WO2025097016A1 - Photoresponsive hydrogels and uses thereof - Google Patents

Abstract

The present invention relates to a photoresponsive hydrogel system. The hydrogel comprises a first macromolecule, a second macromolecule, and a photoinitiator. The first macromolecule has three or more functional groups and the second macromolecule has two or more functional groups. Either the first macromolecule or the second macromolecule has a cleavage site. The hydrogel degrades at the cleavage site upon irradiation with a first light at a first wavelength. The hydrogel crosslinks upon irradiation with a second light at a second wavelength. Also provided is a cell culture comprising the hydrogel and a method for monitoring a cell response in an in vitro injury model.

Description

PHOTORESPONSIVE HYDROGELS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to United States Provisional Application No. 63/595,790, filed November 3, 2023, and the contents of which are incorporated herein by reference in their entireties for all purposes.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This invention was made with government support under grant number 1DP2HL152424-01 awarded by National Institutes of Health. The United States has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to photoresponsive hydrogels and their use in cell cultures as cell or tissue modeling systems, for example, in vitro injury models and in vitro lung models.

BACKGROUND OF THE INVENTION

Fibrosis affects almost every tissue in the body, including pulmonary, dermal, ocular, and cardiac tissues, and is the pathological outcome of misregulated wound healing or chronic inflammation. Fibrotic diseases are defined by the accumulation of excess fibrous connective tissue, causing scarring and organ malfunction. For example, aortic valve disease often leads to valve replacement and causes >28,000 deaths annually, whereas lung fibrosis, specifically idiopathic pulmonary fibrosis (IPF) with unknown cause, currently is uncurable and often fatal. Strikingly, approximately two-thirds of IPF patients die within 5 years and -50,000 new cases are diagnosed annually, a similar number of deaths annually to breast cancer. IPF, amongst other fibrotic diseases, is thought to be initiated by repeated micro-injuries to the lung epithelium and accelerated by the persistence of activated fibroblasts, leading to accumulation of extracellular matrix (ECM) proteins, tissue stiffening, and uncontrolled wound healing with an eventual decline in lung function. However, the disease often is caught in advanced stages, and its exact origins and mechanism accordingly are not well understood or captured by existing model systems, impeding the development of effective therapeutics. Following more than a decade of many unsuccessful clinical trials, the first two pharmaceutical treatments for IPF were approved by the United States Food and Drug Administration (FDA), nintedanib (Ofev and Vargatef) and pirfenidone (Esbriet); these medications slow the progression of the disease yet do not reverse scarred lung architecture. The development of therapeutics for the treatment of IPF and other fibrotic diseases continues to be challenged by a lack of relevant model systems for studying the initiation and early development of the human disease.

There remains a need for model systems to study injury and repair processes for epithelial tissues to provide insights not only for IPF, but also other fibrotic diseases and wound healing, repair, and disease processes in barrier tissues, such as dermal and comeal tissues.

SUMMARY OF THE INVENTION

The present invention relates to photoresponsive hydrogels and uses thereof in in vitro cell culture. The inventors have surprisingly discovered photoresponsive hydrogels responsive to irradiation with a light to switch among different extracellular matrix environments corresponding to different tissue model systems, for example, healthy or injured and diseased states.

The present invention provides a hydrogel. The hydrogel comprises a first macromolecule, a second macromolecule, and a photoinitiator. The first macromolecule has three or more functional groups and the second macromolecule has two or more functional groups. Either the first macromolecule or the second macromolecule has a cleavage site. The hydrogel degrades at the cleavage site upon irradiation with a first light at a first wavelength. The hydrogel crosslinks upon irradiation with a second light at a second wavelength.

The first macromolecule may be functionalized with a cyclooctyne, the second macromolecule may be functionalized with a coumarin-azide, and the first wavelength may be 365-450 nm.

The first macromolecule may be functionalized with a bicyclooctyne (BCN), the second macromolecule may be functionalized with an azide, and the second wavelength may be 450-550 nm.

The first macromolecule may be functionalized with a bicyclooctyne (BCN), the second macromolecule may be functionalized wi th a coumarin-azide, the photoinitiator may be Eosin Y, the first wavelength may be 365-450 nm, and the second wavelength may be 450-550 nm. The first macromolecule may be 4-arm polyethylene glycol (PEG)- bicyclononyne (exo) (BCN-exo) (PEG-4-BCN) or 8-arm PEG-bicyclononyne (exo) (PEG- 8-BCN), and the second macromolecule may be PEG-di-coumarin-azide.

The hydrogel may have a Young’s modulus of 0.5-5kPa.

The hydrogel may have a Young’s modulus of 5-20kPa.

The hydrogel may further comprise an extracellular matrix molecule.

The present invention also provides a cell culture. The cell culture comprises cells, the hydrogel of the present invention, and a culture medium. The cells are seeded onto the hydrogel and grown in the culture of the present invention, (b) irradiating a light to a predetermined area in the hydrogel such that the stiffness of the hydrogel in the predetermined area is changed, and (c) measuring a response of the cells in the predetermined area after step (b). The cells may be epithelial cells, and step (a) may comprise forming an epithelium on the hydrogel. The epithelium may be formed under a submerge culture condition. The epithelium may be formed at an air-liquid interface (ALI).

Step (c) may comprise quantifying proliferation of the cells.

Step (c) may comprise quantifying expression of a biomarker by the cells.

Step (b) may comprise irradiating to a first predetermined area in the hydrogel with the first light at the first wavelength such that the first predetermined area degrades at the cleavage site.

Step (b) may comprise irradiating to a second predetermined area in the hydrogel with the second light at the second wavelength such that the second predetermined area crosslinks.

Step (b) may comprise irradiating to a first predetermined area in the hydrogel with the first light at the first wavelength such that the first predetermined area degrades at the cleavage site; and irradiating to a second predetermined area in the hydrogel with the second light at the second wavelength such that the second predetermined area crosslinks.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-J show design of synthetic basement membrane for photo-injury (PI) model system. A) Hydrogel formed by strain-promoted azide-alkyne cycloaddition (SPAAC) between four-arm PEG-bicyclononyne (PEG-4-BCN) and linear PEG-di- Coumarin-Az (CmPNs). B) Young's modulus (kPa) of synthetic basement membrane mimicking healthy (~5wt%) (‘soft’) and diseased (~9wt%) (‘stiff) tissues. C) Absorbance Spectra of CmPN? and irradiation wavelength (highlighted area centered at 430 nm). D) Hydrogel network injured via degradation of CmPN? by irradiation with 430 nm light. E) Representative images of injured hydrogels with varying time with 100 pm slit on 5wt% hydrogel. F) Quantitative analysis from (E). G) Varying time with 100 pm slit on 9wt% hydrogel. H) Quantitative analysis from (G). I) Representative images of hydrogels with varying the injured size (pm) using slits on 5wt% hydrogels. J) Quantitative analysis of images (I). Data are shown represent mean ± SD (n=3).

FIGS. 2A-E show an effect of ECM compositions on lung epithelial cells under submerged culture conditions on ‘soft’ hydrogels inspired by healthy tissue. A) Schematic approach for development of a healthy epithelium on synthetic photosensitive hydrogel and their phenotypic analysis. B) Assessment of epithelial formation with A549 cells on soft hydrogels using live dye, where Calcein area represents a monolayer formation at day 3 vs. day 10 on PHSRN, AG73, and PHSRN+AG73 hydrogel (confocal z-stack projections, gray (Live cells) stained with Calcein, Scale bar = 100 pm). C) Quantitative analysis of Calcein + area of (B). Significant differences assessed by Student's two-sided t-test, where differences shown for comparison between day timepoints in PHSRN+AG73 conditions (*p < 0.05, **p < 0.01; ***p < 0.001). D) Immunofluorescence demonstrates functional monolayer formation of A549 (confocal z-stack projections, gray (E-cadherin), Scale bar = 50 pm). E) Quantitative analysis of E-cadherin positive cells of (D) relative to total cells (F- actin, not shown). Significant differences assessed by Student's two-sided t-test, where differences shown for comparison between conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3).

FIGS. 3A-C show A) Assessment of epithelial formation of Calu-3 cells on ‘soft’ hydrogels using live/dead dyes, where Calcein area represents a monolayer formation at day 3 vs. day 55 on PHSRN, AG73, and PHSRN+AG73 hydrogel (confocal z-stack projections, Live cells stained with Calcein and dead cells stained with ethidium homodimer (Eth-1 ), Scale bar = 100 pm). B) Quantitative analysis of Calcein + area of (A). C) Quantification was carried out using Velocity software based on live/dead confocal images. Significant differences assessed by Student’s two-sided t-test, where differences shown for comparison between day timepoints in PHSRN+AG73 conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Scale bar = 100 pm. Data are shown represent mean ± SD (n=3).

