High throughput platform for rapid evaluation of cell and tissue response to 3D composite materials

摘要

Introduction: Tissue regeneration occurs naturally in most tissues as cells act to remodel and rebuild damaged tissue by inducing structural and compositional changes, but fails to occur in large defects. Extracellular matrix (ECM) and growth factors (GFs) act synergistically to mediate cell processes critical for regeneration, and a comprehensive understanding is necessary to develop of advanced composite biomaterials. Screening becomes increasingly cost-prohibitive as the experimental space grows larger. In order to identify optimal ECM and GF combinations that promote tissue-specific regeneration, we employ a microscale system to rapidly investigate large experimental spaces efficiently and accurately distinguish between highly growth-permissive and growth-inhibitive 3D composite scaffolds. Materials and Methods: Gaskets for housing hydrogel samples were consrtucted from a 3D-printed mold for creating PDMS arrays. Primary rat neurons were seeded at 1~*106 cells/mL into combinatorial libraries of laminin (LN:0-1500 μg/mL) and fibronectin (FN:0-250 μg/mL) mixed into collagen type Ⅰ gels. Collagen type Ⅰ (COL) was chosen as the substrate because it forms the basis for many tissues in vivo (including peripheral nerve tissue). 3D samples were evaluated for metabolism (Alamar Blue) and then fixed and stained for βⅢ-tubulin and nuclei. Samples were imaged using a Thermo Scientific Cellomics AirayScan VT, image processing and analysis executed through ImageJ and MATLAB, and statistical analysis performed via ANOVA and Tukey's HSD. Results and Discussion: Neurons were embedded in 10μL of 1 mg/mL COL gels containing LN and FN and grown for 3 days. After 1 day, there was no difference in Alamar blue signal, indicating relatively even seeding between all sample groups (not shown). Following immunostaining, Z-stacks were taken and compressed images analyzed for neurite outgrowth. Neurite outgrowth was observed in all samples, and preliminary data indicates that significant neurite outgrowth over COL-only gels was observed in nearly all samples containing LN and/or FN (Figure 1). This platform can applied other cell types to rapidly quantify changes in metabolism, cell death and spreading, in addition to more advanced cell-specific processes such as migration and myelination (not shown). Furthermore, co-culture screenings investigating the neuronal outgrowth and glial migration within the same biomaterial screen would provide "tissue response" to a biomaterial library. Conclusions: This framework can rapidly assay large experimental spaces of composite biomaterials to identify optimal cell- and tissue-specific biomaterials. Ultimately, biomimetic synthetic alternatives to novel synergistic ECM-GF combinations found to promote cell growth and regeneration can be tuned using the same platform prior to downstream animal and clinical studies. Advanced biotherapeutics developed through microscale screening of appropriate cell responses will be cheaper and more effective than those derived using traditional macroscale strategies.