The Combination of Trehalose and Glycerin: An Effective and Non-Toxic Recipe for Clinical Cryopreservation of Human Adipose-Derived Stem Cells

Background: Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPA) protect cells from cryodamage in the process of cryopreservation. Safe and ecient cryopreservation of ADSCs is critical in the clinical application of cell-based therapy. However, most CPAs contain toxic concentrations limiting the possibility of their clinical application. Objective: The aim of this study is to develop a non-toxic xeno-free CPA for ADSCs to achieve high-eciency and low-risk cryopreservation. Methods: We explored the most ecient concentrations in different concentrations of trehalose (0.3M, 0.6M, 1.0M, and 1.25M) and glycerol (10%, 20%, 30% v/v); then evaluated the outcome of the combination of trehalose and glycerol in ADSC cryopreservation, compared to the commonly used CPA, DMSO (10%) + FBS (90%). All samples were slowly freezed and stored in liquid nitrox for 30 days. The effectiveness was evaluated by the cell viability, proliferation, migration and multi-potential differentiation of ADSCs after thawing. Results: Compared to the CPAs with single reagent, 1.0M Tre + 20%Gly group showed signicantly higher eciency in preserving ADSCs activities after thawing, with better outcome in both cell viability and proliferating capacity. Compared to 10%DMSO+90%FBS, ADSCs preserved in 1.0M Tre + 20%Gly group showed similar cell viability, surface markers and multi-potential differentiation but signicantly higher migration capability, indicating that better cell function preservation can be achieved by 1.0M Tre + 20%Gly. Conclusions: 1.0M Tre + 20%Gly can preserve ADSCs with high migration capability and cell viability compared to 10%DMSO+90%FBS and maintain similar stemness and multi-potential differentiation as fresh cells. Our results demonstrate that 1.0M Tre + 20%Gly can achieve highly ecient cryopreservation of ADSCs and is suitable for clinical applications.

e ciency in preserving ADSCs activities after thawing, with better outcome in both cell viability and proliferating capacity. Compared to 10%DMSO+90%FBS, ADSCs preserved in 1.0M Tre + 20%Gly group showed similar cell viability, surface markers and multi-potential differentiation but signi cantly higher migration capability, indicating that better cell function preservation can be achieved by 1.0M Tre + 20%Gly.
Conclusions: 1.0M Tre + 20%Gly can preserve ADSCs with high migration capability and cell viability compared to 10%DMSO+90%FBS and maintain similar stemness and multi-potential differentiation as fresh cells. Our results demonstrate that 1.0M Tre + 20%Gly can achieve highly e cient cryopreservation of ADSCs and is suitable for clinical applications.

Background
Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs) obtained from adipose tissue, which have the self-renew and potential plasticity characteristics [1]. Various studies demonstrated that ADSCs hold great promise for therapeutic use in tissue bioengineering and regenerative medicine due to immunomodulation, anti-in ammatory and angiogenesis properties [2]. However, its tissue acquisition procedure is surgical, which brings the pain and economic burden to the patients. Moreover, the number and the quality of ADSCs reduces with age. It would be more exible and acceptable for ADSC therapy if we can harvest cells from a single surgical procedure at a young age and stored for future use. Therefore, the request for long term ADSC preservation is rapidly surging.
Cryopreservation is a common method to achieve long-term preservation of cells and tissues. Studies have shown that the addition of cryoprotective agent (CPA) avoids damaging effect of intracellular crystallization during freezing and thawing. The most widely used CPA is dimethyl sulfoxide (DMSO) combined with fetal bovine serum (FBS) [3], however, it is not suitable for clinical application due to the toxicity of DMSO and the risk of zoonotic infection induced by FBS [4]. A highly e cient, non-toxic and xeno-free CPA is required for clinical application.
Saccharides are a serial of nontoxic and biodegradable reagents that can be used as cryoprotective agents [5]. As a non-permeable disaccharide, trehalose forms a unique protective lm on the cell surface under severe environmental conditions, such as high temperature, alpine, high osmotic pressure and desiccation [6]. Trehalose has a high glass transition temperature, which can eliminate the formation of intra-and extracellular ice crystals and prevent osmotic shock from ultra-rapid cooling, thereby reducing damage to living cells. However, the use of trehalose in mammalian cells has been limited because these cell membranes are almost impermeable to disaccharides [7][8]. To circumvent this obstacle, various experimental approaches have been tested to improve the e ciency of trehalose [9][10], but still little has been approached in the clinical applications.
Glycerol is a permeable CPA, which can stabilize the cell membrane and improve the viscosity of water inside and outside the cell [12][13]. We hypothesized that the combination of glycerol and trehalose can be more e cient in protecting cells from cryodamage and maintaining cell viability and may be a more e cient formula for clinical cryopreservation of ADSCs. In our study, we rst selected the most optimum concentration of trehalose and glycerol when applied along and then evaluated the effect of the combination of trehalose and glycerol in preserving ADSCs' viability and cell function.

