Fully Synthetic Hydrogels Promote Robust Crypt Formation in Intestinal Organoids

Abstract

Initial landmark studies in the design of synthetic hydrogels for intestinal organoid culture identify precise matrix requirements for differentiation, namely decompression of matrix-imposed forces and supplementation of laminin. But beyond stating the necessity of laminin, organoid-laminin interactions have gone largely unstudied, as this ubiquitous requirement of exogenous laminin hinders investigation. In this work, a fast stress relaxing, boronate ester-based synthetic hydrogel is used for the culture of intestinal organoids, and it is fortuitously discovered that unlike all other synthetic hydrogels to date, laminin does not need to be supplemented for crypt formation. This highly defined material provides a unique opportunity to investigate laminin-organoid interactions and how it influences crypt evolution and organoid function. Via fluorescent labeling of non-canonical amino acids, it is further shown that adaptable boronate ester bonds increase deposition of nascent proteins, including laminin. Collectively, these results advance the understanding of how mechanical and matricellular signaling influence intestinal organoid development.

Keywords: biomaterials; extracellular matrix; intestinal organoids; laminin; stress relaxing hydrogels.

Figure 1.. Boronate ester PEG hydrogel chemistry

a) Schematic of permanent strain promoted azide alkyne cycloaddition (SPAAC) reaction between a dibenzocyclooctyne (gray) and an azide group (green). b) a schematic of the reversible covalent adaptable bond between a fluorophenylboronic acid group (blue) and a nitrodopamine (yellow). c) representation of the boronate ester hydrogel network composed of an 8-arm PEG DBCO, an 8-arm PEG with 2 arms functionalized with azide groups and 6 arms functionalized with fluorophenylboronic acid, and an 8-arm PEG with 2 arms functionalized with azide groups and 6-arms functionalized with nitrodopamine. d) Boronate ester hydrogels relax stress rapidly, with 80% of the network stress relaxed on the order of seconds regardless of macromer weight percent. e) The relaxation time constant, τ, for each condition was calculated by curve fitting the averages of normalized stress data from (d) to a one component Kohlrausch-Williams-Watts function in Matlab. The hydrogels relax faster than Matrigel relax but to the same final extent (n = 3 for 4.2 wt%, 4.5 wt% and Matrigel, n = 1 for 3.8 wt %). f) Boronate ester hydrogel moduli are tuned by varying macromer dweight percent (n = 7 for 3.8, 4.2 wt%, n = 6 for 4.6 wt%, mean ± s.d., *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, one-way ANOVA). g) Intestinal organoid viability was highest in 4.2 wt% hydrogels and was used in subsequent studies (n = 3, ns ≥ 0.05, mean ± s.d., *P ≤ 0.05, **P ≤ 0.01, one-way ANOVA). h-j) Viability of colonies was assessed by visualization of epithelial polarization as marked by F-actin localization (green) Scale bar 100 μm.

Figure 2.. Boronate ester hydrogels promote intestinal organoid differentiation in the absence of supplemental laminin

a) Spherical, intestinal stem cell colonies were isolated from Matrigel and added to a solution containing 8-arm PEG dibenzocyclooctyne (DBCO) and azide functionalized RGD. 8-arm PEG fluorophenylboronic acid (FPBA)/azide and 8-arm PEG nitrodopamine (ND)/azide were simultaneously added, initiating spontaneously polymerization of the hydrogel network. The samples were cultured in differentiation media and after 48 hours the intestinal organoids were fixed. b) The crypt forming efficiency of intestinal organoids cultured in boronate ester hydrogels without exogenous laminin was as robust (− Lam) as when they were cultured in hydrogels with added laminin (+Lam) (+ Lam (n = 5), − Lam (n = 7), Matrigel (n = 3)) ; mean ± s.d., ns ≥ 0.05, one-way ANOVA). c) Intestinal organoid cultured in hydrogels without supplemental laminin (−Lam). d) with supplemental laminin (+Lam) and e) cultured in laminin rich Matrigel. Scale bar 100 μm, white arrows denote location of Paneth cells in crypts. Portions of this figure were created using Biorender.com.

Figure 3.. Laminin-organoid interactions influence measures of crypt architecture

