Table 1.

Experimental studies assessing viability immediately post-thaw or after a period of post-thaw culture

Study	Species	Method of freezing	Concentration at freezing	Method of thawing	Passage number at freezing	Results post-thaw	Method of assessment
Human
Bruder et al. [91]	Human	FBS with 10% DMSO in LN2 (24 h)	NA	NA	NA	Cell recovery after thawing was above 95%	Trypan blue exclusion
Hirose et al. [41]	Human	Cell Banker storage medium, cells cryopreserved at − 150 °C (NA)	5 * 105 cells/mL	Cells were thawed in MEM-α supplemented with 15% FBS	P1	Immediately post-thaw viability was retained post-thaw at about 98%	Fluorescent microscopy: live/dead viability assay kit
Kotobuki et al. [35]	Human	Cell Banker medium, cryopreserved at − 80 °C (NA)	5 * 105 cells/mL	NA	P1	Immediately post-thaw viability was retained post-thaw at above 90%	NucleoCounter (ChemoMetec)
Kotobuki et al. [92]	Human	Cell Banker storage medium (ready-to-use storage medium), then cells stored sequentially: 10 min at 4 °C, 1 h at − 30 °C, 2–3 days at − 80 °C then long-term storage at − 152 °C (0.3–33.6 months)	5 * 105 cells/mL	NA	P1	Immediately post-thaw viability ranged from 71.9 to 100% with average viability about 90%	NucleoCounter (ChemoMetec)
Xiang et al. [93]	Human	30% serum-containing α-MEM with 10% DMSO, 4 °C for 10 min then cooled to − 80 °C at 1 °C/min in a controlled-rate freezer then LN2 (12 months)	1 * 105 cells/mL	Thawed in a 37 °C water bath by shaking lightly for 1 or 2 min	P3	Immediately post-thaw viability ranged from 84.6 to 100%	Flow cytometry—fluorescein diacetate, PI
Zhao et al. [94]	Human (with chronic myeloid leukaemia)	IMDM with 40% FCS and 10% DMSO at 4 °C, beaker with methanol in − 70 °C freezer for 24 h then LN2 (3 or 6 months or 1 year)	1 * 106 cells/mL	37 °C water bath for 2–4 min	P2–3	Immediately post-thaw viability was retained post-thaw at about 90%	Trypan blue exclusion
Heng [30]	Human	Culture medium with 10% DMSO and 0, 10 or 100 microM of Rho-associate kinase (ROCK) inhibitor Y-27632, cooling to − 80 °C for 2 h, then vapour phase of LN2 (1 h)	1.17 * 105 cells/mL	Thawed in a 37 °C water bath	P5	Immediately post-thaw viability dropped to a range about 91.3% to 89.4%; No effect of Y-27632 immediately post-thaw but there was an increase in viability at 24 h post-thaw	Trypan blue exclusion
Liu et al. [28]	Human	13 different freezing media tested with various combinations of different concentrations of serum, DMSO, PEG, trehalose and 1.2-Propanediol, equilibration of cells with freezing media at 4 °C for 10 min, − 80 °C overnight then LN2 (min. 1 week)	1 * 106 cells/mL	Thawed in a 37 °C water bath, shaking gently for 2 min	N/A	A freezing solution composed of 7.5% c(v/v) DMSO, 2.5% (w/v) PEG, 2% bovine serum albumin gave comparable viability (about 82.9%) to 10% DMSO (about 82.7%)	Flow cytometry–PI
Doan et al. [95]	Human	DMEM/F12 with 10% DMSO, incubation, 4 °C for 10 min, − 20 °C for 1 h, − 80 °C for 1 day then LN2 (1 year)	1 * 106 cells/mL	In a water bath at 37 °C	P3	Immediately post-thaw viability was retained post-thaw at 72.95%	Cell Viability Analyzer (Beckman Counter, USA)
François et al. [45]	Human	α-MEM with 30% FBS and 5% DMSO, − 80 °C for 24 h then LN2 (1 week)	NA	NA	Early passage	Immediately post-thaw viability dropped to ≤ 60% (Annexin V/PI) and > 80% (Trypan blue); At 4 h post-thaw viability was between 44 and 61%; viability increased after post-thaw culture	Trypan blue exclusion
Ginis et al. [50]	Human	CryoStor-2, CryoStor-5, CryoStor-10 containing 2%, 5% and 10% DMSO respectively or conventional freezing medium (90% growth medium with 10% FCS, 30% bovine serum albumin and 10% DMSO), pre-cooling on ice for 10 min, slowly cooled to − 5 °C, blast of chilling to − 25 °C, quick return to − 5 °C, cooling to − 60 °C at a rate of 1 °C/min, cooling to − 196 °C at a rate of − 25 °C using programmable cell freezer then LN2 (about 1 month or 5 months)	1 * 106 cells/mL	Thawed fast in a 37 °C water bath with gentle agitation	P2–4	Immediately post-thaw viability after 1-month freezing was about 91.7% and 95.6% and 95.4% for CryoStor-2, CryoStor-5, CryoStor-10 respectively; Immediately post-thaw viability after 5-month freezing was about 72% and 80% for CryoStor-5 and CryoStor-10 respectively	Fluorescence uptake: calcein-AM, ethidium homo-dimer-1
