Jessica Snyder

Bioprinting cell-laden matrigel for radioprotection study of liver by pro-drug conversion in a dual-tissue microfluidic chip

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.

Fabrication of three-dimensional scaffolds using precision extrusion deposition with an assisted cooling device

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 ACs 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 devices 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.

Maskless fabrication of cell-laden microfluidic chips with localized surface functionalization for the co-culture of cancer cells

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.

Surface modification of SU-8 for enhanced cell attachment and proliferation within microfluidic chips.

Advances in micro-electro-mechanical systems (MEMS) have led to an increased fabrication of micro-channels. Microfabrication techniques are utilized to develop microfluidic channels for continuous nutrition supply to cells inside a micro-environment. The ability of cells to build tissues and maintain tissue-specific functions depends on the interaction between cells and the extracellular matrix (ECM). SU-8 is a popular photosensitive epoxy-based polymer in MEMS. The patterning of bare SU-8 alone does not provide the appropriate ECM necessary to develop microsystems for biological applications. Manipulating the chemical composition of SU-8 will enhance the biological compatibility, giving the fabricated constructs the appropriate ECM needed to promote a functional tissue array. This article investigates three frequently used surface treatment techniques: (1) plasma treatment, (2) chemical reaction, and (3) deposition treatment to determine which surface treatment is the most beneficial for enhancing the biological properties of SU-8. The investigations presented in this article demonstrated that the plasma, gelatin, and sulfuric acid treatments have a potential to enhance SU-8s surface for biological application. Of course each treatment has their advantages and disadvantages (application dependent). Cell proliferation was studied with the use of the dye Almar Blue, and a micro-plate reader. After 14 days, cell proliferation to plasma treated surfaces was statistically significantly enhanced (p < 0.00001), compared to untreated surfaces. The plasma treated surface is suggested to be the better of the three treatments for biological enhancement followed by gelatin and sulfuric acid treatments, respectively.

A three-dimensional cell-laden microfluidic chip for in vitro drug metabolism detection

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 drugs 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.

Mesenchymal stem cell printing and process regulated cell properties.

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. Printings 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.

Hetero-cellular prototyping by synchronized multi-material bioprinting for rotary cell culture system.

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 flows internal features and constituent materials 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.

Combined multi-nozzle deposition and freeze casting process to superimpose two porous networks for hierarchical three-dimensional microenvironment

An engineered three-dimensional scaffold with hierarchical porosity and multiple niche microenvironments is produced using a combined multi-nozzle deposition-freeze casting technique. In this paper we present a process to fabricate a scaffold with improved interconnectivity and hierarchical porosity. The scaffold is produced using a two-stage manufacturing process which superimposes a printed porous alginate (Alg) network and a directionally frozen ceramic-polymer matrix. The combination of two processes, multi-nozzle deposition and freeze casting, provides engineering control of the microenvironment of the scaffolds over several length scales; including the addition of lateral porosity and the ratio of polymer to ceramic microstructures. The printed polymer scaffold is submerged in a ceramic-polymer slurry and subsequently, both structures are directionally frozen (freeze cast), superimposing and patterning both microenvironments into a single hierarchical architecture. An optional additional sintering step removes the organic material and densifies the ceramic phase to produce a well-defined network of open pores and a homogenous cell wall material composition. The techniques presented in this contribution address processing challenges, such as structure definition, reproducibility and fine adjustments of unique length scales, which one typically encounters when fabricating topological channels between longitudinal and transverse porous networks.

Localized surface functionalization of polycaprolactone with atmospheric-pressure microplasma jet

Surface properties of biopolymers are crucial for providing topographical and chemical cues to affect cellular behaviors, such as attachment, spreading, viability, proliferation, and differentiation. As an effective surface modification technique, plasma treatment is often applied to enhance surface wettability, adhesion, and biocompatibility of polymers. In this study, an atmospheric-pressure microplasma jet based on dielectric barrier discharge was installed on an automated arm which allows movement in the x–y–z directions at various trajectory presets. Polycaprolactone (PCL) samples were functionalized with helium-oxygen plasma generated by this system and characterized via water contact angle, x-ray photoelectron spectroscopy, and scanning electron microscopy. Mouse osteoblast cells (7F2) were cultured on both treated and native PCL samples and examined by MarkerGene™ Live: Dead/Cytotoxicity and alamarBlue® assaying techniques. The surface and biological characterization results indicate that microplasma treatment improved surface hydrophilicity, as well as cell viability and proliferation. The localized microplasma treatment can lead to the application of bioactive scaffolds with selective surface functionalization.

Evaluating fabrication feasibility and biomedical application potential of in situ 3D printing technology

Purpose This paper aims to present a solid freeform fabrication-based in situ three-dimensional (3D) printing method. This method enables simultaneous cross-linking alginate at ambient environmental conditions (temperature and pressure) for 3D-laden construct fabrication. The fabrication feasibility and potentials in biomedical applications were evaluated. Design/methodology/approach Fabrication feasibility was evaluated as the investigation of fabrication parameters on strut formability (the capability to fabricate a cylindrical strut in the same diameter as dispensing tip) and structural stability (the capability to hold the fabricated 3D-laden construct against mechanical disturbance). Potentials in biomedical application was evaluated as the investigation on structural integrity (the capability to preserve the fabricated 3D-laden construct in cell culture condition). Findings Strut formability can be achieved when the flow rate of alginate suspension and nozzle travel speed are set according to the dispensing tip size, and extruded alginate was cross-linked sufficiently. A range of cross-linking-related fabrication parameters was determined for sufficient cross-link. The structural stability and structural integrity were found to be controlled by alginate composition. An optimized setting of the alginate composition and the fabrication parameters was determined for the fabrication of a desired stable scaffold with structural integrity for 14 days. Originality/value This paper reports that in situ 3D printing is an efficient method for 3D-laden construct fabrication and its potentials in biomedical application.