Preparation and storage of fresh human platelet lysate (HPL), lyophilized platelet lysate (LPL) and 2-year storage counterpart.
Outdated pooled leukocyte-poor platelet concentrate (LPPC) units were collected from the hospital blood bank. Processes were approved by Mahidol University central institutional review board (MU-CIRB2020/180.2307). LPPC was firstly prepared for fresh HPL (fHPL) by freeze-thaw procedure for 2 cycles. LPPCs were frozen at 80°C and thawed at 37°C in a water bath. Clot formation was activated by adding 2M calcium chloride (CaCl2, Merck, Germany), the coagulated product was centrifuged at 4,800 rpm for 30 minutes and removed. The supernatant was collected and filtered with a 0.22 µm sterile filtration membrane (Sartorius, Germany). To prepare fresh lyophilized platelet lysate (fLPL), fHPL were freeze-dried by lyophilizer (LSI; lyophilization system, Inc.). Both fHPL and fLPL were aliquoted and stored at -20°C for 2 years to obtain storage HPL (sHPL) and storage LPL (sLPL), respectively. The preparation of fHPL, fLPL, sHPL and sLPL were processed from the same batch of LPPCs.
Isolation And Culture Of Human Bone Marrow Derived Mesenchymal Stem Cells (Bmscs)
Human bone marrow derived mesenchymal stem cells (BMSCs) were obtained from the Faculty of Medicine, Ramathibodi Hospital according to processes, approved by the Ethical Committee of the Faculty of Medicine, Ramathibodi Hospital, Mahidol University (MURA2017/603). All works were conducted in accordance with the Declaration of Helsinki, donor informed consents were signed and collected before sample collection. BMSCs were isolated from these bone marrow aspirates by Histopaque 1.077 g/mL density gradient centrifugation (Merck, Germany). BMSCs were cultured with growth medium containing Dulbecco’s modified Eagle’s medium low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS, Merck, Germany), 1% Penicillin Streptomycin (Merck, Germany) and 1% of Glutamax (Thermo Fisher Scientific, USA). BMSCs were seeded in T75 cell culture flasks and maintained at 37°C, 5% CO2 with 95% humidity. Growth mediums were changed twice a week. BMSCs at passage 3rd -5th were employed for the experiment in this study.
Cell Proliferation And Population Doubling Time (Pdt) Of Bone Marrow Derived Mesenchymal Stem Cells (Bmscs)
BMSCs at the 3rd passage was seeded on a 24-well flat bottom plate (SPL, Korea) with 5.5 x 103 cells/cm2 density. BMSCs were supplemented with a growth medium containing either 10% of FBS, fHPL, fLPL, sHPL or sLPL. We observed cell morphology and performed daily manual cell counting for up to 7 days. Data were calculated into population doubling time (PDT) using the “cell calculator” tools (doubling-time.com; Roth V., 2006) and presented as mean ± SEM to compare the differences between each medium supplemented group.
Bone Marrow Derived Mesenchymal Stem Cells (Bmscs) Surface Marker Expression: Immunophenotypic Study By Flow Cytometry
BMSCs at the 5th passage was seeded in T25 cell culture flasks (SPL, Korea) with 5.5 x 103 cells/cm2 density. BMSCs were cultured in growth medium supplemented with either 10% of FBS, sHPL or sLPL for 7 days. Cells were harvested and stained with anti-human CD34 PE (BD bioscience, USA), anti-human CD45 PerCP (Miltenyi Biotec, Germany), anti-human CD73 PE/Cy-7 (Biolegend, California), anti-human CD90 APC (Miltenyi Biotec, Germany), anti-human CD105 PE (Biolegend, California) and fixed with 1% paraformaldehyde (GPO, Thailand). Positivity and negativity of all CD markers were examined and analyzed by BDFACS Canto II (BD Bioscience, USA), BDFACSDiva software version 6.1.3.
Tri-lineage differentiation of bone marrow derived mesenchymal stem cells (BMSCs) by alizarin red S, oil red O and alcian blue staining
Tri-lineage differentiation including osteogenic, adipogenic and chondrogenic differentiation of BMSCs were observed by cytochemistry staining. BMSCs at the 5th passage was seeded in 35 mm cell culture dishes (SPL, Korea) with 5.5 x 103 cells/cm2 density. BMSCs were cultured in growth medium supplemented with either 10% of FBS, sHPL or sLPL in combination with specific lineage differentiation medium. For osteogenic differentiation, growth medium was supplemented with 100 nM dexamethasone (Sigma-Aldrich, USA), 50 µg/ml ascorbic acid (Sigma-Aldrich, USA) and 10 mM β-glycerophosphate (Merck, Germany). Alizarin red S (ARS) staining for calcium mineralization was performed. ARS stainings were quantitated at day 7, 14, 21 and 28. Sample absorbance was measured at 405 nm wavelength and analyzed by Skanittm RE version 6.1.1, (Thermo Fisher Scientific, USA). For adipogenic differentiation, 500 µM isobutyl methylxanthine (IBMX, Merck, Germany), 1 µM dexamethasone (Sigma-Aldrich, USA), 10 µM insulin (Sigma-Aldrich, USA), and 200 µM indomethacin (Sigma-Aldrich, USA) were added to the growth medium. Fat droplets were stained with oil red O staining. For chondrogenic differentiation, BMSCs were cultured with StemPro® osteocyte/chondrocyte differentiation basal medium (Thermo Fisher Scientific, USA). Cells were stained using the Alcian blue staining kit (Thermo Fisher Scientific, USA) and proteoglycan synthesis was observed. Cell morphology was observed by an inverted microscope (Olympus, Japan).
Bone morphogenetic protein 2 (BMP-2) and platelet derived growth factor BB (PDGF-BB) measurement by enzyme linked immunosorbent assay for growth factor analysis
Bone morphogenetic protein 2 (BMP-2) and platelet derived growth factor BB (PDGF-BB) in fHPL, fLPL, sHPL and sLPL sample were quantitative analyzed by indirect enzyme-linked immunosorbent assay (ELISA). Recombinant BMP-2 (Biolegend, California) and PDGF-BB (Biolegend, California) were used as standard control. Standard and sample were coated at 4°C, overnight. Excess samples were discarded and washed with 1% tween in phosphate buffer saline (PBS-T). Unbound area was blocked with 1% Bovine serum albumin (BSA, Merck, Germany) in PBS. Primary Antibodies; anti-BMP-2 (Sigma-Aldrich, USA) and anti-PDGF-BB (Merck, Germany), and secondary antibodies with HRP linked; horse anti-mouse IgG (Cell Signaling Technology, USA) and goat anti-rabbit IgG (Abcam, UK) were used to detected sample signal. Detection signals were developed by TMB solution. Sample absorbance was measured at 450 nm wavelength by Skanittm microplate reader and software (Thermo Fisher Scientific, USA).
Statistical Analysis
Results are presented as mean ± standard error of the mean (SEM) obtained from biological differences of BMSCs donors. Figures were representative for the groups. Statistical analysis was performed by using GraphPad Prism software version 8. T-test was applied to compare the mean and statistical significance differences between each growth medium supplement conditions. Data were considered statistical significant at p < 0.05 (*) and p < 0.001 (**).