ExCeL: combining extrusion printing on cellulose scaffolds with lamination to createin vitrobiological models

Abstract

Bioprinting is rapidly developing into a powerful tool in tissue engineering, for both organ printing and the development of in vitro models that can be used in drug discovery, toxicology and in vitro bioreactors. Nevertheless, the ability to create complex 3D culture systems with different types of cells and extracellular matrices integrated with perfusable channels has been a challenge. Here we develop an approach that combines the xurography of a scaffold material (cellulose) with extrusion printing of bioinks onto it, followed by assembly in a layer-by-layer fashion to create complex 3D culture systems that could be used as in vitro models of biological processes. This new method, termed ExCeL, can recapitulate the complexities of natural tissues with a proper 3D distribution of cells, extracellular matrices, and different molecules, while providing the whole structure with mechanical stability, and direct and easy access to the cells, even the ones that are positioned deep in the bulk of the structure, without the need to fix or section the samples. Briefly, the bioprinting of predefined patterns with a feature size of ∼1 mm has been made possible by treating paper with the hydrogel’s crosslinker and printing cell-embedded hydrogel that will solidify immediately upon contact with the paper. These impregnated layers can be used as single layers or in a layer-by-layer approach by stacking them (here up to four layers) for applications such as cell migration and proliferation in 3D structures composed of collagen or alginate. Cells are generally sensitive to the amount of FBS in their culture media and we have shown how FBS amount will effect cell migration. By cutting the paper in certain patterns, printing hydrogel on the remaining parts of it, and stacking the paper in layers, features like embedded channels are formed that will provide cells will better mass transfer in thick structures. This technique provides biologists with a unique tool to perform sophisticated in vitro assays.

A Bioprinted In Vitro Model for Osteoblast to Osteocyte Transformation by Changing Mechanical Properties of the ECM

“…Alginate as a material lends itself well to the ExCeL process, which allows for tuning of the gel stiffness, compared to collagen or gelatin. [25] Additionally, it is also the most commonly used hydrogel in bioprinting processes and has seen success printing with Saos-2 cells. [26,27] These hydrogels crosslink immediately upon contact with the paper and reside on top of it thus giving it mechanical properties independent of the paper.…”

“…A 3D in vitro model was developed using the ExCeL bioprinting technique [25] to study effect of mechanical properties of ECM on osteoblast to osteocyte phenotype transformation. Figure 1 summarizes this process.…”

“…Figure 1 summarizes this process. [25] Additionally, it is also the most commonly used hydrogel in bioprinting processes and has seen success printing with Saos-2 cells. The first condition is referred to further as the low calcium (Low-Ca) content paper, and the second the high calcium (High-Ca) content paper).…”

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges

“…Hydrogels have several uses for tissue engineering. They can be used as a soft 3D ECM‐like environment, [ 115 , 119 , 226 , 227 , 228 , 229 , 230 , 231 , 232 , 233 , 234 , 235 , 236 , 237 , 238 , 239 ] as a 3D matrix filler inside porous scaffolds, [ 240 ] as components of bioinks, [ 34 , 121 , 187 , 222 , 241 , 242 , 243 , 244 , 245 , 246 ] as thin membranes which may be microstructured to produce alignment of cells, [ 126 , 247 ] or as source material to develop porous scaffolds. [ 23 , 248 , 249 ] For the first three uses, cytocompatible gelation is essential, as the cells are introduced into the hydrogel liquid solution before the hydrogel solidifies.…”

A Tailored Biomimetic Hydrogel as Potential Bioink to Print a Cell Scaffold for Tissue Engineering Applications: Printability and Cell Viability Evaluation

“…Besides, the fact that not every hydrogel is repeatable to proceed by the printer leads to the quantification of hydrogel printability, which is sometimes rough and resulting in lack of hydrogel bioink [6,8]. Furthermore, a major obstacle to achieve a better encapsulation of cells and maintain cell viability after 3D printing procedure still present as challenging issues to be resolved [6,10,15]. Due to these facts, the improvement of a printable hydrogel that can maintain a stable structure and facilitate the growth and function of the cell is demanded to escalate the existing approach in tissue engineering [7,12,16].…”