The Effect of Thiol Structure on Allyl Sulfide Photodegradable Hydrogels and their Application as a Degradable Scaffold for Organoid Passaging

Abstract

Intestinal organoids are useful in vitro models for basic and translational studies aimed at understanding and treating disease. However, their routine culture relies on animal-derived matrices that limit translation to clinical applications. In fact, there are few fully defined, synthetic hydrogel systems that allow for the expansion of intestinal organoids. Here, an allyl sulfide photodegradable hydrogel is presented, achieving rapid degradation through radical addition-fragmentation chain transfer (AFCT) reactions, to support routine passaging of intestinal organoids. Shear rheology to first characterize the effect of thiol and allyl sulfide crosslink structures on degradation kinetics is used. Irradiation with 365 nm light (5 mW cm^-2 ) in the presence of a soluble thiol (glutathione at 15 × 10^-3 m), and a photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 1 × 10^-3 m), leads to complete hydrogel degradation in less than 15 s. Allyl sulfide hydrogels are used to support the formation of epithelial colonies from single intestinal stem cells, and rapid photodegradation is used to achieve repetitive passaging of stem cell colonies without loss in morphology or organoid formation potential. This platform could support long-term culture of intestinal organoids, potentially replacing the need for animal-derived matrices, while also allowing systematic variations to the hydrogel properties tailored for the organoid of interest.

Keywords: intestinal organoids; photodegradable hydrogels; tissue engineering.

Figure 1.

Network structure of AFCT-based photodegradable hydrogels. a Structures of the cyclooctyne functionalized PEG macromer, PEG-4DBCO, and the azide functionalized crosslinkers, AS-AA, AS-PA, AS-PhAA containing the allyl sulfide functionality. b Upon mixing, the strained octyne reacts with the azide to form a bond through strain-promoted azide alkyne cycloaddition (SPAAC), resulting in the formation of a hydrogel network incorporating the allyl sulfide functionalities. c The evolution of shear storage (black) and loss (gray) moduli are monitored with shear rheology for AS-PA (solid line), AS-AA (dotted), and AS-PhAA (dashed) crosslinkers. Hydrogels reach a similar shear modulus after 10 minutes. d A frequency sweep of the shear storage (black) and loss (gray) moduli for AS-PA (solid line), AS-AA (dotted), and AS-PhAA (dashed) crosslinked hydrogels shows a lack of frequency dependence, indicative of elastic networks.

Figure 2.

The effect of thiol structure on allyl sulfide hydrogel degradation kinetics. a Reaction mechanisms for AS-PA, AS-AA, and AS-PhAA crosslinkers, resulting in the generation of network tethered thiol species with pKa values of 10.4, 8.8, and 6.6, respectively. b AS-PA (red) and AS-AA (green) hydrogels undergo reverse gelation when degraded with the thiol, 3MMP. AS-PhAA crosslinked hydrogels do not achieve reverse gelation. c Apparent rate constants for photodegradation of crosslinkers AS-PA, AS-AA, and AS-PhAA using 3MMP are shown. Significance determined by one-way ANOVA, N=3, mean ± s.d., *p < 0.05, ***p < 0.001. d. The extent of degradation of AS-PA hydrogels decreases when degraded with thiols of decreasing pKa. The thiols, 3MMP (solid), glutathione (GSH, dash), MTG (dot), and TAA (dot-dash), have pKa values of 10.4, 9.42, 8.8, and 3.6, respectively. e Apparent rate constants for photodegradation of AS-PA crosslinking using 3MMP, GSH, MTG, and TAA are shown. Significance determined by one-way ANOVA, N=3, mean ± s.d., **p < 0.01, ***p < 0.001. Hydrogels were equilibrated with 15 mM soluble thiol and 1 mM lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) at pH 7.4 and exposed to light (365 nm, 5 mW cm⁻²) at t = 0. All rheological plots show a representative trace from N=3.

Figure 3.

