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.
Keywords: applications, human haematopoietic stem/progenitor cells, requirements, standard
1. 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.
This document does not apply to haematopoietic stem cells involved in the Technical management specifications for haematopoietic stem cell transplantation and Cord blood haematopoietic stem cell bank management specifications (Trial).
2. 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.
GB/T 6682 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
T/CSCB 0001 General requirements for stem cells
Pharmacopoeia of the People's Republic of China
National Guide to Clinical Laboratory Procedures
3. TERMS AND DEFINITIONS
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.
3.1. Human haematopoietic stem cells
Stem cells that have the ability to self‐renew and differentiate into all mature blood cell types.
3.2. Human haematopoietic progenitor cells
Progenitor cells that have the ability to differentiate into multiple or a particular lineage of blood cells.
3.3. Haematopoietic colony
Cell clusters or colonies containing recognizable progeny generated from individual haematopoietic progenitor cells cultured in a semi‐solid medium containing the appropriate cytokines.
4. ABBREVIATIONS
BFU‐E: burst‐forming unit‐erythroid
CD: cluster of differentiation
CFU‐GEMM: colony‐forming unit‐granulocyte‐erythroid‐macrophage‐megakaryocyte
CFU‐GM: colony‐forming unit‐granulocyte‐macrophage
EBV: Epstein‐Barr virus
HBV: hepatitis B virus
HCMV: human cytomegalovirus
HCV: hepatitis C virus
HIV: human immunodeficiency virus
HTLV: human T‐cell lymphotropic virus
NOD‐SCID: non‐obese diabetic‐severe combined immunodeficient
STR: short tandem repeat
TP: treponema pallidum
5. TECHNICAL REQUIREMENTS
5.1. Source materials and ancillary materials
5.1.1 The requirements of T/CSCB 0001 shall be followed.
5.1.2 To ensure the safety of the donor and the donated cells, the process for donor evaluation and screening, cell collection, transportation and receipt shall be standardized.
5.1.3 The donor shall be screened for HIV, HBV, HCV, HTLV, EBV, HCMV and TP, and the results shall be documented.
5.2. Primary quality attributes
5.2.1. 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.
5.2.2. Chromosome karyotype
The normal karyotype shall be 46, XY or 46, XX.
5.2.3. Cell viability
Shall be ≥85% before cryopreservation and ≥70% after resuscitation.
5.2.4. Cell markers
The expression of CD34 shall be ≥80% of the cell population.
5.2.5. Colony‐forming unit assays
Number of total colonies shall be ≥10 per 103 cells, and number of CFU‐GEMM colonies shall be ≥1 per 103 cells.
5.2.6. Haematopoietic reconstitution assays
Sixteen weeks post‐transplantation into NOD‐SCID Il2rgnull mice, human CD45+ cell reconstitution levels shall be ≥5%, and human CD45+CD19+ cells, CD45+CD3+ cells, CD45+CD33+ cells and CD45−CD235a+ cells shall be positive in total peripheral blood mononuclear cells of the recipient mice.
5.2.7. Microorganisms
Fungi, bacteria, mycoplasma, HIV, HBV, HCV, HTLV, EBV, HCMV and TP shall be negative.
5.3. Process control
5.3.1 The process of cell expansion, cryopreservation and resuscitation shall follow the requirements of T/CSCB 0001.
5.3.2 The identity of the cells shall match with that of donor cells by STR analysis.
6. TEST METHODS
6.1. Cell morphology
Observe the morphology of cells using a microscope. Cell staining shall be performed following the National Guide to Clinical Laboratory Procedures.
6.2. Chromosome karyotype
The method in the Pharmacopoeia of the People's Republic of China shall be followed.
6.3. Cell viability
The method in Appendix A shall be followed.
6.4. Cell markers
The method in Appendix B shall be followed.
6.5. Colony‐forming unit assays
The method in Appendix C shall be followed.
6.6. Haematopoietic reconstitution assays
The method in Appendix D shall be followed.