Adipose tissue obtained
Abdominal subcutaneous adipose tissues were obtained from 10 healthy donors, age range 40-45 years, who underwent abdominal liposuction. The donors all provided informed consent. This study was approved by the Ethics Committee of Shanghai Ninth People's Hospital and complied with the principles of the Declaration of Helsinki.

Preparation of CPA
For the preparation of CPA, we used the following methods: (i)Trehalose(Tre) group: Trehalose powder (Solarbio, China) was diluted with phosphate buffer saline (PBS) for powder reconstitution, according to the required concentrations, as 0.3M Tre, 0.6M Tre, 1.0 M Tre, and 1.25M Tre, and ltered using a 0.22um lter; (ii)glycerin group (Gly):Glycerin (Hercules, Bio-Rad Laboratories, CA,USA) diluted with PBS to yield the required concentration, as 10%Gly, 20%Gly, 30% Gly; (iii) trehalose and glycerin group: 1.0MTre + 20%Gly: as previously described, trehalose powder was diluted with PBS and then diluted with glycerol according to our needs; (iv)10% DMSO(Sigma-Aldrich, Santa Clara, CA, USA)+ 90% FBS, as positive control; (v) control group: only PBS, without trehalose and glycerin, as negative control.

Cell Cryopreservation and thawing
Approximately 1×10 6 cells were resuspended in 1ml CPAs, and transfer into cryovials (Thermo Fisher, Waltham, MA, USA). The cryovials were frozen in a Nalgene® Mr. Frosty freezing container (Thermo Fisher, Waltham, MA, USA) at a cooling rate of 1 °C /min to -80°C, stored overnight, and transferred into liquid nitrogen for 30 days storing. For thawing, cryovials were placed in a water bath at 37˚C with gentle shake until the ice was completely melted. Thawed ADSCs were rinsed with 10 ml PBS for 2 times and resuspended in 5ml Dulbecco's Modi ed Eagle's Medium (DMEM)/F12 containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin for further assessment.

Assessment of Cell Viability
Trypan blue (Thermo Fisher, Waltham) staining was used to evaluate the cell viability. One ml of resuspended ADSCs was mixed with equal volume of trypan blue and counted by automated cell counter (Thermo Fisher, Waltham, USA).

Assessment of Cell Proliferation
A cell counting kit-8 assay (Beyotime Biotechnology Company, Shanghai, China) was used to assess cell proliferation. Brie y, ADSCs were plated onto 96-well plates (5000 cells/well) and cultured for 24h,48h and 72h. 10ul CCK-8 solution was added to culture medium, and incubated at 37 ℃ for 4 h. The colorimetric was measured at 450 nm with a microplate reader to obtain an optical density (OD) value that represented as ADSCs proliferation.

Assessment of Cell Morphology
The morphology of ADSCs (5×10 5 cells/ml) of different CPAs group was estimated before cryopreservation and 72 h after post-thaw culturing by microscope.

