Nanofibrous microposts and microwells of controlled shapes and their hybridization with hydrogels for cell encapsulation

Abstract

A simple, robust, and cost-effective method is developed to fabricate nanofibrous micropatterns particularly microposts and microwells of controlled shapes. The key to this method is the use of an easily micropatternable and intrinsically conductive metal alloy as a template to collect electrospun fibers. The micropatterned alloy allows conformal fiber deposition with high fidelity on its topographical features and in situ formation of diverse, free-standing micropatterned nanofibrous membranes. Interestingly, these membranes can serve as structural frames to form robust hydrogel micropatterns that may otherwise be fragile on their own. These hybrid micropatterns represent a new platform for cell encapsulation where the nanofiber frames enhance the mechanical integrity of hydrogel and the micropatterns provide additional surface area for mass transfer and cell loading.

Scheme 1. Fabrication of Controllably Micropatterned Alloys, Nanofibrous Membranes, and Nanofiber-Framed Hydrogel Micropatterns

(1) Press PDMS mold on melt alloy and cool to room temperature. (2) Peel off PDMS mold from solidified alloy. (3) Electrospin nanofibers on micropatterned alloy. (4.1) Peel off micropatterned nanofibrous membrane from alloy. (4.2) Cast hydrogel on nanofiber-deposited micropatterned alloy. (5) Peel off nanofiber-framed hydrogel micropatterns.

Figure 1

Microscopic images of micropatterned (a–e, k–o) PDMS molds and (f–j, p–t) alloys. Scale bars: 1000 μm.

Figure 2

(a–j) Microscopic images of free-standing micropatterned nanofibrous membranes. The insert images are magnified micropatterns. Scale bars: 200 μm. (k–p) SEM images of representative micropatterned nanofibrous membranes. Images of n–p are magnified micropatterns. Scale bars: 200 μm. The inset images are individual nanofibers at high resolution. Scale bars: 2 μm.

Figure 3

(a–j) Overlaid fluorescent images of nanofiber (red)-framed hydrogel (green) micropatterns of various geometries. Scale bars: 1000 μm. (See Figure S2 in the Supporting Information for individual channel images.)

Figure 4

Cell encapsulation in nanofiber-framed hydrogel micropatterns. (a) The scheme of two cell encapsulation methods. Method 1:1.1. Disperse non-adherent cells in alginate solution; 1.2. Encapsulate cells in cross-linked nanofiber-framed hydrogel micropatterns. Method 2:2.1. Culture adherent cells on micropatterened nanofibers; 2.2. Place alginate solution on attached cells; 2.3. Encapsulate attached cells in cross-linked nanofiber-framed hydrogel micropatterns. (b–d) The fluorescent images of INS-1 cells (non-adherent model cells) encapsulated in nanofiber-framed hydrogel micropatterns. (b) Red color is alginate hydrogel and (c) the blue color indicates cell nuclei. (e–g) Fluorescent images of encapsulated MDA-MB-231 cells (adherent model cells). (e) Red color is alginate hydrogel and (f) the green color is cells expressing GFP proteins. Scale bars: 400 μm.

Figure 5

hESCs-PPs encapsulation in nanofiber-framed hydrogel micropatterns. (a) Immunostaining of characteristic markers of hESCs-PPs. The green color is PDX1, red color is SOX9, and blue color is cell nuclei. (b, c) Microscopic images (at different magnifications) of hESCs-PPs encapsulated in nanofiber-framed alginate hydrogel micropattern. Scale bars: 100 μm.