Intestinal organoid protocols rely on the use of extracellular scaffolds, typically Matrigel, and upon switching from growth to differentiation promoting media, a symmetry breaking event takes place. During this stage, the first bud like structures analogous to crypts protrude from the central body and differentiation ensues. While organoids provide unparalleled architectural and functional complexity, this sophistication is also responsible for the high variability and lack of reproducibility of uniform crypt‐villus structures. If function follows form in organoids, such structural variability carries potential limitations for translational applications (e.g., drug screening). Consequently, there is interest in developing synthetic biomaterials to direct organoid growth and differentiation. It has been hypothesized that synthetic scaffold softening is necessary for crypt development, and these mechanical requirements raise the question, what compressive forces and subsequent relaxation are necessary for organoid maturation? To that end, allyl sulfide hydrogels are employed as a synthetic extracellular matrix mimic, but with photocleavable bonds that temporally regulate the material's bulk modulus. By varying the extent of matrix softening, it is demonstrated that crypt formation, size, and number per colony are functions of matrix softening. An understanding of the mechanical dependence of crypt architecture is necessary to instruct homogenous, reproducible organoids for clinical applications.
3D organoid models have recently seen a boom in popularity, as they can better recapitulate the complexity of multicellular organs compared to other in vitro culture systems. However, organoids are difficult to image because of the limited penetration depth of high‐resolution microscopes and depth‐dependent light attenuation, which can limit the understanding of signal transduction pathways and characterization of intimate cell‐extracellular matrix (ECM) interactions. To overcome these challenges, phototransfer by allyl sulfide exchange‐expansion microscopy (PhASE‐ExM) is developed, enabling optical clearance and super‐resolution imaging of organoids and their ECM in 3D. PhASE‐ExM uses hydrogels prepared via photoinitiated polymerization, which is advantageous as it decouples monomer diffusion into thick organoid cultures from the hydrogel fabrication. Apart from compatibility with organoids cultured in Matrigel, PhASE‐ExM enables 3.25× expansion and super‐resolution imaging of organoids cultured in synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked via allyl‐sulfide groups (PEG‐AlS) through simultaneous photopolymerization and radical‐mediated chain‐transfer reactions that complete in <70 s. Further, PEG‐AlS hydrogels can be in situ softened to promote organoid crypt formation, providing a super‐resolution imaging platform both for pre‐ and post‐differentiated organoids. Overall, PhASE‐ExM is a useful tool to decipher organoid behavior by enabling sub‐micrometer scale, 3D visualization of proteins and signal transduction pathways.
more » « less- Award ID(s):
- 2033723
- NSF-PAR ID:
- 10446450
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Materials
- Volume:
- 34
- Issue:
- 16
- ISSN:
- 0935-9648
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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