Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells

Accepted Manuscript Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the 1 cryopreservation of human adipose-derived stem cells Chuan Wang, Ran Xiao, Yi-Lin Cao, Hong-Yu Yin PII:

S0006-291X(17)31426-2

DOI:

10.1016/j.bbrc.2017.07.076

Reference:

YBBRC 38182

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 10 July 2017 Revised Date:

0006-291X June 0006-291X

Accepted Date: 13 July 2017

Please cite this article as: C. Wang, R. Xiao, Y.-L. Cao, H.-Y. Yin, Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem 1 cells , Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.07.076. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells1

a

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Chuan Wang a, Ran Xiao a, Yi-Lin Cao a,*, Hong-Yu Yin b,* Research Center of Plastic Surgery Hospital, Chinese Academy of Medical Sciences

and Peking Union Medical College, Beijing, China

Maxillo-facial Surgery Center of Plastic Surgery Hospital, Chinese Academy of

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b

*Corresponding author

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Medical Sciences and Peking Union Medical College, Beijing, China.

Hong-Yu Yin, [email protected], +86 151-0166-0204 Yi-Lin Cao, [email protected], +86 137-0180-2351

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33 Ba-Da-Chu Road, , Shi-Jing-Shan District, Beijing, China, 100144, Beijing, China.

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Abbreviations: ASCs, adipose-derived stem cells; FBS, fetal bovine serum; PL, platelet lysate; DMSO, dimethylsulfoxide; PVP, polyvinylpyrrolidine; GMP, good manufacturing practice; FDA, Food and Drug Administration; DMEM, Dulbecco's Modified Eagle's medium; FITC, fluorescein isothiocyanate; APC, allophycocyanin; MSCs, mesenchymal stem cells

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Abstract Cryopreservation provides an effective technique to maintain the functional properties

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of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can

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introduce risks of viral, prion, zoonose contaminations and evoke immune responses

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after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we

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determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and

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human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS

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and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Keywords adipose-derived stem cells, cryopreservation, cryoprotective agent, platelet lysate

ACCEPTED MANUSCRIPT 1. Introduction

Adipose-derived

stem

cells

(ASCs),

with

their

ability

of

self-renewal,

immunomodulatory properties, and directed differentiation into mature cell types,

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offer unique opportunities in regenerative medicine and tissue engineering applications [1, 2]. However, the clinical application of ASCs requires a large number

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of cells. Unfortunately, in vitro expansion of ASCs does not yield sufficient cell numbers in a short duration. Therefore, wide application of ASCs requires the

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development of safe and efficient protocols for stem cell banking [3]. Cryopreservation can be an ideal choice for banking stem cells in general and the only approach for preserve ASCs while maintaining their viability and functional properties in the long term. The cryopreservation protocols for most cells including

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ASCs use 10% dimethyl sulfoxide (DMSO) in combination with fetal bovine serum (FBS) [4, 5]. Although DMSO is regarded as relatively nontoxic, the clinical use of

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cryopreserved cells treated with DMSO can produce many adverse effects and toxic reactions [6-8]. To replace DMSO, polyvinylpyrrolidine (PVP) and methylcellulose

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have been used as cryoprotectants [9]. However, they are less efficient than DMSO in terms of sustaining ASC viability [10]. Therefore, it is essential to develop cryopreservation protocols either with lower concentrations of DMSO or with nontoxic alternatives to DMSO. ASCs are typically cultured or cryopreserved in the presence of FBS, which provides important nutrients and growth factors for cell growth and reduces the formation of extracellular ice, stabilizes cell membrane, prevents excessive concentration of solutes,

ACCEPTED MANUSCRIPT and minimizes cell dehydration to a tolerable degree. However, due to its xenogeneic origin, the use of FBS introduces the risk of viral, prion, or zoonose contaminations and may cause immune responses after injection [11]. From the point of view of

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regenerative medicine, FBS is required for the adaptation of the cell manufacturing process towards clinical grade Good Manufacturing Practice (cGMP) standards. However, the U. S. Food and Drug Administration (FDA) has established strict

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guidelines against the use of xenoproducts [12], making it necessary to exclude the

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use of FBS from all stages of cell production, including expansion and cryopreservation.

