically read and revised the manuscript.