Mesenchymal Stromal Cell Cryopreservation

Abstract

The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below −80°C and typically below −140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

Introduction

Currently, cryopreservation at temperatures between −80°C and −140°C is the only method able to preserve cell viability, biochemical functions, and long-term characteristics.¹ Cells, tissues, biological fluids, nucleic acids, bacteria, gametes, viruses, and blood can be preserved at these temperatures, but the integrity and viability of these materials is maintained by cryoprotectants (CPAs) and by a progressive decrease in temperature.²

CPA solutions are classified as intra- and extracellular, according to their capacity to cross the plasma membrane. The intracellular cryoprotectant types, such as dimethyl sulfoxide (DMSO), ethylene glycol, 1,2-propanediol, and glycerol are small molecules, whose function is to reduce the extent of cell volume changes during slow freezing and rapid thawing.³

However, the use of extracellular cryoprotectants is restricted, depending on molecular size and biomolecular stabilization. These are long-chain polymers, polypeptides, or carbohydrates able to dehydrate the cell prior to freezing, thus reducing the development of intracellular ice crystals.

Currently, 10% (v/v) DMSO solution with fetal bovine serum (FBS) is considered the most efficient and widespread means of cryopreservation and it has been used for several decades to preserve many types of cells and tissues. DMSO is the most widely accepted cryoprotectant for cell preservation in spite of the fact that its cytotoxicity and negative effect on differentiation have been reported.⁴ Several researchers have highlighted human embryonic stem cell (ESC) pluripotency diminished in a reversible manner beside induction of dose-dependent cell quiescence.^5,6 It has also been reported that DMSO affects the epigenetic system acting on the three DNA methyltransferases (Dnmts) and on five histones by enzyme modification. Following modulation of Dnmts transcription and genome-wide DNA methylation profile, the ESCs and embryonic bodies undergo changes in their phenotypic behavior.⁷

Cell-based therapies are increasing in human and veterinary clinical practice; therefore it is important to identify novel nontoxic cryoprotectants. An alternative freezing method is vitrification, consisting of a physical process based on solidification of a concentrated solution of CPAs during cooling, without crystallization. When material vitrifies, no ice formation occurs, even at cryogenic temperatures. Vitrification does not cause any biological damage usually associated with freezing. In fact, it can markedly improve survival by circumventing ice-induced injury. Furthermore, it seems to be applicable not only to cells but also to tissues and organs.⁸

For this reason, it is fundamental to improving storage conditions that will enable biological characteristics and features of the products used to be preserved.

Generally, cells are frozen by using CPAs such as DMSO. Temperature decrease is gradual to −40°C and then rapid to −196°C. The thawing process occurs rapidly to prevent cell damage caused by water crystallization.⁹

Although this method has been successfully used for storing different types of cells, it is shown to have low protection efficacy on mesenchymal stromal cells (MSCs). Great interest is focused on the preservation of these cells because of their potential application in cell therapy.

Consequently, the aim of this study was to evaluate alternative CPAs, compared to the traditional method based on the use of culture medium with animal serum and DMSO, for the purpose of increasing MSCs viability and replication.

Materials and Methods

Cell cultures

Mesenchymal stromal cells (MSCs), commonly referred to as mesenchymal stem cells, were isolated from sheep, rat, and horse bone marrow and from horse adipose tissue. Equine dermis cells (EDe), used as controls, are registered in the cell culture collection at the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Brescia, Italy (www.izsler.it, Cell Bank code: BS PRC 12). Five samples of each cell line were frozen using different 15 cryogenic media as later described (for a total of 75 vials for each tissue).

Stromal cells isolation from sheep, horse, and rat bone marrow (BMSCs)

Sheep and horse

Bone marrow samples were collected in accordance with institutional animal care facility guidelines. Animals were sedated with detomidine 0.2 mg/animal (Detogesic, Fort Dodge Animal Health S.p.A., Bologna, Italy) and xylazine 220 mg/animal (Megaxilor, Bio98 S.r.l., Milano, Italy). Bone marrow was aspirated from the sternum using 11-gauge jamshidi needles and collected in sterile tubes containing 6000 IU of heparin sodium salt grade IA from porcine intestinal mucosa (Sigma-Aldrich, Milano, Italy). Sample was centrifuged at 1600 g for 10 min at 20°C. The buffy coat was diluted in culture medium (NH Expansion medium, Miltenyi Biotec, Bologna, Italy) and it was carefully stratified on Ficoll-Paque Plus (GE Healthcare, Milano, Italy), in order to isolate mononuclear cells. They were washed twice in culture medium, seeded at a density of 1.0×10⁶ cells/cm² in Eagle's minimum essential medium (MEM, Sigma-Aldrich) and incubated at 37°C in 5% CO₂. Culture medium was replaced at 72-h intervals.