6.7. Microorganisms
6.7.1. Fungi
The method ‘1101 sterility test’ in the Pharmacopoeia of the People's Republic of China shall be followed.
6.7.2. Bacteria
The method ‘1101 sterility test’ in the Pharmacopoeia of the People's Republic of China shall be followed.
6.7.3. Mycoplasma
The method ‘3301 sterility test’ in the Pharmacopoeia of the People's Republic of China shall be followed.
6.7.4. HIV
The nucleic acid test method in WS 293 shall be followed.
6.7.5. HBV
The nucleic acid test method in the National Guide to Clinical Laboratory Procedures shall be followed.
6.7.6. HCV
The nucleic acid test method in WS 213 shall be followed.
6.7.7. HTLV
The nucleic acid test method in the National Guide to Clinical Laboratory Procedures shall be followed.
6.7.8. EBV
The nucleic acid test method in the National Guide to Clinical Laboratory Procedures shall be followed.
6.7.9. HCMV
The nucleic acid test method in the National Guide to Clinical Laboratory Procedures shall be followed.
6.7.10. TP
The nucleic acid test method in WS 273 shall be followed.
7. INSPECTION RULES
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.
7.2. 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.
7.3. Review inspection
Review inspection shall be performed by professional cytological testing institutions/laboratories as necessary.
7.4. 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.
8. INSTRUCTION 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;
Operation manual;
Execution standard number;
Manufacturing address;
Contact information;
Postal code;
Matters that need attention.
Note: Upon user’s requirement, endotoxin test results can be provided.
9. LABELS
The label shall include, but not limited to:
Product name;
Passage number;
Cell number;
Lot number;
Production organization;
Production date.
10. PACKAGE, STORAGE AND TRANSPORTATION
10.1. Package
The appropriate materials and containers shall be selected to ensure maintenance of the primary quality attributes of human haematopoietic stem/progenitor cells.
10.2. Storage
10.2.1 T/CSCB 0001 shall be followed.
10.2.2 Productions should be stored at a temperature below ‐130°C.
10.3. Transportation
10.3.1 T/CSCB 0001 shall be followed.
10.3.2 Cryopreserved cell products shall be transported in dry ice or at temperature below ‐130°C. Non‐cryopreserved cell products shall be transported at temperature between 2 and 8°C.
11. WASTE DISPOSALS
Waste that arises during human haematopoietic stem/progenitor cell production and detection shall be disposed according to the regulations in T/CSCB 0001.
ACKNOWLEDGEMENT
This work was supported by grants from the National Key R&D Program of China 2017YFA0103104, 2018YFA0108400 and 2018YFE0204400; the Strategic Priority Research Program of the Chinese Academy of Sciences XDA16040501, XDA16040502 and XDA16040504; and Guangzhou Health Care and Cooperative Innovation Major Project 201803040005.
APPENDIX A.
(Normative)
Cell Viability Test and Cell Counting
A.1. Instruments
A.1.1 Microscope
A.1.2 Haemocytometer (XB‐K‐25)
A.2. Reagents
Unless otherwise specified, all reagents shall be of analytical purity. The water used in all tests shall be deionized.
A.2.1 Phosphate‐buffered saline: pH 7.4.
A.2.2 Trypan blue solution: concentrated stock solution is diluted to 0.4% (W/V) with phosphate‐buffered saline (A.2.1) for use.
A.3. Procedure
A.3.1 Preparing single‐cell suspension
Collect the cells to be tested and resuspend the cells in phosphate‐buffered saline (A.2.1).
A.3.2 Trypan blue staining
Dilute the cell suspension (A.3.1) in trypan blue solution (A.2.2) at 1:1, and mix well.
A.3.3 Cell counting
Put coverslips on each chamber of a clean haemocytometer (A.1.2). Transfer 10 µL trypan blue/cell suspension (A.3.2) to the edge of the coverslip, allowing the cell suspension to fully fill the chambers under the coverslip without over‐ or underfill. Repeat with the second chamber. Let stand for 30 seconds, and count all cells (stained and unstained) and the stained cells in each chamber under microscope (A.1.1).