FIGS. 4A-B show A) The effect of synthetic ECMs on A549 mesenchymal phenotypic marker expression and proliferation. A549 were cultured over time and showed higher expression of a mesenchymal marker protein (Vimentin) at early times culture. At later times, a lower number of Vimentin positive cells were observed along with the development of sheet of connected cells based on cytoskeletal protein F-actin staining on PHSRN+AG73 condition relative to PHSRN or AG73 alone, demonstrating a synergistic effect of combinations of bioinspired peptides on epithelium formation. B) Quantitative analysis of Vimentin positive cells of (A). Significant differences assessed by Student’s two-sided t-test, where differences shown for comparison between time points (*p < 0.05, **p < 0.01; ***p < 0.001), Scale bar = 50 pm. Data are shown represent mean ± SD (n=3).

FIGS. 5A-B show A) Immunofluorescence staining to determine epithelization of the synthetic ECMs. Calu-3 cells were cultured over time and showed the formation of cellcell junctions with E-cadherin protein expression and the development of net-hke epithelium structure with F-actin protein expression on PHSRN+AG73 condition relative to PHSRN or AG73 alone, demonstrating a synergistic effect of combinations of bioinspired peptides on epithelium formation. B) Quantitative analysis of E-cadherin positive cells of (A). Significant differences assessed by Student’s two-sided t-test, where differences shown for comparison between conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Scale bar = 50 pm. Data are shown represent mean ± SD (n=3).

FIGS. 6A-B show A) Immunofluorescence suggests the limited mesenchymal activity for Calu-3 cells on the synthetic ECMs (confocal z-stack projections, stained for F- actin and Vimentin, Scale bar = 50 pm). B) Quantitative analysis of Vimentin positive cells of (A). Significant differences assessed by Student’s two-sided t-test, where differences shown for comparison between conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3 ).

FIGS. 7A-H show real-time analysis of epithelial growth period and recover)' over time in a wound model. A) Representation of culture period prior to and post photo-injury (PI) on model epithelium. Here, A549 cells were used that had been transduced for constitutive expression of a red fluorescent protein, allowing visualization of cells, and conditional expression of a green fluorescent protein when alpha smooth muscle actin (aSMA) expression was upregulated, allowing assessment of cells exhibiting a mesenchymal-like wound healing phenotype. B) Approach for light-triggered ‘injury'’ to induce damage to the epithelium on hydrogel. C) Epithelium formation with A549 cells on the synthetic PHSRN+AG73 hydrogel over time. D) Quantitative analysis of (C). E) Real- time epithelial cell recovery after photo-injury (50 pm) on hydrogel, (confocal z-stack projections, A549 channel shows all transduced cells in the growth area, aSMA^+ve channel shows cells expressing aSMA, Hydrogel channel shows the hydrogel-based synthetic ECM where injury area is visible with loss of intensity, boundaries of injury noted with dashed lines, Scale bar = 100 pm). F) Quantitative analysis of (E). G) Epithelial cell recovery post photo-injury (100 pm) on hydrogel over time. H) Quantitative analysis of (G). Significant differences assessed by Student's two-sided t-test, where differences shown for comparison between time points in PHSRN+AG73 conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Scale bar = 100 pm. Data are shown represent mean ± SD (n=3).

FIGS. 8A-D show A) Representation of real time analysis of the epithelial cell recovery using reporter A549 cells either in post PI (100 pm) area on the hydrogel culture over time with boundaries of injury noted with dashed lines or B) out of PI area (on a hydrogel with photo-injury but away from the PI area), (confocal z-stack projections A549 channel shows all transduced cells in the growth area, aSMA^+ve channel shows cells expressing aSMA, and Hydrogel channel shows the hydrogel-based synthetic ECM). C) Quantitative analysis of (B). D) Comparison of aSMA protein expression at no PI area on post-PI day 5 (Day 10 culture) between 50 and 100 pm size. Significant differences assessed by Student’s two-sided t-test, where differences shown for comparison between time points in PHSRN+AG73 conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3). Scale bar = 100 pm.

FIGS. 9A-F show that injury leads to global loss of E-cadherin and Ki-67 protein expression in A549. A) Schematic representation for repair and non-repair epithelium on synthetic photosensitive hydrogel and their phenotypic analysis. B) Representative images of A549 showed different responses on the synthetic hydrogel after creating of several sizes of PI in comparison to controls (representative confocal images, cells stained for F-actin and E-cadherin, Hydrogel labeled with fluorophore for visualization of ‘injury’ area observed with loss of intensity, boundaries of ‘injury’ noted with dashed lines, Scale bar = 50 pm). C) Quantitative analysis of cells positive for E-cadherin protein of (B). D) Representative analysis of transepithelial electric resistance (TEER) pre and post injury of A549 (B). E) Immunofluorescent staining of proliferative (Ki-67 positive), mesenchymal (aSMA), and cytoskeleton (F-actin) markers; Hydrogel labeled with fluorophore for visualization of ‘injury’ area observed with loss of intensity, and boundanes of ‘injury’ noted with dashed lines; Scale bar = 50 pm. F) Quantitative analysis of cells positive for Ki-67 of (E). Statistical differences determined Student's two-sided t-test, where differences shown for comparison between conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3).

FIGS. 10A-B show how differences in the size of injury lead to loss of mesenchymal phenotypic marker Vimentin expression in A549. A) Confocal images of A549 showed the cell responses on the photoresponsive synthetic hydrogel with different sizes of photo-injury (PI) (F-actin stained as cytoskeletal marker, Vimentin stained as a mesenchymal marker, Hydrogel labeled with AF647, boundaries of ‘injury’ noted with dashed lines, Scale bar = 50 pm). B) Quantitative analysis of cells positive for Vimentin protein of (A). Statistical differences determined Student’s two-sided t-test, where differences shown for comparison between conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3).

FIGS. 11A-B show that injury leads to loss of E-cadherm protein expression in A549. A) Representative confocal images of A549 showed overall loss of the E-cadherin expression for cells outside of the PI area on a hydrogel that has a PI (no direct injury but on the same hydrogel with a PI of different sizes in comparison to controls) (stained for E- cadherin epithelial marker, F-actin cytoskeletal marker, Hydrogel labeled with AF647, Scale bar = 50 pm). B) Quantitative analysis of cells positive for E-cadherin protein of (A). Statistical differences determined Student’s two-sided t-test, where differences shown for comparison between conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3).

FIGS. 12A-B show that injury leads to apoptotic cells at PI area. A) Confocal images of A549 cells on day 10 (PI D5) captured by LSM 800 using 10X objective after staining with Apoptotic marker Apo- 15 (apoptotic cell death), ethidium homodimer (Eth, dead cells), Hydrogel labeled with AF647, injury border noted with dashed lines. B) Quantitative analysis of (A). Scale bar = 100 pm. Statistical differences determined Student's two-sided t-test, where differences shown for comparison between conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3).

FIGS. 13A-D show that multiple injury leads to apoptotic A549 cells at PI and in areas on a sample with PI away from the PI (No PI area) on day 10 in culture, which is day 5 after PI (PI D5). A) Confocal images post multiple PI captured by LSM 800 using 10X objective after staining with Apoptotic marker Apo- 15 (apoptotic cell death), ethidium homodimer (Eth, dead cells), Hydrogel labeled with AF647, injury boundaries noted with dashed lines, Scale bar = 100 pm. B) Quantitative analysis of (A). C) Confocal images showed dead and apoptotic population out of the PI area post multiple PI D5 (day 10 culture, day 5 after PI). D) Quantitative analysis of (C). Significant differences assessed by Student’s two-sided t-test, where differences shown for comparison between PI sizes in PHSRN+AG73 conditions (*p < 0.05, **p < 0.01; ***p < 0.001). Data are shown represent mean ± SD (n=3).

FIG. 14 shows Calu-3 and HBE cell attachment and epithelium formation at the ALI on well-defined photoresponsive ECM with increasing complexity. Collagen IV coated transwell insert (positive control (Col-IV)) and photoresponsive ECM containing integrin- binding peptide RGDSP (RGDSP) (SEQ ID NO: 1); RGDSP (SEQ ID NO: 1) and proteins Col-IV and lammin-111 (RCL); RGDSP (SEQ ID NO: 1), Col-IV, laminin-111, and polysaccharide hyaluronic acid (RCLH) (SEQ ID NO: 2); and RGDSP (SEQ ID NO: 1), Col-IV, laminin-111, hyaluronic acid, and basement membrane binding (BMB) peptide (RCLHB) (SEQ ID NO: 3).

