General requirements for the production of extracellular vesicles derived from human stem cells

Abstract ‘General requirements for the production of extracellular vesicles derived from human stem cells’ is the first guideline for stem cells derived extracellular vesicles in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the general requirements, process requirements, packaging and labelling requirements and storage requirements for preparing extracellular vesicles derived from human stem cells, which is applicable to the research and production of extracellular vesicles derived from stem cells. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardisation of extracellular vesicles derived from human stem cells.


| SCOPE
This document specifies the general requirements, process requirements, packaging and labelling requirements and storage requirements for preparing extracellular vesicles derived from human stem cells, and the corresponding confirmation method is described.This document applies to the research and production of extracellular vesicles derived from stem cells such as human embryonic stem cells, human mesenchymal stem cells and human induced pluripotent stem cells.

| NORMATIVE REFERENCES
The contents in the following documents constitute indispensable clauses of the document through normative references in the text.
Among them, dated references, only the version corresponding to this date is applicable to this document; for undated references, the latest version (including all amendments) applies to this document.
T/CSCB 0001 General requirements for stem cells T/CSCB 0002 Human embryonic stem cells T/CSCB 0003 Human mesenchymal stem cells T/CSCB 0005 Human-induced pluripotent stem cells

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

| Extracellular vesicle
Particles secreted by cells (spontaneously or induced) with lipid bilayer membrane structure and inability to self-replicate.

| Bulk
The culture supernatant of stem cells (collected once or many times) for preparing extracellular vesicles.

| ABBREVIATIONS
The following abbreviations apply to this document.

| Stem cell collection
Before collecting stem cells for the preparation of extracellular vesicles, ethical approval and informed consent shall be obtained.Meanwhile, the investigation of anamnesis, family history and current health report shall be completed.The detection of infectious disease covers, but is not limited to, HBV, HCV, HIV, HTLV, EBV, HCMV and TP.Other reports, for instance, an official certification of entering and leaving an epidemic hot zone, are also required, if necessary.

| Stem cell reception
Ethical approval documents, informed consent, source of cell lines and isolation methods shall be thoroughly checked when receiving stem cells.

| INFORMATION AND DATA MANAGEMENT
9.1 The whole process of preparation, packaging, labelling and storage of stem cell-derived extracellular vesicles shall be recorded with a traceable information system.The data retention period is no less than 10 years.9.2 When genetic-modified stem cells are used for the preparation of extracellular vesicles, construction details, screening method, exogenous gene sequences, location of integration into the genome, and copy number shall be documented.9.4 The packaging and labelling information of stem cell-derived extracellular vesicles shall be recorded in a timely manner by two staff.9.5 Each preparation and usage of extracellular vesicles shall be recorded in detail in the ledger, and a file must be created.

HBV
5 | GENERAL REQUIREMENTS5.1 The preparation of extracellular vesicles derived from human stem cells shall comply with the ethical review requirements and biosafety operation specifications of the research centre.Note: The preparation process of extracellular vesicles derived from stem cells mainly includes: (a) collection and reception of stem cells; (b) proliferation and induction of stem cells; (c) bulk harvesting; (d) isolation and concentration of extracellular vesicles; and (e) extracellular vesicle harvesting.5.2 The facilities and environmental conditions for preparing extracellular vesicles derived from human stem cells shall accord with the biosafety requirements, meet the conditions for product quality control and avoid microbial contamination and crosscontamination.5.3 According to the preparation process of extracellular vesicles, the primary cell bank, the master cell bank and the working cell bank should be set for the used stem cells, respectively.Note 1: The primary cell bank has uniform storage properties and is suitable for subpackaged cell populations prepared from extracellular vesicles.Note 2: The master cell bank stores subpackaged cell populations cultured from primary cell bank cells to a specific doubling level.Note 3: The working cell bank stores subpackage cell populations cultured from the main cell bank cells to a specific doubling level.5.4 The entire process of preparing extracellular vesicles from stem cell sources shall ensure that the reagents and consumables used, such as culture medium, digestion solution, resuspension solution, elution solution, centrifuge tubes, cryovials, and so on, meet the requirements of sterility, virus-free, mycoplasma-free, and low endotoxin.If necessary, validated processes should be employed for sterilisation, and relevant quality assurance procedures should be established.5.5 After the preparation process of extracellular vesicles derived from stem cells is completed, it should be promptly packaged, labelled, and stored.6 | PROCESS REQUIREMENTS 6.1 | Collection and reception of stem cells

