This topical review with original analysis and empirical results compares cell sensitivity to physical stress during printing. The objective is to frame a reproducible causation between printing environment and printed cell morphology, viability and phenotype stability. Content includes: (1) a topical review classifies the overlap between physical stress vectors during printing and mesenchymal stem cell sensitivities. (2) Original flow analysis frames the feasible range of stress duration and intensity during manufacturing. (3) Preliminary empirical results define cell properties as a function of minimum, mean and maximum stress conditions. The review and analytical characterization serve as an essential precursor to interpret surprising empirical results. Results identify key cell properties are stress-dependent and controllable based on printing process parameter selection. Printing's minimum stress condition preserves cell viability. The maximum stress increases heterogeneity of cell response, induces inelastic ultra-structural distortion of the cell membrane and chromatin, and increases necrotic subpopulations post-printing. The review, analysis and preliminary results support the feasibility of modulating cell properties during fabrication by prescriptively tuning the stress environment. The process control over cell morphology, health and the rate of differentiation is both a direct result of strain during printing and an in-direct result of increased distress signaling from necrotic sub-populations.

Bottom-up tissue engineering requires methodological progress of biofabrication to capture key design facets of anatomical arrangements across micro, meso and macro-scales. The diffusive mass transfer properties necessary to elicit stability and functionality require hetero-typic contact, cell-to-cell signaling and uniform nutrient diffusion. Bioprinting techniques successfully build mathematically defined porous architecture to diminish resistance to mass transfer. Current limitations of bioprinted cell assemblies include poor micro-scale formability of cell-laden soft gels and asymmetrical macro-scale diffusion through 3D volumes. The objective of this work is to engineer a synchronized multi-material bioprinter (SMMB) system which improves the resolution and expands the capability of existing bioprinting systems by packaging multiple cell types in heterotypic arrays prior to deposition. This unit cell approach to arranging multiple cell-laden solutions is integrated with a motion system to print heterogeneous filaments as tissue engineered scaffolds and nanoliter droplets. The set of SMMB process parameters control the geometric arrangement of the combined flow's internal features and constituent material's volume fractions. SMMB printed hepatocyte-endothelial laden 200 nl droplets are cultured in a rotary cell culture system (RCCS) to study the effect of microgravity on an in vitro model of the human hepatic lobule. RCCS conditioning for 48 h increased hepatocyte cytoplasm diameter 2 μm, increased metabolic rate, and decreased drug half-life. SMMB hetero-cellular models present a 10-fold increase in metabolic rate, compared to SMMB mono-culture models. Improved bioprinting resolution due to process control of cell-laden matrix packaging as well as nanoliter droplet printing capability identify SMMB as a viable technique to improve in vitro model efficacy.

A PED (precision extrusion deposition)/replica molding process enables scaffold guided tissue engineering of a heterocellular microfluidic device. We investigate two types of cell-laden devices: the first with a 3D microfluidic manifold fully embedded in a PDMS (polydimethylsiloxane) substrate and the second a channel network on the surface of the PDMS substrate for cell printing directly into device channels. Fully embedded networks are leak-resistant with simplified construction methods. Channels exposed to the surface are used as mold to hold bioprinted cell-laden matrix for controlled cell placement throughout the network from inlet to outlet. The result is a 3D cell-laden microfluidic device with improved leak-resistance (up to 2.0 mL/min), pervasive diffusion and control of internal architecture.