Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers

Abstract

Cell transplantation is a promising therapy for a myriad of debilitating diseases; however, current delivery protocols using direct injection result in poor cell viability. We demonstrate that during the actual cell injection process, mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we hypothesize that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. We use a controlled in vitro model of cell injection to demonstrate success of this acute protection strategy for a wide range of cell types including human umbilical vein endothelial cells (HUVEC), human adipose stem cells, rat mesenchymal stem cells, and mouse neural progenitor cells. Specifically, alginate hydrogels with plateau storage moduli (G') ranging from 0.33 to 58.1 Pa were studied. A compliant crosslinked alginate hydrogel (G'=29.6 Pa) yielded the highest HUVEC viability, 88.9% ± 5.0%, while Newtonian solutions (i.e., buffer only) resulted in 58.7% ± 8.1% viability. Either increasing or decreasing the hydrogel storage modulus reduced this protective effect. Further, cells within noncrosslinked alginate solutions had viabilities lower than media alone, demonstrating that the protective effects are specifically a result of mechanical gelation and not the biochemistry of alginate. Experimental and theoretical data suggest that extensional flow at the entrance of the syringe needle is the main cause of acute cell death. These results provide mechanistic insight into the role of mechanical forces during cell delivery and support the use of protective hydrogels in future clinical stem cell injection studies.

FIG. 1.

Cell viability after syringe needle flow. (A) Photograph of 1% wt/vol, 200 kDa alginate without (top) and with (bottom) crosslinking (1:4 Ca²⁺ ion:G-subunit). (B) HUVEC viability within PBS, 1:4 crosslinked alginate gels, or noncrosslinked alginate solutions with and without ejection through a syringe needle, *p<0.05. † indicates statistical significance between PBS and all ejected crosslinked alginate gels (p<0.05). # indicates statistical significance between ejected 75 kDa crosslinked alginate compared with ejected 147 and 200 kDa crosslinked alginate. HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline. Color images available online at www.liebertonline.com/tea

FIG. 2.

Flow characteristics during syringe needle flow. (A) (Top) Schematic of syringe device, not drawn to scale. (Middle and bottom) Heat map of shear stress during syringe needle flow as obtained by finite element modeling of PBS (middle) and noncrosslinked alginate (bottom). (B) HUVEC viability within PBS, noncrosslinked (75 kDa), or crosslinked (75 kDa, 1:4 crosslinking) alginate exposed to a shear rate of 17,240 S⁻¹ for 5 s. Color images available online at www.liebertonline.com/tea

FIG. 3.

Rheology of alginate hydrogels and solutions. Storage (G′) and loss (G″) moduli of (A) 1:4 and (B) 0.5:4 crosslinked alginate hydrogels and (C) noncrosslinked alginate solutions of varying molecular weight. (D) Shear-thinning and recovery behavior of 1:4 crosslinked alginate hydrogel (200 kDa); inset of recovery period.

FIG. 4.

HUVEC viability after syringe needle flow in various cell carriers. (A) Viability within Newtonian fluids (PBS and glycerol solutions) with and without ejection through a syringe needle, *p<0.05. (B) Viability within PBS and crosslinked alginate hydrogels of varying G′ with and without ejection. Statistical significance is represented by letters above each column, with different letters signifying distinct statistical groups, p<0.05.

FIG. 5.

Viability of HUVEC, hASC, rMSC, and mNPC after syringe needle flow in various cell carriers. (A) Fluorescent images of viable (green, calcein AM) and membrane damaged (red, ethidium homodimer-1) cells postejection in PBS or crosslinked alginate hydrogel (G'=29.6 Pa), scale bars=200 μm. (B) Percent cell viability with and without ejection in PBS or alginate hydrogels of G'=29.6 or 58.1 Pa, *p<0.05. hASC, human adipose stem cells; rMSC, rat mesenchymal stem cells; mNPC, mouse neural progenitor cells. Color images available online at www.liebertonline.com/tea