High cell density and high-resolution 3D bioprinting for fabricating vascularized tissues.

Three-dimensional (3D) bioprinting techniques have emerged as the most popular methods to fabricate 3D-engineered tissues; however, there are challenges in simultaneously satisfying the requirements of high cell density (HCD), high cell viability, and fine fabrication resolution. In particular, bioprinting resolution of digital light processing-based 3D bioprinting suffers with increasing bioink cell density due to light scattering. We developed a novel approach to mitigate this scattering-induced deterioration of bioprinting resolution. The inclusion of iodixanol in the bioink enables a 10-fold reduction in light scattering and a substantial improvement in fabrication resolution for bioinks with an HCD. Fifty-micrometer fabrication resolution was achieved for a bioink with 0.1 billion per milliliter cell density. To showcase the potential application in tissue/organ 3D bioprinting, HCD thick tissues with fine vascular networks were fabricated. The tissues were viable in a perfusion culture system, with endothelialization and angiogenesis observed after 14 days of culture.

High throughput direct 3D bioprinting in multiwell plates.

Advances in three dimensional (3D) bioprinting have enabled the fabrication of sophisticated 3D tissue scaffolds for biological and medical applications, where high speed, high throughput production in well plates is a critical need. Here, we present an integrated 3D bioprinting platform based on microscale continuous optical printing, capable of high throughputin siturapid fabrication of complex 3D biomedical samples in multiwell plate formats for subsequent culture and analysis. Our high throughput 3D bioprinter (HT-3DP) was used to showcase constructs of varying spatial geometries of biomimetic significance, tunable mechanical properties, as well as reproducibility. Live hepatocellular carcinoma 3D tissue scaffolds were fabricatedin situin multiwell plates, after which a functional drug response assay against the chemotherapy drug doxorubicin was performed. Dual cell-type populations involving both live hepatocellular carcinoma as well as human umbilical vein endothelial cells were also printed to demonstrate dual-tissue fabrication capability. This work demonstrates a significant advancement in that the production rate of 3D bioprinted tissue scaffolds with controllable spatial architectures and mechanical properties can now be done on a high throughput scale, enabling rapid generation ofin vitro3D tissue models within conventional multiwell cell culture plates for high throughput preclinical drug screening and disease modeling.

Bioprinting of dual ECM scaffolds encapsulating limbal stem/progenitor cells in active and quiescent statuses.

Limbal stem cell deficiency and corneal disorders are among the top global threats for human vision. Emerging therapies that integrate stem cell transplantation with engineered hydrogel scaffolds for biological and mechanical support are becoming a rising trend in the field. However, methods for high-throughput fabrication of hydrogel scaffolds, as well as knowledge of the interaction between limbal stem/progenitor cells (LSCs) and the surrounding extracellular matrix (ECM) are still much needed. Here, we employed digital light processing (DLP)-based bioprinting to fabricate hydrogel scaffolds encapsulating primary LSCs and studied the ECM-dependent LSC phenotypes. The DLP-based bioprinting with gelatin methacrylate (GelMA) or hyaluronic acid glycidyl methacrylate (HAGM) generated microscale hydrogel scaffolds that could support the viability of the encapsulated primary rabbit LSCs (rbLSCs) in culture. Immunocytochemistry and transcriptional analysis showed that the encapsulated rbLSCs remained active in GelMA-based scaffolds while exhibited quiescence in the HAGM-based scaffolds. The primary human LSCs encapsulated within bioprinted scaffolds showed consistent ECM-dependent active/quiescent statuses. Based on these results, we have developed a novel bioprinted dual ECM 'Yin-Yang' model encapsulating LSCs to support both active and quiescent statues. Our findings provide valuable insights towards stem cell therapies and regenerative medicine for corneal reconstruction.

High-fidelity 3D Printing using Flashing Photopolymerization.

Photopolymerization-based 3D printing has emerged as a promising technique to fabricate 3D structures. However, during the printing process, polymerized materials such as hydrogels often become highly light-scattering, thus perturbing incident light distribution and thereby deteriorating the final print resolution. To overcome this scattering-induced resolution deterioration, we developed a novel method termed flashing photopolymerization (FPP). Our FPP approach is informed by the fundamental kinetics of photopolymerization reactions, where light exposure is delivered in millisecond-scale 'flashes', as opposed to continuous light exposure. During the period of flash exposure, the prepolymer material negligibly scatters light. The material then polymerizes and opacifies in absence of light, therefore the exposure pattern is not perturbed by scattering. Compared to the conventional use of a continuous wave (CW) light source, the FPP fabrication resolution is improved. FPP also shows little dependency on the exposure, thus minimizing trial-and-error type optimization. Using FPP, we demonstrate its use in generating high-fidelity 3D printed constructs.

3D Printable Non-Isocyanate Polyurethanes with Tunable Material Properties.

