From laboratory to clinic: Bridging regulatory and manufacturing gaps in stem cell-based therapies

Table 3 Critical quality attributes and assays for advanced therapy medicinal product manufacturing

Attribute	Example assays/methods	Guideline references
Identity	Flow cytometry (cell surface markers), PCR for unique genes	EMA/ICH guidance, pharmacopoeias
Purity	Sterility testing, mycoplasma PCR, endotoxin (LAL assay)	Ph.Eur./USP
Viability	Dye exclusion (trypan blue, 7-AAD), metabolic activity assays	CBER/EMA guidance
Potency	Functional bioassays (e.g., MSC-mediated T-cell suppression)	Case-specific; often ICH Q6B
Genetic stability	Karyotype, array CGH, NGS for mutations	CBER draft guidance, ISSCR recommendations
Tumorigenicity	Soft agar colony formation, injection into immunodeficient mice[23]	WHO TRS 878, FDA draft guidance
Viral safety	Filtration of spent media, adventitious virus testing panels	ICH Q5A(R2)[27]
Exosome-specific	Particle size analysis (NTA), exosome marker proteins (CD63, CD81)[16]	MISEV2018 standard

PCR: Polymerase chain reaction; EMA: European Medicines Agency; ICH: International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; Ph.Eur.: European Pharmacopoeia; USP: United States Pharmacopeia; 7-AAD: 7-aminoactinomycin D; CBER: Center for Biologics Evaluation and Research; MSC: Mesenchymal stem cell; CGH: Comparative genomic hybridization; NGS: Next-generation sequencing; ISSCR: International Society for Stem Cell Research; WHO: World Health Organization; FDA: Food and Drug Administration; NTA: Nanoparticle tracking analysis; MISEV2018: Minimal Information for Studies of Extracellular Vesicles 2018.