FIG. 15 shows Calu-3 and HBE epithelial marker expression (Tubulin, MUC5AC) at the ALI on well-defined photoresponsive ECM with increasing complexity. Col IV coated transwell insert (positive control (Col-IV)) and photoresponsive ECM containing integrin-binding peptide RGDSP (RGDSP) (SEQ ID NO: 1); RGDSP (SEQ ID NO: 1) and proteins Col-IV and laminin-111 (RCL); RGDSP (SEQ ID NO: 1), Col-IV, laminin-111, and polysaccharide hyaluronic acid (RCLH) (SEQ ID NO: 2); and RGDSP (SEQ ID NO: 1), Col-IV, laminin-111, hyaluronic acid, and basement membrane binding (BMB) peptide (RCLHB) (SEQ ID NO: 3).

FIG. 16 shows example response of HBEs and Calu-3 to photo-injury. Filling of injury is observed with differential expression of epithelial markers in injured vs. noninjured areas and samples. Here, day 3 post photo-injury (PI D3) is shown, which is at day 31 in ALI culture (stained for Tubulin (for HBE cells) and MUC5AC (for Calu-3) epithelial markers and F-actin cytoskeletal marker, Hydrogel labeled with AF647, ‘injury’ denoted with dashed lines, Scale bar = 50 pm).

FIGS. 17A-G show an overview of motivation and hydrogel design for ECM damage followed by stiffening and approach for developing a healthy to damaged epithelium for probing cell responses with photoresponsive system. A) In vitro conditions were designed to mimic the healthy formation of an epithelium followed by acute injury and stiffening associated with maladaptive wound healing. B) Representation of epithelial layer formation with submerge or air-liquid interface (ALI) culture on photoresponsive hydrogel and characterize. C) Mimicking of damaged (either single or multiple) epithelial tissue using irradiation of light in vitro model to degrade matrix for characterizing the cells response to wound. D) Example of hydrogels to mimic ECM formed via strain-promoted azide-alkyne click chemistry (SPAAC) reaction of PEG-8-BCN with PEG-2-CmAz, a photodegradable linker. E) The concentration of BCN groups and the ratio between BCN: Azide groups was varied to achieve a range of matrix stiffness, as measured by Young’s moduli. Here, a concentration of 10 mM BCN end groups and a 3: 1 ratio was selected for having a modulus in the range of typical healthy lung tissue while optimizing the concentration of free BCN groups for subsequent stiffening. F) Components, Eosin Y as a Photoinitiator and PEG-4-SH as an example of a co-initiator, were selected for secondary crosslinking and stiffening of the hydrogel. G) Eosin Y was chosen due to the absorbance of light being at a different wavelength range than PEG-2-CmAz, enabling the independent control of the injury (e.g., 430 nm light) and stiffening (e.g., 530 nm light) processes.

FIGS. 18A-G show that secondary photopolymerization of hydrogels increases storage modulus for in situ gels. A) Gels were formed by SPAAC reaction of PEG-8-BCN, PEG-2-CmAz, and 2mM of bioinspired peptide for 3500s. Stiffening components also were included in the hydrogel precursor solution. Stiffening was then triggered by 4mW/cm² at 530 nm (denoted with Tight bulb’) to react free BCN groups with Eosin Y and co-initiator. Storage modulus (G’) was used to track relative modulus changes. Loss modulus (G”) was constant around 0 Pa for all conditions. B) Evaluation of conditions for the maximum extent of secondary stiffening was done via in situ formation and stiffening of the gels on a parallel plate rheometer. C) Thiol functional groups were identified as a promising co-initiator for use with Eosin Y compared to other co-initiator pairs (e.g., Eosin Y and TEOA). D) Increasing the ratio of BCN:Az groups in the hydrogel precursor solution and therefore the amount of free BCN in the formed hydrogel was evaluated as a handle for controlling the degree of stiffening possible. E) The functionality of the co-initiator (e.g., mono, di, tetrafunctional thiols) was increased to evaluate the effect on the magnitude of stiffening. F) Quantification of final storage moduli of hydrogels with different functionality of thiol co- initiator. G) Concentration of the co-initiator was increased to evaluate the effect on stiffening. Approximately 6.6 mM was chosen as the co-initiator concentration that minimizes variability but maximizes the extent of stiffening.

FIGS. 19A-E show stiffening of equilibrium swollen photoresponsive hydrogels. A) Formation and stiffening protocol of equilibrium swollen hydrogels. B) Extent of increase in Young’s moduli with one light illumination of 10, 30, or 90 mins compared to a control gel that was not swelled in stiffening components nor exposed to light. C) Young’s moduli with multiple light illuminations for times of 10- and 30-min intervals and swelling in PEG- 4-SH, Eosin Y, and 0 mM or 6.6 mM of PEG-4-BCN. D) Final Young’s moduli of gels undergoing different intermediate swelling conditions and light illumination intervals. E) Volume changes of hydrogels associated with different stiffening workflows.

FIG. 20 show's an A549 layer retained with different stiffening cycles (stiffening process with different lengths (minutes [min]) and numbers of light cycles) relative to control (soft).

FIG. 21 show's a 3D printed photomask holder example (left) and its use in transwell insert plate (right).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to photoresponsive hydrogels useful for providing in vitro tissue model systems, for example, in vitro injury models, in vitro tunable air-liquid interface (ALI) lung models, and culture platforms for mimicking tissues having different matrix stiffness. The inventors have surprisingly discovered a photoresponsive hydrogel comprising two macromolecules each having multiple functional groups, for example, one macromolecule having three or more functional groups, including a cyclooctyne, and another macromolecule having two or more functional groups, including an azide, and the stiffness of the hydrogel may be adjusted by irradiation with a light at a desirable w'av elength. The present invention provides a human model system for hypothesis testing, probing patient-specific responses for informing treatment options, and more broadly evaluation of therapeutic.

The inventors have established an in vitro culture system using photosensitive materials that mimic the extracellular matrix (ECM), particularly the basement membrane, for creating epithelial layers and triggering injuries to the underlying matrix with light (e.g., synthetic ECM photo-degradation, and/or photo-stiffening) for studying and understanding wound healing responses and mechanisms. Key inventive concepts include 1) generation of a light-responsive synthetic basement membrane with tunable well-defined bioinspired mechanical properties and biochemical content for controlled complexity and promoting relevant cellular functions; 2) integration of this material into air-liquid interface (ALI) culture system for creation of functional epithelium with a range of epithelial cell types; and 3) methods for triggering controlled photo-injury (matrix erosion, matrix stiffening, novel photomasking technique in transwell cultures) in the presence of cells (e.g., epithelial cells, monocytes, macrophages, lymphocytes, dendritic cells, natural killer (NK) cells, fibroblasts; range of tissue origins; cell lines and primary cells) and following cellular responses to these over time where the size of injury is controlled by the resolution of light and the type of injury (matrix degradation, matrix stiffening) is controlled by the light wavelength.

The technology' according to the present invention provides the ability to study cell responses to different sizes, frequencies, and durations of injuries and to ask questions about the effects of matrix composition and mechanics in conjunction with injury on cellular responses. The technology provides new tools for controlled cell cultures and enables new mechanistic studies of injury, repair, and disease processes for developing more effective treatment strategies.

The present invention allows tuning of the composition of the well-defined matrix for controlling its biophysical and biochemical properties for mimicking different tissue types and states (e.g., healthy to diseased lung or other barrier tissues) and then triggering of injuries to the matrix through the engineering of its underlying molecular structure (e.g., degradation of the matrix, and/or stiffening of the matrix) and applying cytocompatible doses of light. Since light is used as the trigger, injuries of any shape or size can be created when and where desired in cell culture for probing cell responses. The 3D pnnted receptacle for holding a photomask at the surface of cells in culture allows easy and accessible control of where light is shined on cells in culture without disturbing the underlying cell layer or requiring more sophisticated light setups (e.g., focused lasers, digital light processing (DLP) technology). The present invention allows the formation of epithelial layers in submerged culture and at the ALI all with well-defined bioinspired soft materials.

Traditionally, scratch assays have been used for studying cell responses to injury in cell culture. These methods broadly use mechanical force or similar to create a “scratch” or void on the surface where cells are cultured and then monitor cells filling in this void space as a measure of healing. Variations of this assay include: i) scratching the surface on which cells are being cultured with a sharp instrument (e.g., pipette tip), removing cells in that region and creating a ‘scratch’; ii) using a laser or microjet to ablate regions; or iii) covering a region of the culture surface prior to cell seeding so that a ‘scratch’ region without cells is revealed upon barrier removal, each providing different levels of uniformity and sizes of injury. While these each have different levels of spatiotemporal precision, their approach to injury is non-specific in nature. The inventors have discovered molecular engineering approaches in conjunction with light to specifically cause damage to the underlying matrix and then probe cell response.

