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Tissue Engineering

Tissue Engineering - Poster Session B

Poster K19 - Development and Characterization of DLP-Printed Hydrogel Constructs for Intestinal Epithelial Cell Culture Models

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The small intestinal epithelium (SIE) is difficult to model in vitro due to its uneven topography. Villi–which are small, fingerlike projections on the surface of the SIE–are an important feature that allow for efficient nutrient absorption and waste removal, and are host to a variety of cell types, including intestinal stem cells and differentiated SIE cells. Recreating the villous morphology of the SIE using in vitro models is crucial towards understanding SIE behavior and gastrointestinal disease progression and therapeutics. Unfortunately, traditional cell culture techniques involve growing cells on flat, hard tissue culture plastic, yielding SIE models that are not physiologically accurate.

Recent advances in 3D printing has allowed for the creation of tissue culture scaffolds that better represent the villous topography of the SIE. Molding and extrusion processes have allowed for the creation of villous scaffolds, however these methods can be labor and/or time intensive. Conversely, digital light printing (DLP) has allowed previous investigators to 3D print hydrogels with fine structures.

In this study, we focused on the creation and characterization of a villous hydrogel to better replicate the native microenvironment of the SIE. To do so, we 3D printed hydrogels using digital light printing (DLP). The hydrogels were characterized using microscopy and mechanical testing. To assess their capability to serve as intestinal scaffolds, we completed biocompatibility studies using Caco-2 cells, and found that these scaffolds can serve as more accurate models to study the SIE in vitro.

Materials and

Methods:  Villous hydrogels were designed using Solidworks, a 3D computer-aided design (CAD) software. Villi were designed as 200-micron diameter columns extruded 500-microns high off of the surface of a flat base with a diameter of 8.5 milimeters (Figure 1 A-B). We printed hydrogels utilizing either polyethylene glycol-diacrylate (PEGDA) or gelatine methacrylate (GelMA) photoink solutions. We purchased four formulations of PEGDA photoinks from Cellink: PEGDA, PEGDA 200, PEGDA 500, and PEGDA Start. The GelMA photoink was formulated in-house, using 0.5% w/v LAP photoinitiator, 2% w/v methacrylated hyaluronic acid (HAMA), 0.5% w/v benzophenone-9 (R1800) photoabsorber, 0.5% w/v xanthan gum (XG), and 10% w/v GelMA. Both photoinks were used to print hydrogels using a LumenX+ DLP 3D printer (Cellink). The hydrogels were then imaged using a Keyence VHX-7000 digital microscope to measure villi height and radius (Figure 2). Compression testing was performed to assess the mechanical properties of the hydrogels. Before assessing the biocompatibility of the gels, we first leached the hydrogels of excess, autofluorescent photoink via shaking and PBS exchange. We then sterilized our samples under UV light for 24 hours in a 12-well plate before seeding Caco-2 cells at a density of 750,000 cells/well. To assess hydrogel biocompatibility, we cultured the cells for 2 days, we used live/dead staining and imaged the cells on the gels using a Nikon fluorescent microscope. Statistical analysis on morphology was completed using Graphpad Prism. A one-way ANOVA with Tukey’s HSD test was used.

Results, Conclusions, and Discussions: Hydrogels with standing villi were successfully printed with PEGDA Start, PEGDA 500, and PEGDA 200 Cellink Photoinks. The difference in average villi diameter between photoinks was not significant (Figure 3A), but the average villi height of the PEGDA 200 was significantly different (Figure 3B). This could be explained by imaging limitations when using the digital microscope; we visually observed that the height of the villi changed as the hydrogels began to dry on the microscope platform. The hydrogels were then assessed via mechanical testing. Although gel stiffness was expected to increase with the molecular weight, the mechanical testing results were inconclusive due to the presence of the villi, which seemed to inhibit the compressing . Additional mechanical testing on test coupon hydrogels without villi will be performed in the future. After 2 days of culture atop the hydrogel, the Caco-2 cells were viable and able to attach to the surface of the synthetic polymer. We hypothesize that attachment and proliferation will be increased by incorporation of extracellular matrix (ECM) proteins collagen-1 and laminin. Adding these ECM proteins to the PEGDA hydrogels is the next step and eventually, we will spatially distribute these ECM proteins to mimic the gradients that have been previously reported in the SIE. This should yield greater attachment and differentiation when using more complex cell lines.

We also printed hydrogels using our in-house GelMA formula. While we were able to crosslink the gels using the LumenX printer, additional modifications to the gel formula and adjustments to the LumenX settings are required to successfully print villi on the gels. We hypothesize that increasing the concentration of HAMA, GelMA, or XG, and increasing the irradiance intensity or exposure duration of the LumenX could increase hydrogel stiffness.

To conclude, the material makeup, mechanical properties, and surface characteristics of tissue culture scaffolds are important factors to consider when creating in vitro cell culture models. Tissue microenvironment features, including the ECM, influence differentiation and functionality of SIE cells. We have successfully created hydrogels to begin tackling the multifaceted challenge of modeling the microenvironment of the SIE.

Acknowledgements (Optional):