Currently, human platelet lysate (PL) represents the most promising natural alternative to FBS in supporting mesenchymal stem cells (MSC) expansion and

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maintaining their functional properties [13, 14]. PL is generated from ordinary platelet units by a simple freeze-thaw procedure and contains a variety of growth factors, enzymes, and proteins to support cell attachment, growth, and proliferation [13, 15].

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Different aspects of PL production, safety, quality control, and activity in vitro and in

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vivo have been widely reviewed [16, 17]. It has also been conceivably documented that ASCs retain their capacity for multipotential differentiation during and after expansion in medium supplemented with PL [18]. However, little is known about the effect of PL as a cryoprotectant on the susceptibility of ASCs to cryopreservation. Therefore, we performed a quantitative evaluation to compare the effects of various combinations of cryoprotectants (DMSO, PL, and FBS) on human ASCs in term of cell viability, proliferation potential, phenotype, and differentiation potential. Based

ACCEPTED MANUSCRIPT on the comparison, we determined the ideal cryoprotectant for use in efficient human ASC preservation. The findings from this study would affect the establishment of standardized cryopreservation protocols for human ASCs for future clinical

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applications.

2. Materials and Methods

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2.1 Culturing of human ASCs

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Adipose tissue was obtained by liposuction from 3 patients (age range: 24–36 years; average: 29.3 years). All human tissue collection was approved by the Ethical Committee of Plastic Surgery Hospital and was carried out in accordance with the approved guidelines. Tissue samples (200–300 mL) were washed 2–3 times in

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phosphate-buffered saline (PBS) and centrifuged for 5 min at 350 g at room temperature. The middle layer of fat granule was supplemented with 0.075% Type I collagenase (Sigma, USA) and digested with 200 rpm rocking for 40–60 min at 37°C.

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The digests were then again centrifuged for 5 min at 350 g at room temperature. Next,

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the supernatant was aspirated, and the pellet was resuspended in PBS and passed through an 80-µm cell strainer. Thereafter, the filtrate was centrifuged for 5 min at 350 g at room temperature and the pellet was resuspended in Mesenchymal Stem Cell Medium (Sciencell, USA) in a 10-cm diameter petri dish. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Fresh cells were expanded until passage 3 and some of them were cryopreserved at the end of passage 2.

ACCEPTED MANUSCRIPT 2.2 Cryopreservation About 1 × 106 ASCs at passage 2 were suspended in 1 mL of the cryoprotective agents (CPAs) and loaded into a cryovial. The CPAs were combinations of DMSO

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(Sigma, USA), FBS (Gibco, USA), and PL (AventaCell BioMedical, USA) at different concentrations as follows: 1, 100% FBS; 2, 99% FBS + 1% DMSO; 3, 98% FBS + 2% DMSO; 4, 97% FBS + 3% DMSO; 5, 96% FBS + 4% DMSO; 6, 95% FBS

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+ 5% DMSO; 7, 94% FBS + 6% DMSO; 8, 93% FBS + 7% DMSO; 9, 92% FBS + 8%

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DMSO; 10, 91% FBS + 9% DMSO; 11, 90% FBS + 10% DMSO; 12, 100% PL; 13, 99% PL + 1% DMSO; 14, 98% PL + 2% DMSO; 15, 97% PL + 3% DMSO; 16, 96% PL + 4% DMSO; 17, 95% PL + 5% DMSO; 18, 94% PL + 6% DMSO; 19, 93% PL + 7% DMSO; 20, 92% PL + 8% DMSO; 21, 91% PL + 9% DMSO; and 22, 90% PL +

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10% DMSO. All the cryovials were cryopreserved at a cooling rate of 1 degree/min down to -80°C overnight and then transferred to liquid nitrogen (-196°C) the next day. After more than 1 month of cryopreservation, the cells were thawed rapidly at 37°C,

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their viability was evaluated, and the cells were subcultured to passage 3 to assess

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their proliferation, phenotype, and differentiation properties.

2.3 Cell viability assays The viability of both fresh and cryopreserved ASCs was determined using the Muse™ Cell Analyzer with the Muse™ Count & Viability Reagent (EMD Millipore Corporation, USA). Briefly, roughly 1 × 106 cryopreserved cells were thawed rapidly at 37°C, centrifuged for 5 min at 350 g, and resuspended in 1000 µL low-glucose

ACCEPTED MANUSCRIPT Dulbecco's Modified Eagle's medium (DMEM, HyClone, USA). About 1 × 106 fresh cells were resuspended in 1000 µL low-glucose DMEM as well. Approximately 20 µL of each cell suspension was then mixed with 380 µL of the Muse™ Count & Viability

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Reagent and incubated in the dark at room temperature for 5 min. The cells were then analyzed on the Muse™ Cell Analyzer, and the cell viability preserved in various

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CPAs was determined.

