Array of Biodegradable Microraftsfor Isolation and Implantation of Living, Adherent Cells

Abstract

A new strategy for efficient sorting and implantation of viable adherent cells into animals is described. An array of biodegradable micro-structures (microrafts) was fabricated using a polydimethylsiloxane substrate for micromolding poly(lactic-co-glycolic acid) (PLGA). Screening various forms of PLGA determined that the suitability of PLGA for microraft manufacture, biocompatibility and in vitro degradation was dependent on molecular weight and lactic/glycolic ratio. Cells plated on the array selectively attached to the microrafts and could be identified by their fluorescence, morphology or other criteria. The cells were efficiently dislodged and collected from the array using a microneedle device. The platform was used to isolate specific cells from a mixed population establishing the ability to sort target cells for direct implantation. As a proof of concept, fluorescently conjugated microrafts carrying tumor cells stably expressing luciferase were isolated from an array and implanted subcutaneously into mice. In vivo bio-luminescence imaging confirmed the growth of a tumor in the recipient animals. Imaging of tissue sections from the tumors demonstrated in vivo degradation of the implanted microrafts. The process is a new strategy for isolating and delivering a small number of adherent cells for animal implantation with potential applications in tissue repair, tumor induction, in vivo differentiation of stem cells and other biomedical research.

Fig. 1

A conceptual scheme to isolate and deliver adherent cells for direct animal implantation. (a) Biodegradable microraft array. (b) Cells plated on the array selectively attach to the surface of microrafts. (c) A microraft with targeted cells is dislodged from the array and collected. (d) The microraft is transferred using a syringe needle. (e) The microraft is implanted. (f) The implanted cells grow in vivo while the microrafts degrade into metabolizable small molecules.

Fig. 2

Fabrication of an array of biodegradable microrafts by a drain coating process. (a) Schematic of the fabrication process. (i) A PDMS microwell array (shown in gray) is fabricated by standard soft lithography. (ii) A polymer solution (shown in dark red) is added to the PDMS microwell array. (iii) The dewetting of polymer solution from PDMS results in an isolated convex polymer solution in each well. (iv) Evaporation of solvent results in concave polymer microrafts inside each well. (b) Setup of the drain coating process. The PDMS microwell array is hung vertically in a glass bottle. To improve visualization in this example, PLGA #10 (40 wt% in GBL) was mixed with rhodamine B (0.001 wt% of polymer). (c) SEM image of an array of biodegradable microrafts (PLGA #10, 100 µm square, 20 µm inter-raft gap). Inset shows a close-up of a disrupted section of an array.

Fig. 3

In vitro degradation of biodegradable microrafts made from PLGA #1–#5 in PBS at 37 °C. (a) Brightfield images of microrafts at different time points over a 20 day period. (b) SEM images of microrafts after 20 days in PBS at 37 °C. Scale bar is 100 µm.

Fig. 4

Cell culture and imaging on biodegradable microrafts made from PLGA #4. Brightfield (a) and SEM (b) images of H1299 cells growing on biodegradable microrafts after 48-h in culture. Brightfield (c) and fluorescence (d and e) images of four HeLa cells loaded with fluorescent dyes. Cytoplasmic staining utilized CellTracker Orange CMTMR (d), and nuclear staining was performed with Hoechst 33342 (e). The size of microrafts is 100 µm.

Fig. 5

Cell sorting with biodegradable microrafts made from PLGA #3. To enhance visualization, PLGA #3 was conjugated with TF3 dye. (a) Brightfield (top) and fluorescence (bottom) images of a region of an array containing colonies of H1299 cells possessing fluorescent cytoplasm after 72-h culture. (b) Images of the colony seen in “a” 4 h after release and collection. (c) Images of the same colony 72 h after isolation. The fluorescent daughter cells are seen to be dividing and growing off the isolated microraft. The size of microrafts is 100 µm.

Fig. 6

Implantation of AsPc-1-Luc cells in mice using biodegradable microrafts (PLGA #3). The PLGA #3 was conjugated with TF3 dye. (a) AsPC-1-Luc cells on PLGA microrafts. Left: AsPc-1-Luc cells cultured on the array for 48 h. Right: microrafts possessing cells were released from the array and collected in a separate Petri dish. The size of the microrafts was 200 µm. (b) In vivo imaging of xenograft tumor growth in a mouse after transplantation of 500 microrafts. (c) Photomicrographs of an H&E stained tissue section. The tumor was harvested from a mouse 3 months after implantation. A tumor mass at the lower of the image was surrounded by subcutaneous tissue displaying invading tumor cells and inflammatory cell infiltrate (100×). (d) Microscopic imaging of tissue sections harvested from tumors at 1 month (left) and 3 months (right) post-implantation. The remnants of PLGA microrafts were discerned by both brightfield and fluorescence imaging. Scale bar in (d) = 200 µm.