Human Spinal Cord GABAergic Neural Progenitor Cell

Abstract

Full text

‘Human spinal cord GABAergic neural progenitor cell’ is the latest set of guidelines on human spinal cord GABAergic neural progenitor cells in China, jointly drafted and agreed upon by experts from the Standard Committee of Chinese Society for Cell Biology. This standard specifies requirements for human spinal cord GABAergic neural progenitor cells, including the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage and transportation requirements and waste disposal requirements. This standard is applicable for the quality control of human spinal cord GABAergic neural progenitor cells, whether derived from human tissues or differentiated/transdifferentiated from stem cells. It was originally released by the Chinese Society for Cell Biology on 28 October, 2024. We hope that the publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, thereby accelerating the international standardisation of human spinal cord GABAergic neural progenitor cells for various applications.

Scope

1

This document specifies the general requirements, technical requirements, detection methods, inspection rules, instructions for use, labelling, transportation and storage of human spinal cord GABAergic neural precursor cells.

This document is applicable to the detection of human spinal cord GABAergic neural precursor cells that are isolated from human tissues or obtained through stem cell differentiation or transdifferentiation.

Normative References

2

The contents in the following documents constitute indispensable clauses of this document through normative references in the text. Among them, for dated references, only the version corresponding to the date is applicable to this document; for undated references, the latest version (including all amendments) is applicable to this document.

Pharmacopoeia of the People's Republic of China (2020 edition, Volume III)

National clinical laboratory operating procedures

WS 213 diagnosis of hepatitis C

WS 273 diagnosis of syphilis

WS 293 diagnostic criteria for AIDS and HIV infection

Terms and Definitions

3

The following terms and definitions apply to this document.

Human Spinal Cord GABAergic Neural Progenitor Cell

3.1

Cells with the potentials to differentiate into GABA interneurons and astrocytes, which can be obtained through direct isolation from spinal cord tissues, directional differentiation from human pluripotent stem cells, or transdifferentiation.

Human Spinal Cord GABAergic Interneuron

3.2

An inhibitory interneuron located in the human dorsal spinal cord that regulates the balance of excitation and inhibition within neural networks.

Abbreviations

4

The following abbreviations apply to this document.

• EBV: Epstein Barr virus;
• HBV: Hepatitis B virus;
• GABA: γ—aminobutyric acid;
• GAD: Glutamic acid decarboxylase;
• HCMV: Human cytomegalovirus;
• HCV: Hepatitis C virus;
• HIV: Human immunodeficiency virus;
• HTLV: Human T‐lymphotropic virus;
• hPSC: Human pluripotent stem cell;
• STR: Short tandem repeat;
• TP: Treponema pallidum .

General Requirements

5

Raw Materials

5.1

The acquisition of raw materials and their use in research shall comply with domestic recognised ethics and local laws and regulations.

According to the purpose of use, cell research and production organisations shall establish corresponding donor evaluation criteria.

Process and Information Management

5.2

The key factors affecting product quality in the process of raw material acquisition, preparation, detection, transportation and storage of cells should be recorded.

The minimum retention time of records shall be specified to ensure the integrity of records and the safety of record storage.

Technical Requirements

6

Cell Morphology

6.1

Under the condition of suspension culture, it has a dense neurosphere‐like morphology, good transparency and smooth and regular edges.

Cell Marker Proteins

6.2

The positive rate of human spinal cord marker HOXB4 should be ≥ 80%, the positive rate of human spinal cord GABAergic neural precursor cell marker ptf1a should be ≥ 80%, and the positive rate of ascl1 should be ≥ 80%.

Cell Viability

6.3

The survival rate of unfrozen cells shall be ≥ 80%, and the survival rate of frozen cells after resuscitation shall be ≥ 50%.

Cell Function Indicators

6.4

When human spinal cord GABAergic neural precursor cells continue to form adult spinal cord GABAergic interneurons in culture, the positive rate of GABA should be ≥ 70%, and the positive rate of gad65/67 (glutamate decarboxylase 65/67, the key enzyme of GABA synthesis) should be ≥ 70%.

According to the whole cell patch clamp electrophysiological detection of human spinal cord GABAergic interneurons, more than 50% of the cells should have the tonic discharge pattern characteristic of spinal cord GABA neurons.

Karyotype

6.5

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

Microorganisms

6.6

Fungi, bacteria, mycoplasma, HIV, HBV, HCV, HTLV, EBV, HCMV, TP shall be negative.

Bacterial Endotoxin

6.7

Endotoxin should be < 5.0 EU/mL.

2STR Identification

6.8

It shall conform to the human standard STR map, be consistent with the donor cells, and be free of impurity cell contamination.

Inspection Method

7

Cell Morphology

7.1

Under the condition of in vitro culture, the cell structure can be observed using light microscope.

Cell Marker Proteins

7.2

The method is described in Appendix A.