a) Intestinal organoids largely interact with laminin via the α6β4 integrin. When either an α6 integrin subunit blocking antibody (+ Anti α6) or a β4 integrin subunit blocking antibody (+Anti β4) are added to media, organoid interactions with laminin are inhibited. b) the crypt forming efficiency of organoids in boronate ester hydrogels dramatically decreases, whether exogenous laminin has been added to the matrix (+ Lam) or not (−Lam) (− Lam (n = 7 hydrogels) ,+ Lam (n = 5 hydrogels, Matrigel (n = 3 hydrogels)) and are from Fig 2. +Anti α6 − Lam (n = 7 hydrogels), +Anti β4 − Lam (n = 6 hydrogels), +Anti α6 + Lam (n = 5 hydrogels), + Anti β4 + Lam (n = 5 hydrogels), mean ± s.d. ns ≥ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, one-way ANOVA). c) For organoids with blocked laminin interactions that still formed crypts, the crypts were smaller (n = 1041 crypts analyzed for Matrigel, n = 110 crypts analyzed for − Lam, 15 for + Lam, 22 for + Anti α6 − Lam, 31 for + Anti β4 − Lam, 31 for + Anti α6 + Lam, and 22 for + Anti β4 + Lam, each from at least 3 hydrogels per condition, mean ± s.d., ns ≥ 0.05, ****P ≤ 0.0001, one-way ANOVA) and d) there were fewer crypts per colony (n = 109 organoids analyzed for Matrigel, n = 32 organoids analyzed for − Lam, 30 for + Lam, 28 for + Anti α6 − Lam, 34 for + Anti β4 − Lam, 21 for + Anti α6 + Lam, and 10 for + Anti β4 + Lam, each from at least 3 different hydrogels, violin plot, ns ≥ 0.05, ****P ≤ 0.0001, one-way ANOVA). e) Organoids cultured in Matrigel averaged more crypts per organoid but had overall shorter crypts, marked by Paneth cells (red with white arrows pointing to them) residing in their crypts. f) The majority of colonies cultured in boronate ester hydrogels without (−Lam) or g) with (+Lam) formed budded intestinal organoids. h) The majority of colonies grown in boronate ester hydrogels with anti α6 antibodies or i) anti β4 blocking antibodies remained cystic with no Paneth cells. A much smaller population of colonies encapsulated in hydrogels j) with anti α6 antibodies or k) with anti β4 antibodies formed fewer and smaller crypts compared to conditions without blocking antibodies. l,m) However, some organoids that were treated with blocking antibodies formed buds without Paneth cells (Lyz − Bud +) or n,o) had Paneth cells without budding (Lyz + Bud −). Scale bar 100 μm, white arrows point to Paneth cells in crypts. p) However, these populations were not seen in conditions without blocking antibodies. In this analysis, we define a crypt as a budding feature with at least one Paneth cell present. Scale bar 100 μm, white arrows point to Paneth cells in crypts (− Lam (n = 6 hydrogels), + Lam (n = 5 hydrogels),+ Anti α6 + Lam (n = 2 hydrogels), + Anti β4 + Lam (n = 3 hydrogels), + Anti α6 − Lam (n = 3 hydrogels), and + Anti β4 − Lam (n = 2 hydrogels), data presented as means). Portions of this figure were created using Biorender.com.

Figure 4.. FAK inhibitor decreases crypt forming efficiency and influences crypt architecture

a-d) Intestinal organoids were encapsulated in boronate ester hydrogels with or without supplemental laminin (+/− Lam) and with or without the FAK inhibitor (+/− FAK) Scale bar 100 μm. Organoids were immunostained for the presence of crypts, marked by Paneth cells (red). e) When intestinal organoids were cultured in boronate ester hydrogels with a FAK inhibitor, crypt forming efficiency decreased regardless of whether laminin was added to the matrix (+ Lam) or not (−Lam) (n = 7 hydrogels for − FAK − Lam, 3 for + FAK − LAM, 5 for − FAK + LAM and 4 for + FAK + Lam, − FAK condition data from Fig 3, mean ± s.d., ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, one-way ANOVA). f) Similarly, the number of crypts per colony decreased (n = 32 organoids analyzed for − FAK − Lam and 77 for + FAK − Lam, both from 3 hydrogels per condition, − FAK condition data from Figure 3, violin plot, ****P ≤ 0.0001, one-way ANOVA) and g) the size of colonies decreased (n = 110 crypts analyzed for − FAK − Lam and 36 for + FAK − Lam, both from 3 hydrogels per condition, − FAK condition data from Figure 3, mean ± s.d., ****P ≤ 0.0001, one-way ANOVA).

Figure 5.. Intestinal organoids deposit laminin in boronate ester hydrogels.