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2.4 Cell proliferation assay

The fresh ASCs and the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO at passage 3 were seeded into 96-well plates (Corning Inc) at a density of 2.5 × 103 cells/well and incubated in

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low-glucose DMEM plus 10% FBS. Cell Counting kit-8 (DOJINDO, Japan) was then used after 4 hours to determine cell attachment on days 0 (0 hours), 1 (24 hours), 3, 5,

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and 7.

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2.5 Cell phenotyping

The fresh ASCs and the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO at passage 3 were stained with monoclonal antibodies (Biolegend, USA) with either fluorescein isothiocyanate (FITC) or allophycocyanin (APC) fluorochromes before flow cytometry analysis. These antibodies included CD44 (FITC), CD73 (FITC), CD90 (FITC), CD105 (APC), CD14 (FITC), CD19 (FITC), CD34 (FITC), CD45 (FITC), and HLA-DR (FITC). The

ACCEPTED MANUSCRIPT cells were then resuspended in PBS and centrifuged at 350 g for 10 min. Next, 5 µL of monoclonal antibodies was added to each sample containing 50 µL of cell suspension in PBS. After 30 min of incubation at room temperature in the dark, the

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cells were washed twice with PBS and analyzed using flow cytometry (FACS) (BD FACS Aria II, USA). Each measurement was conducted on a single cell population using a minimum of 10000 events per sample. Data analysis was conducted using

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Flow Jo 7.6.2 software.

2.6 Assays for determining cell differentiation potential

Differentiation was induced in the fresh ASCs and in ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO at

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passage 3. To induce adipogenic and osteogenic differentiation, the ASCs were grown to 80% confluency in 6-well plates (Corning Inc), and the culture medium was then replaced with adipogenic differentiation medium (high-glucose DMEM containing 10%

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FBS, 100 U/mL penicillin, 0.1 g/mL streptomycin, 0.25 mM IBMX, 5 µM

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rosiglitazone, 1 µM dexamethasone, 66 µM biotin, 34 µM D-pantothenate, and 200 nM human insulin) and osteogenic differentiation medium (Cyagen Biosciences Inc, USA), respectively. To induce chondrogenesis, 1 × 106 ASCs in pellet form were cultured in chondrogenic induction medium (Cyagen Biosciences Inc, USA). After 7 days in adipogenic induction medium, 14 days in osteogenic induction medium, and 28 days in chondrogenic induction medium, the cells were stained with Oil Red O, Alizarin Red, and Alcian Blue, respectively.

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2.7 Statistical analysis All results are presented as mean ± standard deviation (SD). Statistical analysis was

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performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). Student’s t-test and analysis of variance (ANOVA) were used to analyze all the results.

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P < 0.05 was considered statistically significant.

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3. Results 3.1 Effects of cryopreservation on ASC viability

In this set of experiments, the fresh and post-thaw viability of the ASCs were summarized in Figure 1. In both the FBS and PL groups, the viability of ASCs

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improved with an increase in DMSO concentration. In the absence of DMSO protection, ASC activity was 14.3 ± 1.57% in the 100% FBS group and 18.43 ± 2.32% in the 100% PL group. When the DMSO concentration was increased to 1%, the

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activity of ASCs also significantly increased (55 ± 2.42% in 99% FBS and 1% DMSO

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group; 64.46 ± 2.08% in 99% PL and 1% DMSO group). The cell activity in the 95% FBS and 5% DMSO group reached 86.57 ± 5.07%, which was similar to that of the fresh ASC group (95.6 ± 0.41%). In the 97% PL and 3% DMSO group, ASC viability was 85.2 ± 2.69%, which was also similar to that of the fresh control group (93.87 ± 2.11%). Moreover, the cell survival rates of the traditional 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO groups were similar.

ACCEPTED MANUSCRIPT 3.2 Comparison of the proliferation rate between cryopreserved and fresh ASCs We also determined cellular proliferation rate and found that the cryopreserved and fresh ASCs displayed a similar absorbance in cultures from day 0 to day 7 (Figure 2).

