Development of multilayered cell-hydrogel composites using an acoustic focusing technique

Abstract

Multilayered composites, composed of mammalian cells arranged in a hydrogel, have been prepared using an acoustic focusing technique. Acoustic focusing is a simple, nonchemical technique that allows for the fast arrangement of cells in matrices where the control of cell geometry is beneficial. Breast cancer cells (MDA-MB231) were dispersed in a 30 wt % solution of poly(ethylene glycol) diacrylate (PEGDA) of molecular weight 400 at a density of 5 x 10(6) cells/mL of PEGDA solution. An ultrasonic field was used to organize the cells before polymerization of PEGDA. Disk-shaped hydrogel composites, typically 1 cm in diameter and 2-mm thick were prepared based on a PEGDA solution volume of 130 microL. At an acoustic frequency of 2.32 MHz, composites having cells positioned within concentric cylindrical shells interspersed with zones of cell-free hydrogel were produced. The cells were located in annuli approximately 80-microm thick and about 300 microm apart. The structure and viability of the cells within these constructs were studied using a fluorescent LIVE/DEAD assay. The viability of the cells was on the order of 50%. For the conditions used in this study, cell death was primarily attributed to exposure of cells to the PEGDA solution prior to polymerization, rather than adverse effects of polymerization or the sound field itself.

Figure 1

Schematic of cylindrical acoustic chamber.

Figure 2

Arrangement of cells at radial positions emanating from a cylindrical sound source

Figure 3

Bright-field image of microtome slice of inner rings composed of cancer cells arranged in 30 wt % PEGDA disc at a concentration of 1 × 106 cells per ml of fluid. Scale bar is equal to 300 μm. Distortions and the tear in the image was the result of the methacrylate embedding process.

Figure 4

Fluorescent images of live (a) and dead cells (b) after acoustic focusing in a PEGDA solution, following by UV light photopolymerization. The frequency of the sound field was 2.32 MHz and the cell initial concentration was 5 × 106 cells/ml. The LIVE/DEAD assay was applied 40 hr after encapsulation and incubation. Scale bar is equal to 500 μm.

Figure 5

Fluorescent images of living (a) and dead (b) cells exposed to no sound field or UV-light in cell growth media; images of living (c) and dead (d) cells exposed to 20 min of 2.32 MHz ultrasound at and 4 V_RMS with no UV-light. Images indicated that there was no significant cell death from application of the sound field. The cell concentration was equal to 5 × 10⁶ per ml of fluid. The scale bar is equal to 500 μm.