A biofabrication technology for generating multiscale channels in hydrogels for complex 3D in vitro co-cultures


Seijas-Gamardo, Adrián (Complex Tissue Regeneration department (CTR), MERLN Institute )


Native tissues are characterized by its 3D organization and distribution of cells, with specific cell-cell and cell-extracellular matrix (ECM) interactions dictating tissue function. The spatial distribution of cells and ECM in tissues is not arbitrary. There are specifically located cell populations for generating interconnected lumen structures, creating a fundamental structure-function relationship that determines the role of numerous tissues. Therefore, the capability to mimic this 3D environment is key for a correct in vitro modeling of tissues and for future tissue engineering applications.
Here we present a templating strategy using a thermally responsive polymer and we show the fabrication, in one step, of a network of interconnected channels within a hydrogel. While other polymers have been previously described for similar applications, we uniquely show that our template can create a defined 3D polymer scaffold to which cells can adhere, leading to the subsequent formation of a channel network that directly incorporates cells. This approach is based on a family of oxazoline polymers (POXA) that have been specifically designed to have a uniquely tunable range of lower critical solubility temperature (LCST), above which it remains insoluble and below which it is triggered to dissolve.
We were able to produce fibers with our POXA polymer with diameters as small as 5µm, up to the millimeter scale using melt electrowriting (MEW). We recorded stereomicroscope time lapses of their dissolutions while decreasing the temperature in a controlled way from 37°C to 4°C. Additionally, we monitored its water contact angle at different temperatures, confirming their change in stability and solubility. By using a customized fabrication method, we could include microchannels in a set of hydrogels with different crosslinking mechanisms: collagen, PEGDA, polyacrylamide and fibrin. Additionally, we were able to culture Schwann cells (SC) and HUVECs on top of these micro-scale fibers and transfer them to a hydrogel where the polymer would be dissolved, leaving cells growing in specific patterns inside of microchannels. With our method, we could fabricate in vitro models for vasculature by culturing HUVECs on the lumen of the channels and other cell types such as fibroblasts embedded in the surroundings. Allowing us to control the arrangement of the vascular channel as well as their interactions with the environment. We also showed how SC could migrate along these microchannels, as they would do in vivo, this is a key mechanism during tissue innervation, guiding axonal growth towards other tissues. Similarly, we cultured sensory neurons (nociceptors) derived from human iPSCs and showed how their axons could grow along the microchannels. Potentially becoming a new tool for nerve repair and tissue innervation studies or drug testing in clinical applications.
Here, we demonstrate the value and versatility of this novel templating technology to generate complex 3D cell culture models. We believe that this system will allow the tissue engineering field to fabricate more realistic in vitro models considering cellular arrangement and EMC interactions.


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