Fabrication of three-dimensional porous cell-laden hydrogel for tissue engineering

Abstract

For tissue engineering applications, scaffolds should be porous to enable rapid nutrient and oxygen transfer while providing a three-dimensional (3D) microenvironment for the encapsulated cells. This dual characteristic can be achieved by fabrication of porous hydrogels that contain encapsulated cells. In this work, we developed a simple method that allows cell encapsulation and pore generation inside alginate hydrogels simultaneously. Gelatin beads of 150-300 microm diameter were used as a sacrificial porogen for generating pores within cell-laden hydrogels. Gelation of gelatin at low temperature (4 degrees C) was used to form beads without chemical crosslinking and their subsequent dissolution after cell encapsulation led to generation of pores within cell-laden hydrogels. The pore size and porosity of the scaffolds were controlled by the gelatin bead size and their volume ratio, respectively. Fabricated hydrogels were characterized for their internal microarchitecture, mechanical properties and permeability. Hydrogels exhibited a high degree of porosity with increasing gelatin bead content in contrast to nonporous alginate hydrogel. Furthermore, permeability increased by two to three orders while compressive modulus decreased with increasing porosity of the scaffolds. Application of these scaffolds for tissue engineering was tested by encapsulation of hepatocarcinoma cell line (HepG2). All the scaffolds showed similar cell viability; however, cell proliferation was enhanced under porous conditions. Furthermore, porous alginate hydrogels resulted in formation of larger spheroids and higher albumin secretion compared to nonporous conditions. These data suggest that porous alginate hydrogels may have provided a better environment for cell proliferation and albumin production. This may be due to the enhanced mass transfer of nutrients, oxygen and waste removal, which is potentially beneficial for tissue engineering and regenerative medicine applications.

Figure 1

Schematics of the fabrication process for porous cell-laden alginate hydrogel. First, gelatin microspheres were prepared by adding 10% gelatin solution at 1 mL min⁻¹ into mineral oil under stirring at 600 rpm and by gelling in ice bath. Cells were mixed with the alginate solution and varying gelatin microsphere volume ratios. This solution was then molded into uniform disk-shaped hydrogels using Ca²⁺-containing agarose molds.

Figure 2

Scanning electron micrographs showing alginate hydrogels (labeled as A) with or without gelatin beads (labeled as G). Alginate hydrogel (2% w/v) without gelatin beads; (a) alginate has no macroscopic pores and (b) intrinsic porous structure with submicron size pores. Alginate hydrogel with 50% volume fraction of gelatin beads before (c) and after (d) pore generation. Alginate hydrogel with 80% volume fraction of gelatin beads before (e) and after (f) pore generation.

Figure 3

(a) Compressive moduli of porous alginate hydrogels. Values were determined from the 5 to 10% strain region of the stress–strain curve (n = 5, mean ± SD). (b) Permeability of porous alginate hydrogel. Permeability was measured by the water column method with 120 cm H₂O pressure difference. Water permeability increased about 500 times for porous alginate hydrogel. Highly porous alginate hydrogel (gel 80) showed high water flow rate and permeability (n = 3, mean ± SD). For both experiments, one-way ANOVA showed a significantly different trend across the groups (p < 0.01), whereas between group comparisons were done by Student's paired t-test, * p < 0.05; ** p < 0.01.

Figure 4

Fluorescence microscopic images of cell distribution in the hydrogels; cell membrane was labeled with a fluorescent dye (green). Distribution of cells immediately after gelling in the nonporous alginate hydrogel at day 0 (a) and after 7 days in culture (c); in porous gel 80 at day 0 (b) and after 7 days in culture (d). Dashed lines in (b) and (d) illustrate the boundaries of pores in porous gel 80 samples. Cells were closely associated with each other and distributed in the alginate walls at day 0. Cells in the nonporous gel 0 hydrogel remained rounded even after day 7 whereas those in porous gel 80 samples show cell to cell contact and spreading. 3D movie images of confocal microscopy of cell distribution in porous and nonporous hydrogels are available in the supplementary data at stacks.iop.org/BF/2/035003/mmedia.

Figure 5

Cell viability of HepG2 liver cells for 9 days in culture. (a) Bar graphs showing cell viability and (b) fluorescence microscope images showing live (green) and dead (red) cells on days 0 and 7. Cell viability on different days was not significantly different within all the hydrogels (for each porosity condition, three hydrogel discs were evaluated for cell viability. For each hydrogel disk, at least six different images were counted, one-way ANOVA, p > 0.05).

Figure 6

Cell proliferation of HepG2 by mitochondrial activity assay (WST-1) in porous alginate hydrogels. Data were normalized to control alginate hydrogel for each measurement. Mitochondrial activity significantly increased in gel 80 compared to control gel 0 condition after day 5 (mean ± SD, n = 6, Student's paired t-test, ** p < 0.01).

Figure 7

HepG2 spheroid formation after 30 days in culture. (a) Microscopic view of HepG2 spheroids in different alginate hydrogels after 30 days in culture. The spheroids occupied more hydrogel area in gel 50 and gel 80 as compared to gel 0 and gel 30. (b) Bar graph showing spheroid coverage in the hydrogels; hepatic spheroids occupied smaller area in gel 0 and gel 30 compared to higher porosity conditions (mean ± SD, n = 10 images, one-way ANOVA, p < 0.001). Spheroids occupied significantly higher area in gel 50 and gel 80 samples compared to gel 0 (Student's paired t-test, ** p < 0.01). (c) Histogram showing the % spheroid area (μm²) occupied by each size range compared to the total spheroid area; area occupied by larger size spheroids increased with porosity.

Figure 8

Analysis of albumin secretion by HepG2 spheroids encapsulated in porous and nonporous alginate. Spheroids encapsulated in hydrogels showed higher albumin secretion throughout the culture period. Higher porosity conditions enhanced albumin secretion further compared to nonporous gel 0. From day 5, gel 80 showed a significant difference compared to a nonporous condition (gel 0) (n = 3, mean ± SD, Student's paired t-test, ** p < 0.01, * p < 0.05).