Spheroid model for functional osteogenic evaluation of human adipose derived stem cells

摘要

Introduction: Three dimensional (3D) spheroids of bone cells allow better cellular interactions contributing to cell differentiation towards osteogenic lineage than the current two dimensional (2D) monolayer cell culture. The aim of this study was to form an in vitro 3D osteogenic cell culture model from human adipocyte derived stem cells (hASCs) using a conjugate of a recombinant protein, elastin-like polypeptide (ELP), with a charged polyelectrolyte, polyethyleneimine (PEI). With our previous studies showing successful spheroid evaluations of proliferation and differentiation in 3T3-L1 adipocytes and H35 rat hepatoma, we hypothesized that hASCs cultured as 3D spheroids would differentiate more toward osteoblastic lineage when compared to the traditional 2D monolayer. Methods: ELP expression, purification, chemical modification with PEI and its coating (0.5 mg/cm2) atop tissue culture polystyrene (TCPS) surface was performed as described elsewhere. hASCs isolated from IRB-approved elective liposuction aspirates were seeded onto uncoated and ELP-PEI coated TCPS surfaces of 24-well cell culture plates (50,000 cells/well) and cultured in DMEM/F12 with 10% FBS for 3 days of acclimation followed by supplementation with osteogenic cocktail containing DMEM, 50 μM L-ascorbic acid, 10 mM β-glycerophosphate, 0.05 nM dexamethasone. 10% FBS, and 1% penicillin/streptomycin for 3 weeks. Assays for viability (Live/Dead), total protein content (BCA total protein assay), osteogenic differentiation (Alkaline phosphatase activity, ALP), maturation (Osteocalcin), and mineralization (Alizarin red staining) were performed using manufacturers' protocols. Statistical evaluation was performed with ANOVA and Games-Howell post hoc test. Results: hASC cells displayed monolayer features during acclimation on uncoated TCPS surface while ELP-PEI surface showed cell aggregates during the first 24 h and formed spheroids later. Live/Dead assay showed high number of live cells on coated and uncoated TCPS surfaces (＞ 90%) (Fig.1) on day 23. The total protein content had an increasing trend in 2D monolayer (data not shown) indicating cells were metabolically active and proliferating. However, the 3D spheroids showed early plateau in total protein content values, which was due to contact inhibited growth arrest. The ALP activity normalized to total protein content was greater on days 8,15 and 22 for 3D spheroids than 2D monolayer indicating higher differentiation of hASCs into osteoblastic lineage (Fig. 2a). Similarly, the normalized osteocalcin was greater on days 8,15 and 22 for 3D spheroids than the 2D monolayer (Fig 2.b,c). Alizarin red staining on day 23 showed negligible amount of mineralization in 2D monolayer while the 3D spheroids had greater Alizarin red staining (0.19 ± 0.14 nM/μg protein) indicating higher mineralization activity. Significant statistical differences were seen between 3D spheroids and 2D monolayer in ALP activity, osteocalcin, and mineralization indicating differences in cellular response to ELP-PEI coated and uncoated TCPS surfaces. Conclusion: Osteogenic differentiation, maturation, and mineralization were considerably greater in 3D spheroids formed using hASCs cultured atop ELP-PEI coated surfaces than the 2D monolayer formed on uncoated surfaces indicating 3D spheroids to be a better culture technique. 3D in vitro culture of osteogenic cells may serve as an alternative to 2D culture by providing better understanding of cellular functions and interactions in bone tissue engineering.