Requirements for human natural killer cells

Abstract ‘Requirements for Human Natural Killer Cells’ is the latest set of guidelines on human NK cells in China, jointly drafted and agreed upon by experts from the Standards Committee of Chinese Society for Cell Biology. This standard specifies requirements for the human natural killer (NK) cells, including the technical requirements, test methods, test regulations, instructions for use, labeling requirements, packaging requirements, storage and transportation requirements, and waste disposal requirements of NK cells. This standard is applicable for the quality control of NK cells, derived from human tissues, or differentiated/transdifferentiated from stem cells. It was originally released by the Chinese Society for Cell Biology on 30 August, 2022. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human NK cells for applications.

Regeneration, Chinese Academy of Sciences, Beijing, China.Email: wangjinyong@ioz.ac.cnWe hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human NK cells for applications.

| SCOPE
This document specifies requirements for the human natural killer (NK) cells, including the technical requirements, test methods, test regulations, instructions for use, labeling requirements, packaging requirements, storage and transportation requirements, and waste disposal requirements of NK cells.This standard is applicable for the quality control of NK cells, derived from human tissues, or differentiated/transdifferentiated from stem cells.

| NORMATIVE REFERENCES
The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document.
For dated references, only the edition cited applies.For undated references, only the latest edition (including all amendments) applies.National Guide to Clinical Laboratory Procedures, Fourth Edition.

| TERMS AND DEFINITIONS
The following terms and definitions apply to this document.

| Non-specific killing
Non-specific killing refers to direct killing of tumour cells or virus-infected cells without the necessities of somatic gene rearrangement, expression of specific antigen-recognition receptors, and pre-sensitization.

| Antibody-dependent cellular cytotoxicity, ADCC
ADCC refers to the mechanism by which NK cells expressing FcγRIII (CD16) can recognize the Fc segment of antigen-bound antibody, thus killing the antibody-bound target cells.

| Immunomodulation of natural killer cells
NK cells interact with other immune cells by releasing a large number of cytokines and chemokines and regulate the immune state and immune function of the body.

| ABBREVIATIONS
The following abbreviations are applicable for this document.

| Cellular markers
The immunophenotyping of NK cells described as follows: The expressions of CD56 and CD45 shall be≥90% of the cell population, and the expression of CD16 shall be ≥10% of the cell population.

| Cell purity
The expressions of CD3 shall be ≤5% of the cell population.

| Cell viability
The cell viability shall be≥70% prior to cryopreservation, and ≥ 50% after thawing but before the application.

| Microorganisms
Cells shall be negative for fungi, bacteria, mycoplasma, chlamydia, and viruses.

| Identification of cell STR
NK cells isolated from human tissues, differentiated or transdifferentiated from stem cells shall be subjected to STR assay and typing identification, without cross-contamination between samples.

| Cell morphology
Observe the morphology of cells grown under 2D conditions in vitro using a microscope.

| Chromosome karyotype
The method in the Pharmacopoeia of the People's Republic of China shall be followed.
Note: Determine the appropriate karyotype result based on the characteristics of the target sample.Normal karyotype shall be 46, XX, or 46, XY.

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

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

| Cellular activity and function
The detection method for granzyme and perforin in activated NK cells shall be tested according to the method in Appendix B.
The detection method for the release of interferon in activated NK cells shall be tested according to the method in Appendix B.
The cytotoxicity assay of NK cells shall be carried out according to the method in Appendix C.
The ADCC detection of NK cells shall be tested according to the method in Appendix D.

| Cell viability
The method 'Cell viability test' in the T/CSCB 0002 shall be followed.
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.TRANSPORTATION

| Package
The appropriate materials and containers shall be selected to ensure maintenance of the primary quality attributes of NK cells.

WASTE DISPOSAL
The waste generated during the production and testing of human natural killer cells shall be in accordance with the waste cell management documents, strict implementation of management standards, and detailed records.effector-to-target cell ratio (e.g., E:T = 2 Â 10 5 : 6 Â 10 5 ).The corresponding reagents for each experiment shall be added to each well and allowed to incubate for 1 h in an incubator with humidified atmosphere (5% CO 2 and 37 C).

B.4.3 Flow cytometry analysis
The expression levels of CD107a (degranulation ability), granzyme B, and perforin in NK cells shall be detected separately according to the flow cytometry assay procedure.Among them, CD107a shall be extracellular staining, and both Granzyme B and Perforin shall be intracellular staining.

