(T1430-05-32) The Printability of an Alginate-Based Hydrogel and Its Compatibility with NIH-3T3 Cells

Purpose: Since taking medication via the oral route is ranked among patients as the most convenient, there is a valid effort to create a model that would simulate absorption of drugs in in-vitro conditions. These models could be ideally applicable in the preclinical phase, which could lead to the increase of the safety of newly developed drugs. However, the in-vivo absorption is a complex process with a great number of variables. The general idea is to create a 3D villi-like structure of a bilayer architecture – the layer of enterocytes (Caco-2 cells at first) would be cultivated on top of 3D printed layers of fibroblasts, serving as a support, thus making the environment more “human-like”. 

Methods: Fabrication of villi-like constructs was achieved with 3D bioprinter (BIOX, Cellink, Sweden). Parameters such as temperature, printing pressure, infill density, nozzle diameter and the alginate-gelatin hydrogel composition was investigated. Cells encapsulated in the hydrogel were observed with laser scanning confocal microscopy (Olympus Fluoview 1000-FV, Olympus, Japan). In order to determine the cell viability, cells were stained with calcein-am (Thermo Fisher, Waltham, USA), dead cells were stained with ethidium homodimer-1 (Thermo Fisher, Waltham, USA). Cell viability was also determined quantitatively using AlamarBlue assay (resazurin sodium salt, Sigma-Aldrich, USA). The cell morphology was studied by staining cells with Texas Red dye (Sigma Aldrich, USA) which colors cell cytoskeleton and Hoechst 33342 (Sigma Aldrich, USA) revealing cell nucleus. 

Results: Hydrogel composition 3.2% alginate, 2% gelatin showed good fabrication properties, constructs were well defined. However, cell-laden hydrogel of the same composition had different mechanical properties making the printed villi or grid rather melted, unable to hold the wanted structure. After establishing the cell encapsulation process, we observed the survival of the NIH-3T3 cells encapsulated in the hydrogel. The Live/Dead assay was performed and stained cells were observed with the light scanning confocal microscopy. We were able to confirm that cells can live and proliferate in the alginate-gelatin hydrogel for a minimum of 10 days. Furthermore, we focused on the NIH-3T3 cell morphology which when adhered naturally occur in star-like shape, characteristic for fibroblasts. The morphology of NIH-3T3 cells encapsulated in the hydrogel was rather circular. This may be attributed to the fact, that alginate-based hydrogels lack the number of adhesive sites to which cells can bind. As per the approach mentioned in the "Purpose" section, we determined if the co-culture of Caco-2 cells and NIH-3T3 cells is possible. We found out that the Caco-2 cells can grow and proliferate on top of the NIH-3T3 cells.

Conclusion: We are able to 3D bioprint structures simillar to villi present in the small intestine. However, bioprint of cell-laden hydrogels is not ideal and need further optimization. The process of cell encapsulation was established. Caco-2 cells (simulate absorption) are able to grow on NIH-3T3 cells (simulate support layer). NIH-3T3 cells are capable of surviving both the encapsulation process and the incubation for a minimum of 10 days.

References: [1] Madden, L. R.; et al. Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME/Tox Functions. iScience 2018, 2, 156–167.​
[2] Shahiwala, A. Formulation approaches in enhancement of patient compliance to oral drug therapy. Expert Opinion on Drug Delivery 2011, 8 (11), 1521–1529.​
[3] Press, B.; Grandi, D. Permeability of Intestinal Absorption: Caco-2 Assay and Related Issues. Current Drug Metabolism 2008, 9, 893–900.​
[4] Caliari, S. R.; Burdick, J. A. A practical guide to hydrogels for cell culture. Nature Methods 2016, 13, 405–414.

Acknowledgements: The financial support from the Specific University Research (MSMT), of the Czech Science Foundation (project No. GX19-26127X).

Graphically summarized experimental procedure of construct preparation.

3D bioprinted villi-like structures with printing parameters. (a) 3D model of villi-like construct, (b) and (c) 3D bioprinted villi-like structure, grid dimensions: 10.5x10.5x0.2 mm, villus dimensions: 1x1x1 mm, distance between each villi: 1.5 mm.

(a) NIH-3T3 cells ecapsulated in an alginate-based hydrogel day 1, (b) NIH-3T3 cells encapsulated in the hydrogel day 10, (c) NIH-3T3 cells cultured on a Petri dish. Cells were stained with calcein am labeling live cells, observed with laser scanning confocal microscopy - excitation: 495 nm, emission: 515 nm.