a) Intestinal organoids cultured in boronate ester hydrogels deposited laminin (scale bar 100 μm), b) with more laminin in crypt regions compared to the organoid body (n = 29 organoids analyzed for crypt and 19 for body, both from 3 hydrogels per condition, mean ± s.d., ****P ≤ 0.0001, unpaired t-test). c) The percent of crypt surface that was covered by laminin was similarly greater than the percent of organoid body that was covered by laminin (n = 25 organoids analyzed for crypt and 15 for body, both from 3 hydrogels per condition, mean ± s.d., ****P ≤ 0.0001, unpaired t-test). d) A variation in the boronate ester hydrogel formulation that has only permanent bonds and no adaptable bonds was implemented for organoid culture. This system does not contain the 8-arm PEG nitrodopamine (ND)/azide and only contains the 8-arm PEG DBCO and 8-arm PEG fluorophenylboronic acid (FPBA) /azide arms. e) In this system, the DBCO and azide groups form covalent bonds via the SPAAC reaction (gray and green, respectively), and because there is no ND present (yellow), the FPBA is free and the matrix is not stress relaxing, but elastic. f) Rheology illustrates that boronate ester hydrogels without ND present (−ND) do not relax stress (blue) versus the original boronate ester hydrogels with ND present (+ND) do relax stress rapidly (black) (Data for +ND from Figure 1d of this manuscript, n = 3 for both conditions). g) The modulus of the elastic condition (−ND) was matched to what has historically been reported as ideal for intestinal organoid differentiation in elastic hydrogels (~600 Pa). The modulus for +ND is from Figure 1f of this manuscript and is the 4.2 wt% condition (n = 7 for + ND and n = 3 for − ND, * P ≤ 0.05, unpaired, two tailed t-test). h) Organoids were encapsulated in the elastic (− ND) and stress relaxing (+ND) forms of the boronate ester hydrogels, with (+Lam) or without (−Lam) supplemental laminin. Organoids grown in the elastic hydrogel (−ND) had decreased crypt forming efficiency when laminin was not present, but the crypt formation was rescued when laminin was added to the matrix (+ ND − Lam (n = 7 hydrogels), − ND − Lam (n = 3 hydrogels), − ND + Lam (n = 3 hydrogels), +ND condition data from Fig 2, mean ± s.d., *P ≤ 0.05, ***P ≤ 0.001, one-way ANOVA). i-k) Organoids were immunostained for lysozyme, DAPI and f-actin to evaluate for the presence of crypts as marked by Paneth cells (red) in +ND and −ND hydrogels (both without Lam). Scale bars 100 μm. l) The percentage of organoid colonies with associated laminin was lower in the elastic (−ND) compared to the stress relaxing (+ND) hydrogels, (+ ND (n = 3 hydrogels), − ND (n = 4 hydrogels), mean ± s.d., *P ≤ 0.05, unpaired t-test) and m) The thickness of the secreted laminin near organoid crypts was thinner in elastic (−ND) compared to the stress relaxing (+ND) hydrogels (n = 29 organoids for + ND and 19 for − ND both from 3 hydrogels per condition, mean ± s.d. ***P ≤ 0.001, unpaired t-test). n,o) These laminin interactions were evaluated through the immunostaining of intestinal organoids for Laminin (Red), DAPI and F-actin in stress relaxing (+ ND) and elastic (−ND) hydrogels. Scale bars 100 μm.

Figure 6.. Stress relaxation allows for increased intestinal organoid nascent protein deposition.

a) Intestinal organoids were cultured in boronate ester hydrogels in methionine free base media that was supplemented with an alkyne containing methionine analog. The organoid uptakes this analog and secretes proteins containing alkyne groups. These alkyne groups can be fluorescently labeled with an azide fluorophore through a copper catalyzed SPAAC reaction. b) Deposited protein from intestinal organoids cultured in stress relaxing boronate ester hydrogels (+ND) was labeled with a 405 azide fluorophore (blue) and stained with CellMask Plasma Membrane Green. c) to ensure that only extracellular protein was analyzed (white) Scale bar 100 μm. d) Organoids had more deposited protein in the crypt region compared to the body of the organoid (n = 11 organoids analyzed from 3 independent biological experiments, mean ± s.d., **P ≤ 0.01, unpaired t-test). e) The percent of crypt surface covered with deposited protein was greater in the crypt regions (n = 12 organoids analyzed for crypt and 11 for body, both from 3 independent biological experiments, mean ± s.d., **P ≤ 0.01, unpaired t-test). f) The elastic (−ND) boronate ester hydrogel formulation without the 8-arm PEG nitrodopamine/azide was again implemented to evaluate the influence bond adaptability on nascent protein deposition. Nascent protein was fluorescently labeled blue and organoids were stained with CellMask Plasma Membrane Green g) to allow for visualization of extracellular protein (white) Scale bars 100 μm. h) A lower percentage of organoids in the elastic (−ND) hydrogels deposited matrix than those in stress relaxing (+ND) hydrogels (n = 3 independent biological experiments, mean ± s.d., unpaired t-test), and i) the organoids in the stress relaxing (+ND) hydrogels deposited a thicker matrix (n = 14 organoids analyzed for + ND and 13 for − ND, both from 3 independent biological experiments, mean ± s.d., ****P ≤ 0.0001, unpaired t-test). j) Similarly, the percentage of the organoid surface covered in ECM was greater in the stress relaxing (+ND) compared to the elastic (−ND) hydrogel (n = 11 organoids analyzed for + ND and 13 for − ND, both from 3 independent biological experiments, mean ± s.d., ****P ≤ 0.0001, unpaired t-test).