Isolation and cultivation of cells
The hCS-MSCs were isolated from cadaver eyes obtained as leftover material from Norwegian Eye Bank (following DSAEK operation). The processing and use of the human tissue are in accordance with the directives of the Helsinki Declaration and all tissue harvesting was approved by the Regional Committee for Medical and Health Research Ethics (REK 2017/418) in Norway. The hCS-MSCs isolation was performed following our own methodology described by Nagymihaly et al.59. Briefly, the corneal disc was separated from the corneal scleral ring, placed in a Petri dish with Dulbecco’s Phosphate Buffered Saline (DPBS) 1X, and the epithelial and Descemet’s membranes were removed by scratching under a stereomicroscope, rinsed and cut in approximately 10–12 pieces. The tissue pieces were then transferred to a microplate of 6 wells (Corning®, Axygen®, Merck/Sigma-Aldrich, MO, USA) placing 1 or 2 pieces in each well with 800 µL of Dulbecco’s modified eagle medium (DMEM) low glucose with Glutamax™ supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco®, Thermo Fisher Scientific, MA, USA) and 1% antibiotic/antimycotic solution (Merck/Sigma-Aldrich, MO, USA) (complete DMEM) and incubated at 37 °C 5% CO2 for 24 h. After the first day in culture, the wells were carefully filled up with 2–3 mL of pre-warmed complete DMEM, and the culture media was changed every 3–4 days. The adherent hCS-MSCs from the attached tissue pieces were trypsinized, filtered with a strainer with at least 70 µm pore size, and cryopreserved in liquid nitrogen at -196 °C until further use, approximately 25–30 days after the isolation day. The isolated hCS-MSCs were phenotypically characterized by flow cytometry using a Becton Dickinson (BD) FACS Canto II (BD biosciences, USA) and BD Stemflow Human MSC Analysis Kit (BD biosciences, USA) prior to the experiments.
The hCS-MSCs were cultured in complete DMEM or in Minimum Essential Medium (MEMα, Gibco®, Thermo Fisher Scientific, MA, USA) supplemented with 5% (pooled) human platelet lysate (HPL) (Cook Regentec, IN, USA), 1 mM sodium pyruvate (100 mM, 07555 A20, Thermo Fisher Scientific, MA, USA), 2 mM L-Glutamine (200 mM, Merck/ Sigma-Aldrich, MO, USA) and 5 μg/mL gentamycin (50 mg/mL, Merck/ Sigma-Aldrich, MO, USA) (complete MEM), and the media was changed every 2–3 days during cell expansion. The cells were then trypsinized by TrypLE Express Enzyme (Gibco®, Thermo Fisher Scientific, MA, USA) at approximately 80% confluency to avoid trans-differentiation and losing their MSC phenotype59.
Storage of hCS-MSCs by applying different cryoprotective agents
The hCS-MSCs were frozen using two different CPAs: standard 10% DMSO (Merck/Sigma-Aldrich, MO, USA) in complete DMEM or MEM, and glycerol (Merck/Sigma-Aldrich, MO, USA) at three different concentrations (10, 15, and 20%) in complete DMEM or MEM (see Table 1 for the experimental setup of the media used).
Three primary hCS-MSCs donors were counted with a Bürker chamber using Tryphan Blue to visualize the live cells and, subsequently, approximately 7.5·105–1·106 cells/mL were cryopreserved in media containing DMSO or glycerol. The hCS-MSCs cryopreserved in DMSO were gently mixed and immediately transferred into − 80 °C inside a freezing container (Mr. Frosty; 1 °C/min cooling rate), while the hCS-MSCs cryopreserved with glycerol were gently mixed in the cryovial and left on the bench for 20 min prior to transferring into − 80 °C inside the Mr. Frosty. All the cryovials were transferred after a few days into liquid nitrogen for at least two weeks storage.
Cell surface marker phenotype characterization of the hCS-MSCs
The immunophenotype characterization of the hCS-MSCs was carried out using a BD Accuri™ C6 personal flow cytometer (Becton Dickinson, NJ, USA). FITC, PE and APC- conjugated antibodies (Biolegend, CA, USA) were obtained, against cell surface- expressed proteins: CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD117, CD146, CD166, CD184 (Sup. Table 1).
