To seed cells within scaffolds, the sterilized scaffolds were pre-wet in MEM over night, and submerged in 1 ml of hMSC suspension (passages 3C4, approximately 16 104 cells/ml) in 1.5 ml Eppendorf tubes. induced to differentiate into osteogenic or adipogenic lineages could be classified into the three lineages (stem, Rabbit polyclonal to USP20 adipogenic, osteogenic) with 80% precision and sensitivity, within 72 hours. Using this framework, the augmentation of osteogenesis by scaffold design exerted by porogen leached scaffolds was also profiled within 72 hours with ~80% high sensitivity. Furthermore, by employing 3-D SC-35 organizational metrics, differential LDN-214117 osteogenesis induced by novel electrospun fibrous polymer mats incorporating decellularized matrix could also be elucidated and predictably modeled at just 3 days with high precision. We demonstrate that 3-D SC-35 organizational metrics can be applied to model the stem cell state in 3-D scaffolds. We propose that this methodology can robustly discern minute changes in stem cell states within complex 3-D architectures and map single cell biological readouts that are critical to assessing population level cell heterogeneity. and three dimensional stem cell culture to guide tissue regeneration in response to external stimuli [1C3]. Stem cell processes stimulated by external cues are commonly assessed using end point biochemical assays and histological analysis [4, 5]. Widely used methodologies for profiling cells cultured in 3-D scaffolds involve harvesting cells to detect gene expression changes using flow-cytometry, micro-arrays, PCR and immuno-assays [4, 5]. These approaches are time consuming and require cells to either be experimentally maintained for the entire time span necessary to fully attain the endpoint state (differentiation, apoptosis, transformation) or necessitate the removal of the cells from their 3-D niche for processing cell response would offer several key advances as compared to present methods, as it would allow characterizing cells without disrupting the organization, offer early and timely detection, and a quantitative estimation of cell heterogeneity. In this study we advance a high content image informatics methodology that employs high content analysis of the true three dimensional SC-35 organization in tandem with machine learning approaches to classify emergent cell states when cells are cultured in 3-D scaffolds including hydrogels, electrospun mats and porogen leached scaffolds. 2. Materials and Methods 2.1. Cell culture Human mesenchymal stem cells (hMSCs) were obtained from Texas A&M University (College Station, TX). Cells were cultured in a humidity-controlled environment under 5% CO2 and 37C and fed every 3 to 4 4 days with basal growth media (BA) consisting of Alpha Minimum Essential medium (MEM) with L-glutamine (LifeTechnologies) supplemented with fetal bovine serum (FBS, 10% v/v, Atlanta Biologicals) and penicillin-streptomycin (0.1% v/v, LifeTechnologies). Cells LDN-214117 were received at passage 1 and used for up to 5 passages. Osteogenic differentiation (OS) was induced by culturing hMSCs in BA media supplemented with 0.5 mM LDN-214117 L-ascorbic acid-2-phosphate, 0.2 M dexamethasone (dex), and 20 mM -glycerophosphate. Adipogenic differentiation (AD) was induced with BA media supplemented with 1 M dexamethasone, 50 M indomethacin, 10 g/ml insulin, and 100 M 3-isobutyl-1-methyl-xanthine. Cells were allowed to adhere overnight in basal growth media, followed by a media change with appropriate induction media. All culture reagents were purchased from Sigma-Aldrich unless otherwise specified. 2.2. Preparation of porogen leached scaffolds Cylindrical scaffolds (8 mm diameter by 2 mm in height) were fabricated from a tyrosine-derived polycarbonate designated as E1001(1k) using a combination of lyophilization and particulate leaching and characterized as described previously [12C14]. This particular polymer composition was selected from a large library LDN-214117 of tyrosine-derived polycarbonates and was used to create bone regeneration scaffolds with both macro- and micropores having a size 212C450 mm and < 20 mm, respectively, and a compressive modulus of 2 MPa [14]. The scaffolds were sterilized by ethylene oxide gas sterilization using Anprolene AN74i (Andersen Products). To seed cells within scaffolds, the sterilized scaffolds were pre-wet in MEM over night, and submerged in.