Biocompatible PEGDA Resin for 3D Printing

Abstract

We report a non-cytotoxic resin compatible with and designed for use in custom high-resolution 3D printers that follow the design approach described in Gong et al., Lab Chip 17, 2899 (2017). The non-cytotoxic resin is based on a poly(ethylene glycol) diacrylate (PEGDA) monomer with avobenzone as the UV absorber instead of 2-nitrophenyl phenyl sulfide (NPS). Both NPS-PEGDA and avobenzone-PEGDA (A-PEGDA) resins were evaluated for cytotoxicity and cell adhesion. We show that NPS-PEGDA can be made effectively non-cytotoxic with a post-print 12-hour ethanol wash, and that A-PEGDA, as-printed, is effectively non-cytotoxic. 3D prints made with either resin do not support strong cell adhesion in their as-printed state; however, cell adhesion increases dramatically with a short plasma treatment. Using A-PEGDA, we demonstrate spheroid formation in ultra-low adhesion 3D printed wells, and cell migration from spheroids on plasma-treated adherent surfaces. Given that A-PEGDA can be 3D printed with high resolution, it has significant promise for a wide variety of cell-based applications using 3D printed microfluidic structures.

Keywords: 3D printing; biocompatibility; cytotoxicity; microfluidics; resin; spheroid.

Figure 1:

3D printed NPS-PEGDA (left) and A-PEGDA (right) substrates for cellular cytotoxicity and adherence testing. Samples are still attached to their glass substrates from 3D printing.

Figure 2:

Spheroid migration characterization.

Figure 3:

(a) Cell viability for NPS-PEGDA as a function of wash time for water and ethanol. The result for unwashed A-PEGDA is also shown (corresponding error bar is at the left of the dashed green line). Data were normalized against a TCPS control seeded at the same density. Error bars indicate standard deviation of replicates (n≥3). (b)-(e) Stained EA.hy926 cells for (b) TCPS (control), (c) as-printed NPS-PEGDA (i.e., no washing) (d) NPS-PEGDA washed in ethanol for 24 hours, and (e) as-printed A-PEGDA. Scale bars are 100 μm.

Figure 4:

(a) Cell viability of EA.hy926 cells adhered to plasma-treated A-PEGDA and NPS-PEGDA. Data are shown as the fractional surface coverage of the cells normalized to a TCPS control. Error bars indicate standard deviation. The average and standard deviation are, respectively, 0.959 and 0.092 for A-PEGDA and 0.848 and 0.052 for NPS-PEGDA. Images of adhered and stained EA.hy926 cells for plasma-treated (b) unwashed A-PEGDA and (c) washed NPS-PEGDA, and (d) washed NPS-PEGDA with no plasma treatment. Scale bars are 100 μm.

Figure 5:

Characterization of 3D growth and migration. a-d) migration of cells from spheroids in flat printed plates, where: a) workflow that includes transfer from low-adhesion seeding and growth well to a 3D printed A-PEGDA well; b) phase-contrast images of spheroids 24hr post-transfer to the plates (NT = no plasma treatment) where zoom images show migrated cells out of the spheroid (yellow arrow); and quantifications of cell migration according to Fig. 2 where c) is the migration area and d) is the migration area normalized to the spheroid area. Significance is based on the Kruskal-Wallis non-parametric test with sample size of 3 (TC-PS), 7 (PEGDA NT), and 3 (PEGDA + Plasma); bars indicate mean and standard deviation; points are observed individual values. e) 3D growth in printed A-PEGDA micro 6-well device. The red box shows 3D spheroid growth in epithelial cell (A549) culture.

Figure 6:

Endothelial (EA.hy-926) 3D cell growth in V-slope flat-bottom A-PEGDA printed devices with varying bottom diameters of 700 μm (a,d,g), 200 μm (c,e,h), and 100 μm (c,f,i). CAD designs (a-c) and representative phase microscopy images (d-i). Zoom images are shown to illustrate 3D networks that formed.

Figure 7:

Endothelial (EAhy)-fibroblast(HFL1) coculture shows enhanced 3D formation on a low-adherence A-PEGDA device.