2.1 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.
2.2 ADSCs Isolation and Culture
ADSCs were obtained by collagenase digestion as previously described [14]. Isolated ADSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 containing 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin (GE Healthcare Life Sciences, Freiburg, Germany), and incubated at 37°C in an atmosphere containing 5% CO2 and 95% humidity. Every 2–3 days to change the complete medium changes. On reaching 80% confluence, the cells were passaged by trypsin-EDTA (Sigma, St. Louis, MO) and subcultured for three passages(P3).
2.3 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 filtered using a 0.22um filter; (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.
2.4 Cell Cryopreservation and thawing
Approximately 1×106 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 Modified Eagle's Medium (DMEM)/F12 containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin for further assessment.
2.5 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).
2.6 Assessment of Cell Proliferation
A cell counting kit-8 assay (Beyotime Biotechnology Company, Shanghai, China) was used to assess cell proliferation. Briefly, 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.
2.7 Assessment of Cell Morphology
The morphology of ADSCs (5×105cells/ml) of different CPAs group was estimated before cryopreservation and 72 h after post-thaw culturing by microscope.
2.8 Assessment of Cell Migration
Post-thaw ADSCs were plated in 6-well plates (5×105 cells/well) and incubated at 37℃ until 90% confluence. 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.
2.9 Assessment of Cell Markers
For flow cytometric analysis, post-thaw P3 ADSCs and fresh P3 ADSCs were incubated with monoclonal antibodies for CD44 (PE, clone555479; BD Biosciences, USA), CD29 (PE, clone555443; BD Biosciences), CD34(PE, clone555822; BD Biosciences) and CD31(PE, clone555446; BD Biosciences). Unstained cells to exclude autofluorescence and to control the intensity of background. The cells were subsequently washed with PBS and analysed on a FACS Aria-flow cytometer (Becton‐Dickinson, San Jose, CA).
2.10 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].
2.11 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 significant at p < 0.05.