Melt electrowitten sandwich scaffold technique using Sulforhodamine B to monitor stem cell behavior.

BACKGROUND 3-dimensional (3D) printing using melt electrowriting (MEW) technology is a recently developed technique to produce biocompatible micron-level mesh scaffolds layer-by-layer that can be seeded with cells for tissue engineering. Examining cell behavior, such as growth rate and migration, can be problematic in these opaque 3D scaffolds. A straight-forward and quantitative method was developed to examine these cellular paramenters on poly-e-caprolactone (PCL) multi-layered MEW scaffolds developed as components of the annulus fibrosus region of bioengineered intervertebral discs. Experiment: The anti-adhesion protein, bovine serum albumin (BSA), was used to coat plasticware in order to improve mesenchymal stem cell (T0523) adhesion to MEW scaffolds. Cells were seeded on circular MEW discs (cMEW) as defined growth starting points sandwiched between two test template scaffolds investigated at varying pore sizes. Cell expansion, growth, and migration were quantitated utilizing the protein specific dye sulforhodamine B (SRB). Live cell imaging combined with image analysis were used to examine cell motility and expansion on scaffolds. RESULTS After one coating of BSA, cells remained nonadherent for the duration of the study with cell spheroids formed and enlarging over 21 days and becoming entangled in MEW scaffold pores. Cells grown on the 250 µm pore size scaffolds exhibited a doubling time of 7 days, while the 400 µm pore size scaffolds time was 11.5 days. CONCLUSIONS BSA-coating of petri-dishes prevented surface adhesion of cells to vessel surfaces and promoted spheroid formation which encouraged attachment to the PCL scaffolds. Batch printed cMEW scaffolds were useful as a defined starting point for quantitative assays that successfully measured cell migration, expansion and proliferation on test scaffolds. The SRB assay was shown to be a useful and straightforward way to quantitate cell numbers in multilayered MEW scaffolds. A pore size of 250 μm exhibited the fastest cell growth, spread and expansion.