Rat

Two-month-old rats were humanely sacrificed as described in National Welfare Animals Guidelines. Their femurs were used as the bone marrow source. Femurs were isolated, cleaned from surrounding tissues and immersed in Phosphate Buffered Solution (PBS) containing 500 IU/ml penicillin, 125 μg/ml streptomycin, and 1 μg/ml amphotericin B (each from Squibb, Roma, Italy). Limbs were then transferred to another beaker containing saline and a higher concentration of penicillin (2500 IU/ml), streptomycin (625 μg/ml), and amphotericin B (51 μg/ml). Bone marrow was collected using 10 ml syringes with 18G needle containing 2 ml of growth medium specific for stem cells (NH Expansion medium, Miltenyi Biotec), and 1 ml of heparin (6000 IU/ml). After collection, the bone marrow was centrifuged at 1600 g for 10 min at 4°C. The cell pellet was diluted into 7 ml of culture medium and carefully stratified on Ficoll-Histopaque 1077 g/l (GE Healthcare).¹⁰ Mononuclear cells were collected, washed twice in culture medium, seeded at a density of 1.0×10⁶ cells/cm² and incubated at 37°C in 5% CO₂. At 72-h intervals, fresh culture medium was replaced and the cell culture was incubated to confluence.

Stromal cells isolation from adipose tissue (ASCs)

Approximately 60 grams of adipose tissue samples were collected under sterile conditions from the intra-abdominal fat deposits of adult horses within one h after slaughtering.

Tissue was minced and washed in PBS containing 1000 IU/ml penicillin and 100 μg/ml streptomycin. Subsequently, tissue was treated with 7.5 mg/ml type I collagenase (Sigma-Aldrich, Milano, Italy) at 37°C. The cell suspension was filtered through a 50 μm nylon mesh and centrifuged at 250 g for 10 min at 20°C. The cell pellet was diluted in Dulbecco's modified Eagle's medium low glucose (Sigma–Aldrich) containing 10% (v/v) fetal bovine serum (FBS, Mascia Brunelli S.p.A., Milano, Italy), 1000 IU/ml penicillin, and 100 μg/ml streptomycin. Finally, cells were diluted at 5.0×10⁴ cells/cm² in culture medium, seeded in tissue culture flasks, and incubated at 37°C in 5% CO₂. After 72 h intervals, the medium was replaced.

MSCs subcultivation

Subcultures were performed when cells reached 80%–90% confluence. The cell monolayer was treated with Trypsin-EDTA (ethylenediaminetetraacetic acid) (Sigma-Aldrich) solution following IZSLER's standardized protocols. The cell suspension, after dilution in culture medium, was incubated at 37°C in 5% CO₂. A maximum of four serial passages were carried out. EDe were propagated in Eagle's minimum essential medium in Earle's Balanced Salt Solution added with penicillin (1000 IU/ml), streptomycin (500 μg/ml), amphotericin B (2 μg/ml), and 10% (v/v) FBS.

Cryopreservation media

Five batches of each cell type were frozen in the following cryogenic media (CM):

• CM1: culture medium+20% (v/v) FBS, 10% (v/v) DMSO (Sigma-Aldrich)

• CM2: CS2 (BioLife Solutions, Bothell, WA, ready to use) medium containing 2% (v/v) DMSO

• CM3: CS5 (BioLife Solutions, ready to use) medium containing 5% (v/v) DMSO

• CM4: CS10 (BioLife Solutions, ready to use) medium containing 10% (v/v) DMSO

• CM5: culture medium+20% (v/v) FBS, 5% (v/v) DMSO, 60 mM Trehalose (Sigma-Aldrich)

