Sequential Photodegradation of Hydrogels to Spatiotemporally Control in Vitro Crypt Fission

The inner lining of mammalian intestine is composed of an epithelial layer that is responsible for digestion, absorption of nutrients, and overall protection of the intestine. For its efficient functioning, the epithelial layer is organized in a crypt-villus structure. During its normal functioning, the epithelial cells experience multiple stresses which requires their frequent turnover. Intestinal stem cells (ISCs) in the base of the crypts continuously divide and differentiate to replenish the epithelium[1].

Crypt fission is the process of a crypt dividing into two daughter crypts which is required during development, homeostasis, and crypt regrowth after injury. During development, fission widens and elongates the intestinal tract[2]. Fission in adult crypts is more of a repair mechanism allowing for homeostasis. In healthy adults, intestinal crypts can be injured due to damaged tissue from inflammation, intestinal resection, or toxic injury. It has been shown that crypt fission is increased in patients who suffer with inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease. In healthy tissue, crypt fission rates are low, and division is mainly symmetric; however, in diseased tissue, crypt fission occurs at a much higher rate with increased asymmetry. It is important to understand how crypt fission is mechanistically initiated and progresses during crypt regeneration under different physiological conditions.

The mechanisms of crypt fission and the roles of different epithelial cells in this process are not comprehensively understood due to a lack of in vitro models. The goal of this project is to create an in vitro model that simulates crypt fission that is comparable to that observed in vivo fission. This includes the mechanical and biochemical cues that drive crypt fission.

Using a Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) reaction to spontaneously form a polyethylene(glycol) (PEG) hydrogel, we encapsulate ISC derived organoids in 3D. Then, 405 nm light is utilized to degrade specific regions of the hydrogel, directing crypt formation and ideally, crypt fission. Preliminary data show bifurcation of crypts occurring at desired dimensions, and the system is being optimized to ensure optimal crypt length and width during the fission process. The system also allows differentiation of stem cells into secretory cells as observed in vivo crypts.

Ongoing studies indicate that the cell nuclei might play an active role in the process of crypt formation. Since variations in epithelium shape can dictate nuclear forces, I developed an analytic method to quantify cell shape, nuclear deformation, and protein expression which can identify pathways critical in crypt fission. Using immunostaining and integrin staining to identify specific biochemical pathways, we will observe extracellular matrix (ECM) deposition from the intestinal organoids as well as location of critical differentiated cell types, such as Paneth cells and Lgr5-GFP+ cells. Thus, this model provides a platform to study in vitro what factors regulate crypt fission and intestinal regeneration.

[1] Z. Zhao et al., Nature Reviews Methods Primers, vol. 2, no. 1, Dec. 2022.

[2] G. M. Jowett and E. Gentleman, Cell Stem Cell, vol. 28, no. 9, pp. 1505–1506, Sep. 2021.