The effect of aromatic thiols on allyl sulfide hydrogel degradation kinetics. a. When equilibrated with the aromatic thiol, 4MPAA, AS-PhAA (blue) hydrogels degrade rapidly, while AS-PA (red) and AS-AA (green) hydrogels experience limited degradation. b Apparent rate constants for photodegradation of crosslinkers AS-PA, AS-AA, and AS-PhAA using 4MPAA are shown. Significance determined by one way ANOVA, N=3, mean ± s.d., **p < 0.01. c AS-PhAA crosslinked hydrogels equilibrated with 4MPAA degrade more rapidly at pH 5 (dash) than at pH 7.4 (solid). d Apparent rate constants for photodegradation of AS-PhAA using 4MPAA at pH 5 and pH 7.4 are shown. Significance determined by t test, N=3, mean ± s.d., **p < 0.01. Hydrogels equilibrated with 15 mM soluble thiol and 1 mM LAP at pH 7.4 (or pH 5) were exposed to light (365 nm, 5 mW cm⁻²) at t = 0. All rheological plots show a representative trace from N=3.

Figure 4.

The effect of thiol oxidation state and initiator type on allyl sulfide hydrogel degradation kinetics. a AS-PA hydrogels equilibrated with glutathione (GSH, black) and glutathione disulfide (GSSG, grey) display similar degradation kinetics when degraded using 1mM LAP and 365nm light (5 mW cm⁻²). b Apparent rate constants for photodegradation of AS-PA using GSH and GSSG are shown. Significance determined by t test, N=3, mean ± s.d.. c Degradation is slower using GSH and LAP at higher wavelengths, such as 405 nm light (solid black). However, using 405 nm light and the photoinitiator eosin Y, degradation is more rapid with GSH (black dot), while GSSG (grey dot) exhibits little degradation. d Apparent rate constants for photodegradation of AS-PA using thiols, GSH and GSSG, and photoinitiators, LAP and eosin y, are shown. Significance determined by one way ANOVA, N=3, mean ± s.d., ***p < 0.001. Hydrogels equilibrated with 15 mM soluble thiol and 1 mM photoinitiator at pH 7.4 were exposed to light (365 nm or 405/436, 5 mW cm⁻²) at t = 0. All rheological plots show a representative trace from N=3.

Figure 5.

Allyl sulfide photodegradable hydrogels support the expansion and repetitive passaging of intestinal organoids. a Shear rheology of swollen allyl sulfide hydrogels shows a correlation between macromer concentration and shear storage modulus. b The efficiency of colony formation from single encapsulated ISCs shows a dependence on the macromer concentration. c Allyl sulfide photodegradation was used to repetitively passage ISC colonies (p1, p2, p3), which maintained polarized epithelium, as seen by DAPI (blue) and f-actin (green) staining. Scale bars 50μm. d Relative mRNA expression for intestinal genes was similar in organoids grown in allyl sulfide photodegradable hydrogels compared to Matrigel controls. Significance determined by one-way ANOVA, N = 3, mean ± s.d., ***p < 0.001. e Colonies were formed at a slightly decreasing rate upon repetitive passaging. Open circles represent average measurements from one gel replicate, while bars represent the average of the three gel replicates.

Figure 6.

Allyl sulfide photodegradation maintains stem cell differentiation potential. Allyl sulfide hydrogels laden with colonies were degraded to release the encapsulated colonies, which were encapsulated into Matrigel and cultured with differentiation media (lacking CHIR99021 and valproic acid). a After 3 days, buds had protruded off of the colonies to form crypts, which immunostained for lysozyme (red), an indicator of Paneth cells and a mature crypt, as well as DAPI (blue) and f-actin (green). b Staining for EdU (red, 24 hour pulse) and DAPI (blue) showed proliferative cells residing in the crypt ends. Scale bars 50μm. c A low percentage of organoids formed from ISC colonies expanded in allyl sulfide gels and transplanted into Matrigel formed crypts containing lysozyme (22.5 ± 1.0 %, N = 3) and EdU positive cells (27.5 ± 0.9 %, N = 4). Mean ± s.d..

Scheme 1.

Light initiated radical network degradation. a Thiyl radicals react with an allyl sulfide to form a radical intermediate that subsequently undergoes beta scission, resulting in the fragmentation of crosslinks and the regeneration of a thiyl radical and allyl sulfide. b A soluble thiol species (red) exchanges with the allyl sulfide, resulting in a cleaved crosslink and the generation of a network tethered thiyl radical. The released network thiyl radical undergoes chain transfer to a soluble thiol species, which can initiate further exchange reactions, amplifying the degradation process.