• CM6: culture medium+20% (v/v) FBS, 2,5% (v/v) DMSO, 30 mM Trehalose

• CM7: culture medium+20% (v/v) FBS, 264 mM Trehalose

• CM8: culture medium+5% (v/v) DMSO, 20% (v/v) FBS, 500 mM Trehalose

• CM9: culture medium+7% (v/v) DMSO, 5% (v/v) hydroxy ethyl starch (HES, Sigma-Aldrich), 2% (v/v) bovine serum albumin (BSA, Sigma-Aldrich)

• CM10: culture medium+5% (v/v) DMSO, 4% (v/v) HES, 4% (v/v) FBS

• CM11: culture medium+10% (v/v) DMSO, 12% (v/v) HES, 8% (v/v) FBS

• CM12: culture medium+30 μM Caspase Inhibitor z-VAD-fmk (Promega, Milano, Italy), 10% (v/v) DMSO, 20% (v/v) FBS

• CM13: vitrification medium (1 M DMSO, 1 M acetamide (Sigma-Aldrich), 3 M propylene glycol (Sigma-Aldrich)

• CM14: vitrification medium added with 20% (v/v) DMSO

• CM15: culture medium added with 90% (v/v) FBS, 10% (v/v) DMSO

A total of 75 vials of each cell line were frozen.

Freezing

Sheep, rat, and horse BMSCs, horse ASCs, and EDe, enzymatically disaggregated, were centrifuged at 125 g for 10 min at 4°C and cell pellets were diluted in culture medium devoid of serum and re-centrifuged in the same conditions. Each cell pellet was cryopreserved in cell media aforementioned, except for CM13 and CM14, at the mean cell concentration of 1.0×10⁶ cells/ml.

Cryovials were stored at 4°C for 60 min and frozen by a gradual reduction of temperature at −1°C/minute to −40°C. From −40°C to −70°C the decrease rate was −10°C/min faster. The process was performed in a controlled rate freezer (CryoMed, Forma Scientific, Marietta, OH), which was programmed to the parameters for the temperature diminution. When the cryovials reached −70°C, they were moved to the liquid nitrogen vapor phase of a storage tank.

Vitrification

200 μl of vitrification medium (CM13 and CM14) was added to each cell suspension sample. This procedure was performed in direct contact with liquid nitrogen. Special care was taken to ensure that the temperature of vitrified samples did not exceed −130°C to prevent recrystallization or devitrification.¹¹

Thawing method

Sample cryovials were thawed 5 days after freezing. They were immediately immersed in water at 37°C and replaced with appropriate media, with a substitution step for CM12. Thawed cell suspensions were diluted in culture medium and centrifuged (125 g, 10 min, 4°C) twice in order to eliminate cryoprotective agents. Finally, cells were seeded in 6-well plates and incubated in culture medium at 37°C in 5% CO₂. Cells cryopreserved with CM12 were seeded in culture medium containing 30 μM caspase inhibitor z-VAD-fmk and incubated at 37°C in 5% CO₂.

Cell viability and cell death assays

Cell viability and death were evaluated after thawing each vial (5 for each tissue) at different time points: 0 (T0), 24 (T24), and 48 (T48) hours after seeding, respectively. MSCs were counted with Cellometer^® Automated Cell Counters (Nexcelom Bioscience, USA) with Trypan Blue (Sigma-Aldrich, Italy) staining. Finally, data were analyzed and the mean value for each tissue and species, cryopreserved in the same medium, was calculated.

Statistical analysis

Differences among cryogenic media at three time points were checked on the cell lines of equine origin, by two-way ANOVA. The significance threshold was set at p<0.01 (Prism 2.01, GraphPadSoftware, San Diego, CA).

Results

Cell culture

BMSCs and ASCs adhered to the surface of the flask, 24 hours after seeding and after replication; cells showed a typical fibroblastic morphology reaching subconfluence in 10 and 7 days, respectively.

Cryogenic media and methods

Viability of cells, frozen in media either containing low concentrations or completely deprived of DMSO (CM2, CM3, CM5, CM6, and CM7), appeared unsatisfactory. In fact, cell survival and replication capacity decreased as soon as 24 h post-thaw. In particular, horse BMSCs viability was lower than 10%; therefore this approach and media were discarded from any other subsequent tests. Rat and sheep stem cells demonstrated a high viability when cryopreserved with CM1. Results obtained with BMSCs, ASCs, and EDe, frozen with CM1 (culture medium with 20% (v/v) FBS and 10% (v/v) DMSO), CM4 (CS10, with 10% (v/v) DMSO), and CM8 (medium containing 5% (v/v) DMSO, 20% (v/v) FBS, and 500 mM trehalose), showed a high percentage of viability and replication capacity with the exception of BMSCs, which showed a significant reduction (p<0.0001) of cell viability on T24 (Table 1).

