Speaker
Description
Introduction
Polymer-based hydrogels serve as excellent mimics of the extracellular matrix, enabling the generation of 3D in vitro tissue models.1 To improve the ability of these models to replicate tissue in vivo, there is great interest in enhancing model complexity.2 Towards this goal, the utilization of photoresponsive chemistries (e.g. polymerization/degradation) permits precise user-defined control over hydrogel properties to match tissue structure in vivo. Furthermore, the synergy between photoresponsive chemistries and biofabrication platforms (e.g. 3D extrusion bioprinting, photolithography) permits tissue models to achieve clinically relevant size scales.3 Unlike photopolymerizable bioinks, the generation of photodegradable bioinks has remained cost prohibitive and synthetically challenging, ultimately limiting the achievable complexity of in vitro tissue models. Here, we discuss our use of photodegradable chemistries to probe mechanisms involved in organoid development. We then present an inexpensive and easily accessible photodegradable bioink based on radical induced thiol-maleimide cleavage4 that enables photodegradation of large, cell-laden constructs to generate clinically relevant tissue constructs.
Methods
Intestinal organoids were encapsulated in photoresponsive hydrogels containing either allyl sulfide or nitrobenzyl ether functionalized polymers. A laser scanning confocal microscope was used to apply patterned light (405nm) to organoid-laden hydrogels. Live imaging and immunohistochemistry were used to evaluate organoid behavior following hydrogel photopatterning. Gelatin maleimide (GelMal) was synthesized by reacting a maleimide functionalized small molecule with gelatin using EDC/NHS chemistry. GelMal hydrogels were formed with varying concentrations of a multifunctional thiol. Shear rheology (1 Hz, 1 rad/s) was used to assess hydrogel mechanical properties following thiol gelation, and during photodegradation (400-500 nm, 30 mW cm-2). Centimetre-scale GelMal hydrogel constructs were then formed using an extrusion bioprinter and photodegraded using bulk irradiation.
Results
Bulk and spatially defined irradiation of organoid-laden hydrogels revealed that organoid development is dependent on hydrogel stiffness and epithelial shape, providing variables to direct organoid form and function. With the desire to apply photodegradation reactions to large-scale organoid constructs, we sought to develop a scalable photodegradable bioink. The addition of a multifunctional thiol crosslinker to GelMal induced the formation of thiol-maleimide crosslinks, where hydrogel mechanical properties were tuned using the thiol crosslinker concentration. Following GelMal hydrogel formation, a photoinitiator was added to generate radicals upon exposure to UV light, which cleaved thiol-maleimide crosslinks to facilitate controlled photodegradation. Photoshear rheology revealed rapid GelMal degradation in response to light exposure that was dependent on photoinitiator concentration and light dose. Centimeter-scale constructs were fabricated using extrusion bioprinting and fully degraded within minutes, highlighting the opportunity to utilize photodegradation reactions and biofabrication techniques to generate large-scale tissue architectures.
Discussion
We highlight the importance of photodegradation as a method to explore complex biological mechanisms and introduce the need for scalable photodegradable platforms to enhance the physiological relevance of tissue models.
References
MW Tibbitt, et al., Biotechnology and bioengineering, 103.4 (2009): 655-663
FM Yavitt, et al., ACS biomaterials science & engineering 8.11 (2022): 4634-4638.
L Moroni, et al., Trends in biotechnology, 36.4 (2018): 384-402.
TS Hebner, et al., Advanced Science 11.25 (2024): 2402191.
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