Single-step laser-based fabrication and patterning of cell-encapsulated alginate microbeads

Abstract

Alginate can be used to encapsulate mammalian cells and for the slow release of small molecules. Packaging alginate as microbead structures allows customizable delivery for tissue engineering, drug release, or contrast agents for imaging. However, state-of-the-art microbead fabrication has a limited range in achievable bead sizes, and poor control over bead placement, which may be desired to localize cellular signaling or delivery. Herein, we present a novel, laser-based method for single-step fabrication and precise planar placement of alginate microbeads. Our results show that bead size is controllable within 8%, and fabricated microbeads can remain immobilized within 2% of their target placement. Demonstration of this technique using human breast cancer cells shows that cells encapsulated within these microbeads survive at a rate of 89.6%, decreasing to 84.3% after five days in culture. Infusing rhodamine dye into microbeads prior to fluorescent microscopy shows their 3D spheroidal geometry and the ability to sequester small molecules. Microbead fabrication and patterning is compatible with conventional cellular transfer and patterning by laser direct-write, allowing location-based cellular studies. While this method can also be used to fabricate microbeads en masse for collection, the greatest value to tissue engineering and drug delivery studies and applications lies in the pattern registry of printed microbeads.

Figure 1

Schematic of single-step laser-based bead fabrication and deposition process. (a) System overview, (b) alginate droplet ejection, and (c) in situ crosslinking of microbead.

Figure 2

Laser-fabricated microbeads; (a) Single 100-µm diameter microbead with encapsulated M231 cells. (b) Single 350-µm-diameter microbead with encapsulated M231 cells. (c) Single 500-µm-diameter microbead with encapsulated M231 cells. Representative pattern of (d) 100-µm-diameter microbeads with encapsulated cells and (e) 350-µm-diameter microbeads with encapsulated cells. Scale bars are 200 µm.

Figure 3

Influence of laser beam size on fabricated bead diameter. Error bars are one standard deviation.

Figure 4

3-D ApoTome z-stack images of laser-fabricated microbeads (a) with rhodamine dye dissolved in alginate and (b) with GFP-fluorescent M231 cells.

Figure 5

Grid patterns of adjacent microbeads with (a) 100-µm-diameter microbeads in a grid with 100-µm spacing between microbead centroids and (b) 150-µm-diameter microbeads in a grid with 175-µm spacing between microbead centroids. Scale bars are 200 µm.

Figure 6

10×10 array of microbeads. Scale bar is 500 µm.

Figure 7

Live-dead stain of M231 cells showing viability on the print ribbon (day 0), and in representative printed microbeads after 1, 3, and 5 days in culture (green live, red dead). Scale bars are 200 µm.

Figure 8

Time-lapse images of a single microbead over 7 days, illustrating that cells encapsulated inside of microbeads grow in size, while maintaining their relative position. Over time in culture, the cells are able to survive and grow, yet are immobilized within the alginate bead, showing no evidence of either migration or proliferation. Scale bars are 100 µm.

Figure 9

Schematics and images of hybrid cell/microbead array on glass cover slip shown immediately after printing (a, c, e) and after one day (b, d, f). Schematics (a, b) show the desired alternating checkerboard pattern of M231 cells (green) encapsulated in microbeads and fibroblasts (red) printed directly on the common substrate. Phase contrast images (c, d) and fluorescent images (e, f) show the printed hybrid cell/microbead construct immediately after printing (c, e), and that after 1 day in culture (d, f) the M231 cells remain encapsulated and localized within the microbeads, while growth of fibroblasts is unrestricted. Scale bar is 500 µm.

Figure 10

Proposed mechanism of laser-based bead fabrication by droplet ejection and in situ crosslinking.