Quality by design strategy of human mesenchymal stem/stromal cell drug products for the treatment of knee osteoarthritis

Table 1 The definition of critical quality attributes for mesenchymal stem/stromal cell-based therapy according to the defined Quality Target Product Profile

QTPP	CQA	Quality control	Acceptable standards	Ref.
Cell characteristics	Morphology	Microscopic visual inspection	Shuttle shaped in wall attached state	[90]
	Cell viability	Cell count and viability detection	Cell viability ≥ 90% before product distribution	[91]
	MSC identity	Specific positive antigen expression ≥ 95%	Flow cytometry: CD105+, CD73+, CD90+	[92]
		Specific negative antigen expression ≤ 2%	Flow cytometry: CD45-, CD34-, CD14- or CD11b-, CD79α- or CD19-, HLA-DR-	[92]
	PDT	PDT calculation formula	8 hours < PDT < 48 hours	[93]
	Cell differentiation	Osteogenic differentiation	Color reaction with Alizarin Red S	[94]
		Adipogenic differentiation	Color reaction with Oil Red O	[95]
		Chondrogenic differentiation	Color reaction with Alcian Blue	[96]
	Cell cycle	PI staining	G0/G1% > 70%
Product quality	Purity	Sterility testing for each lot	No detectable	[97]
		Mycoplasma testing for each lot	No detectable	[98]
		Endotoxin testing for each lot	≤ 0.5 CFU/mL	[98]
	Genetic stability	Karyotype	23 pairs of chromosomes	[99]
		STR	Each cell passage is from the same source
	Biological safety	Human telomerase activity	No or weak expression	[100]
		MSC soft agar cloning	No detectable	[101]
Therapeutic potency	In vivo effect	Co-culture with T cells	Inhibition of T cell growth	[102]
	Paracrine action	The secretion of bioactive molecules	PGE-2, IL-10	[103]

QTPP: Quality Target Product Profile; CQA: Critical quality attribute; MSC: Mesenchymal stem/stromal cell; PDT: Population doubling time; STR: Short tandem repeat; CFU: Colony forming unit; PGE-2: Prostaglandin E2; IL-10: Interleukin-10.