Comparative cytocompatibility of multiple candidate cell types to photoencapsulation in PEGNB/PEGDA macroscale or microscale hydrogels

Abstract

The encapsulation of live cells into photopolymerized hydrogel scaffolds has the potential to augment or repair tissue defects, establish versatile regenerative medicine strategies, and be developed as well-defined, yet tunable microenvironments to study fundamental cellular behavior. However, hydrogel fabrication limitations constrain most studies to macroscale hydrogel scaffolds encapsulating millions of cells. These macroscale materials possess regions of heterogeneous photopolymerization conditions and are therefore poor platforms to identify the response of individual cells to encapsulation. Recently, microfluidic droplet-based hydrogel miniaturization and cell encapsulation offers high-throughput, reproducible, and continuous fabrication. Reports of post-encapsulation cell viability, however, vary widely among specific techniques. Furthermore, different cell types often exhibit different level of tolerance to photoencapsulation-induced toxicity. Accordingly, we evaluate the cellular tolerance of various encapsulation techniques and photopolymerization parameters for four mammalian cell types, with potential applications in tissue regeneration, using polyethylene glycol diacrylate or polyethylene glycol norbornene (PEGNB) hydrogels on micro- and macro-length scales. We found PEGNB provides excellent cellular tolerance and supports long-term cell survival by mitigating the deleterious effects of acrylate photopolymerization, which are exacerbated at diminishing volumes. PEGNB, therefore, is an excellent candidate for hydrogel miniaturization. PEGNB hydrogel properties, however, were found to have variable effects on encapsulating different cell candidates. This study could provide guidance for cell encapsulation practices in tissue engineering and regenerative medicine research.

Fig 1.

Schematic of cell encapsulation within PEGNB or PEGDA hydrogel forming solutions via either flow-focusing (a) or cross-flow microfluidic channels (b). Once formed, PEGNB droplets are collected for off-chip batch photopolymerization (a) or polymerized on-chip by UV exposure while flowing through a double layer nitrogen jacketed channel device (b).

Fig 2.

(a) Fluorescence images, and (b) size distribution of PEGNB/PEGDA microgels produced by photopolymerizing microfluidically generated droplets over a range of relative aqueous:oil flow rate ratios. Cellladen PEGNB microgels (c) are fluorescently labeled with Rhodamine B. Scale bar, 200 μm.

Fig 3.

Cell tolerance to variable photopolymerization parameters under bulk conditions. (a) A schematic of bulk photopolymerization in which cells are encapsulated within macroscale PEG hydrogels. (b) Cell viability in PEGNB or PEGDA hydrogels following photopolymerization under varying UV intensity while holding exposure time constant at 20 seconds. (c) Cell viability in PEGNB or PEGDA hydrogels with varying UV exposure times while holding intensity constant at 100 mW/cm². (d) Cell viability in PEGNB or PEGDA hydrogels formed under UV exposure at 100 mW/cm² for 20 seconds with varying photoinitiator concentrations.

Fig 4.

Cell response to changes in hydrogel surface area. (a) Fluorescent images of A549s encapsulated within 10 wt% PEGDA or PEGNB hydrogel discs or hydrogel thin films. Scale bar, 100 μm (b) Quantified initial cell viability of different polymerization schemes.

Fig 5.

Cell-dependent tolerance to encapsulation into PEGNB or PEGDA bulk hydrogel discs with varying macromer concentrations. (a) A549, (b) 3T3 fibroblast, (c) MSC, (d) β-cell.

Fig 6.

Cell-dependent tolerance to microfluidic encapsulation into PEGNB or PEGDA microgels with varying macromer concentrations. (a) A549, (b) 3T3 fibroblast, (c) MSC, (d) β-cell.

Fig 7.

Response of 4 cell types to encapsulation into 10 wt % PEGDA/PEGNB bulk or microgels over 2-week period. Given cell viability is 0% in PEGDA microgels on day 14, results are normalized to a 100% decrease from day 1.

Fig 8.

Relative performance of 4 cell types to encapsulation into 10 wt % PEGDA bulk or microgels on day 1 and day 14, normalized to corresponding PEGNB gels. On day 14, cell viability in PEGDA microgels, which is 0% for all the cases, is considered as 100% decrease compared to that in PEGNB.

Fig 9.

Relative response of 4 cell types to micro encapsulation into 10 wt % PEGDA or PEGNB microgels on day 1 and day 14, which is normalized to corresponding bulk gels. Cell viability in PEGDA microgels on day 14 is considered as 100% decrease from that in bulk gel.