Melt Electrowritten Sandwich Scaffold Technique Using Sulforhodamine B to Monitor Stem Cell Behavior.

Three-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 straightforward and quantitative method was developed to examine these cellular parameters on poly-ɛ-caprolactone (PCL) multilayered MEW scaffolds developed as components of the annulus fibrosus region of bioengineered intervertebral discs. The anti-adhesion protein, bovine serum albumin (BSA), was used to coat plasticware to improve mesenchymal stem cell (T0523) adhesion to MEW scaffolds. Cells were seeded on circular MEW (cMEW) discs 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 3D scaffolds. 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, whereas the 400 μm pore size scaffolds time was 11.5 days. BSA coating of tissue culture dishes prevented surface adhesion of cells to vessel surfaces and promoted spheroid formation that 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. Impact statement In this article, a new, useful, and straightforward method to quantitate cell numbers on three-dimensional (3D) melt electrowritten (MEW) scaffolds is presented. By using the sulforhodamine B assay on bovine serum albumin-coated dishes cell migration, expansion and proliferation in 3D printed MEW test scaffolds were quantitatively measured. Printed circular MEW (cMEW) scaffolds sandwiched between two MEW test scaffolds (Fig. 3) were used as defined cellular growth starting points with a particular pore size of 250 μm displaying the fastest cell growth and migration. This MEW sandwich technique could potentially be used to quantitate cell numbers and migration in other 3D multilayered MEW scaffold systems.