Transwell-based culture conditions have been established for the culture of epithelial cells, including human bronchial epithelial (HBE), at the ALI for the formation of a functional epithelium. For well-defined cultures, the transwell insert is coated with a harvested and purified protein (e.g., Collagen IV) to promote desired cell attachment and function. Recently, synthetic hydrogels that integrate harvested de-cellularized tissues have been integrated within ALI cultures to allow control of matrix stiffness while promoting cell attachment and function. The inventors have developed a synthetic matrix for 1) controlling stiffness of the microenvironment to mimic different types of healthy to diseased tissues; 2) allowing triggered injury; and 3) permitting well-defined presentation of specific ligands (e.g., peptides inspired by ECM proteins to bind cell receptors and cell-secreted ECM; specific ECM proteins and polysaccharides) for controlled complexity and the formation of a functional epithelium at the ALI. The present invention provides a system that is modular and well defined and can be tuned to work well with both cell lines and primary cells. While the focus has been on two-dimensional (2D) culture of epithelial cells, the system according to the present invention, also is permissive to three-dimensional (3D) culture.

Hydrogels are increasingly used as culture platforms for mimicking tissues and studying cellular responses to stimuli. Given the importance of matrix stiffness in cell functions and the knowledge that matrix stiffness changes with disease, a number of hydrogel systems have been developed to allow changes in matrix stiffness in the presence of cells, including use of light-switchable moieties and proteins and photopolymerization and enzyme-mediated reactions. The inventors have developed the technology that uniquely allows the initiation of an “injury” (matrix erosion and/or maladaptive stiffening) to the hydrogel-based synthetic matrix under cytocompatible conditions and in ALI cultures 1) using one wavelength of visible light to trigger hydrogel degradation and erosion and 2) using another wavelength of visible light and biologically inert building blocks to trigger in situ hydrogel crosslinking and increase modulus.

Microinjuries to the lung microenvironment, amongst other barrier tissues, can induce maladaptive wound healing processes, which are hypothesized to lead to a range of maladies including fibrous or cancer recurrence. These events are associated with changes in the biophysical properties of the cellular microenvironment, which greatly impact cellular functions. To accurately study onset and progression of these conditions, it is necessary to design systems that mimic the mechanical properties of relevant microenvironments, allow culture of human cells including primary cells and have dynamic properties for mimicking injury, repair, and potentially maladaptive wound healing processes. To replicate this process, dynamic biophysical properties in the hydrogel would be beneficial to study the cellular responses to injury and repair processes, from matrix degradation to stiffening associated with fibrosis. Photoresponsive hydrogels have spatiotemporal control, preciseness, and reproducibility. This is because the chemistries used to impart photoresponsiveness from the molecular level up are controllably responsive to light intensity and wavelength. Therefore, the degradation or stiffening of the hy drogel is highly tunable. Further, using visible light as a trigger is advantageous over UV light as it can penetrate further into different types of tissues including synthetic tissue mimics and poses less threat of biological toxicity. The inventors have fabricated a platform that mimics the biochemical and biophysical properties of relevant tissues and the changes in biophysical properties associated with injury and maladaptive wound healing, from injury to subsequent stiffening, to study the cellular responses, particularly epithelial responses at the ALI, to these dynamic changes for mechanistic insights and toward the development of more effective therapeutic strategies.

The term “hydrogel” as used herein refers to a water-swollen crosslinked network of macromolecules.

The term “macromolecule” as used herein refers to a large molecule made of connected subunits, with ~ 3 or greater subunits (e.g., monomers) per large molecule. The macromolecule may comprise a polymer, peptide, protein, nucleic acid, polysaccharide or a combination thereof. Exemplary macromolecules include polymers, peptides, proteins, polysaccharides and combinations thereof. The term “peptide” as used herein refers to a compound consisting of two or more amino acids linked in a short chain (2 to 50 amino acids).

The term “protein” as used herein refers to a compound consisting of a long chain of ammo acids, for example, 50 or more amino acids.

The term “polysaccharide” as used herein refers to a carbohydrate whose molecules consist of a number of sugar molecules bonded together. Examples of polysaccharides include hyaluronic acid and dextran.

The term “polymer” as used herein refers to a macromolecule composed of repeating subunits (monomers). Examples of polymers include polyethylene glycol (PEG) and polyvinyl alcohol (PVA).

The term “functional group” as used there in refers to an atom or group of atoms within a molecule that imparts the molecule's characteristic chemical properties especially chemical reactions. Examples of the functional groups include a cyclooctyne (e.g., bicyclooctyne (BCN)), an azide (e.g., coumarm-azide (CmPNs or CmAz)), or variants thereof. A molecule having a functional group is also referred to a functionalized molecule.

The terms “photoresponsive,” “photosensitive,” “responsive to light,” and “responsive to irradiation with light” are used herein interchangeably and refer to molecules and materials that exhibit a property change upon the application of light.

The term “stiffness” as used herein refers to the extent to which an object resists deformation in response to an applied force and is a term used within the art synonymously with terms such as elasticity and inversely with terms such as compliance. The stiffness may be quantified by measurement of Young’s modulus using conventional techniques, where the specific stiffness of a material can be calculated based on Young’s modulus and how the stiffness is measured (e.g., material axial stiffness k = material cross-sectional area (A) times Young’s modulus (E) divided by length (L)).

The term “degradation” as used herein refers to the process by which a material’s structure breaks down and related property changes (e.g., decrease in crosslinks leading to decrease in Young’s modulus).

The term “stiffening” and “crosslinking” are used herein interchangeably and refer to the process by a which linkages are formed in a material’s structure and related property changes (e.g., increase in crosslinks leading to increase in Young’s modulus). The term “extracellular matrix molecule” as used herein refers to macromolecules found in the microenvironment of cells. These macromolecules may provide a variety of functions including structural support, guiding cell-cell interactions, and presenting biochemical moieties that influence cellular responses.

The term “submerge culture condition” as used herein refers to cell culture in liquid medium.

The term “air-liquid interface (ALI)” as used herein refers to cell culture where cells are grown with their basal (bottom) surfaces in contact with liquid medium and the apical (top) of the cellular layer in contact with air. A cell culture in which cells are grown at an ALI may be used as an in vitro lung model.

The term “bioinspired” as used herein refers to macromolecules, materials, and processes inspired by or based on biological structures or processes.

The present invention provides a hydrogel. The hydrogel comprises a first macromolecule, a second macromolecule, and a photoinitiator. The first macromolecule has three or more functional groups and the second macromolecule has two or more functional groups. Either the first macromolecule or the second macromolecule has a cleavage site. The hydrogel degrades at the cleavage site upon irradiation with a first light at a first wavelength. The hydrogel crosslinks upon irradiation with a second light at a second wavelength.

The first macromolecule may be functionalized with a cyclooctyne, for example, a bicyclooctyne (BCN). The first macromolecule may be 4-arm polyethylene glycol (PEG)- bicyclononyne (exo) (BCN-exo) (PEG-4-BCN) or 8-arm PEG-bicyclononyne (exo) (PEG- 8-BCN).

The second macromolecule may be functionalized with an azide, for example, a coumarin-azide (Coumarin-PEG-azide (CmPN3) or CmAz).

Either the first macromolecule or the second macromolecule may comprise a linker having the cleavage site. The second macromolecule may be a linker having the cleavage site. Any cleavable or responsive linker may be used in the conjunction with the coumarinazide to allow triggering of degradation with other wavelengths of light or in response to enzymes. Such a linker may have a nitrobenzyl group for cleavage with 350-405 nm light, thrombin for cleavage of a peptide linker, or a matrix metalloproteinase cleavage of a peptide linker (e.g., GK(az)GVPLSLYSGGK(az)G (SEQ ID NO: 4)). Other light responsive moieties and enzyme responsive linkers include LOV2 protein, Sortase ligation, tyrosinase ligation, and photo-polymerization of other functional groups.

In the hydrogel, the first macromolecular may be functionalized with a cyclooctyne, the second macromolecule may be functionalized with a coumarin-azide, and the first wavelength may be about 365-450 nm, 420-440 nm or 425-435 nm. The stiffness of the hydrogel may decrease due to the degradation.

In the hydrogel, the first macromolecule may be functionalized with a bicyclooctyne (BCN), the second macromolecule may be functionalized with an azide, and the second wavelength may be about 450-550 nm, 520-540 nm, or 525-535 nm. The photoinitiator may be Eosin Y. The hydrogel may further comprise a co-initiator. The co-initiator may increase crosslinking of the hydrogel. The co-initiator may be a thiol having one or more functional groups, preferably a tetra functional thiol. The stiffness of the hydrogel may increase due to the crosslinking.

