Microporous cell-laden hydrogels for engineered tissue constructs

Abstract

In this article, we describe an approach to generate microporous cell-laden hydrogels for fabricating biomimetic tissue engineered constructs. Micropores at different length scales were fabricated in cell-laden hydrogels by micromolding fluidic channels and leaching sucrose crystals. Microengineered channels were created within cell-laden hydrogel precursors containing agarose solution mixed with sucrose crystals. The rapid cooling of the agarose solution was used to gel the solution and form micropores in place of the sucrose crystals. The sucrose leaching process generated homogeneously distributed micropores within the gels, while enabling the direct immobilization of cells within the gels. We also characterized the physical, mechanical, and biological properties (i.e., microporosity, diffusivity, and cell viability) of cell-laden agarose gels as a function of engineered porosity. The microporosity was controlled from 0% to 40% and the diffusivity of molecules in the porous agarose gels increased as compared to controls. Furthermore, the viability of human hepatic carcinoma cells that were cultured in microporous agarose gels corresponded to the diffusion profile generated away from the microchannels. Based on their enhanced diffusive properties, microporous cell-laden hydrogels containing a microengineered fluidic channel can be a useful tool for generating tissue structures for regenerative medicine and drug discovery applications.

Figure 1

Schematic of the fabrication process for cell-laden hydrogels containing micropores and a microchannel. (a) Sample preparation: Sucrose crystals (50–200 wt%), cells (10⁷ cells ml⁻¹) and agarose (6 wt%) solution were mixed at 40 °C. (b) Device fabrication: The mixture was cured within a PDMS cylindrical mold containing a microneedle connected between two metal tubes on PDMS side walls. (c–d) Fabrication of the microengineered hydrogels: When the mixture was confined in the PDMS mold, a microneedle was removed from the PDMS mold to create the microchannel for the microvascularized structure. (e–f) Cell culture in a device: Hepatic cells encapsulated within microporous agarose gels were cultured for 5 days within a fluidic device that could provide continuous medium perfusion.

Figure 2

Micrographs of sucrose crystals embedded in agarose gels. (A) Phase contrast images of sucrose mixtures (0–300 wt%) after natural cooling (from 40 °C to 25 °C). (B) Phase contrast images of sucrose mixtures (0–200 wt%) after rapid cooling (from 40 °C to 4 °C). (C) Phase contrast images of hydrogels derived from the formulation with 100 wt% sucrose after dissolution (37°C).

Figure 3

Microporosity and mechanical stiffness of hydrogels. (A) Images of microporosity: Phase contrast images of sucrose crystals (0–200 wt%, a–c), sucrose crystals dissolved within agarose gels (d–f), and cross-section images of agarose gels containing micropores (g–i). Confocal microscope images (j–l) and SEM images (m–o) of microporosity within agarose gels. (B) Microporosity in agarose gels with different sucrose concentrations. The percentage of the microporosity is directly proportional to sucrose concentrations. (C) Mechanical stiffness of agarose gels with sucrose concentrations. Compressive moduli were inversely proportional to sucrose concentrations. Every quantification of the above data was performed five times for each condition.

Figure 4

Diffusion profiles in agarose gels containing the microchannel and micropores. (A) Phase contrast images of a microchannel within agarose gels. (B) Phase contrast and fluorescent images of diffusion profiles in the agarose microchannels containing different micropores. These diffusion profiles of FITC-dextran (0.25 mM, 20 kDa) were evaluated under static conditions without medium perfusion. (C) The experimental and theoretical diffusion profiles of the fluorescent dye in the agarose microchannel with different sucrose concentrations (0–200 wt%) as a function of channel distances. (D) The characterization of diffusion coefficient within agarose microchannels containing different sucrose concentrations (0–200 wt%). All experiments and quantification of the above data were performed five times for each condition.

Figure 5

The viability of hepatic cells cultured within agarose gels containing different microporosities without medium perfusion. (A) Fluorescent images of the cell viability at initial time (a), after culturing for 5 days in 0 wt% (b), 100 wt% (c), and 200 wt% (d) sucrose mixtures. (B) The viability of cells near the surfaces (500 μm deep from the surface) in agarose gels with different sucrose concentrations (0–200 wt%). Cells were cultured within microporous agarose gels for 5 days in vitro. (C) The viability of cells cultured for 5 days as a function of the distance away from the agarose gel surface. All quantification of the above data was performed three times for each condition.

Figure 6

The viability of hepatic cells exposed to continuous medium perfusion from a microchannel within agarose gels. Cells were cultured in the agarose gel microchannel for 5 days in vitro. (A) Phase contrast (a, c, e) and fluorescent images (b, d, f) of cells on the cross-sections in agarose gels with different sucrose concentrations (0–200 wt%). The viability of the cells cultured for 5 days within the agarose gel channel with the medium perfusion (B) and PBS perfusion (C). The cell viability was analyzed and quantified as a function of the distance away from the microchannel surface. All quantification of the above data was performed three times for each condition.