Table 6.

Characterization Test for MSC-based Product for comparability assessment.

MSC product characterization	Method/Read-out parameter	Advantages	Disadvantages	Recommendation level
Viability (fresh & post-thaw)	Hemocytometer/viable cell count.	Simple, rapid, cheap.	1.High inter-user deviation; 2.Time-consuming for large number of samples; 3.Only detect live and dead cells.	Low +
	Automated cell-counting instruments/viable cell count.	High precision and high sample throughput.	1.Relatively expensive; 2.Only detect live and dead cells.	High +++
	Flow Cytometry-based Assay/viable, apoptotic, and necrotic cell count.	1.Detect viable, apoptotic and necrotic cells; 2.Can evaluate cryopreservation impact on MSCs.	1.Relatively expensive; 2.Time-consuming.	High +++
Senescence	Beta-galactosidase assay/Senescence cell count.	Can detect senescent cells in the MSCs after long term in vitro expansion.	1.High deviation; 2.Time-consuming.	Middle ++
Growth rate	Cell counting/Population doubling time (PDT).	1.Can evaluate replicative capability of long term in vitro expanded MSCs and cryopreserved MSCs; 2.No extra analytic instrument required.	1.Data obtained after harvest; 2.Time-consuming. 3.Cost for cultivation.	High +++
Functional Characterization
Immunosuppression (MSC-mediated immunosuppression)	Machine learning Morphological profiling method/IFN-γ-stimulated MSCs [13].	1.Distinguish subpopulations of Interferon-γ-stimulated MSCs; 2.Machine calculation.	need special software and devices	High +++
Immunosuppression (MSC-mediated Inhibition of T-cell activation)	Flow Cytometry-based Assay/Expression level of PDL-1 surface protein (%) (IFN-γ-stimulated & resting MSCs).	1.PDL-1 is a well-known mechanism for T cells inhibition; 2.Direct measurement of MSCs activation marker; 3.Simple testing system, do not need third party responder cells; 4.Mimic inflammatory environment in vivo.	1.Narrow detection range (0–100%); 2.Do not directly address the effect on T cells; 3.Both resting and primed MSCs express PDL-1.	High +++
	Flow Cytometry-based Assay/Expression level of IDO intracellular Protein (%) (IFN-γ-stimulated & resting MSCs).	1.IDO is the key factor of MSCs for inhibition of activated T cells; 2.Mimic inflammatory environment in vivo; 3.Use resting MSCs (no IDO expression) as internal control.	1.Intracellular protein stain; 2.Narrow detection range (0–100%); 3.Does not directly measure the effect on T cells.	High +++
	Quantitative Flow Cytometry-based Assay/IDO immunosuppressive protein binding value (IPBv) [15,17] (IFN-γ-stimulated & resting MSCs).	1.IDO is the key factor of MSCs for inhibition of T cells proliferation; 2.quantitative measurement of intracellular IDO expression level of primed MSCs; 3.Mimic inflammatory environment in vivo; 4.Use resting MSCs (no IDO expression) as internal control; 5.Simple testing system, do not need third party responder cells; 6.Can apply to comparability and stability studies of therapeutic MSC product manufacturing.	1.Relatively quantitative measurement via a calibration plot; 2.Requires high flow cytometry skill; 3.Does not directly address the effect on T cells.	High +++
Immunosuppression Inhibition of PBMC/T cell Proliferation	Cell proliferation assay/Suppression rate.	1.Directly measure MSC's effect on activated T cell proliferation; 2.Most popular biological assay for immunosuppression of MSCs.	1.Need third party T cells; 2.Complex contact-dependent and contact -independent mechanism of effectors (MSCs) and responder cells (T cells); 3.Individual variability of different MSCs and responder cells; 4.Narrow detection range (0–100%).	High +++