In the hydrogel, the first macromolecule may be functionalized with a bicyclooctyne (BCN), the second macromolecule may be functionalized with a coumarin-azide, the first wavelength may be about 365-450 nm, 420-440 nm or 425-435 nm, and the second wavelength may be about 450-550 nm, 520-540 nm, or 525-535 nm. The photoinitiator may be Eosin Y. The hydrogel may further comprise a co-initiator. The co-initiator may increase the crosslinking. The co-initiator may be a thiol having one or more functional groups, preferably a tetra functional thiol. The stiffness of the hydrogel may decrease due to the degradation of the hydrogel. The stiffness of the hydrogel may increase due to the crosslinking of the hydrogel. The desirable stiffness of the hydrogel may be achieved by irradiation of the hydrogel with the first light at the first wavelength and the second light at the second wavelength sequentially or simultaneously.

In the hydrogel, the first macromolecule may be 4-arm polyethylene glycol (PEG)- bicyclononyne (exo) (BCN-exo) (PEG-4-BCN) or 8-arm PEG-bicyclononyne (exo) (PEG- 8-BCN), and the second macromolecule may be PEG-di-coumarin-azide.

The hydrogel may have a Young’s modulus of about 0.01-100 kPa, 0.01-50 kPa, 0.01-15 kPa, 0.01-10 kPa, 0.01-5 kPa, 0.01-4 kPa, 0.01-3 kPa, 0.01-2 kPa, 0.01-1 kPa, 0.01- 0.5 kPa, 0.01-0.1 kPa, 0.1-100 kPa, 0.1-50 kPa, 0.1-15 kPa, 0.1-10 kPa, 0.1-5 kPa, 0.1-4 kPa, 0.1-3 kPa, 0.1-2 kPa, 0.1-1 kPa, 0.1-0.5 kPa, 0.5-100 kPa, 0.5-50 kPa, 0.5-15 kPa, 0.5- 10 kPa, 0.5-5 kPa, 0.5-4 kPa, 0.5-3 kPa, 0.5-2 kPa, 0.5-1 kPa, 1-100 kPa, 1-50 kPa, 1-15 kPa, 1-10 kPa, 1-5 kPa, 1-4 kPa, 1-3 kPa, 1-2 kPa, 2-100 kPa, 2-50 kPa, 2-15 kPa, 2-10 kPa, 2-5 kPa, 2-4 kPa, 2-3 kPa, 3-100 kPa, 3-50 kPa, 3-15 kPa, 3-10 kPa, 3-5 kPa, 3-4 kPa, 4- 100 kPa, 4-50 kPa, 4-15 kPa, 4-10 kPa, 4-5 kPa, 5-100 kPa, 5-50 kPa, 5-20 kPa, 5-15 kPa, 5-10 kPa, 10-100 kPa, 10-50 kPa, 10-20 kPa, 10-15 kPa, 15-20 kPa, or 50-100 kPa. In one embodiment, the hydrogel may have a Young’s modulus corresponding to that of a healthy tissue (e.g., a healthy lung tissue) at, for example, about 0.5-5 kPa, 0.5-4 kPa, 0.5-3 kPa, 0.5-2 kPa, 0.5-1 kPa, 1-5 kPa, 1-4 kPa, 1-3 kPa, 1-2 kPa, 2-5 kPa, 2-4 kPa, 2-3 kPa, 3-5 kPa, 3-4 kPa, 4-100 kPa, 4-50 kPa, 4-10 kPa, or 4-5 kPa. In another embodiment, the hydrogel may have a Yong’s modulus corresponding to that of a lung tissue (e.g., fibrotic lung tissue) at, for example, about 5-20 kPa, 5-15 kPa, 5-10 kPa, 10-20 kPa, 10-15 kPa, or 15-20 kPa.

The hydrogel may further comprise an extracellular matrix molecule. The amino acid sequence of the extracellular matrix molecule may be found in extracellular matrix. The extracellular matrix molecule may be a peptide, protein, or polysaccharide. The extracellular matrix molecule may be a bioinspired peptide, protein, or polysaccharide. The extracellular matrix molecule may promote cell binding or impart bioactivity.

The extracellular matrix molecule may be selected from the group consisting of laminin inspired AG73 peptide having the amino acid sequence of RKRLQVQLSIRT (SEQ ID NO: ), fibronectin inspired RGDS-PHSRN (PHSRN) peptide having the amino acid sequence of PHSRNGGGGGGGGGGRGDSPG (SEQ ID NO: 6), fibronectin/vitronectin/collagen I inspired RGDS peptide having the sequences of RGDSP (SEQ ID NO: 1), basement membrane binder (BMB) peptide having the amino acid sequence of IS AFLGIPFAEPPMGPRRFLPPEPKKP (SEQ ID NO: 7), collagen IV (Col- IV) protein, laminin-111 (LN) protein, fibronectin (FN) protein, hyaluronic acid (HA) polysaccharide, and a combination thereof.

Other extracellular matrix molecules include 1) laminin inspired peptides such as YIGSR (SEQ ID NO: 8), IKVAV (SEQ ID NO: 9), IKLLI (SEQ ID NO: 10); 2) collagen I and IV inspired peptides such as GFOGER (SEQ ID NO: 11) and variants thereof, DGEA (SEQ ID NO: 12), and FYFDLR (SEQ ID NO: 13); 3) other ECM protein inspired peptides such as perlecan inspired peptide TWSKVGGHLRPGIVQSG (SEQ ID NO: 14), elastin inspired peptide VAPG (SEQ ID NO: 15), tenascin-inspired peptide VAEIDGIEL (SEQ ID NO: 16); 4) ECM binding peptides such as fibronectin binder TLQPVYEYMVGV(- COOH) (SEQ ID NO: 17), HA binder KTKATVLIKNKQKSKNALKQKIVLLSK (SEQ ID NO: 18), Collagen IV heparin binding domain TAGSCLRKFSTM (SEQ ID NO: 19), and heparan sulfate binder RRRPKGRGKRRREKQRPTDCHL (SEQ ID NO: 20); and 5) ECM biomacromolecules such as other collagens, laminins, entactins, perlecan, chondroitin sulfate, secreted protein acidic and rich in cysteine (SPARC), or fragments of these ECM components.

For each hydrogel of the present invention, a cell culture is provided. The cell culture comprises cells, the hydrogel, and a culture medium. The cells are grown in the culture medium. The cells may have been seeded onto the hydrogel. The cells may be primary cells or from cell lines. The cells may be of any tissue origin (e.g., lung, skin, eye, intestine). The cells may be epithelial cells, keratinocytes, monocytes, macrophages, lymphocytes, dendritic cells, natural killer (NK) cells, or fibroblasts. The epithelial cells may be A549 cells, Calu-3s cells, keratinocytes, or human bronchial epithelial (HBE) cells. Where the cells are epithelial cells, the cells may form an epithelium on the hydrogel. The epithelium may be formed under a submerge culture condition. The epithelium may be formed at an air-liquid interface (ALI). A cell culture in which an epithelium is formed at an ALI may be used as in an in vitro lung model. The stiffness of the hydrogel in the cell culture may be adjusted to correspond to that of a healthy or injured tissue such that an in vitro healthy or injury model may be generated for characterization of the morphology, function and behavior of the cells.

For each cell culture of the present invention, a method for monitoring a cell response in an in vitro injury model is provided. The method comprises growing the cells in the cell culture of the present invention. The cells may be primary cells or from cell lines. The cells may be of any tissue origin (e.g., lung, skin, eye, intestine). For example, the cells may be epithelial cells, keratinocytes, monocytes, macrophages, lymphocytes, dendritic cells, natural killer (NK) cells, or fibroblasts. The epithelial cells may be A549 cells, Calu- 3s cells, keratinocytes, or human bronchial epithelial (HBE) cells. Where the cells are epithelial cells, the cells may form an epithelium on the hydrogel. The epithelium may be formed under a submerge culture condition. The epithelium may be formed at an air-liquid interface (ALI). A cell culture in which an epithelium is formed at an ALI may be used as in an in vitro lung model. The method further comprises irradiating a light to a predetermined area in the hydrogel to change the stiffness of the hydrogel in the predetermined area. The stiffness of the hydrogel in the predetermined area may be changed to correspond to that of a healthy or injured tissue such that an in vitro healthy or injury model is generated for characterization of the morphology, function and behavior of the cells. The method may comprise irradiating the light for a predetermined time to achieve a desirable stiffness of the hydrogel in the predetermined area. For example, the irradiation may last about 0.1-90 minutes, 0.1-30 minutes, 0.1-10 minutes, 0.1-1 minutes, 0. 1-0,5 minutes, 1-90 minutes, 1-30 minutes, 1-10 minutes, 10-90 minutes or 10-30 minutes. The method may further comprise irradiating two or more lights at the same or different wavelengths to the same or different predetermined areas.