Briefly, 1·105 dissociated cells were pipetted into individual 1.5 mL tubes (Corning® Axygen®, Merck/Sigma-Aldrich, MO, USA), rinsed and pelleted in cold fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin (Thermo Fisher Scientific, MA, USA) in DPBS (1X)). Antibody solutions (1:40) were added to the individual tubes with the cells, gently resuspended and incubated on ice for 30 min. Once completed, the hCS-MSCs were washed twice in cold FACS buffer, centrifuged (3 min, 500 RCF g, 4 °C) then resuspended in 0.2 mL buffer before measurement. The immunophenotype characterization was performed with all the primary hCS-MSCs cryopreserved with cryopreserved with 10% DMSO and 10% glycerol, in complete DMEM and MEM, respectively. The data are presented as mean ± standard deviation (SD) values.
Quantification of hCS-MSCs
The hCS-MSCs cryopreserved with the different CPAs were rapidly thawed in a water bath after 2–4 weeks in liquid nitrogen and counted using a Bürker chamber by Trypan blue to calculate the survival rate ((cells alive after thawing/cells alive before freezing) × 100). Subsequently, the live cells were transferred into 48 well plates to study the cell adhesion after 6 h incubation, and the proliferation assay from day 1 to day 4. At the end of each time-point, the hCS-MSCs were rinsed twice with DPBS (1X) and lysed by 300 μL of mammalian-protein extraction reagent (Thermo Fisher Scientific, MA, USA). Once all samples were collected, the relative number of attached cells was quantified by measuring the lactate dehydrogenase (LDH) activity using the Cytotoxicity Detection KitPLUS LDH (Roche Applied Science, Penzberg, Germany). The samples were measured spectrophotometrically at 492 nm using a Victor 3 microplate reader (PerkinElmer, Waltham, MA, USA), and the number of cells were calculated using the calibration curve prepared with known cell numbers. All the experimental conditions were performed in triplicate.
Human CS-MSCs trilineage differentiation
Human CS-MSCs cryopreserved with 10% glycerol were differentiated to three lineages using adipogenesis, chondrogenesis and osteogenesis kits (Gibco, Stem- Pro, Thermo Fisher Scientific, MA, USA). The kit media were replaced every 3–4 days for a period of 2–3 weeks in a 48-well plate. Once the differentiation period ended, the media were removed and the cells were fixated in 4% formalin solution. Alizarin Red S (Merck/Sigma-Aldrich), Alcian Blue solution (Merck/Sigma-Aldrich), and HCS LipidTOX (Invitrogen, Thermo Fisher Scientific, MA, USA) were used to stain fixated hCS-MSCs for osteogenesis, chondrogenesis, and adipogenesis, respectively. Images were taken using an EVOS FL fluorescent microscope or a Zeiss upright light microscope.
Cell surface covering, cell spreading and image analysis
In parallel to the cell adhesion and proliferation assays, 50 000 cells were placed in culture in T-25 flasks to quantify the surface coverage at days 1, 2, 3 and 4 through brightfield microscopy images. Moreover, 20 000 cells were seeded in duplicates in a 48 well plate, and after 6 h incubation, the attached hCS-MSCs were rinsed twice with DPBS (1X), fixed with 10% formalin for 10 min, washed 3 times, and the cells were stained with Vimentin (MA5-16,409, Rabbit Monoclonal, 1:200, Thermo Fisher Scientific, MA, USA) and DAPI (D1306, dilution 1:1000, Thermo Fisher Scientific, MA, USA). Images were taken by an EVOS FL fluorescent microscope (Thermo Fisher Scientific, MA, USA), and further analyzed using ImageJ 1.51w software (NIH, Bethesda, MD, USA) to determine cell area and surface coverage.
Statistical analysis
All the data are presented as mean values ± SD. ANOVA with multiple comparisons HSD-Tukey test was used to determine statistically significant differences (Graphpad Prism 9, GraphPad Software, Inc., San Diego, CA). Statistical significance was set at p-value < 0.05.
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- Source: https://www.nature.com/articles/s41598-024-65469-4