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The ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO,

3.3 Effects of cryopreservation on ASC phenotype

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and 97% PL and 3% DMSO showed a proliferation rate similar to that of fresh ASCs.

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Flow cytometry analysis revealed that the fresh and cryopreserved ASCs were positive for CD44, CD73, CD90, and CD105 and negative for CD14, CD19, CD34, CD45, and HLA (figure 3). The ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO expressed similar patterns of

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cell surface marker expression as the fresh ASCs.

3.4 Effects of cryopreservation on ASC differentiation potential

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The fresh and cryopreserved ASCs were induced to differentiate into adipocytes,

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osteocytes, and chondrocytes, and the representative results are shown in Figure 4. To assess the adipogenic, osteogenic, and chondrogenic differentiation potential of the ASCs, the fresh ASCs and ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO were stained with Oil red O, Alizarin Red, and Alcian blue after induction. All the cells were clearly inducible to the specified phenotypes. Oil red O staining evidenced adipogenic differentiation by red deposits in vacuoles. Alizarin Red staining showed the formation of red calcium

ACCEPTED MANUSCRIPT deposits as a marker of osteogenic differentiation. Pellet sections of chondro-induced samples stained with Alcian Blue showed a strong blue signal. The fresh ASCs and ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and

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97% PL and 3% DMSO were all inducible for differentiation into adipocytes, osteocytes, and chondrocytes.

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4. Discussion

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Cryopreservation can provide a large number of available cells for further experimental or clinical application [5]. Conventional cryopreservation protocols using 10% DMSO and FBS ensure high cell viability for some research purposes after thawing, but limit the therapeutic applicability of the cells by introducing the risk of

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undesirable side effects [19-21]. Clinical-grade cryobanking of stem cells preferentially requires defined xeno-free cryopreservation medium to avoid the risks of transmitting infectious diseases or prions or exposure to antigens that may induce

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immunological reactions [22, 23]. Although FBS can provide important nutrients and

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growth factors for cell growth and reduce the formation of extracellular ice, stabilize the cell membrane, prevent excessive concentration of solutes, and minimize cell dehydration to a tolerable degree, it introduces the risk of viral, prion, zoonose contaminations and may evoke immune responses after injection [11]. Thus, it is important to exclude the use of FBS from all stages of cell production, including expansion and cryopreservation. Human PL prepared by various release strategies has been established as a suitable alternative to FBS as culture medium supplement,

ACCEPTED MANUSCRIPT enabling efficient propagation of human cells under animal serum-free conditions for multiple applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet-derived products for inducing

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human cell proliferation and differentiation has also uncovered a considerable variability in human platelet lysate preparations, which limits the comparability of results [13-16]. So far, the effect of PL as a cryoprotectant on the susceptibility of

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ASCs to the cryopreservation process has not been extensively studied.

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Additionally, numerous strategies aimed to completely remove DMSO from cryopreservation solutions have been already published. However, a large number of cells die without DMSO protection during cryopreservation, and some other cryoprotectants such as PVP and methylcellulose are less efficient than DMSO for

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sustaining ASC viability [10].

Several recent reports have suggested that cells can be successfully cryopreserved with minimal concentrations of DMSO [8, 24-29]. For example, Liseth et al [28]

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concluded that the post-thaw survival for CD34+ cells was almost identical when

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frozen with either 4% or 5% DMSO but further lowering the concentration of DMSO to 2% had an adverse effect on post-thaw cell survival. Sreedhar et al [30] suggests that post-thaw ASC viability and adipogenic and osteogenic differentiability can be maintained even when they are frozen in a minimal concentration of 2% DMSO. Kar Wey Yong et al

[31] suggested that 5% DMSO is an ideal CPA for efficient

long-term cryopreservation of human ASCs. Thus, we felt it important to determine the most suitable DMSO concentration for use in ASCs cryopreservation. In this study,

ACCEPTED MANUSCRIPT we examined the activity of fresh ASCs and ASCs cryopreserved with different combinations of the CPAs, DMSO, FBS, and PL. In the FBS and PL groups, the viability of the ASCs improved with increasing DMSO concentration. In the absence

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of DMSO protection, ASC activity was very low; in contrast, increase in DMSO concentration to 1% significantly increased the activity of ASCs. This result indicated that DMSO plays a very important role in the cryopreservation of ASCs. We also