B.4.4 Gating
Gate the target population of cells based on particle size and transparency, excluding cell debris and other irrelevant particles.The gating of positive staining cells shall be determined by the fluorescence intensity using isotype controls as a reference.
Both positive and negative experimental controls shall be set up for gating and the following analysis.NK cell populations shall be defined according to CD3 À CD45 + CD56 + , followed by analysis of positive proportions of CD107a, granzyme B, and perforin from NK cells.

B.5 | ANALYSIS OF RESULTS
Analyse the results using software according to manufacturer's instructions.The target cells such as K562 cells shall be labelled by CFSE according to the instructions.
Then, the cells in the 96-well culture plate shall be collected, resuspended with PBS + 2% FBS solution, and incubated with 7-AAD staining solution at 2-8 C for 15 min protected from Funding information the National Key R&D Program of China, Grant/Award Numbers: 2020YFA0112404, 2021YFA1101604, 2018YFA0108400, 2018YFE0204400; the National Science Fund for Distinguished Young Scholars of the National Natural Science Foundation of China, Grant/Award Number: 81925002; the Young Scientists Fund of the National Natural Science Foundation of China, Grant/Award Number: 82100128; the Youth Innovation Promotion Association CAS, Grant/Award Number: 2020084 derived from human tissues, or differentiated/transdifferentiated from stem cells.It was originally released by the Chinese Society for Cell Biology on 30 August, 2022.
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.T/CSCB 0002 Requirements for human embryonic stem cells.Pharmacopoeia of the People's Republic of China, 2020 Edition Three.
NK cells are isolated from human tissues or obtained by differentiation of stem cells or transdifferentiation of other cell types.It belongs to the intrinsic lymphocytes and has three functions, non-specific killing, antibody-dependent cellular cytotoxicity, and immune-modulation.

ADCC
Tandem Repeat.5 | TECHNICAL REQUIREMENTS 5.1 | Cell morphology NK cells are cultured in vitro in a suspended state.After activation, their volume shall become larger, and the cell shape shall become irregular.
NK cells shall kill the cells whose HLA-I expression is down-regulated or lost.After activation, NK cells shall release granzyme, perforin, and interferon.Combined antibodies can enhance the cytotoxicity of NK cells.Note: For example, NK cells have cytolytic activity against solid tumour cells (ovarian cancer cells) and haematological tumour cells (myeloid leukaemia tumour cells) with down-regulated expression of HLA-I molecules.
Unqualified cells, remaining discarded cells, or donations in the research and production of human retinal pigment epithelial cells shall be disposed of legally, properly, and ethically.AUTHOR CONTRIBUTIONSWang J, Hao J, Zhao T, and Ma A contributed to conception and design.Niu S, Xia C, Huang D, Wang L drafted and revised the manuscript.Hu H, Yu S, Wu N, Dong Z, Zhou J, Zhang Y, Wu J, Yu J, Wang C, Fu B, Cao J, Liang L, Xu L, Chen L and Zhou Q critically read and revised the manuscript.A.4.2 Gating Gate the target population of cells based on particle size and transparency, excluding cell debris and other irrelevant particles.The gating of positive staining cells shall be determined by the fluorescence intensity using isotype controls as a reference.Both positive and negative experimental controls shall be set up for gating and the following analysis.A.5 | ANALYSIS OF RESULTS Analyse the results using software according to manufacturer's instructions.APP E NDIX ( NORMAT IVE) B : ANALYSIS OF CD107A, PERFORIN, AND GRANZYME B EXPRESSION LEVELS IN ACTIVATED NK CELLS (FLOW CYTOMETRY) with 2% FBS is recommended for use as a buffer to dilute or wash nucleated cells B.2.3 Relevant antibodies mentioned in context and corresponding isotype control antibodies B.2.4 Prepare the following solutions according to the relative requirements for flow cytometry: wash solution, and antibody dilution solution B.2.5 Target cells, such as K562 cells (leukaemia cell line established from a female patient with chronic myelogenous leukaemia) B.3 | SAMPLE STORAGE The wash solution and stained samples shall be stored at 2-8 C. Antibodies shall be stored according to the manufacturer's instructions.B.4 | TESTING PROTOCOL B.4.1 Effector cell and target cell collection Effector NK cells and target cells, such as K562 cells, shall be collected separately.Single-cell suspensions shall be prepared and counted.B.4.2 Coincubation of effector cells with target cells NK cells were incubated with or without target cells at 1:3 3 PBS with 2% FBS is recommended for use as a buffer to dilute or wash nucleated cells C.2.4 CFSE assay kit C.2.5 Target cells, such as K562 cells C.3 | TESTING PROTOCOL C.3.1 CFSE-labelled target cells