Mamidi et al. [33]	Human	90% FBS with 10% DMSO, programmable slow freezing unit then vapour phase of LN2 (long-term storage)	3 * 106 cells/2 mL vial	Thawed in a 37 °C water bath, shaking gently for 1–2 min	P3 and then characterized at P4–6 (with another freezing at passage 4)	Viability was about 80% upon thawing then > 95% after subsequent plating (3 passages post-thaw)	Trypan blue exclusion; flow cytometry—7-AAD
Matsumura et al. [26]	Human	COOH-PLLs 7.5% (w/w) at pH of 7.4 OR 10% DMSO in DMEM without FBS, − 80 °C freezer (1 week or 24 months)	1 * 106 cells/mL	Thawed in a 37 °C water bath with gentle shaking	P3–5	Cryopreservation for one week with PLL (0.5–0.8) did not affect the viability at 0 h and 6 h post-thaw; Cryopreservation for 24 months with PLL (0.65) provides protection comparable to 10% DMSO	Trypan blue exclusion
Chinnadurai et al. [20]	Human	Freezing media, − 80 °C then LN2 (NA)	5 * 106 cells/mL	Quickly thawed (1–2 min)	P3–5	Immediately post-thaw viability dropped to about 87% (trypan blue) and 71.5% (flow cytometry)	Trypan blue exclusion; flow cytometry—Annexin V, PI
Holubova et al. [69]	Human	60% α-MEM medium with 30% pHPL and 10% DMSO, programmable controlled rate freezer at rate 1 °C/min to − 80 °C then LN2 (1,3,6,7 and 8 months)	1 * 106 cells/mL	NA	P3	Immediately post-thaw viability is 70–90%	Flow cytometry—7-AAD staining
Moll et al. [38]	Human	4 °C human blood type AB plasma containing 10% DMSO, frozen to − 80 °C using rate-controlled cell freezing device (NA)	1–2 * 106 cells/mL	NA	P2–4	Viability reduced twofold by cryopreservation when exposed to human serum (cell count and PI incorporation)	Cell counter and analyser system (CASY-TT); flow cytometry–Annexin V, PI
Verdanova et al. [25]	Human	15 different freezing solutions containing various concentrations of DMSO (0, 1, 5, 10 and 100%) in the presence or absence of sericin at 1 or 5%, cooling to − 80 °C at a rate 1 °C/min in a CoolCell container then LN2 (72 h)	1.4 * 105 cells/mL	In a 37 °C water bath as quickly as possible	P1–3	Highest viability (24 h post-thaw) was obtained using standard freezing medium (10% DMSO and 25% FBS in culture medium); Viability of cells (24 h post-thaw) frozen in culture medium containing 10% DMSO and 1% sericin was not significantly different from standard freezing medium	Fluorescent microscopy—DAPI
Al-Saqi et al. [66]	Human	10% DMSO in Mesencult-XF or STEM-CELLBANKER at 4 °C, cryovials on ice then moved to − 80 °C with a cooling rate − 1 °C/min for 24 h then then LN2 (NA)	0.5–1 * 106 cells/mL	Thawed in a 37 °C water bath for 1 or 2 min	P3	No difference in viability immediately post-thaw between two freezing media; CELLBANKER (85.6%) and 10% DMSO (86%); No significant difference in viability between non-cryopreserved and cryopreserved using both media	Fluorescence-based live/dead assay immediately post-thaw; flow cytometry—PI (two passages post-thaw)
Luetzkendorf et al. [40]	Human	5% human albumin and 10% DMSO, automatized process in a programmable freezer then LN2 (21–51 days)	1.8 * 108 in cryopreservation bags	Thawed at	P3–4	Immediately post-thaw viability was retained at > 90% viability using both methods for 4 donors out of 5	Trypan blue exclusion; flow cytometry: 7-AAD
Pollock et al. [67]	Human	60% plasmalyte A, 20% of 25% HAS and 20% DMSO (final concentration of DMSO was 10% by volume), controlled rate freezer then LN2 (30–45 days)	1–10 * 106 cells/mL	Thawed quickly in a 37 °C	P1–6	Immediately post-thaw viability was retained at > 80% for almost all samples	Fluorescent microscopy—Acridine orange, PI
