Effects of storage media, supplements and cryopreservation methods on quality of stem cells

Table 1 Comparison of different protocols used during cryopreservation of hematopoietic stem cells

HSC source	Storage period and temperature	Cryopreservation	Viability post freezing	Engraftment in days	Results	Ref.
< 600 x 106 cells/mL autologous PBSC	5-15 yr, -150 °C	10% DMSO and 23.3% Plasma Lyte A	66.4%	12	Viable CD34+ cells or CFU-GM is a reliable predictor of rapid engraftment	[13]
< 300 x 106 cells/mL autologous PBSC	< 6 mo, -80 °C	3.5% DMSO, 1% HSA and 2.5% HES	72%	14	Low DMSO conc allows successful engraftment and reduces toxicity (8%); Similar engraftment after combination of DMSO with or without HES and HSA	[115]
< 100 x 106 cells/ mL autologous PBSC	< 6 mo, -80 °C	5% or 10% DMSO, autologous plasma, 5% ACD	85%	14	19.1% infusion-related toxicity in the 10% DMSO group vs 6.8% in the 5% DMSO group, lowering DMSO results in reduction in infusion toxicity and lower costs with a similar hematopoietic reconstitution	[116]
Autologous PBSC	< 11 yr, -80 °C	3.5% DMSO + 1% HSA and 2.5% HES vs 6% DMSO + 6% HES	no significant change	11-12	Uncontrolled-rate freezing and cryopreservation with 5% DMSO/HES at −80 °C supports hematopoietic reconstitution comparable to that of controlled-rate freezing and liquid nitrogen storage	[117]
< 4000 x 106 cells/mL autologous PBSC	1-98 wk, -80 °C	3.5% DMSO, 2.5% HES and 1% HSA	60.8%	11-20	Reduction in DMSO concentration decreases transfusion-related adverse events. PBPCs cryopreserved in low DMSO/HES/HSA at -80°C allow successful engraftment	[24]
50 x 106 cells/mL autologous PBSC and BM	PB: 35 mo (26-78); BM 16 mo (27-71), -90 C	5% DMSO, 6% HES and 4% HSA in RPMI1640	93%		DMSO-associated toxicity during infusion, storage of HSCs at -90°C in DMSO/HES/HSA did not cause loss of cell numbers, viability, and clonogenic activity	[118]
Autologous PBSC	Controlled rate freezing at -186 °C	5% or 10% DMSO and 6% HES		10-20	Two patients who received components cryopreserved with DMSO alone experienced serious neurological toxicity, none of the recipients who received components frozen in DMSO/HES experienced serious infusion-related toxicity, better hematopoietic recovery in presence of HES independent of DMSO concentration	[14]
100 x 106 cells/mL – 200 x 106 cells/mL autologous PBSC	5-6 yr, controlled rate freezing at -160 °C	2%-10% DMSO, 10% ACD	73% with 5% DMSO	10-14	Cryopreservation using 5% instead of 10% DMSO improves CD34 + cell and leukocyte viability, but has only minor effects on supernatant levels of leukocyte- and platelet-derived soluble mediators	[61]
75 x 106 cells/mL - 250 x 106 cells/mLautologous PBSC	32-180 d, controlled rate freezing, -196 °C	5% or 10% DMSO	84%-95%	10-14	The use of 5% instead of 10% DMSO was associated with a decrease in side effects, cryopreservation with 5% DMSO followed by storage in nitrogen is a simple, highly standardized, and safe procedure for cryopreservation of autologous stem cell graft	[119]
UCB	1-2 mo, uncontrolled vs controlled rate freezing at -90 °C	5% or 10% DMSO	Uncontrolled 84.2%; controlled 92.5%		Best recovery of UCB cells when controlled-rate freezing and 5% DMSO were combined	[120]
15 x 106 cells/mL UCB	> 2 wk, controlled rate freezing at -170 C	5%, 10% or 20% DMSO and 2% HSA or autologous plasma	89%		Optimal conditions for cryopreservation were 10% DMSO and 2% HSA with fast addition and removal of DMSO	[121]
800 x 106 cells/mL UCB	10 yr, controlled rate freezing at -196 °C	10% DMSO and 5% Dextran	83.7%		Long term storage of UCB units does not affect the quality of the HSCs	[122]
Autologous BM	4 mo, -80 °C	5% DMSO and 6% HES	82.2%	21	BM cells can be rapidly and inexpensively cryopreserved in DMSO/ HES, without need for rate-controlled freezing or storage in liquid nitrogen	[123]
20 x 106 cells/mL BM or 17 x 106 cells/mL PBSC	Controlled rate freezing at -196 °C	10% DMSO or 0.25-1 mol/L TH with or without 0.25 IU/mL insulin (I)	DMSO: 33%TH: 32%; TH/I: 30%		DMSO-cryopreserved cells exhibited the best median viability-rate after thawing. Comparable results could be achieved with trehalose 0.5 mol/L with/without insulin	[45]
200 x 106 cells/mL autologous BM or PBSC	BM: 11.8 yr vs PB: 33 d controlled rate freezing at -196 °C	10% Medium 199 , 80% autologous plasma and 10% DMSO	BM: 81.5%; PBSC: 68.0%		BM can be cryopreserved for more than a decade without apparent loss of progenitor activity in comparison to short-term cryopreserved PBSC	[124]

HSC: Hematopoietic stem cell; DMSO: Dimethyl sulfoxide; CFU-GM: Colony Forming Unit-Granulocyte/Macrophage; ACD: Acid citrate dextrose; RPMI: Roswell Park Memorial Institute Medium; HES: Hydroxyethyl starch; HSA: Human serum albumin; BM: Bone marrow; UCB: Umbilical cord blood; TH: Trehalose.