Human neural stem cells

Abstract ‘Human neural stem cells’ jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.

This document specifies the technical requirements, test methods, test regulations, instructions for use, labelling, packaging, storage, transportation and waste disposal requirements for hNSCs.This standard is applicable for the quality control of hNSCs derived from primary tissue and differentiated from pluripotent stem cells.

| NORMATIVE REFERENCES
The following content constitute 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
The following terms and definitions apply to this document.

| Human neural stem cells
Cells in the human central nervous system, which have self-renewal ability and the potential to differentiate into astrocytes, neurons and oligodendrocytes.

| Abbreviations
The following abbreviations are applicable for this document.

| Cell morphology
In two-dimensional culture, the nucleus of hNSCs is indistinguishable, cells exhibit a columnar morphology in single-or multi-layers under a microscope.In three-dimensional culture, cells can form compact and transparent spheres spontaneously.

| Chromosome karyotype
The karyotype shall be normal as 46, XX or 46, XY.

| Characteristics of differentiated cells
hNSCs shall be able to further differentiate into neurons and glial cells (astrocytes and/or oligodendrocytes).
Cell morphology: cells shall contain bipolar or multipolar neurons with long neurites.

| Cell morphology
Observe the morphology of cells using an optical microscope.

| Chromosome karyotype
The method in the Pharmacopoeia of the People's Republic of China (2020) shall be followed.

| Cell viability
The method in Annex A shall be followed.

| Cell markers
The method in Annex B or C shall be followed.

| Fungi
The '1101 Sterility Inspection Method' in Pharmacopoeia of the People's Republic of China (2020) (Volume IV) shall be followed.

| Bacteria
The '1101 Sterility Inspection Method' in Pharmacopoeia of the People's Republic of China (2020) (Volume IV) shall be followed.

| Mycoplasma
The '3301 Mycoplasma Inspection Method' in Pharmacopoeia of the People's Republic of China (2020) (Volume IV) shall be followed.

| HIV
The nucleic acid test method in WS 293 shall be followed.

| HBV
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| HCV
The nucleic acid test method in WS 213 shall be followed.

| HTLV
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| EBV
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| HCMV
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| TP
The nucleic acid test method in WS 273 shall be followed.

| In vitro differentiation capacity
The method in Annex B or C shall be followed.

| INSPECTION RULES
6.1 | Sampling method 6.1.1Cells produced from the same production cycle, same production line, same source, same passage and same method are considered to be the same batch.
6.1.2Three smallest units of packaging shall be randomly sampled from the same batch.
6.2 | Quality inspection and release 6.2.1 Each batch of products shall be subject to the qualify inspection before release, and inspection reports shall be attached.

6.2.2
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 or laboratories as necessary.

| Decision rules
6.4.1 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.6.4.2Products 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:  The appropriate materials and containers shall be selected to ensure the maintenance of the primary quality attributes of hNSCs.

| Storage
9.2.1 T/CSCB 0001 shall be followed.9.2.2 Products shall be stored in liquid nitrogen at a temperature below À130 C.

| Transportation
Ws 213 Diagnosis of hepatitis C Ws 273 Diagnosis for syphilis Ws 293 Diagnosis for HIV/AIDS T/CSCB 0001 General requirements for stem cells Pharmacopoeia of the People's Republic of China (2020) National Guide to Clinical Laboratory Procedures 3 | TERMS, DEFINITIONS AND ABBREVIATIONS

EBV
cytomegalovirus HIV: human immunodeficiency virus hNSC: human neural stem cell HTLV: human T-lymphotropic virus STR: short tandem repeats TP: treponema pallidum 4 | TECHNICAL REQUIREMENTS 4.1 | Raw materials and ancillary materials 4.1.1The collection of raw materials shall be in accordance with the domestically legal and ethical requirements.4.1.2The requirements of T/CSCB 0001-2020 shall be followed.4.1.3The donors shall be negative for HIV, HBV, HCV, HTLV, EBV, HCMV and TP.
4.3.1The process of cell cryopreservation, cell thawing and other cell manufacturing shall follow the requirements of T/CSCB 0001-2020.4.3.2The STR test results of cell products shall be consistent with STR of the donor cell.
code; n.Matters that need attention.Note: Provide endotoxin content according to user's requirement.
9.3.1 T/CSCB 0001-2020 shall be followed.9.3.2Cryopreserved cell products shall be transported in dry ice or below À130 C. 10 | WASTE DISPOSAL 10.1 Wastes generated during hNSCs production and testing shall follow the waste cell management documents, strictly implement the management standards and make detailed records.10.2 Any disposal of unqualified cells, leftover cells for disposal or donations during the research and production of hNSCs shall be conducted properly in accordance with appropriate legal and ethical requirements.
supported by the National Key Research and Development Program of China, Grant/Award Number: 2019YFA0110800, Project for Young Scientists in Basic Research, Grant/Award Number: YSBR-041; National Natural Science Foundation of China, Grant/Award Number: 31970821; Joint Funds