Assessment of Cell Migration
Post-thaw ADSCs were plated in 6-well plates (5×10 5 cells/well) and incubated at 37℃ until 90% con uence. Next, cells were switched in medium without serum and used a 200ul pipette tip to create a scratch of the cell monolayer and removed debris and smoothed the edge of the scratch by PBS. Cells were photographed immediately (0 h), 12 h and 24 h later. The level of migration was assessed by the ratio of the closure area to the initial wound area: migration area (%) = (A0-An)/A0×100, where A0 represents the area of initial wound area and An represents the residual area of the wound at the end point (t=n h). Data analysis was performed with Image J software.

Assessment of Multi-potential Differentiation
The multi-potential differentiation of ADSCs was assessed based on their differentiation into adipogenic, osteogenic, and chondrogenic cells by using a Human Adipose-derived Stem Cell Adipogenic Differentiation kit, Osteogenesis Differentiation kit and Chondrogenic Differentiation kit (all from Oricell, Cyagen) according to the manufactural protocols [15].

Statistical Analysis
All data were collected from at least three independent replications. Numeric data are presented as the mean ± standard deviation (SD). Statistical analyses were performed by GraphPad Prism8 software (version 6.01 software). Group differences was analyzed using a bilateral student's t-test or one-way analysis of variance(ANOVA), and differences were considered signi cant at p < 0.05. (63±0.65%) alone showed signi cantly higher than other concentration (Figure 1). We tried to combine 1.0M Tre and 20% Gly to cryopreserve ADSCs. The results of the viability of post-thaw ADSCs preserved in 1.0M Tre + 20%Gly (77±1.72%) showed no difference to that of 10% DMSO+90%FBS(75±0.37%, p=0.2583), signi cantly higher than that of cells preserved in 1.0M Tre alone (p 0.05) and 20%Gly (p 0.01) alone.
3.2 Effect of CPA on the proliferation of ADSCs after cryopreservation: The CPA of 1.0M Tre + 20%Gly signi cantly improves the proliferation of post-thaw ADSCs.
3.3 Effect of CPA on the morphology of ADSCs after cryopreservation: ADSCs were cryopreserved in 1.0M Tre + 20%Gly exhibit a similar morphology compared with fresh cells.
The morphology of ADSCs preserved with 1.0M Tre + 20%Gly, 10%DMSO + 90%FBS was documented after 72-hours culturation, compared to that of fresh ADSCs. The results showed that there was no visible difference in the shape and cell density of these three groups (Figure 3).
3.4 Effect of CPA on the migration of ADSCs after cryopreservation: The CPA of 1.0M Tre + 20%Gly signi cantly improves the migration of post-thaw ADSCs.
3.5 Effect of CPA on surface markers of ADSCs after cryopreservation: Cells cryopreserved in 1.0M Tre + 20%Glyexpress the same surface markers as fresh cells.
3.6 Effect of CPAs on the multi-lineage differentiation potential of ADSCs after cryopreservation: Cells cryopreserved in 1.0M Tre + 20%Glyexhibit the same multi-lineage differentiation potential as fresh cells.