Table 1.

Mean Percentage and Standard Deviation of Cell Viability after Thawing

	Freezing Medium	Cell Viability at Thawing	Cell Viability after Thawing
			24 Hour	48 Hour
Equine dermis control	CM1	95±1.5%	100±0.33%	100±0%
	CM4	90±2.8%	95±2.4%	100±0%
	CM8	90±4.1%	90±3.6%	95±2.7%
Equine ASCs	CM1	84±3.9%	85±2.2%	88±3.7%
	CM4	80±6.4%	82±5.9%	85±4%
	CM8	70±2.6%	73±3.3%	75±3.4%
Equine BMSCs	CM1	33±5.9%	20±7.6%	38±4.6%
	CM4	85±6.7%	30±6.5%	31±5.7%
	CM8	82±2.7%	22±6.2%	12±7.1%
Sheep BMSCs	CM1	90±1.99%	95±1.97%	95±1.17%
	CM4	82±0.99%	72±3.88%	83±3.07%
	CM8	32±1.5%	46±1.47%	45±1.67%
Rat BMSCs	CM1	95±2.27%	95±2.24%	95±3.11%
	CM4	90±6.24%	85±3.86%	85±3.25%
	CM8	75±2.91%	72±4.62%	70±3.85%

CM1, culture medium added with 20% (v/v) FBS and 10% (v/v) DMSO; CM4, CS10 (Biolife solution, ready to use) medium containing 10% (v/v) DMSO; CM8, culture medium added with 5% (v/v) DMSO, 20% (v/v) FBS, and 500 mM trehalose.

ASCs, adipose stem cells; BMSCs, bone marrow stem cells.

The overall results indicated that the best CPAs were CM1 and CM4, both containing 10% (v/v) DMSO except for BMSCs, which were always characterized by low viability (30%) 24 h after thawing regardless of the cryopreservation medium. Viability was similar to that observed after freezing in CM1.

In order to improve equine BMSC viability, cells frozen in CM1 were compared to those frozen in CM9, CM10, CM11, CM12, CM13, CM14, and CM15. EDe cells were used as the reference control.

Results showed Ede ability to adhere to the plastic surface and to replicate with the media containing hydroxy ethyl starch (CM9, CM10, and CM11). In fact, at T48, these cells reached the same values as those stored in control medium (CM1). ASCs showed better viability with CM1, CM9, and CM11 than CM10 (p<0.0001) on T0. This parameter decreased after thawing, until a complete loss of viability at T48. The presence of HES in the cryopreservation solution enabled BMSCs survival during freezing (Table 2).

Table 2.

Results of Cells Cryopreserved with HES Compared to Medium Containing 10% DMSO (Mean Value and Standard Deviation)

	Freezing Medium	Cell Viability at Thawing	Cell Viability after Thawing
			24 Hour	48 Hour
Equine dermis control	CM1	93±2.93%	100±0.2%	100±0%
	CM9	91±3.18%	95±4.35%	100±0%
	CM10	87±5.96%	90±4.49%	100±0%
	CM11	94±6.13%	100±0.34%	100±0%
Equine ASCs	CM1	93±1.83%	80±3.27%	90±3.01%
	CM9	89±2.21%	45±2.93%	0%
	CM10	78±3.15%	38±5.18%	0%
	CM11	95±5.61%	80±6.39%	0%
Equine BMSCs	CM1	34±1.643%	25±1.63%	18±1.55%
	CM9	0%	0%	0%
	CM10	0%	0%	0%
	CM11	0%	0%	0%

CM1, culture medium added with 20% (v/v) FBS and 10% (v/v) DMSO; CM9, culture medium added with 7% (v/v) DMSO, 5% (v/v) hydroxy ethyl starch (HES) and 2% (v/v) bovine serum albumin (BSA); CM10, culture medium added with 5% (v/v) DMSO, 4% (v/v) HES, and 4% (v/v) FBS; CM13, vitrification medium (1 M DMSO, 1 M acetamide, 3 M propylene glycol).

ASCs, adipose stem cells; BMSCs, bone marrow stem cells.