Where the hydrogel comprises a first macromolecular functionalized with a cyclooctyne and a second macromolecule functionalized with a coumarin-azide, the method may comprise irradiating to a predetermined area of the hydrogel with a light at a wavelength of, for example, about 365-450 nm, 420-440 nm or 425-435 nm, such that the hydrogel in the predetermined area may degrade. The stiffness of the hydrogel in the predetermined area may decrease due to the degradation.

Where the hydrogel comprises a first macromolecule functionalized with a bicyclooctyne (BCN), a second macromolecule functionalized with an azide, and a photoinitiator, the method may comprise irradiating to a predetermined area of the hydrogel with a light at a wavelength of, for example, about 450-550 nm, 520-540 nm, or 525-535 nm, such that the hydrogel in the predetermined area may crosslink. The photoinitiator may be Eosin Y. The hydrogel may further comprise a co-initiator. The co-initiator may increase the crosslinking. The co-initiator may be a thiol having one or more functional groups, preferably a tetra functional thiol. The stiffness of the hydrogel in the predetermined area may increase due to the crosslinking.

Where the hydrogel comprises a first macromolecule functionalized with a bicyclooctyne (BCN), a second macromolecule functionalized with a coumarin-azide, and a photoinitiator, the method may comprise irradiating to a first predetermined area of the hydrogel with a first light at a first wavelength of, for example, about 365-450 nm, 420-440 nm or 425-435 nm, such that the first predetermined area may degrade. The stiffness in the first predetermined area may decrease due to the degradation. The method may further comprise irradiating a second predetermined area of the hydrogel with a second light at a second wavelength of, for example, about 450-550 nm, 520-540 nm, or 525-535 nm, such that the second predetermined area may crosslink. The photoinitiator may be Eosin Y. The hydrogel may further compnse a co-initiator. The co-initiator may be a thiol having one or more functional groups, preferably a tetra functional thiol, or other known co-initiators (e.g., triethanol amine, triethyl amine). The co-initiator may increase the crosslinking. The stiffness of the hydrogel in the second predetermined area may increase due to the crosslinking.

According to the method, after the irradiating, the hydrogel may have a Young’s modulus of about 0.01-100 kPa, 0.01-50 kPa, 0.01-15 kPa, 0.01-10 kPa, 0.01-5 kPa, 0.01-4 kPa, 0.01-3 kPa, 0.01-2 kPa, 0.01-1 kPa, 0.01-0.5 kPa, 0.01-0.1 kPa, 0.1-100 kPa, 0.1-50 kPa, 0.1-15 kPa, 0.1-10 kPa, 0.1-5 kPa, 0.1-4 kPa, 0.1-3 kPa, 0.1-2 kPa, 0.1-1 kPa, 0. 1-0.5 kPa, 0.5-100 kPa, 0.5-50 kPa, 0.5-15 kPa, 0.5-10 kPa, 0.5-5 kPa, 0.5-4 kPa, 0.5-3 kPa, 0.5-2 kPa, 0.5-1 kPa, 1-100 kPa, 1-50 kPa, 1-15 kPa, 1-10 kPa, 1-5 kPa, 1-4 kPa, 1-3 kPa, 1-2 kPa, 2-100 kPa, 2-50 kPa, 2-15 kPa, 2-10 kPa, 2-5 kPa, 2-4 kPa, 2-3 kPa, 3-100 kPa, 3-50 kPa, 3-15 kPa, 3-10 kPa, 3-5 kPa, 3-4 kPa, 4-100 kPa, 4-50 kPa, 4-15 kPa, 4-10 kPa, 4-5 kPa, 5-100 kPa, 5-50 kPa, 5-20 kPa, 5-15 kPa, 5-10 kPa, 10-100 kPa, 10-50 kPa, 10-20 kPa, 10-15 kPa, 15-20 kPa, or 50-100 kPa. In one embodiment, the hydrogel may have a relatively low Young’s modulus corresponding to that of a healthy tissue (e.g., a healthy lung tissue) at, for example, about 0.5-5 kPa, 0.5-4 kPa, 0.5-3 kPa, 0.5-2 kPa, 0.5-1 kPa, 1-5 kPa, 1-4 kPa, 1-3 kPa, 1-2 kPa, 2-5 kPa, 2-4 kPa, 2-3 kPa, 3-5 kPa, 3-4 kPa, 4-100 kPa, 4- 50 kPa, 4-10 kPa, or 4-5 kPa. In another embodiment, the hydrogel may have a relatively high Young’s modulus corresponding to that of a lung tissue (e.g., fibrotic lung tissue) at, for example, about 5-20 kPa, 5-15 kPa, 5-10 kPa, 10-20 kPa, 10-15 kPa, or 15-20 kPa.

The method may further comprise measuring a response of the cells in the predetermined area after the irradiating. The method may further comprise quantifying proliferation of the cells, for example, using a biomarker such as Ki-67. The method may further comprise quantifying expression of a biomarker by the cells. The biomarker may be an epithelial biomarker (e.g., E-cadherin) or a mesenchymal biomarker (e.g., aSMA, Vimentin). The biomarker may be a functional epithelium (e.g., tubulin for cilia, MUC5AC, or MUC5B for mucus) or an epithelial cell barrier property (e.g., transepithelial electrical resistance (TEER)). The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

Example 1. Photoresponsive system especially for studying cell responses to injury, repair, and disease processes

1. Synthetic basement membrane for photo-injury model

We aimed to formulate a photodegradable hydrogel to study lung epithelial cell response to injuries simulated by selective degradation of crosslinks within the bioinspired synthetic ECM. The hydrogel was formed by reacting 4-arm polyethylene glycol (PEG)- bicyclononyne (exo) (BCN-exo) (PEG-4-BCN) with photodegradable PEG-di-coumarin- azide (CmPNs) via strain promoted azi de-alkyne cycloaddition (SPAAC) (FIG. 1A). For promoting cell attachment and function, here, ImM of each bioinspired peptides, laminin inspired AG73 and fibronectin inspired RGDS-PHSRN (PHSRN), were also reacted to form each hydrogel via azide reactive handles. The weight percentage of the total PEG monomers crosslinked to form the hydrogel was varied to adjust the modulus of the hydrogel to mimic different tissue types, including healthy to diseased lung tissue. Rheological measurements were then done to measure the moduli of the equilibrium swollen hydrogels. Here, 5 wt% PEG inclusion was used to mimic healthy lung tissue (-2 kPa), and 9 wt% PEG inclusion was found to mimic fibrotic lung tissue (~10 kPa) (FIG. IB). The CmPN? crosslinker included in these hydrogels then is used to selectively degrade the synthetic ECM to mimic injury. The absorbance spectra of the crosslinker, CmPNs (FIG. 1C), spans the UV to visible such that the hydrogel readily degrades with low cytocompatible doses of light in the wavelength in range of -350 nm - 475 nm. Tuning in this range can be used to modulate the rate and mode of degradation (e.g., surface erosion when irradiated with a highly-absorbed wavelength vs. bulk degradation when irradiated with a less-absorbed wavelength). A light source of 430 nm centered illumination spectra was used to degrade the surface of the hydrogels to simulate injuries. The changes in the network occur with the degradation of CrnPNs via the coumarin group to coumarin methyl ester degradation within the crosslinker (FIG. ID). Selective degradation of these hydrogels was done by using a photomask held in a 3D printed holder, allowing close proximity to the cells in culture without disrupting the cells. Here, the photomask had a clear line surrounded by a black background (‘slit’) that was varied in size to change the width of the simulated injury; however, photomasks with other printed features can be used. Orthogonal images taken by confocal microscopy of the hydrogels enabled visualization and quantification of the degraded area (FIGS. IE, G, I). The width of the ‘slit’ on the photomask does not affect the depth of the injury (FIG. 1 J), nor does the weight percent of the hydrogel (FIGS. IF, H). Time was found to be the best way to control the depth of degradation. The hydrogel was exposed to the light for increasing amounts of time to increase the depth of the 'injury' to the synthetic ECM.