Page 8/17
Adipose-derived stem cells (ADSCs), capable of self-renewal and multi-lineage differentiation, are considered as an ideal kind of stem cells for regenerative medicine [19][20]. Various studies showed that ADSCs have multiple functions in clinical applications, such as cartilage and bone repair, skin wound healing, neuronal regeneration, heart regeneration, and immune disorder treatment [21][22]. Long-term preservation of ADSCs is crucial for clinical application of cell-based therapy. Cryopreservation is the commonly used in storing cells, but the cryodamage during the process of freezing and thawing is a threaten to cell viability [23]. CPAs help to minimize the formation of ice crystals and other cryodamage by reducing the crystallization process of water and increasing the viscosity of the solution [24][25][26]. At present, the most widely used CPA is DMSO combined with FBS. However, it is not suitable for clinical application due to toxicity of DMSO and the risk of zoonotic infection induced by FBS [27][28][29][30]. Thus, the main purpose of current researches is to develop a non-toxic xeno-free CPA to t the requirement of clinical application.
Recently, there have been many researches on new CPA for ADSCs, however, with limitations. The current research mainly involves two categories. One is to reduce the concentration of DMSO. Shu et al reported that using 0.5M DMSO combined with 0.2M trehalose achieved high e ciency for cryopreservation of ADSCs [31]. López et al reported 3.5%DMSO + 3.5%EG + 0.25M trehalose + 2% PVA + 5% coll + 0.1 mM EGTA had better outcome in cell viability preservation compared to 10%DMSO + 90%FBS [32]. However, these recipes still contain DMSO, which was not suitable for clinical application.
The other way is to replace DMSO with other non-toxic reagents. Trehalose and glycerol are nontoxic and economic reagents for cryopreservation [34][35][36][37]. However, mammalian plasma membrane is nonpermeable to trehalose, which limits the e ciency of trehalose [38][39]. These reagents, when used separately, cannot reach the same outcome as 10%DMSO + 90%FBS [40]. Though some several solutions have been reported to increase the trehalose permeable [33], they are complex and di cult for clinical translation.
In this research, we evaluated the e ciency of the combination of trehalose and glycerol. The results demonstrated that the viability of ADSCs preserved with the combination of 1.0M Tre and 20%Gly reached 77%, signi cantly higher than that with either agent alone. Viability and proliferation rates of cell preserved with 1.0M Tre and 20%Gly were similar as cells preserved with 10%DMSO + 90%FBS. The migration of ADSCs with 1.0M Tre and 20%Gly was signi cantly higher than that with 10%DMSO + 90%FBS, indicating that the combination of 1.0M Tre and 20%Gly had better effect in preserving cell functions.
Glycerol and trehalose, as one permeable and one non-permeable CPA respectively, provide protection via different mechanisms. When combined together, these two reagents could complete each other and provide better protection. Moreover, glycerol may help trehalose penetrate the cell membrane, improving the e ciency of trehalose.
Glycerol is a permeable CPA that can reduce the water crystallization process, inhibit the growth of ice crystal and reduce damage to the cell structure and function. Cell viability was reduced in cells cryopreserved in glycerin alone compared with that of cells cryopreserved in 10%DMSO + 90%FBS. However, when glycerin was combined with trehalose, the cryopreservation e ciency was signi cantly improved. The increased e ciency was attributed to the fact that glycerol itself functions as a CPA. We also speculated that glycerol may help trehalose penetrate the cell membrane, improving the e ciency of trehalose.
Previous studies have shown that the damage was mainly related to the process of freezing. The duration of cryopreservation has minor in uence on cell viability [41]. Martinetti et al showed reported that there was no difference in the viability of CD34 + cells cryopreserved for 20 days and for 3 months.
Thus, in this research, we evaluated the outcome at 30 days of preservation.

Conclusion
The combination of trehalose and glycose can achieve higher e ciency in ADSC cryopreservation than single reagent CPAs. ADSCs preserved with 1.0M Tre + 20%Gly showed high cells viability, proliferation, migration, and multi-potential differentiation. As a non-toxic xeno-free CPA, 1.0M Tre + 20%Gly may have a promising clinical application prospect.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics declarations
Ethics approval and consent to participate The donors of abdominal subcutaneous adipose tissues all provided informed consent. This study was approved by the Ethics Committee of Shanghai Ninth People's Hospital and complied with the principles of the Declaration of Helsinki. Consent to participate is not applicable.

Consent for publication
Not applicable Effect of CPAs on the proliferation ability of post-thaw ADSCs. The proliferation of post-thaw ADSCs preserved in 1.0M Tre + 20%Gly showed no difference to that of 10%DMSO+90%FBS, signi cantly higher than 1.0M Tre and 20%Gly alone. Values are expressed as the mean ± SD; N=10. *p<0.05; **p<0.01; **p<0.001; ns, no signi cant difference.   Expression of the surface markers after cryopreservation with different CPAs. The CPA of 1.0M Tre + 20%Gly can maintain the surface markers of ADSCs.