Requirements for human embryonic stem cells

Abstract ‘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.

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 REFEREN CE S
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.

| 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. 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.

| Primary quality attributes
Primary quality attributes shall meet the requirements in Table 1.

| Process control
The process of cell expansion, cryopreservation and resuscitation shall follow the requirements of T/CSCB 0001-2020.

| 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.

| 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.

| Mycoplasma
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.

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.
Chromosome karyotype 46, XX or 46, XY Cell viability ≥90% before cryopreservation and ≥60% after resuscitation 1. Cells produced from the same production cycle, same production line, same source, same passage and same method are considered to be the same batch.
2. Three smallest units of packaging shall be randomly sampled from the same batch.

| Quality inspection and release
1. Each batch of products shall be subject to the qualify inspection before release, and inspection reports shall be attached.
2. 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
1. 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.
2. 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.

| IN S TRUC TI ON S FOR USAG E
The instructions for usage shall include, but not limited to: 14. Matters that need attention.

| L AB EL S
The label shall include but not limited to:

| Package
The appropriate materials and containers shall be selected to ensure maintenance of the primary quality attributes of human embryonic stem cells.
2. Productions should be stored in liquid nitrogen.
2. Cryopreserved cell products shall be transported in dry ice or liquid nitrogen. Technology.

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest are disclosed.

A.1 Instruments and equipment
A.

A.3.2 Amplification of STR loci using fluorescent primers
Perform amplification according to the manufacturer's instructions.

A.3.3 Capillary electrophoresis of PCR products
Capillary electrophoresis testing and analysis shall be performed according to the manufacturer's instructions for the genetic sequence analyzer.

A.4 Result analysis
Alignment of the obtained STR typing map with the human standard STR map for comparison.

B.2 Reagents
Unless otherwise stated, all reagents used shall be analytical grade. The water used for testing shall be deionized water.

B.4 Calculation and analysis
Cell viability is calculated according to equation (1): In the equation:

S--viability of cells M--total number of cells D--number of stained cells
The viability of cells is the mean of two duplicate samples. Two independent cell viability tests shall be performed on the same sample. The mean value of two independent viability tests is recorded as the viability of cells.

B.5 Accuracy
The absolute difference value between the two independent tests, under the same conditions, shall not exceed 10% of their arithmetic mean.

D.3.3 Teratoma sampling and processing
Six-10 weeks after transplantation with human embryonic stem cells (avoid allowing teratomas to grow beyond 15% of the mouse body weight), euthanize the mice. Dissociate the teratomas in the mice and cut the teratomas into small blocks (the volume of each block shall not exceed 5 × 5 × 2 mm). Fix the teratoma blocks with 4% paraformaldehyde at 4°C overnight.

D.3.4 Paraffin sectioning and HE staining
Embed the fixed samples in paraffin and section the paraffinembedded tissue blocks into slices of 4-10 μm in thickness. Float the slices onto clean glass slides. Perform haematoxylin and eosin (HE).
Record the staining results by photography under a microscope.

D.4 Analysis of results
Teratomas containing tissues and cells from all three embryonic germ layers (eg, glandular epithelial tissue from the endoderm, cartilage tissues from the mesoderm, and neural tissues from the ectoderm) indicate that the human embryonic stem cells possess pluripotency.