2. Development of healthy epithelium on photoresponsive hydrogel

A basement membrane mimicking hydrogel system was established using photoresponsive PEG-4-BCN for developing a healthy lung epithelium (FIG. 2A). To evaluate the effects of integrin-binding peptides on A549, we utilized fibronectin (PHSRN) and laminin (AG73) mimicking peptides in hydrogel either alone or combinedly. Cell proliferation was measured based on the percentage of the Calcein-positive area with live- dead confocal images over time (FIG. 2B), where initial attachment of A549 cells was quantified using bright field images. A synergistic effect was observed with an extensive surface coverage of hydrogel using % Calcein+area in PHSRN+AG73 conditions, compared to PHSRN and AG73 alone conditions (FIG. 2C). However, no significant difference was observed in A549 viability over time between single or combined peptide hydrogel conditions; still, the cell proliferation rate differed. A minimal cell attachment was observed with PHSRN and AG73 alone by day 1, where a combination of peptides led to significant enhancement of cell attachment with the spreading of A549 on the PHSRN+AG73-hydrogel. Note that PHSRN+AG73 hydrogel had >90% surface coverage w ith the formation of a monolayer on day 10 culture, where surface coverage remained <80% on PHSRN & AG73 hydrogel. Furthermore, additional Calu-3 cells were cultured on hydrogel to test the hypothesis on peptide combination effects in cellular attachment and proliferation. Similarly, Calu-3 cells were processed for live-dead staining and monitored over time using a bright field, imaged by confocal microscopy, and quantitatively analyzed (FIG. 3). Similar trends were noticed where a combination of peptides (PHSRN+AG73) showed a significant monolayer surface coverage by day 55 and initial attachment on day 1 compared to the single peptide condition, suggesting a synergistic effect in cellular attachment and proliferation. These observations supported the relevance of cell-matrix interaction with more surface functionality in promoting epithelial cell proliferation and phenotypic maintenance, where we continued to study a better understanding of epithelial mechanisms with cell-matrix interaction.

To further phenotypic characterization, additional samples were stained for the epithelial marker E-cadherin (an adherens junction protein expressed during growth), that are integral in cell adhesion and maintains epithelial phenotype of cells and mesenchymal marker Vimentin (expressed in cytoskeletal component). We performed confocal imaging and quantitative analysis. Noted that E-cadherin protein expression was higher on day 10 than 3 culture, and a large number of E-cadherin positive cells was perceived when A549 cells were cultured on PHSRN+AG73-hydrogel and statistically higher than PHSRN and AG73 alone (FIGS. 2D, E). Furthermore, samples were assessed with a Vimentin marker, and a notable low count of Vimentin positive cells was observed when cells were cultured on PHSRN+AG73-hydrogel, with statistically decreased positive counts over time and compared to PHSRN and AG73 (FIG. 4), indicating maintenance of epithelial phenotype. Further, we examined E-cadherin and Vimentin expression of Calu-3 cells over time culture, and a slightly different trend was observed in epithelial and mesenchymal properties. The expression of E-cadherin protein was higher in PHSRN and PHSRN+AG73 than AG73 conditions, where no expression of Vimentin protein was noticed for all the conditions either on day 3 or 55 culture (FIGS. 5, 6), suggesting epithelial phenotype with non-mesenchymal properties of Calu-3 over time. These exciting observations further supported maintaining epithelial properties in regulating monolayer formation and a better in vitro system for further wound healing studies.

3. In vitro injury model for real-time monitoring of epithelial cell response

To investigate the monolayer formation and then response to injury in real-time, we cultured epithelial cells on PHSRN+AG73 hydrogel using a stable reporter cell line (A549 cells engineered to constitutively express a red fluorescent protein and conditionally express a green fluorescent protein when alpha smooth muscle actin was upregulated) and demonstrated how cells responded to epithelial injury (FIGS. 7A, B). Cells were monitored over time by confocal microscopy, where >90% surface coverage was observed on day 10 culture, and a negative expression of aSMA (alpha smooth muscle actin) was indicated a non-mesenchymal, epithelial-like phenotype for cells when cultured on PHSRN+AG73- hydrogel (FIGS. 7C, D). Next, we developed a controlled epithelial injury hydrogel system and assessed the effects of epithelial response to injury with irradiation with 430 nm light. Epithelial wounds varied in size (as a width), where a similar depth of injury using 4 minutes of irradiation w as considered for a better understanding of the response of the epithelial cells to wound, and a photomask was used to avoid the creation of unwanted regions of injury on the monolayer. A significant expression of aSMA protein was observed at the post-injury (PI) injury area in 50 and 100 pm cases over time (FIGS. 7E-H). However, the expression of aS MA protein remained significantly higher at an out-of-injury area over day 5 PI compared to day 0 PI control (FIG. 8A), interestingly a large width (where cells weren’t able to fill the gap of wound area over time) of injury created by 100 pm size of slit showed a significant number of aSMA positive cells at without injury area comparatively lower number of positive cells in a less width (where cells filled the gap of wound area over time) of injury created by 50 pm slit on day 5 PI (FIG. 8D), suggesting mesenchymal phenotype enhancement for epithelial cells when injury width was increased. These findings indicated that A549 cells could maintain epithelial phenotype with a minimum aSMA positive cells during a healthy epithelium formation, whereas they can switch to a mesenchymal phenotype with a significant aSMA positive cells when injured epithelium on photoresponsive hydrogel. These observations further support the global phenotypic loss of epithelial-like cells during injury, which may influence maladaptive healing processes depending on injury size and contribute disease processes.

4. Epithelial injury leads to global loss of phenotypic marker expression

To further a better understanding of epithelial cell response to wound repair, we culture similarly non-transduced A549 cells on hydrogel and characterized by immunofluorescence with and without photo-injury samples after day 10 culture (FIG. 9A). We varied injury sizes using 50, 100 and 200 pm slits, where depth of injury was similar with 4 minutes of irradiation for all the conditions and control samples continued to grow for 10 days. A549 cells were stained with epithelial (E-cadherin) and mesenchymal (Vimentin) markers and showed distinct localization of adherens junction protein marker, E-cadherin at injury area when created a small size wound by 50 pm slit on healthy epithelium at day 5 post-injury (total 10 days culture) (FIG. 9B). On the other hand, we observed a minimum of E-cadherin-positive cells at the injury site when using 100 and 200 pm slits for large size of wound creation (FIG. 9C), suggesting loss of cell-cell signals at injury when a large wound create on epithelium. In context, the expression of mesenchymal marker Vimentin was significantly increased in small wound (using 50 pm slit) areas than in large wounds (using 100 and 200 pm slit) (FIGS. 10A, B), indicating cell migration with a reducing gap between cell-cell signals at small injury areas where cells can recover and re- epithelize of the wound by switching from epithelial-mesenchymal and mesenchymal- epithelial phenotype and increasing gap between cell-cell connection can enter to the stress phase and fail to recover the wound area, maybe because of cell apoptosis and death. Additionally, to better understand the mechanism of epithelial phenotypic switch in postinjury effects, transepithelial electrical resistance (TEER) measurements of the epithelial layer were conducted using a commercial system (EVOM 2, World Precision Instruments) using chopstick electrodes method. We evaluated TEER values on the monolayer of A549 over time, exhibiting epithelial resistance >500 Ohm cm², suggested healthy epithelium formation on photoresponsive hydrogel. In contrast, TEER values were significantly decreased by <200 Ohm cm² on damaged epithelium created by photo-injury on day 6 (PI DI), where was less physiological value <20 Ohm cm² in 100 pm slit condition. We continued the culture after injur}' until PI D5 (day 10 culture), followed by the measurement of epithelial resistance, where TEER values slightly increased in both 50 and 100 pm slits conditions compared to PI DI, but a significantly higher value was detected on the injury epithelium with 50 pm slit on PI D5 compared to either 100 pm slit on PI D5 or 50 pm slit on PI DI (FIG. 9D). This result recommended a healthy epithelium formation on our hydrogel system while maintaining the phenotypes and a significant loss of epithelial resistance because injury size increases and the entire population is affected.

To investigate the entire population phenotype after injury, the expression of E- cadherin protein was analyzed far away from the photo-injury (as a no PI) area of epithelium, and a significant dropped of E-cadherin positive numbers was detected when injury was created using all sizes of 50, 100 and 200 pm slit compared to control, where a non-significance difference was observed between 50 and 100 pm slit conditions on day 5 PI culture; additionally, a significantly lower expression of E-cadherin with a minimum number of positive cells was noted in 200 pm slit condition compared to 50 and 100 pm slit (FIG. 11). These results suggested that injury of any size affects the wound area and the entire epithelial population, where cells might recover and repair the wound with a phenotypic switch in small injury effects later in time. Furthermore, we processed the extra samples with staining of cell proliferation markers (e.g., Ki-67) and mesenchymal markers (e.g., aSMA) to broadly understand the entire cell population growth mechanism of the wound response. A high number of Ki-67 - positive and aSMA-negative cells were analyzed on control samples with an entire population, suggesting the proliferative stage of A549 cells without mesenchymal phenotypes. While Ki-67 -positive cells decreased in counts at the wound area with 50 and 100 pm condition after day 5 injury and a significant dropped of Ki-67-positive cells was noted in 100 pm slit compared to 50 pm slit, and a few aSMA-positive cells were observed at 50 pm injury area, but no positive at 100 pm (FIGS. 9E, F). The results suggested that A549 cells can proliferate and migrate with recovery at the wound area when a small injury occurs on the epithelium,

5. Multiple injuries enhance A549 cell apoptosis

Increased injury size is associated with increased apoptosis (FIG. 12). Further, the system allows the introduction of multiple injunes to the same location or different locations and over time, where increased apoptosis is observed with multiple injuries even with a smaller size of injury (FIG. 13).