Green chemistry-based non-isocyanate polyurethanes (NIPU) are synthesized and 3D-printed via rapid, projection photopolymerization into compliant mechanisms of 3D structure with spatially-localized material properties. Trimethylolpropane allyl ether-cyclic carbonate is used to couple the unique properties of two types of reaction chemistry: (1) primary diamine-cyclic carbonate ring-opening conjugation for supplanting conventional isocyanate-polyol reactions in creating urethane groups, with the additional advantage of enabling modular segment interchangeability within the diurethane prepolymers; and (2) thiol-ene (click) conjugation for non-telechelic, low monodispersity, quasi-crystalline-capable, and alternating step-growth co-photopolymerization. Fourier Transform Infrared Spectroscopy is used to monitor the functional group transformation in reactions, and to confirm these process-associated molecular products. The extent of how these processes utilize molecular tunability to affect material properties were investigated through measurement-based comparison of the various polymer compositions: frequency-related dynamic mechanical analysis, tension-related elastic-deformation mechanical analysis, and material swelling analysis. Stained murine myoblasts cultured on NIPU slabs were evaluated via fluorescent microscopy for "green-chemistry" affects on cytocompatibility and cell adhesion to assess potential biofouling resistance. 3D multi-material structures with micro-features were printed, thus demonstrating the capability to spatially pattern different NIPU materials in a controlled manner and build compliant mechanisms.

3D-Printing of Functional Biomedical Microdevices via Light- and Extrusion-Based Approaches.

3D-printing is a powerful additive manufacturing tool, one that enables fabrication of biomedical devices and systems that would otherwise be challenging to create with more traditional methods such as machining or molding. Many different classes of 3D-printing technologies exist, most notably extrusion-based and light-based 3D-printers, which are popular in consumer markets, with advantages and limitations for each modality. The focus here is primarily on showcasing the ability of these 3D-printing platforms to create different types of functional biomedical microdevices-their advantages and limitations are covered with respect to other classes of 3D-printing, as well as the past, recent, and future efforts to advance the functional microdevice domain. In particular, the fabrication of micromachines/robotics, drug-delivery devices, biosensors, and microfluidics is addressed. The current challenges associated with 3D-printing of functional microdevices are also addressed, as well as future directions to improve both the printing techniques and the performance of the printed products.

3D-printed biomaterials with regional auxetic properties.

Tissue engineering is replete with methods for inducing and mediating cell differentiation, which are crucial for ensuring proper regrowth of desired tissues. In this study, we developed a 3D-printed, non-positive Poisson's Ratio (NPPR) scaffold intended for future use in stretch-mediated cell differentiation applications, such as in muscle and tendon regeneration. We utilized dynamic optical projection stereolithography (DOPsL) to fabricate multi-layered, cell-laden NPPR scaffolds - these scaffolds can not only support aggregate cell growth, but can also be printed with locally-tunable force-displacement properties at length scales appropriate for tissue interaction. These NPPR multilayered mesh scaffolds can be embedded into highly elastic hydrogels in order to couple a reduced NPPR behavior to a normally Positive Poisson's Ratio (PPR) solid bulk material. This hybrid structure may potentially enable induced 'auxetic' behavior at the single-cell scale while tuning the Poisson's Ratio to a more isolated value. This would be uniquely suited for providing stretch-mediated effects for various cell-types within the tendon-to-muscle tissue transition.

Continuous Optical 3D Printing of Green Aliphatic Polyurethanes.

Photosensitive diurethanes were prepared from a green chemistry synthesis pathway based on methacrylate-functionalized six-membered cyclic carbonate and biogenic amines. A continuous optical 3D printing method for the diurethanes was developed to create user-defined gradient stiffness and smooth complex surface microstructures in seconds. The green chemistry-derived polyurethane (gPU) showed high optical transparency, and we demonstrate the ability to tune the material stiffness of the printed structure along a gradient by controlling the exposure time and selecting various amine compounds. High-resolution 3D biomimetic structures with smooth curves and complex contours were printed using our gPU. High cell viability (over 95%) was demonstrated during cytocompatibility testing using C3H 10T1/2 cells seeded directly on the printed structures.

Direct 3D-printing of cell-laden constructs in microfluidic architectures.

Microfluidic platforms have greatly benefited the biological and medical fields, however standard practices require a high cost of entry in terms of time and energy. The utilization of three-dimensional (3D) printing technologies has greatly enhanced the ability to iterate and build functional devices with unique functions. However, their inability to fabricate within microfluidic devices greatly increases the cost of producing several different devices to examine different scientific questions. In this work, a variable height micromixer (VHM) is fabricated using projection 3D-printing combined with soft lithography. Theoretical and flow experiments demonstrate that altering the local z-heights of VHM improved mixing at lower flow rates than simple geometries. Mixing of two fluids occurs as low as 320 µL min(-1) in VHM whereas the planar zigzag region requires a flow rate of 2.4 mL min(-1) before full mixing occurred. Following device printing, to further demonstrate the ability of this projection-based method, complex, user-defined cell-laden scaffolds are directly printed inside the VHM. The utilization of this unique ability to produce 3D tissue models within a microfluidic system could offer a unique platform for medical diagnostics and disease modeling.