6. 2 |
Proliferation and induction of stem cells 6.2.1 The proliferation of stem cells shall be carried out in a class A microorganism-free environment (see Appendix A).Humanderived stem cells intended for the preparation of extracellular vesicles shall be tested for fungi, bacteria, mycoplasma, HBV, HCV, HIV, HTLV, EBV, HCMV and TP.Stem cells for allogeneic usage shall be free from all the above-mentioned pathogenic microorganisms, and autologous usage shall be free from fungus, bacteria and mycoplasma contamination.Stem cells containing infectious viruses shall be operated in the dedicated area.The contaminated zone shall be sterilised, and contagion risk must be eliminated after each operation.6.2.2 Based on the cell types, key quality attributes shall be measured according to T/CSCB 0001, T/CSCB 0002, T/CSCB 0003 and T/CSCB 0005 standards.6.2.3 Stem cells used for the preparation of extracellular vesicles shall be cultured and proliferated according to the verified standard operating procedures.6.3 | Harvest of conditioned medium6.3.1 Once stem cells reach the stable growth stage, the medium should be refreshed and collected regularly according to cell status.6.3.2In the process of harvesting the crude solution (6.3.2), a culture medium with clearly defined components should be used, and substances with unclear components such as animal serum and platelet lysate should be avoided.Unclear substances should not be used during the bulk harvesting stage for extracellular vesicles intended for clinical trial purposes.If reagents with components that are not fully defined, such as animal serum, platelet lysate, and pituitary extract, are used in the culture medium during the bulk harvesting stage, the potential effects caused by these reagents should be considered.Extracellular vesicles should be removed from the relevant reagents in advance.6.3.3The bulk shall be packaged separately into autoclaved, virusfree, mycoplasma-free and low-endotoxin containers under sterile conditions.The storage period shall not exceed 48 h at 4 C, and long-term preservation shall be carried out at À80 C. Importantly, it is better to minimise freeze-thaw cycles.6.4 | Isolation and concentration of extracellular vesicles 6.4.1There are many techniques for isolating and concentrating extracellular vesicles from a conditioned medium, including, but not limited to, differential centrifugation, precipitation with polyethylene glycol, density gradient centrifugation, size exclusion chromatography, ultrafiltration and immunoaffinity chromatography.Notably, heterologous contamination shall be avoided.6.4.2Clear process control parameters for the separation and concentration of extracellular vesicles shall be established to ensure the safety, efficacy, and stability of extracellular vesicle preparations.For example, a protocol for isolating extracellular vesicles by differential centrifugation is available in Appendix B. 6.4.3Microbial and chemical contamination shall be controlled during the entire process of extracellular vesicle isolation and concentration.6.5 | Harvest of extracellular vesiclesVerified operating procedures shall be established, and the reagents used shall have clearly defined compositions.7 | PACKAGING AND LABELLING7.1 The vesicles shall be packaged under the same environmental conditions as vesicle isolation with containers made of vesiclefriendly materials by a validated procedure.7.2 Each minimum package shall have a unique identification for traceability.The required information is as follows: The storage period of stem cell-derived extracellular vesicles should not exceed 48 h at 4 C. Long-term preservation should be performed at À80 C, and repeated freeze-thaw cycles should be avoided.

9. 3
The preparation process of stem cell-derived extracellular vesicles shall be recorded in detail.The contents of the record are as follows: a. Patch number; b.Stem cell attribute, including donor information, cell line, isolation method, and generation; c.Stem cells quality, including microbial test results, cell morphology, karyotype, cell viability, and protein markers; d.Manufacturer and equipment; e.The information of reagents and supplies used in the production, including name, batch number, and lot number; f. Preparation process and parameters; g.Environmental parameters, including temperature, humidity, and cleanliness; h.Manufacture date.