Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues.

Cell-laden microgels have been used as tissue building blocks to create three-dimensional (3D) tissues and organs. However, traditional assembly methods can not be used to fabricate functional tissue constructs with biomechanical and structural complexity. In this study, we present directed assembly of cell-laden dual-crosslinkable alginate microgels comprised of oxidized and methacrylated alginate (OMA). Cell-laden OMA microgels can be directly assembled into well-defined 3D shapes and structures under low-level ultraviolet light. Stem cell-laden OMA microgels can be successfully cryopreserved for long-term storage and on-demand applications, and the recovered encapsulated cells maintained equivalent viability and functionality to the freshly processed stem cells. Finally, we have successfully demonstrated that cell-laden microgels can be assembled into complicated 3D tissue structures via freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting. This highly innovative bottom-up strategy using FRESH 3D bioprinting of cell-laden OMA microgels, which are cryopreservable, provides a powerful and highly scalable tool for fabrication of customized and biomimetic 3D tissue constructs.

Large volume syringe pump extruder for desktop 3D printers.

Syringe pump extruders are required for a wide range of 3D printing applications, including bioprinting, embedded printing, and food printing. However, the mass of the syringe becomes a major challenge for most printing platforms, requiring compromises in speed, resolution and/or volume. To address these issues, we have designed a syringe pump large volume extruder (LVE) that is compatible with low-cost, open source 3D printers, and herein demonstrate its performance on a PrintrBot Simple Metal. Key aspects of the LVE include: (1) it is open source and compatible with open source hardware and software, making it inexpensive and widely accessible to the 3D printing community, (2) it utilizes a standard 60 mL syringe as its ink reservoir, effectively increasing print volume of the average bioprinter, (3) it is capable of retraction and high speed movements, and (4) it can print fluids using nozzle diameters as small as 100 µm, enabling the printing of complex shapes/objects when used in conjunction with the freeform reversible embedding of suspended hydrogels (FRESH) 3D printing method. Printing performance of the LVE is demonstrated by utilizing alginate as a model biomaterial ink to fabricate parametric CAD models and standard calibration objects.

3D Printing PDMS Elastomer in a Hydrophilic Support Bath via Freeform Reversible Embedding.

Polydimethylsiloxane (PDMS) elastomer is used in a wide range of biomaterial applications including microfluidics, cell culture substrates, flexible electronics, and medical devices. However, it has proved challenging to 3D print PDMS in complex structures due to its low elastic modulus and need for support during the printing process. Here we demonstrate the 3D printing of hydrophobic PDMS prepolymer resins within a hydrophilic Carbopol gel support via freeform reversible embedding (FRE). In the FRE printing process, the Carbopol support acts as a Bingham plastic that yields and fluidizes when the syringe tip of the 3D printer moves through it, but acts as a solid for the PDMS extruded within it. This, in combination with the immiscibility of hydrophobic PDMS in the hydrophilic Carbopol, confines the PDMS prepolymer within the support for curing times up to 72 h while maintaining dimensional stability. After printing and curing, the Carbopol support gel releases the embedded PDMS prints by using phosphate buffered saline solution to reduce the Carbopol yield stress. As proof-of-concept, we used Sylgard 184 PDMS to 3D print linear and helical filaments via continuous extrusion and cylindrical and helical tubes via layer-by-layer fabrication. Importantly, we show that the 3D printed tubes were manifold and perfusable. The results demonstrate that hydrophobic polymers with low viscosity and long cure times can be 3D printed using a hydrophilic support, expanding the range of biomaterials that can be used in additive manufacturing. Further, by implementing the technology using low cost open-source hardware and software tools, the FRE printing technique can be rapidly implemented for research applications.

Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.

We demonstrate the additive manufacturing of complex three-dimensional (3D) biological structures using soft protein and polysaccharide hydrogels that are challenging or impossible to create using traditional fabrication approaches. These structures are built by embedding the printed hydrogel within a secondary hydrogel that serves as a temporary, thermoreversible, and biocompatible support. This process, termed freeform reversible embedding of suspended hydrogels, enables 3D printing of hydrated materials with an elastic modulus <500 kPa including alginate, collagen, and fibrin. Computer-aided design models of 3D optical, computed tomography, and magnetic resonance imaging data were 3D printed at a resolution of ~200 µm and at low cost by leveraging open-source hardware and software tools. Proof-of-concept structures based on femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains were mechanically robust and recreated complex 3D internal and external anatomical architectures.