Chinnadurai et al. [68]	Human	IFNɣ, caspase inhibitor Z-VAD-FMK or 3-methyl adenine pre-licensing 48 h prior to cryopreservation, 5% human serum albumin, 5%, 20%, 40%, 90% hPL in aMEM with 10% DMSO OR CryoSOfree DMSO-free cryopreservation medium, cooling rate 1 °C/min then step-down freezing using a 7-step program in CryoMed controlled-rate freezer then LN2 (NA)	5–10 * 106 cells/mL	In a 37 °C water bath for 1 min	P2–6	The addition of various concentrations of human platelet lysate did not significantly enhance MSC recovery and viability; IFNɣ pre-licensing prior to cryopreservation enhances thawed MSC survival	Trypan blue exclusion; flow cytometry—7-AAD
Gramlich et al. [18]	Human	CryoStor CS5 media, − 80 °C for 90 min then vapour phase of LN2 (7–30 days)	1 * 106 cells per mL	In a 37 °C water bath	P3–5	Immediately post-thaw viability was retained at > 95% (viability only marginally reduced after thawing)	TUNEL staining; Fluorescent microscopy—Hoechst, PI
Lechanteur et al. [34]	Human	40% PBS + 40% of HSA solution (20%) + 20% DMSO added under agitation at 4 °C, automated cryofreezer with a 9-step program to − 160 °C then vapour phase of LN2 (NA)	2 * 106 cells/mL	Freezing bag is protected in sterile plastic bag and thawed in a 37 °C water bath for a few min	P3	Immediately post-thaw viability ranged from about 50% to 90% with about 14% decrease in viability	Trypan blue exclusion
Yuan et al. [52]	Human (BM-MSC engineered to express TRAIL)	5% DMSO, 30% FBS in alpha-MEM OR human albumin with 0.5–20% DMSO, isopropanol freezing box overnight in, − 80 °C freezer then LN2 (1–3 weeks)	1 * 106 cells/mL or 5 * 106 cells/mL or 10 * 106 cells/mL	In a water bath at 37 °C with gentle shake for 2 min	P5	Significantly reduced immediately post-thaw viability with 0% DMSO (5.16%); Immediately post-thaw viability increased with increased DMSO% in the freezing; 15% and 20% DMSO gave reduced viability (about 70.6% and 64.1% respectively) immediately post-thaw solution up to 10%; at 5% DMSO same viability obtained for different cell concentrations	Flow cytometry—Annexin V, DAPI
Other species
Carvalho et al. [44]	Rat	DMEM with 10% FBS and 5% DMSO, cells incubate at room temperature for 15 min then vials cooled at 3 °C/min, 5 °C/min, 10 °C/min during 15, 45, 10 min respectively until − 80 °C using programmable freezing device then LN2 (1 month)	1 * 107 cells/mL	Thawed in a 37 °C water bath with constant gentle shaking	Frozen down after 4 weeks in culture	Immediately post-thaw viability dropped to about 90.58% (trypan blue) and 66.25% (flow cytometry)	Trypan blue; flow cytometry—Annexin V, 7-AAD
Liu et al. [29]	Rat, mouse and calf	14 different freezing solutions tested with various combinations of different concentrations of serum, DMSO, PEG, trehalose and 1.2-Propanediol, equilibration for 15 min at 4 °C, − 80 °C overnight then LN2 (min. 1 week)	1 * 106 cells/mL	Thawed in a 37 °C water bath with gentle shaking for 2 min	NA	There were variations between species with respect to cell viability—Mouse MSCs were more robust than rat and bovine MSCs; Reduced DMSO (5%) with 2% PEG, 3% trehalose and 2% albumin gave higher immediately post-thaw viability (91.5% [mouse]) to 10% DMSO (75.3% [mouse])	Trypan blue exclusion
Naaldijk et al. [27]	Rat	Cryoprotectant consisted of hydroxyethyl starches of different mean molecular weights [MW = 109, 209, 309, 409, 509, 609 kDa] and/or DMSO, then cells were frozen according to one of seven different freezing protocols (NA)	1 * 105 cells/0.5 mL	Thawed in a 37 °C water bath	P1–3	Immediately post-thaw viability was approximately 85%; viability after 3 days of thawing was lower	Trypan blue exclusion
Davies et al. [42]	Rat	10% DMSO in 90% FBS, then vials incubated for 1 h at 4 °C, 2 h at − 20 °C, overnight at − 80 °C then LN2 (NA)	1 * 106 cells/mL	Thawing in a 37 °C RS Galaxy S + incubator for about 5 min	P1	Immediately post-thaw viability was retained post-thaw at > 90%; But lower viability was obtained after in vitro expansion of cryopreserved cells	Trypan blue exclusion