Repeat steps A.3.2 to A.3.3 once.
A.3.4 Calculating the viability
A.4. Calculation
The cell viability can be calculated with the following formula (A.1):
S = (M‐D)/M × 100% (A.1)
Note:
S—cell viability
M—total cell number
D—stained cell number
Calculate the average viability of two repeats. This result is the average cell viability.
A.5. Precision
Under the same condition, the absolute deviation of the two repeats should not exceed 10% of the arithmetic mean.
APPENDIX B.
(Normative)
Cell markers and flow cytometry analysis
B.1. Instruments
B.1.1 Flow cytometer
B.1.2 Horizontal centrifuge
B.1.3 Electronic balance
B.2. Reagents
All reagents in this method are of analytical purity. Unless otherwise specified, the water used in all tests is the level 1 water as specified in GB/T 6682.
B.2.1 Phosphate‐buffered saline (PBS): pH 7.4.
B.2.2 Bovine serum albumin (BSA): purity ≥98%
B.2.3 Anti‐human CD34 antibody and isotype control.
B.2.4 Prepare solutions needed for flow cytometry assay using electronic balance (B.1.3): washing buffer, fixation buffer, blocking/permeabilization buffer and antibody dilution buffer.
B.3. Procedure
B.3.1 Sample preparation
Harvest single cells by centrifugation at 300 g for 5 min. Discard the supernatant. Wash the cell samples with an appropriate volume of wash solution, then collect samples by centrifuging at 300 g for 5 min, discard the supernatant and repeat 2 times.
B.3.2 Antibody incubation
Divide the cell suspension into two equal aliquots for isotype control and test. Dilute and stain the cells with anti‐human CD34 antibody (labelled as sample group) and isotype control (labelled as negative control group) separately according to their instructions/manuals. Wash the cells with washing buffer twice, collect samples by centrifuging at 300 g for 5 min, and discard the supernatant.
B.3.3 Flow cytometry analysis
Resuspend the cells with the washing solution, and then transfer the cell suspension to the tube through a 40‐μm filter, and test sample on the flow cytometer according to the application manual.
B.3.4 Gating strategies
First, exclude events of debris, dead cells and untargeted cell populations by drawing a gate (gate 1) according to estimated cell size (FSC) and granularity (SSC). Next, by comparing the test and isotype control, place the gate for positive staining population (gate 2) to exclude the cells not labelled with fluorescent antibodies. Isotype antibodies should be used as negative control.
B.4. Result analysis
The results of flow cytometry analysis are analysed comprehensively with appropriate software following its user manual.
APPENDIX C.
(Normative)
Colony‐forming unit assays
C.1. Instruments
C.1.1 Microscope
C.1.2 Haemocytometer
C.2. Reagents
C.2.1 Medium for colony‐forming unit assays: Iscove’s Modified Dulbecco’s Medium (IMDM), methylcellulose, bovine serum albumin, foetal bovine serum, β‐mercaptoethanol, L‐glutamine, supplemented with stem cell factor (SCF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin‐3 (IL‐3), erythropoietin (EPO) and other cytokines. Store the medium at −20°C and avoid light. The medium shall be thawed overnight at 4°C before use and shall be used within 1 week.
C.2.2 IMDM: Store at 4°C and avoid light.
C.2.3 Acetic acid with methylene blue: 3%.
C.2.4 Trypan blue solution: 0.4%.
C.3. Procedure
C.3.1 Nucleated cell counting
Collect the cells and resuspend the cells in IMDM. Dilute the cell suspension in 3% acetic acid with methylene blue at 1:50, and mix well. Draw up 10 μl of the diluted sample and examine the density of the nucleated cells according to the method in Appendix A. Determine the concentration of the nucleated cells in suspension using the following calculations: the nucleated cells per ml = average count per square × 50 (dilution factor) × 104.