Cell Viability

7.3

The method is described in Appendix B.

Cell Function Indicators

7.4

GABA and gad65/67 are examined according to the method in Appendix B. Refer to the method in Appendix C to test the electrophysiological function.

Karyotype

7.5

According to the ‘preparation and quality control of animal cell matrix for the production and verification of biological products’ in the Pharmacopoeia of the People's Republic of China (2020 edition, Volume III).

Microorganisms

7.6

Bacteria and Fungi

7.6.1

The ‘1101 sterility test method’ in the Pharmacopoeia of the People's Republic of China (2020 Edition, Volume III) shall be followed.

Mycoplasma

7.6.2

The ‘3301 Mycoplasma test method’ in the Pharmacopoeia of the People's Republic of China (2020 edition, Volume III) shall be followed.

HBV

7.6.3

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

HCV

7.6.4

HCV shall be tested in accordance with the WS 213 nucleic acid method.

HIV

7.6.5

HIV shall be tested in accordance with the WS 293 nucleic acid method.

HTLV

7.6.6

HTLV shall be tested by the nucleic acid method in the National Guide to Clinical Laboratory Procedures.

EBV

7.6.7

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

HCMV

7.6.8

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

TP

7.6.9

TP shall be tested in accordance with the WS 273 nucleic acid method.

Bacterial Endotoxin

7.7

The ‘1143 bacterial endotoxin test method’ in the Pharmacopoeia of the People's Republic of China (2020 Edition, Volume III) shall be followed.

Inspection Rules

8

Sampling Method and Quantity

8.1

In a production cycle, products prepared from the same batch, source, generation and method are one batch.

Randomly select three minimum packaging units from the same batch of products.

Qualification Rate Inspection

8.2

Each batch of products shall be inspected and attached with inspection report.

Inspection items shall include all items specified in Section 6.

Recheck and Inspection

8.3

Fitting for purpose, the professional cell testing institution/laboratory should carry out recheck and inspection.

Judgement Rules

8.4

All the ex‐factory inspection items comply with the provisions of Section 6 and are judged as qualified products. If one or more items fail to meet the requirements of this document, it will be judged as unqualified.

All the rechecked inspection items meet the provisions of Section 6 and are judged as qualified products; if one or more items fail to meet the requirements of this document, it will be judged as unqualified.

Instructions for Use

9

It shall at least include the following contents:

• Name;
• Generation;
• Quantity;
• Production date;
• Production batch number;
• Production organisation;
• Storage conditions;
• Transportation conditions;
• Contact information;
• Method of use;
• Executive standard number;
• Production address;
• Postal code;
• Precautions.

Note: Provide endotoxin results according to user requirements.

Label

10

It shall at least include the following contents:

• Name;
• Generation;
• Quantity;
• Production batch number;
• Production organisation;
• Date of manufacture.

Transportation and Storage

11

Transportation

11.1

According to the use requirements of cells, select appropriate transportation methods and conditions to ensure the biological characteristics, safety, stability and effectiveness of cells.

Factors such as cell characteristics, containers carrying cells, transportation routes, transportation conditions, transportation equipment, transportation methods, transportation risks and safeguard measures shall be considered for the transportation of cells.

The control of transportation conditions shall include but not limited to temperature range, oscillation, pollution‐free, equipment performance and appropriate packaging, etc.

Corresponding accompanying inspection documents and technical guidance documents shall be provided according to user requirements

Packages in transit should be checked if necessary, and new freezing sources (such as dry ice or liquid nitrogen) should be added if possible to maintain the appropriate transport temperature.

Storage

11.2

Optimised freezing procedures and methods should be adopted to minimise damage to cells during freezing and thawing to ensure that their normal functions are not affected by freezing and thawing.

The cryopreservation information of cells shall be recorded, including but not limited to:

• Name;
• Batch number;
• Quantity;
• Passage times;
• Freezing date;
• Cryopreservation solution components;
• Operator name.

Cell storage conditions shall be recorded, including but not limited to:

• Storage conditions;
• Storage date;
• Storage life;
• Storage personnel.

Author Contributions

Hong Chen and Jia Xu contributed to conception and design. Baoyang Hu, Xiaoqing Zhang, Tongbiao Zhao, Aijin Ma, Jie Hao, Tianqing Li and Boqiang Fu revised the manuscript. Lixiang Ma, Yan Liu, Peng Xiang, Kun Qian, Xiaohua Han, Yajie Li, Lijun Zhu, Qiyuan Li, Qiang Wei, Tingting Wu, Lei Wang, Jiani Cao, Ka Li, Hongling Zhao and ShuaiShuai Niu critically read and revised the manuscript.

Funding

This work was supported by grants from the National Key R&D Program of China (Grant/Award Numbers: 2023YFC3605100 and 2023YFC2308600) and the National Natural Science Foundation of China (Grant/Award Numbers: 82472621 and 82171422).