Table 1 Experimental studies assessing viability immediately post-thaw or after a period of post-thaw culture
From: The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review
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 |