C.3.2 Viable cell counting
Draw up 10 μl of the cell suspension, and determine the concentration of the viable cells in suspension using the haemocytometer (C.1.2) and microscope (C.1.1), according to the method in Appendix A.
C.3.3 Cell plating
Adjust the concentration of cell suspension to 1 × 104 cells per ml. Drop up 0.5 ml of the cell suspension and dilute cells to 5 ml of the medium for colony‐forming unit assays. Vortex the tube to mix the contents thoroughly. Let stand for several minutes to allow the bubbles to rise to the top. Draw up the media containing cells into the syringe with blunt‐end needle, and dispense a volume of 1.1 ml into each 35‐mm dish. Rotate the dish to allow the medium to attach to the wall of the dish on all sides.
C.3.4 Colony formation
Incubate at 37°C, in 5% CO2 with ≥95% humidity for 14–16 days.
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.
APPENDIX D.
(Normative)
In vivo haematopoietic reconstruction transplantation experiment
D.1. Instruments
D.1.1 Microscope
D.1.2 Centrifuge
D.1.3 Flow cytometer
D.1.4 Haemocytometer
D.2. Reagents
D.2.1 Phosphate‐buffered saline (PBS): pH 7.4.
D.2.2 Disodium ethylenediaminetetraacetic acid solution: 1%
D.2.3 Red blood cell lysis buffer.
D.2.4 Antibodies and isotype controls. For antibody storage conditions, please refer to product instructions.
D.3. Procedure
D.3.1 Sample preparation
Harvest single cells by centrifugation (D.1.2) at 300 g for 5 min. Discard the supernatant. Collect the cells and resuspend the cells. Determine the concentration of the viable cells in suspension, according to the method in Appendix A. Adjust the concentration of cell suspension to 5 × 106 cells per ml.
D.3.2 Cell transplantation
5.0 × 105 cells were injected into sublethally irradiated (200‐300 cGy) NOD‐SCID Il2rgnull mice at 6 to 8 weeks of age via the tail vein, setting up a blank control group that was injected with an equal volume of phosphate buffer to the cell suspension. The number of mice per group was ≥8.
D.3.3 Peripheral blood cell collection and antibody labelling
After 16 weeks of cell injection, collect the anticoagulated peripheral blood of transplanted mice, lyse the red blood cells, and label the human CD45 antibody, CD19 antibody, CD3 antibody, CD33 antibody, CD235a antibody or the corresponding isotype control, according to the method in Appendix A. After cell incubation and washing, perform flow cytometry (D.1.3) to analyse the percentage of human CD45+ cells, human CD45+ CD19+ cells, human CD45+ CD3+ cells, human CD45+ CD33+ cells and human CD45−CD235a+ cells.
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.
Nan X, Zhang B, Hao J, et al. Requirements for human haematopoietic stem/progenitor cells. Cell Prolif.2022;55:e13152. doi: 10.1111/cpr.13152
This standard is drafted complying with the regulations in GB/T 1.1‐2020 China.
This standard is proposed by the Chinese Society for Stem Cell Research, Chinese Society for Cell Biology.
This standard is under the jurisdiction of the Chinese Society for Cell Biology.
Xue Nan, Bowen Zhang, Jie Hao, Wen Yue are contributed equally.
Contributor Information
Aijin Ma, Email: maaj@btbu.edu.cn, Email: zhoujx@ihcams.ac.cn, Email: tbzhao@ioz.ac.cn, Email: peixt@bmi.ac.cn.
Jiaxi Zhou, Email: zhoujx@ihcams.ac.cn.
Tongbiao Zhao, Email: maaj@btbu.edu.cn, Email: zhoujx@ihcams.ac.cn, Email: tbzhao@ioz.ac.cn, Email: peixt@bmi.ac.cn.
Xuetao Pei, Email: peixt@bmi.ac.cn.