Quantitative criteria to benchmark new and existing bio-inks for cell compatibility

Abstract

Recent advancements in 3D bioprinting have led to the fabrication of more complex, more precise, and larger printed tissue constructs. As the field continues to advance, it is critical to develop quantitative benchmarks to compare different bio-inks for key cell-biomaterial interactions, including (1) cell sedimentation within the ink cartridge, (2) cell viability during extrusion, and (3) cell viability after ink curing. Here we develop three simple protocols for quantitative analysis of bio-ink performance. These methods are used to benchmark the performance of two commonly used bio-inks, poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), against three formulations of a novel bio-ink, Recombinant-protein Alginate Platform for Injectable Dual-crosslinked ink (RAPID ink). RAPID inks undergo peptide-self-assembly to form weak, shear-thinning gels in the ink cartridge and undergo electrostatic crosslinking with divalent cations during curing. In the one-hour cell sedimentation assay, GelMA, the RAPID inks, and PEGDA with xanthan gum prevented appreciable cell sedimentation, while PEGDA alone or PEGDA with alginate experienced significant cell settling. To quantify cell viability during printing, 3T3 fibroblasts were printed at a constant flowrate of 75 μl/min and immediately tested for cell membrane integrity. Less than 10% of cells were damaged using the PEGDA and GelMA bio-inks, while less than 4% of cells were damaged using the RAPID inks. Finally, to evaluate cell viability after curing, cells were exposed to ink-specific curing conditions for five minutes and tested for membrane integrity. After exposure to light with photo-initiator at ambient conditions, over 50% of cells near the edges of printed PEGDA and GelMA droplets were damaged. In contrast, fewer than 20% of cells found near the edges of RAPID inks were damaged after a 5-minute exposure to curing in a 10 mM CaCl2 solution. As new bio-inks continue to be developed, these protocols offer a convenient means to quantitatively benchmark their performance against existing inks.

Non‐Newtonian Polymer–Nanoparticle Hydrogels Enhance Cell Viability during Injection

“…Technology development is needed to enhance the delivery, protection, and encapsulation of cells to increase the viability and local retention of cells when injected. Previous works have shown that hydrogels are promising alternatives to PBS for administration of cell therapies, as they enhance local retention and improve the viability of cells during injection when compared to injections performed in PBS . It has been proposed that hydrogel “plug flow,” a phenomena caused by the rheology of these materials, protects the cells from the shear and extensional forces during injection from a syringe and needle .…”

“…To date, researchers have fit this seemingly contradictory and complex design envelope for cell delivery by developing i) covalently bound static materials such as hydrogels injected as low viscosity precursors that gel in situ to form a localized cell environment; and ii) dynamic supramolecular systems such as hydrogels comprising host–guest moieties, peptide‐functionalized polymers, and peptide amphiphiles that employ shear‐thinning to flow through needle. Supramolecular hydrogels are promising because they shear‐thin and do not require in situ polymerizations …”

“…Given the non‐Newtonian behavior of the 1:5 HPMC:NP hydrogel, we hypothesized that the yield stress of the hydrogel would serve to prevent any cell sedimentation after mixing. A settling assay was performed as reported by Dubbin et al . A maximum projection confocal fluorescence image of hMSCs in a 1:5 HPMC:NP gel is shown in Figure c showing that they did not settle over the course of 30 min of incubation at 37 °C.…”

Methods To Assess Shear-Thinning Hydrogels for Application As Injectable Biomaterials

“…6,36 For bioprinting applications, injection parameters are also important in modulating both the resolution and stability of printed structures, as well as cellular viability. 37,38 For these reasons, it is important to engineer and tune hydrogels for injection and to understand how various design parameters influence these outcomes, using both quantitative and qualitative techniques. In this methods paper, we detail techniques useful for exploring the mechanical properties that enable shear-thinning and self-healing and demonstrate protocols that can be employed to ensure consistent hydrogel injection and retention.…”

Injectable supramolecular polymer–nanoparticle hydrogels enhance human mesenchymal stem cell delivery

“…Hydrogels are an excellent medium to create homogenous cell suspensions and prevent cell aggregation on the syringe 37. In an attempt to uniformly suspend the therapeutic cells in the liquid solution and avoid highly variable cell concentrations clinicians must vigorously mix the cell suspensions before injection.…”