When EDe and BMSCs, cryopreserved with caspase inhibitor z-VAD-fmk (CM12), were thawed, a decrease in the percentage of cell viability was observed. Moreover, in this case, no live EDe cells were detected at T24. In contrast, results obtained during ASCs thawing, showed 98% viability, and 48 h later it decreased to 76% (p<0.0001), indicating CM1 as the most useful for preservation of this cell line. On the other hand, BMSCs viability and growth increased using CM12 with a statistical significance (p<0.0001), versus CM1 (Table 3). The vitrification method appeared completely unable to preserve viability of all kind of cells (Table 4). Finally, five samples of each cell line were cryopreserved with FBS added with 10% (v/v) DMSO (CM15). In this last trial, not only Ede and ASCs reached confluence at T48, but also more than 50% of equine BMSCs survived freezing and they were characterized by cell growth and replication between T0 to T48 (p<0.0001) (Table 5).

Table 3.

Cell Freezing with Caspase Inibitor z-VAD-fmk (Mean Value and Standard Deviation)

	Freezing Medium	Cell Viability at Thawing	Cell Viability after Thawing
			24 Hour	48 Hour
Equine dermis control	CM1	95±1.28%	95±0.92%	97±1.81%
	CM12	94±0.48%	0%	0%
Equine ASCs	CM1	98±0.25%	90±0.65%	88±0.5%
	CM12	98±0.2%	79±0.54%	76±0.68%
Equine BMSCs	CM1	39±3.23%	35±0.66%	20±1.15%
	CM12	51±1.63%	45±0.86%	40±2.92%

CM1, culture medium added with 20% (v/v) FBS and 10% (v/v) DMSO; CM12, culture medium added with 30 μM caspase inhibitor z-VAD-fmk, 10% (v/v) DMSO, and 20% (v/v) FBS.

ASCs, adipose stem cells; BMSCs, bone marrow stem cells.

Table 4.

Cell Vitrification (Mean Value and Standard Deviation)

	Freezing Medium	Cell Viability at Thawing	Cell Viability after Thawing
			24 Hour	48 Hour
Equine dermis control	CM1	98±1.58%	100±0%	100±0%
	CM13	12±4.26%	0%	0%
	CM14	8±1.29%	0%	0%
Equine ASCs	CM1	98±2.67%	90±6.2%	90±2.36%
	CM13	72±3.07%	0%	0%
	CM14	58±3.35%	0%	0%
Equine BMSCs	CM1	39±2.36%	32±2.13%	29±1.04%
	CM13	29±5.35%	0%	0%
	CM14	28±2.45%	0%	0%

CM1, culture medium added with 20% (v/v) FBS and 10% (v/v) DMSO; CM13, vitrification medium (1 M DMSO, 1 M acetamide, 3 M propylene glycol); CM14, vitrification medium added with 20% (v/v) DMSO.

ASCs, adipose stem cells; BMSCs, bone marrow stem cells.

Table 5.

Cells Cryopreserved with Medium Added with 90% FBS (v/v) in Comparison to CM1 (Mean Value and Standard Deviation)

	Freezing Medium	Cell Viability at Thawing	Cell Viability after Thawing
			24 Hour	48 Hour
Equine dermis control	CM1	96±0.45%	100±0.16%	100±0%
	CM15	98±0.48%	100±0.44%	100±0%
Equine ASCs	CM1	98±0.77%	90±0.63%	100±0%
	CM15	98±0.44%	90±0.7%	100±0.13%
Equine BMSCs	CM1	36±0.16%	34±0.48%	32±0.64%
	CM15	56±0.41%	65±0.4%	80±0.67%

CM1, culture medium added with 20% (v/v) FBS and 10% (v/v) DMSO; CM15, culture medium added with 90% (v/v) FBS, 10% (v/v) DMSO.

ASCs, adipose stem cells; BMSCs, bone marrow stem cells.

