Hepatocyte cryopreservation: Is it time to change the strategy?

Table 1 Summary of freeze-rate comparison studies

Species	Cryoprotectant	Freeze rate	Storage temperature	Ref.
Human	DMSO	-1°C/min	-80°C	[55]
Rat	DMSO	-1°C/min to -38°C (with cooling shock) then liquid nitrogen	Liquid nitrogen	[61]
Dog, monkey, human	DMSO	-1.9°C/min from 4°C to -30°C, then -30°C from -30°C to -150°C	Liquid nitrogen	[57]
Rat	DMSO	-38°C/min	Liquid nitrogen	[59]
		-2°C/min
		Slow variable
		Optimized variable rate
Human	DMSO	-1.9°C/min from 4°C to -30°C, then -30°C from -30°C to -150°C	Liquid nitrogen	[51]
Rat	DMSO	Cooling in 10 min down to 0°C, 8 min at 0°C, in 4 min down to -8°C, in 0.1 min down to -28°C, in 2 min down to -33°C, in 2 min up to -28°C, in 16 min down to -60°C, in 4 min down to -100°C (variable rate)	Liquid nitrogen	[60]
Human	DMSO	Variable rate	Liquid nitrogen	[6]
Dog, monkey, human	DMSO	-1.9°C/min from 4°C to -30°C, then -30°C from -30°C to -150°C	Liquid nitrogen	[58]
Rat	DMSO	Variable rate	Liquid nitrogen	[39]
Pig	DMSO	Optimized[59]	Liquid nitrogen	[22]
		Modified variable

DMSO: Dimethylsulfoxide.

Table 2 In vitro evaluation of post-thawing quality of hepatocytes

Hepatocyte in vitro model	Cryopreservation protocol	Parameters evaluated: impairment following C/T	Parameters evaluated: no impairment following C/T	Ref.
Rat and human	Pre-incubation	Plating	CYP induction	[17]
	-20°C, -70°C, liquid nitrogen
Porcine	Slow freezing protocol up to -80°C	Trypan blue exclusion test		[23]
		Plating
		Ammonia clearance
Rat	Slow freezing protocol up to -80°C	Trypan blue exclusion test		[25]
		Plating
		Ammonia clearance
Human	20% DMSO, 40% FCS	Trypan blue exclusion test	ATP	[63]
	Slow freezing protocol	Plating	Urea synthesis
		LDH release
		MTT
Porcine	Freezing boxes or slow freezing protocol	CYP	Plating	[72]
		Glycogen synthesis
		Glycogenolysis
		Gluconeogenesis
Rat and mouse	Slow freezing protocol	Plating		[41]
		Uptake of neutral red
		Protein synthesis
Porcine	Immediate cryopreservation	Protein synthesis	Trypan blue exclusion test	[30]
	Serum free	Gluconeogenesis
		CYP activity
		Urea synthesis
Rat (monolayer culture post-thawing)	Not available	Protein synthesis		[61]
Human	Storage in liquid nitrogen		Conjugation and secretion of biliary acids	[75]
Human, rat, rabbit, dog and monkey	Slow freezing protocol		CYP activity	[77]
			Phase 2 enzymes
Human, rat and mouse	Slow freezing protocol	Phase 2 enzymes	Phase 1 enzymes	[78]
Monkey	Slow freezing protocol	LDH release	Phase 1 enzymes	[79]
		Plating	Phase 2 enzymes
Human	Storing at -80°C	Cytosolic enzymes: glutathione S-transferase	CYP activity	[55]
Rat	Slow freezing protocol	CYP induction		[60]
Human	Slow freezing protocol	CYP induction		[80]
Rat	Slow freezing protocol		CYP induction	[81]
Human	Not available	CYP induction		[82]
Human	Not available	CYP induction		[83]

C/T: Cryopreservation/thawing; FCS: Fetal calf serum; LDH: Lactate dehydrogenase; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; CYP: Cytochrome P450.