Requirements for human haematopoietic stem/progenitor cells

Abstract ‘Requirements for human haematopoietic stem/progenitor cells’ is the first set of guidelines on human haematopoietic stem/progenitor cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, inspection methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements for human haematopoietic stem/progenitor cells, which is applicable to the quality control for human haematopoietic stem/progenitor cells. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human haematopoietic stem/progenitor cells for applications.


| SCOPE
This document specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements of human haematopoietic stem/progenitor cells.
This document is applicable for the production and testing of human haematopoietic stem/progenitor 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 DEFINITI ON S
For the purpose of this document, the terms and definitions in T/ CSCB 0001, T/CSCB 0002 and following terms and definitions apply to this document.

| Human haematopoietic stem cells
Stem cells that have the ability to self-renew and differentiate into all mature blood cell types.

| Human haematopoietic progenitor cells
Progenitor cells that have the ability to differentiate into multiple or a particular lineage of blood cells.

| Haematopoietic colony
Cell clusters or colonies containing recognizable progeny generated from individual haematopoietic progenitor cells cultured in a semisolid medium containing the appropriate cytokines. institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human haematopoietic stem/progenitor cells for applications.

| Cell morphology
Cells grown under suspended conditions shall be round and uniform in size. Cells shall be round, and exhibit high nuclear-to-cytoplasmic ratio, light blue cytoplasm and no granular structure in the cytoplasm after the Wright-Giemsa staining.

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

| Cell markers
The expression of CD34 shall be ≥80% of the cell population.

| Colony-forming unit assays
Number of total colonies shall be ≥10 per 10 3 cells, and number of CFU-GEMM colonies shall be ≥1 per 10 3 cells.

| Cell morphology
Observe the morphology of cells using a microscope. Cell staining shall be performed following the National Guide to Clinical Laboratory Procedures.

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

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

| Cell markers
The method in Appendix B shall be followed.

| Colony-forming unit assays
The method in Appendix C shall be followed.

| Haematopoietic reconstitution assays
The method in Appendix D shall be followed.

| Fungi
The method '1101 sterility test' in the Pharmacopoeia of the People's Republic of China shall be followed.

| Bacteria
The method '1101 sterility test' in the Pharmacopoeia of the People's Republic of China shall be followed.

| Mycoplasma
The method '3301 sterility test' in the Pharmacopoeia of the People's Republic of China shall be followed.

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

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

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

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

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

| IN S PEC TI ON RULE S
7.1 | Sampling method 7.1.1 Cells produced from the same production cycle, same production line, same source, same passage and same method are considered to be the same batch.
7.1.2 Three smallest units of packaging shall be randomly sampled from the same batch.

| Quality inspection and release
Each batch of cell preparation shall be subject to the quality inspection before release. The quality inspection items shall include all the attributes specified in 5.2. The inspection reports shall be attached.

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

| Decision rules
Products that pass all requirements in 5.2 for the quality inspection and quality review inspection are considered to be qualified.
Products that do not meet these criteria should be considered unqualified.

| IN S TRUC TI ON FOR USAG E
The instructions for usage 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 haematopoietic stem/progenitor cells.

| WA S TE D IS P OSAL S
Waste that arises during human haematopoietic stem/progenitor cell production and detection shall be disposed according to the regulations in T/CSCB 0001.

A.2 | Reagents
Unless otherwise specified, all reagents shall be of analytical purity.
The water used in all tests shall be deionized.

A.3.1 Preparing single-cell suspension
Collect the cells to be tested and resuspend the cells in phosphate-

A.4 | Calculation
The cell viability can be calculated with the following formula

A.5 | Precision
Under the same condition, the absolute deviation of the two repeats should not exceed 10% of the arithmetic mean.

A PPE N D I X B (N O R M ATI V E)
Cell markers and flow cytometry analysis

B.3.1 Sample preparation
Harvest single cells by centrifugation at 300 g for 5 min. Discard

B .4 | R E S U LT A N A LYS I S
The results of flow cytometry analysis are analysed comprehensively with appropriate software following its user manual.

A PPE N D I X C (N O R M ATI V E)
Colony-forming unit assays

C.4 | Analysis of results
Count and evaluate the colonies including BFU-E, CFU-G/M/GM and CFU-GEMM using the microscope and the gridded scoring dish.

A PPE N D I X D (N O R M ATI V E)
In vivo haematopoietic reconstruction transplantation experiment

D.3.4 Result analysis
Using flow cytometry software to analyse the results of flow cytometry, the percentage of human CD45 + cells in the PBMCs is not less than 5%.
Unlabelled PBSCs from transplanted mice were used as negative control 1, and antibody-labelled PBMCs from transplanted mice were used as biological control 2.
Firstly, according to the FSC and SSC, the target cell group 1 was gated, and the dead cells, platelets, red blood cells and cell fragments were excluded. According to the fluorescence intensity of negative control 1, the positive gate was delimited, and the positive cell group 2 was gated based on group 1.
Following the above gate setting principles, human CD45 + CD19 + cells, human CD45 + CD3 + cells, human CD45 + CD33 + cells and human CD45 − CD235a + cells were detected. The tested positive rate minus the positive rate of control 2 is the actual positive rate of the tube. If the value is greater than 1%, it is considered positive.