Table 4.

CAR-T cell release tests in different CAR-T cell production methods.

Parameter	Retroviral and lentiviral vector-based method	Transposon/transposase-based method	mRNA electroporation-based method
Safety	#Gram stain, 7–14 day culture#Mycoplasma (qPCR) #Endotoxin level (cell lines PTC) #Copies of transgene insertion (qPCR) #RCR/RCL (for RCR: marker-rescue cell culture assay; for RCL: co-culture on C8166 cells with amplification and indicator phases)	##Gram stain, 7–14 day culture #Mycoplasma (qPCR) #Endotoxin level (LAL test)	#Gram stain, 7–14 day culture #Mycoplasma (qPCR) #Endotoxin level (LAL test)
Purity	#%CD3+T cells (flow cytometry) #%CAR-T cells (flow cytometry) #Residual tumor burden (flow cytometry) #Residual beads (microscopy)	#%CD3+T cells (flow cytometry) #%CAR-T cells (flow cytometry) #Residual AAPCs (flow cytometry)	#%CD3+T cells (flow cytometry)
Identity	#%CAR+T cells (flow cytometry)	ND	ND
Potency	#In vitro CTL or IFN-γ secretion (chromium release assay & ELISA)	ND	ND
References	151	149	113

AAPC, artificial antigen-presenting cells; CAR, chimeric antigen receptor; CTL, cytotoxic T lymphocyte; IFN, interferon; RCL, replication-competent lentivirus; RCR, replication-competent retrovirus, PTC, point to consider; qPCR, quantitative PCR; LAL, limulus amoebocyte lysate; AAPCs, artificial antigen-presenting cells.

ND, not detected.