Requirements for human cardiomyocytes

Abstract ‘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 REFEREN CE S
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.

| TERMS AND DEFINITI ON S
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.

| ABBRE VIATIONS
The following abbreviations are applicable for this document. 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.

| 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.

| 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.

| 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.

| 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.

| Membrane potential
The method in Appendix C shall be followed.

| Field potential
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.

| Sampling method
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.
2. Three smallest units of packaging shall be randomly sampled from the same batch.

| Quality inspection and release
1. Each batch of products shall be subject to qualify inspection before release, and inspection reports shall be attached.
2. 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
1. 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.
2. 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.

| IN S TRUC TI ON FOR USAG E
The instructions for usage shall include, but not limited to

A.2 | Reagents
Unless otherwise stated, all reagents used shall be analytical grade.
The water used for testing shall be deionized water.

| Package
The appropriate materials and containers shall be selected to ensure maintenance of the primary quality attributes of human cardiomyocytes.
2. Cryopreserved cell products shall be transported in dry ice or liquid nitrogen.

| WA S TE D IS P OSAL
T/CSCB 0001-2020 shall be followed.

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest are disclosed.

A.4 | Calculation and analysis
Cell viability is calculated according to equation (1): In the equation:

S-viability of cells M-total number of cells D-number of stained cells
The viability of cells is the mean of two duplicate samples. Two independent cell viability tests shall be performed on the same sample. The mean value of two independent viability tests is recorded as the viability of cells.

A.5 | Accuracy
The absolute difference value between the two independent tests, under the same conditions, shall not exceed 10% of their arithmetic mean.

B.3 | Sample storage
The wash solution and fixed samples shall be stored at 2-8°C. The fixing solutions shall be aliquoted, sealed, labelled and stored at or below -20°C. Antibodies shall be stored according to the manufacturer's instructions.

B.4 | Test protocol B.4.1 | Sample preparation and fixation
Collect samples by centrifuging single-cell suspensions at 250 g for

B.4.2 | Blocking and permeabilization
Resuspend the fixed sample (B.4.1) with the blocking/permeabilization solution and aliquot the cells into two independent samples, which will be used as a testing sample and an isotype control sample respectively. Incubate the samples on ice for 20 minutes and then wash the samples with the wash solution.

B.4.3 | Antibody incubation
Incubate the samples with the diluted antibodies or corresponding isotype controls according to the manufacturer's instructions.

B.5 | Analysis of results
Analyse the results using software according to manufacturer's instructions.

A PPE N D I X C
(Normative appendix) Detection of membrane potential (patch clamp)

C.3.3 | Patch-clamp conduction
Use the manipulator to touch the cell membrane with the pipette tip.
In whole-cell measurements, changes in membrane potential shall be observed in current-clamp mode.

C.4 | Data acquisition and analysis
With software integrated in patch-clamp experimental system, action potentials shall be recorded. Three general types of cardiac action potential are presented in Figure A1, which shall be divided into depolarization and repolarization. Time in millisecond (ms) shall be on the X-axis and membrane potential in millivolts (mV) shall be on the Y-axis.

(Normative appendix)
Detection of field potential (microelectrode arrays) and sealed by water.

D.4 | Data acquisition and analysis
Typical waveform of cardiac field potential is presented in Figure A2.
Time in millisecond (ms) shall be on the X-axis and field potential in microvolts (mV) shall be on the Y-axis.

E.4 | Data acquisition and analysis
Periodic variation of calcium signal is presented in Figure A3. Time in millisecond (ms) shall be on the X-axis and standard fluorescence intensity in F/F0 shall be on the Y-axis, F0 represents resting fluorescence intensity.