Discussion

Recently, it has been proven that MSCs have the ability to differentiate towards several phenotypes, including osteo-, adipo-, chondro-, and tenocytic lineages.^12–14 They can be expanded ex vivo and they are a promising tool in regenerative medicine. In order to control cell batches, it is important to have systems that sustain long-term storage and preserve their viability and features. Currently, the most efficient cryopreservation reagent is culture medium containing 10% (v/v) DMSO and a different percentage of FBS. Although scientists have long considered using natural or synthetic substances to find better CPAs, unfortunately, no efficient alternative has been developed to date.⁴

During routine IZSLER's laboratory activities, this decrease of horse BMSCs and high sensitivity was observed after the freezing/thawing process. For this reason, it was our purpose to identify a cryogenic medium able to reduce mortality of this cell line. In order to better understand cell sensitivity, survival ability of equine, rodent, and ovine BMSCs and equine ASCs, these were evaluated following the freeze/thaw procedure. Our target was to investigate the ability of different cryogenic media and in this regard, 14 solutions were compared to the specific cell culture medium, added with 10% DMSO and 20% FBS (CM1).

At the beginning of this research, rodent, ovine, and equine stem cells were included in the trial; at first rat and sheep cells demonstrated high performances in terms of viability and replication with CM1. For this reason, these cell lines were excluded from the study, because the authors focused their attention on equine stem cells, in particular on BMSCs, characterized by either poor viability, low replication after freeze/thaw process, or great significance in cell-based therapies.

Results about equine cells highlighted different behavior related to either the cell line or analyzed medium. The first step showed EDe strong tolerance to the major part of the considered media, with the exception of CM13 and CM14, used for vitrification. These cells are able to survive the freeze/thaw process when stored with CM12, but their viability decreased at T24. On the other hand CM1, CM4, CM8, CM9, CM10, CM11, and CM15 represented an efficient tool for preserving cells and their features, ensuring cell replication after cryopreservation procedure.

The same evaluation was performed for ASCs. These cells were characterized by a good viability and replication capacity when cryopreserved with CM1, CM4, CM8, and CM12. CM9, CM10, CM13, and CM14, which guaranteed a high percentage of live cells at thawing, decreased to 0% at T48. Finally, the best results were obtained with CM15, in fact it allowed cell survival and replication, demonstrating the suitability of this medium.

BMSCs have a lower resistance to intracellular damages caused by ice crystals. Even if they were stored with different solutions, these cells were incapable of replicating after thawing. In this study, BMSCs survival never went above 50%, with three exceptions: CM4, CM8, and CM15.

CM4 and CM8 permitted survival of a great number of cells, but they did not preserve cell replication ability, indeed at T48 all cells died. In conclusion, CM15 was the only medium able to guarantee either cell survival or significant replication with a pick between T24 and T48, respectively.

Another main finding was represented by the vitrification process; in particular, this procedure is normally performed in order to preserve oocytes and organs.^15,16 As described by Cetinkaya et al., vitrification represents the most suitable process to preserve tissues from freeze damage.⁸ During our study comparing various media, it was noticed that vitrification was efficacious to maintain cell viability, but it was completely unable to preserve cell features. In particular, cells lost their growth and replication abilities. This finding does not recommend vitrification when cell culture freezing is necessary.

It is of paramount importance to determine an alternative cryopreservation solution suitable for cells, in particular for BMSCs.

The main outcome of this trial was the identification of a medium composed only by FBS and DMSO, as the most convenient instrument able to guarantee not only cell viability but also their replication. Moreover, this medium permitted cell growth even after thawing and seeding in culture plates.

It is the authors' opinion, as described by others, that FBS plays a central role as a buffer of osmotic pressure and as a cell membrane protector; in fact it reduces the risk of ice recrystallization damage during freezing and thawing.¹⁷ Moreover, DMSO is considered a remarkable tool, able to protect the structural integrity of cell membranes.¹⁸

MSCs are gaining a relevant role in clinical medical care according to their successful long-term preservation, with the maintenance not only of their viability, but also of their own features, such as differentiation capacity, playing a key role for the future approach in cell therapy. Accordingly, the identification of a nontoxic cryoprotectant agent, able to maintain high levels of cell viability, is a basic tool in the field of cell biology.

In conclusion, a future development will consider the evaluation of a greater number of samples for cryopreservation, in order to standardize the technique and the correct procedure, increasing the cell viability percentage and preserving original cell characteristics.

Footnotes

Acknowledgments

The authors thank Mrs. Annalisa Ghizzardi, Dr. Elisabetta Razzuoli, and Dr. Leonardo J. Vinco for their practical support and collaboration.

Author Disclosure Statement

This study was funded by Italian Ministry of Health (PRC2007011). No competing financial interests exist.

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