Renzi et al. [31]	Sheep, horse and rat	13 different freezing media tested with various combinations of different concentrations of FBS, DMSO, Trehalose, hydroxyethyl starch, bovine serum albumin and Caspase inhibitor z-VAD-fmk, 4 °C for 60 min, gradual reduction of temperature − 1 °C/min to − 40 °C, − 10 °C/min to − 70 °C in a controlled rate freezer then vapour phase of LN2 (5 days)	1 * 106 cells/mL	Thawed in a 37 °C water bath	P4	No DMSO or low DMSO gave very poor viability; The best viability was obtained when using FBS with 10% DMSO	Trypan blue exclusion (evaluated at 0, 24 and 48 h post-thaw)
Li et al. [96]	Dog	DMEM with 10% FBS and 10% DMSO, 4 °C for 1 h, − 20 °C for 2 h, − 80 °C for 10.5 h then LN2 (1 month)	1 * 106 cells/mL	Thawed at 37 °C	P4	Immediately post-thaw viability was retained post-thaw at 90.1%	Trypan blue exclusion
Zhu et al. [46]	Dog	DMEM containing 10% FBS and 10% DMSO, 4 °C for 1 h, − 20 °C for 2 h, − 80 °C for 10.5 h then LN2 (3 years)	1 * 106 cells/mL	Thawed in at 37 °C	P4	No significant difference in cell viability	Trypan blue exclusion
Edamura et al. [36]	Dog	Cryoprotectant solution with or without 10% DMSO and 10% FBS, biofreezing vessel at − 80 °C in a freezer (7 days)	1 * 106 cells/mL	Thawed in a 37 °C water bath for 1 min	P1	DMSO and FBS-free freezing gave higher viability (about 99%); DMSO and FBS containing freezing media gave lower viability (about 89.7%)	Trypan blue exclusion
Nitsch et al. [97]	Monkey	Freezing medium containing 0,1,5,10 or 15% DMSO (v/v), controlled rate freezer using an optimised freezing rate then − 150 °C freezer (1 week)	1 * 106 cells/mL	In a 37 °C water bath	P9	Immediately post-thaw viability was about 80% for the different DMSO concentrations; Highest viability 24 h post-thaw for cells frozen with 5 or 10% DMSO	Trypan blue exclusion
Lauterboeck et al. [49]	Monkey	Three different freezing solutions tested (2 of them xeno-free) containing different concentrations of DMEM, DMSO and/or FBS, methylcellulose, poloxamer-188, α-tocopherol, cell suspension equilibrated for 10, 30 or 60 min then placed in controlled rate freezer using one-step freezing protocol or two-step freezing protocol then − 150 °C (at least 24 h)	1 * 106 cells/mL	In a 37 °C water bath for 90 s	NA	Viability maintained after thawing	Automatic cell counter
Ock and Rho [51]	Pig	ADMEM solution supplemented with 10% FBS and 1% penicillin–streptomycin with 40%, 20% or 10% DMSO, controlled rate programmable freezing device at − 1 °C/min from 25 °C to − 80 °C then then LN2 (< 1 month)	2 * 106 cells/mL	In a 37 °C water bath for 1 min	P5	There was a significant difference between fresh and cells cryopreserved with 10% (about 77.6%) or 20% DMSO (about 67%); No significant difference between fresh and cells cryopreserved with 5% DMSO (about 83.9%)	Trypan blue exclusion
Romanek et al. [98]	Pig (BM-MSC treated with a high hydrostatic pressure (HHP) before freezing)	10% DMSO, 2 h at − 20 °C then LN2 (up to 4 weeks)	NA	37 °C water bath with gentle shaking	NA	Significant difference between cells treated with HHP and control immediately post-thaw (about 75.2%–81.7%); No difference in viability at 8 days post-thaw (about 81.6%–82.1%)	Trypan blue exclusion
Mitchell et al. [32]	Horse	Six different freezing solutions tested (20% serum [autologous equine serum, commercial equine serum or FBS], 10% DMSO and 70% media OR 95% serum and 5% DMSO), − 80 °C freezer for 24 h then liquid phase of LN2 (2–5 days)	10 * 106 cells/mL	In a 35 °C water bath with gentle agitation	P3–6	Immediately post-thaw viability was retained at about 80–90% regardless of the cryopreservation formulation	Flow cytometry—Fluorescein diacetate, PI

Details on the experimental cryopreservation processes taken by different research groups. This table aims to provide the individual freezing protocols outlined in the extracted papers alongside the concentration and passage of cells at the point of cryopreservation and the process of thawing