The objective of this paper is to introduce a novel cell printing and microfluidic system to serve as a portable ground model for the study of drug conversion and radiation protection of living liver tissue analogs. The system is applied to study behavior in ground models of space stress, particularly radiation. A microfluidic environment is engineered by two cell types to prepare an improved higher fidelity in vitro micro-liver tissue analog. Cell-laden Matrigel printing and microfluidic chips were used to test radiation shielding to liver cells by the pro-drug amifostine. In this work, the sealed microfluidic chip regulates three variables of interest: radiation exposure, anti-radiation drug treatment and single- or dual-tissue culture environments. This application is intended to obtain a scientific understanding of the response of the multi-cellular biological system for long-term manned space exploration, disease models and biosensors.

3D human cancer models provide a better platform for drug efficacy studies than conventional 2D culture, since they recapitulate important aspects of the in vivo microenvironment. While biofabrication has advanced model creation, bioprinting generally involves extruding individual cells in a bioink and then waiting for these cells to self-assemble into a hierarchical 3D tissue. This selfassembly is time consuming and requires complex cellular interactions with other cell types, extracellular matrix components, and growth factors. We therefore investigated if we could directly bioprint pre-formed 3D spheroids in alginate-based bioinks to create a model tissue that could be used almost immediately. Human breast epithelial cell lines were bioprinted as individual cells or as preformed spheroids, either in monoculture or co-culture with vascular endothelial cells. While individual breast cells only spontaneously formed spheroids in Matrigel-based bioink, pre-formed breast spheroids maintained their viability, architecture, and function after bioprinting. Bioprinted breast spheroids were more resistant to paclitaxel than individually printed breast cells; however, this effect was abrogated by endothelial cell co-culture. This study shows that 3D cellular structure bioprinting has potential to create tissue models that quickly replicate the tumor microenvironment.

Abstract

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.

In the field of biofabrication, tissue engineering and regenerative medicine, there are many methodologies to fabricate a building block (scaffold) which is unique to the target tissue or organ that facilitates cell growth, attachment, proliferation and/or differentiation. Currently, there are many techniques that fabricate three-dimensional scaffolds; however, there are advantages, limitations and specific tissue focuses of each fabrication technique. The focus of this initiative is to utilize an existing technique and expand the library of biomaterials which can be utilized to fabricate three-dimensional scaffolds rather than focusing on a new fabrication technique. An expanded library of biomaterials will enable the precision extrusion deposition (PED) device to construct three-dimensional scaffolds with enhanced biological, chemical and mechanical cues that will benefit tissue generation. Computer-aided motion and extrusion drive the PED to precisely fabricate micro-scaled scaffolds with biologically inspired, porosity, interconnectivity and internal and external architectures. The high printing resolution, precision and controllability of the PED allow for closer mimicry of tissues and organs. The PED expands its library of biopolymers by introducing an assisting cooling (AC) device which increases the working extrusion temperature from 120 to 250 °C. This paper investigates the PED with the integrated AC's capabilities to fabricate three-dimensional scaffolds that support cell growth, attachment and proliferation. Studies carried out in this paper utilized a biopolymer whose melting point is established to be 200 °C. This polymer was selected to illustrate the newly developed device's ability to fabricate three-dimensional scaffolds from a new library of biopolymers. Three-dimensional scaffolds fabricated with the integrated AC device should illustrate structural integrity and ability to support cell attachment and proliferation.

The utilization of the microfabrication technique to fabricate advanced computing chips has exponentially increased in the last few decades. Needless to say, this fabrication technique offers some unique advantages to develop micro-systems. Though many conventional microfabrication techniques today uses very harsh chemicals, the authors believe that the manipulation of system components and fabrication methods may aid in the utilization of the microfabrication techniques used in fabricating computer chips to develop advanced biological microfluidic systems. Presented in this paper is a fabrication approach in which popular fabrication methods and techniques are coupled together to develop an integrated system that aids in the fabrication of cell-laden microfluidic systems. This system aims to reduce the uses of harsh chemicals and decreases the lengthy fabrication time. Additionally, this integrated system will enable the printing of cells as the microfluidic chip is being fabricated. To demonstrate the unique capabilities of the integrated system, an advanced microfluidic chip is being fabricated and investigated. The advanced chip will feature the investigation of cancer cells in a co-cultured microfluidic environment. The investigations presented demonstrate co-cultures in a microfluidic chip, advanced cell printing with localized surface enhancement, cell integration, and full additive fabrication of a microfluidic chip.

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.

Three-dimensional tissue platforms are rapidly becoming the method of choice for quantification of the heterogeneity of cell populations for many diagnostic and drug therapy applications. Microfluidic sensors and the integration of sensors with microfluidic systems are often described as miniature versions of their macro-scale counterparts. This technology presents unique advantages for handling costly and difficult-to-obtain samples and reagents as a typical system requires between 100 nL to 10 µL of working fluid. The fabrication of a fully functional cell-based biosensor utilizes both biological patterning and microfabrication techniques. A digital micro-mirror (photolithographic) system is initiated to construct the tissue platform while a cell printer is used to precisely embed the cells within the construct. Tissue construct developed with these technologies will provide an early diagnostic of a drug's potential use. A three-dimensional interconnected microfluidic environment has the potential to eliminate the limitations of the traditional mainstays of two-dimensional investigations. This paper illustrates an economical and an innovative approach of fabricating a three-dimensional cell-laden microfluidic chip for detecting drug metabolism.