3D Printing of Neural Tissues Derived from Human Induced Pluripotent Stem Cells Using a Fibrin-Based Bioink

Abstract

3D bioprinting offers the opportunity to automate the process of tissue engineering, which combines biomaterial scaffolds and cells to generate substitutes for diseased or damaged tissues. These bioprinting methods construct tissue replacements by positioning cells encapsulated in bioinks into specific locations in the resulting constructs. Human induced pluripotent stem cells (hiPSCs) serve as an important tool when engineering neural tissues. These cells can be expanded indefinitely and differentiated into the cell types found in the central nervous systems, including neurons. One common method for differentiating hiPSCs into neural tissue requires the formation of aggregates inside of defined diameter microwells cultured in chemically defined media. However, 3D bioprinting of such hiPSC-derived aggregates has not been previously reported in the literature, as it requires the development of specialized bioinks for supporting cell survival and differentiation into mature neural phenotypes. Here we detail methods including preparing base material components of the bioink, producing the bioink, and the steps involved in printing 3D neural tissues derived from hiPSC-derived neural aggregates using Aspect Biosystems’ novel RX1 printer and their lab-on-a-printer (LOP) technology.

3D Bioprinting Pluripotent Stem Cell Derived Neural Tissues Using a Novel Fibrin Bioink Containing Drug Releasing Microspheres

“…Bioink was prepared prior to printing as previously described (Abelseth et al, 2018). NPCs at a concentration of 1 million cells/mL were thawed and resuspended in the bioink composed of 20 mg/mL of fibrinogen (Sigma, St. Louis, MO, United States), 0.5% w/v of alginate (120,000-190,000 g/mol, M/G ratio 1.56) (Sigma, St. Louis, MO, United States), and 0.3 mg/mL of genipin (Sigma, St. Louis, MO, United States) dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, United States), along with 0.5 mg of microspheres in tris-buffered saline (TBS) with phenol red (Sigma, St. Louis, MO, United States) when appropriate.…”

“…Our lab uses the Aspect Biosystems RX1 printer with its novel microfluidic Lab-on-a-Printer technology due to its ability to protect the cells within the bioink from shear stress during printing -enabling us to maximize cell viability (Beyer et al, 2016;Bsoul et al, 2016). Our own group developed a novel fibrin-based bioink for printing hiPSC-derived neural aggregates that both maintained their viability and differentiated into mature neural tissues after 46 days of culture (Abelseth et al, 2018). This same formulation was also used to print dissociated hiPSC-derived neural progenitor cells (NPCs) that could be matured into spinal cord-resembling tissues upon treatment with specific small molecules (De La Vega et al, 2018a).…”

Bioprinting Neural Systems to Model Central Nervous System Diseases

“…2-20 mPa s [29] • Compatible with other biofabrication strategies involving extrusion of thermoplastics [30] • Limited structural integrity and shape fidelity [27,31] • Reported cell numbers varied per deposited drop [26] • Low cell densities (<1 × 10 6 ; 2100 cells mm −1 (physiological) vs 20 cells mm −1 (printed)) [30] ≈300 µm [30] P2-4 rat primary retinal ganglion cells (RGCs) [30] Calcein AM/SYTOX: 1.2fold increased survivability (printed vs nonprinted) if growth medium was added to the bioink formulation (postprinting) [30] Extrusionbased 200 µm [32] hiPSC-derived spinal progenitor cells (sNPCs) and oligodendrocyte progenitor cells (OPCs) [32] Calcein AM/EthD:5, 15, 30 min of exposure to nonhumidified environment: ≈98%, ≈45%, ≈0% [32] • Wide range of viscosity (30 mPa s to 60 kPa s) [33] • Achieved in situ reprogramming and differentiation [34] • High cell densities (8 × 10 7 cells mL −1 ) [34a] • Shown formation of intercellular connections in neuronal networks [35] • High density of cell aggregates are printable, microfluidic-chip enabled complex prints [36] • Easy accessibility: RepRap hardware [37] • Embedding bioprinting strategies (e.g., FRESH) [38] • Poor structural integrity and shape fidelity in low-viscosity inks, and potential cell damage from shear forces while extruding [27,31] • Slow printing speed may lead to gel dehydration thus requiring strategies to control the printing environment [32] ≈410 µm (nozzle diameter) [34b] Adult fibroblasts [34b] VB-48/propidium Iodide (PI):…”

3D Printed Neural Regeneration Devices

“…Moreover, depending on the specific requirement for the printing process, the printer can be customized. For instance, to reduce shear stress on cells during extrusion, Willerth et al have integrated microfluidic channels into the printhead, which allowed a separate flow of cells in bioinks and the associated cross‐linker . This enabled low viscous flow, resulting in successful neural differentiation from printed human induced pluripotent stem cells (iPSCs).…”

“…To overcome this limitation, RGD peptides have been covalently bonded with alginate to improve cell‐adhesion when alginate is used as a cell‐laden hydrogel . Similarly, to ensure neuronal differentiation and neurite extension, alginate has been admixed with fibrinogen, which was then cross‐linked by a mixture of cross‐linking reagents (i.e., chitosan, calcium chloride, thrombin, and genipin) …”