Requirements for human cardiomyocytes.

'Requirements for human cardiomyocytes', jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human cardiomyocytes in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packing requirements, storage requirements, transportation requirements and waste disposal requirements for human cardiomyocytes, which is designed to normalize and standardize human cardiomyocyte research and production. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that the publication of this guideline will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human cardiomyocytes for applications.

SCOPE

This document specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packing requirements, storage requirements, transportation requirements and waste disposal requirements for human cardiomyocytes. This standard is applicable for the quality control of human cardiomyocytes.

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

Pharmacopoeia of the People's Republic of China

National Guide to Clinical Laboratory Procedures

T/CSCB 0001‐2020 General requirements for stem cells

T/CSCB 0002‐2020 Requirements for human embryonic stem cells

TERMS AND DEFINITIONS

The following terms and definitions apply to this document.

Human cardiomyocytes

Cardiac muscle cells with excitability, conductivity, autorhythmicity and contractility, which is particularly rich in myofibril, striae and mitochondria.

Membrane potential

The potential difference between the inside and outside of the cell caused by ion migration, which is determined by concentration gradients of ions across the membrane or by membrane permeability to each type of ion.

Field potential

Changes in extracellular local potential in the formation of synchronous discharge of cell population.

Calcium transient

Rapid oscillation of cytoplasmic calcium concentration caused by action potential or other reasons.

ABBREVIATIONS

The following abbreviations are applicable for this document.

EBV: Epstein‐Barr virus

HBV: hepatitis B virus

HCMV: human cytomegalovirus

HCV: hepatitis C virus

HIV: human immunodeficiency virus

HTLV: human T‐lymphotropic virus

STR: short tandem repeat

TP: treponema pallidum

TECHNICAL REQUIREMENTS

Primary quality attributes

Cell morphology

With two‐dimensional culture, human cardiomyocytes shall be adherent growth and exhibit spindle‐shaped, short column‐shaped or irregular shaped. Most of them shall be mononuclear, while some of them can be coenocytic occasionally.

Cell viability

≥90% before cryopreservation and ≥60% after resuscitation.

Cell markers

cTnT‐positive rate ≥70.0% and α‐actinin‐positive rate ≥70.0%.

Membrane potential or field potential

Shall exhibit typical waveform.

Calcium flux

Shall exhibit periodic variation of calcium signal.

Contractility

Shall exhibit rhythmical mechanical contraction spontaneously or induced.

Pharmacological action

The beat frequency shall elevate with isoproterenol treatment while decline when treated with carbachol.

Microorganisms

Shall be negative for fungi, bacteria, mycoplasma, HIV, HBV, HCV, HTLV, EBV, HCMV and TP.

Process control

The process of cell expansion, differentiation, cryopreservation and resuscitation shall follow the requirements of T/CSCB 0001‐2020. Cell STR shall be consistent with the donor’s identity.

TEST METHODS

Cell morphology

Observe the morphology of cells grown in 2D condition under microscope.

Cell viability

The method in Appendix A shall be followed.

Cell markers

The method in Appendix B shall be followed.

The method in Appendix C shall be followed.

The method in Appendix D shall be followed.

Calcium flux

The method in Appendix E shall be followed.

Contractility

Observe the contractility under microscope.

Drug reactivity

Observe the contractility under microscope under the treatment of isoproterenol (1 µM) or carbachol (1 µM) and count beats per minute.

Microorganisms

The method for microbiological detection referred in T/CSCB 0001‐2020 shall be followed.

INSPECTION RULES

Sampling method

Cells produced from the same production cycle, same production line, same source, same passage and same method are considered to be the same batch.

Three smallest units of packaging shall be randomly sampled from the same batch.

Quality inspection and release

Each batch of products shall be subject to qualify inspection before release, and inspection reports shall be attached.

The quality inspection items shall include all the attributes specified in 5.2.

Review inspection

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

Decision rules

Products that pass all requirements in 5.2 for the quality inspection for release are considered to be qualified. Products that fail to pass one or more requirements in 5.2 for the quality inspection for release are considered to be unqualified.

Products that pass all requirements in 5.2 for the quality review inspection are considered to be qualified. Products that fail to pass one or more requirements in 5.2 for the review inspection are considered to be unqualified.

INSTRUCTION FOR USAGE

The instructions for usage shall include, but not limited to

Product name;

Passage number;

Cell number;

Production date;

Lot number;

Production organization;

Storage conditions;

Shipping conditions;

Contact information;

Operation manual;

Execution standard number;

Manufacturing address;

Postal code;

Matters that deserve attention.

Note: indicate endotoxin content if required

LABELS

The label shall include but not limited to

Product name;

Passage number;

Cell number;

Lot number;

Production organization;

Production date.

PACKAGE, STORAGE AND TRANSPORTATION

Package

The appropriate materials and containers shall be selected to ensure maintenance of the primary quality attributes of human cardiomyocytes.

Storage

T/CSCB 0001‐2020 shall be followed.

Productions should be stored in liquid nitrogen.

Transportation

T/CSCB 0001‐2020 shall be followed.

Cryopreserved cell products shall be transported in dry ice or liquid nitrogen.

WASTE DISPOSAL

T/CSCB 0001‐2020 shall be followed.

CONFLICT OF INTEREST

No potential conflicts of interest are disclosed.

AUTHOR CONTRIBUTIONS

SH, TZ, JH and BF contributed to conception and design. MY, WL, JC and LW drafted and revised the manuscript. AM, ZZ, HY, ZS, FL, FC, PL, XP, PX, JY, YZ, YZ, QL, JZ, JW, YP, HZ, LL and NC critically read and revised the manuscript.