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found that ASC viability with 95% FBS and 5% DMSO was similar to that of the

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fresh ASCs. In the PL groups, the use of 3% DMSO could achieve the effect of 5% DMSO concentration in the FBS group. There was also no significant difference in the viability of the ASCs in the 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO groups. Therefore, we concluded that the combination of

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97% PL and 3% DMSO showed the best potential for cryopreserving ASCs for clinical applications. We also determined ASC proliferation rates among the different groups and found that the fresh ASCs and ASCs cryopreserved with 90% FBS and 10%

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DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO showed similar

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proliferation rates in cultures from day 0 to day 7. This result suggested that the use of a combination of 97% PL and 3% DMSO as a cryoprotectant can also maintain the proliferation rate of the cryopreserved ASCs. Further, to determine the effect of cryopreservation on ASC phenotype, we conducted flow cytometry analysis. The results showed that the fresh ASCs and ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO were positive for CD44, CD73, CD90, and CD105 and negative

ACCEPTED MANUSCRIPT for CD14, CD19, CD34, CD45, and HLA. These results indicated that the cryopreserved ASCs expressed a similar pattern of cell surface markers as the fresh ASCs, which is in accordance with previous results reported by Gonda et al. [32]. In

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addition, according to Dominici et al. [33], ASCs possess the characteristics of MSCs and therefore express mesenchymal-associated markers (CD90, CD105, and CD73) but do not express hematopoietic-associated markers (CD14, CD19, CD34, CD45,

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were not affected by the cryopreservation process.

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and HLA DRDPDQ). Taken together, these data suggest that the ASC phenotypes

Moreover, the results of the present study showed that the cryopreserved ASCs maintained their capabilities of differentiating into adipocytes, osteocytes, and chondrocytes, thus meeting the minimum criteria for characterizing human MSCs, as

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suggested by Dominici et al. [33]. Taken together, our findings indicate that cryopreserved ASCs, particularly ASCs preserved in 97% PL and 3% DMSO, can be potentially used in various clinical applications such as bone and cartilage

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regeneration due to their ability to maintain normal viability, proliferation, and

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differentiation potential. Our results thus highlight the potential for the combination of 97% PL and 3% DMSO for use as an ideal CPA for ASC cryopreservation. Conflict of interest

The listed authors have no conflict of interests. Acknowledgments This work was supported by the Foundation of Special Fund for Scientific Research in the Public Interest (201502016).

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Figure legends

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Figure 1. Effect of cryopreservation on ASC viability. (A) Cell viability of fresh ASCs and ASCs cryopreserved in 0–10% DMSO + 90–100% FBS. (B) Cell viability of fresh ASCs and ASCs cryopreserved in 0–10% DMSO + 90–100% PL. (*** P <

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0.001)

Figure 2. Effect of cryopreservation on ASC proliferation. Fresh and cryopreserved ASCs displayed similar absorbance in cultures from day 0 to day 7, thus indicating

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that the proliferative potential of the ASCs was maintained after cryopreservation.

Figure 3. Fresh and cryopreserved ASCs highly expressed positive markers (CD44, CD73, CD90, and CD105) but did not express hematopoietic lineage markers (CD14, CD19, CD34, CD45, and HLA-DR).

Figure 4. Differentiation assay of fresh and thawed ASCs. Adipogenic cultures contain consistent red deposits in vacuoles (first row, Oil Red O staining). Osteogenic

ACCEPTED MANUSCRIPT induction can be observed by the formation of red calcium deposits (second row, Alizarin Red staining). Chondrocyte induction is shown as robust fiber tracts of collagen matrix (third row, Alcian Blue staining). Images in column 1 represent fresh

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cells. Images in column 2 represent cells that were cryopreserved in media containing 10% DMSO and 90% FBS. Images in column 3 represent cells that were cryopreserved in media containing 5% DMSO and 95% FBS. Images in column 4

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represent cells that were cryopreserved in media containing 3% DMSO and 97% PL.

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Figure1

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Figure2

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Figure3

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Figure4

ACCEPTED MANUSCRIPT Highlights 95% FBS with 5% DMSO can cryopreserve ASCs efficiently




97% PL with 3% DMSO can cryopreserve ASCs efficiently




PL can replace FBS as an ideal cryoprotective agent to cryopreserve ASCs




PL can preserve ASC viability, proliferation, phenotype, and differentiation

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