6. ALI cultures of a range of epithelial cells on well-defined bioinspired ECMs probing response to injury

Moving to the ALI allows the formation of a functional epithelium more rapidly than in submerged culture with model Calu-3 epithelial cells. Further tuning of the bioinspired synthetic basement membrane composition for increased complexity allows the effective and consistent culture of Calu-3 and HBE at the ALI with the culture system (FIGS. 14, 15), as well as other cell types including keratinocytes, monocytes, and macrophages (not shown). Here, additional peptides to more broadly bind integrins and cell- secreted ECM were integrated along with harvested, purified specific basement membrane ECM proteins and polysaccharides. To our knowledge, there are few reports of primary epithelial cell cultures at the ALI on such well-defined molecularly engineered ECMs. Epithelial cell response to photo-injury at the ALI then can be probed with the system (FIG. 16), as shown above in submerged cultures.

7. Photostiffening followed by photo-injury

To probe responses to matrix damage followed by matrix stiffening, a PEG-based strain-promoted azide-alkyne cycloaddition hydrogel was formed using a coumarin- containing crosslinker to enable injury mimetic properties (FIG. 17). The stiffening mimetic properties were then enabled by incorporation of excess BCN groups during hydrogel formation and triggered crosslinking reactions via photoinitiation of Eosin Y. The biomechanical properties of this hydrogel-based synthetic ECM were modulated to mimic injury via selective photodegradation by photomasking and subsequent photostiffening to mimic the injury cascade. Assessment of these mechanical changes was done by rheometry and confocal imaging. Epithelial cells were cultured on these hydrogels at the air-liquid interface and were then subjected to injury, stiffening, and injury-stiffening, and cellular responses were assessed via immunostainmg and imagining. The use of this photoresponsive hydrogel-based synthetic ECM platform to probe cellular response to injury and fibrosis aims to provide insights into cellular response to dynamic biomechanical changes.

Hydrogels were formed using PEG-8-BCN and CmPNa at a 3: 1 ratio of BCN groups to azide groups. Here, the functionality of the PEG was increased from 4 in our earlier photo-injury model to 8 to accommodate both photodegradation, bioactive groups, and photostiffening. This composition forms a hydrogel with a Young’s Modulus at the lower end of the range of healthy lung tissue. 2 mM of bioadhesive peptides were also included in this hydrogel formulation, 1 mM of AG73 and 1 mM of PHSRN, to create this synthetic basement membrane mimic. Eosin Y and co-initiator molecules together upon photopolymerization with green light allow significant increases the modulus of the SPAAC initially formed hydrogel (FIG. 18). Thiol containing molecules can be used as a co-initiator with Eosin Y for stiffening. Increasing the functionality of this thiol increases the stiffening of the gel. Concentration of this thiol co-initiator and concentration of free cyclooctyne also increase stiffening. Stiffening solutions that were used resulted in final concentrations of 6.6 mM thiol groups from PEG-4-SH and 0.5 mM Eosin Y. Multiple mechanisms of crosslinking were occurring with polymerization: these include mono-thiolyne, di-thiolyne, and homo BCN polymerization. Additional BCN groups diffused into the gels resulted in increases in swelling which offsets the increase in crosslinking/stiffening.

This stiffening system was transferred successfully from in situ formed to equilibrium swollen hydrogels (FIG. 19). The time and intensity of the green light was tuned to maximize extent of stiffening while minimizing light exposure time. This was found to be 30 mins. Stiffening can be stopped and started with light being stopped and started. Injury and followed by stiffening can be done with blue and green light, respectively.

A549 human alveolar basal epithelial cells formed an epithelial layer at the ALI with the hydrogel system used for stiffening (FIG. 20). In principle, this approach can be used i th other epithelial cells. These hydrogels were then stiffened with the epithelial layer on top. Morphological changes were observed, with on-going analyses of phenotypic responses.

8. Conclusions

A photoresponsive hydrogel -based synthetic ECM platform to mimic injury and stiffening cascade, including at the ALI, is useful to understand cellular responses for mechanistic studies and will be relevant for future use in the development of more effective therapeutic approaches. This PEG-BCN, CmPNv and peptide-based hydrogel functions well as a synthetic ECM with tunable stiffness and biochemical content for promoting relevant cellular functions, including formation of an epithelium, and is capable of dynamic biomechanical changes with matrix degradation triggered with one wavelength of light and matrix stiffening triggered with another wavelength of light, initiator Eosin Y, and building block and co-initiator (e.g., PEG-4-SH). Specifically, these hydrogels can be degraded by blue light (e.g., 430 nm) separately from the stiffening process to trigger matrix degradation and induce injury, and green light (e.g., 530 nm) can be used to trigger dynamic stiffening. This engineered photoresponsive hydrogel network can be used to mimic microinjuries to the epithelium and subsequent stiffening (e.g., associated with fibrosis or cancer reoccurrence).

9. Methods

9.1 Development of lung epithelium formation on photoresponsive hydrogel Reactive handles and functionalized polymer and peptide building blocks were synthesized by published known methods. (Jessica et al., The Journal of Organic Chemistry 2018 83 (14), 7500-7503, DOI: 10.1021/acs.joc.7b02329; Badeau et a/., Nature Chem 10, 251-258 (2018), https://doi.org/10.1038/nchem.2917; Schieber et n/., Angew Chem Int Ed Engl. 2012 Oct 15;51(42): 10523-7. doi: 10.1002/anie.201202876. Epub 2012 Sep 20. PMID: 22996637; DeForest et al., Nature Mater 14, 523-531 (2015). https://doi.org/10. 1038/nmat4219.;.LeV alley et al., ACS Appl Bio Mater, vol. 3, no. 10, pp. 6944-6958, Oct 19 2020, doi: 10.1021/acsabm.0c00823). Photoresponsive PEG-4- BCN(exo) hydrogels (10-100pL) were first prepared with CmPNs using PHSRN, AG73, or PHSRN+AG73 integrin binding peptides in 12 well transwell insert (Coming #3460) and sterilely incubated overnight for full gelation. All the hydrogels were hydrated and washed three times with sterile 500 iL of PBS, and a final wash was with 500 pL of DMEM-F12 before seeding of the non-transduced or transduced A549 cells culture. ~ 80-90% confluent A549 cells were trypsinized from the tissue culture flask and seeded onto the hydrogels in 12 well transwell inserts at 200,000 cells per insert. Cells were submerged in DMEM-F12 growth media over 10 days. DMEM-F12 media was changed every 2-3 days over time culture. This protocol also was utilized with other transwell insert sizes with success, scaling volumes accordingly. Samples were irradiated through a photomask using a 3D printed insert (FIG. 21) with appropriate wavelengths and intensities of light (see examples in Results).

9.2 ALI cultures on photoresponsive hydrogel

Calu-3s and HBEs were cultured following published protocols, particularly from the lab of Dr. Scott Randell at UNC-Chapel Hill. Cell seeding densities, timing of transition from submerged to ALI culture, hydrogel thickness, and hydrogel biochemical content as noted in Results were tuned to achieve formation of an effective lung epithelium.

9.3 In situ stiffening

A master mix of PEG-8-BCN, PHSRN, AG73, and PBS added at ratios set stiffening components such as Eosin Y disodium salt and thiol coinitiators were added as well. This was left to mix for 1 hr to react the peptides with the BCN groups (on tube rack w rapped in aluminum Foil on rocking plate). Then a part of this master mix for the gel was moved from the master mix into a new tube and 3.3 mM of CmPNs was added. IOUL of hydrogel solution was then placed on the center of a UV quartz rheometer bottom plate and a stainless steel smooth 8 mm geometry lowered to al40 nm gap. 1 rad/s and 1% strain, within linear viscoelastic region for formed hydrogel, w as applied and the modulus measured overtime as the hydrogel gelled. Light mineral oil was added at 250-300s to prevent drying. Light was turned on at 3500s, 450 nm, 4 mW/cm² for stiffening. This is at a point where initial gelation was typically considered completed.

9.4 Equilibrium swollen stiffening

Equilibrium gels were formed by first preparing a master mix of PEG-8-BCN, PHSRN, AG73, and PBS. A portion of this was then removed and 3.3 mM CmPNs was added to this solution. This was then vortexed and centrifuged before being added to a syringe mold, a 1 mL syringe with the top cut off. A second syringe plunger was then inserted into this cut-off side to seal the gel into the mold overnight. Gels were then removed from the molds and placed into an untreated 24 well plate with 1 mL of DI water. They were then swelled for 24 hours. Water was then replaced with PBS for 24 more hours. Starting modulus of the hydrogel was then measured at 6 rad/s 1% oscillation strain. After measurement, the gels were placed back in PBS until all could be measured. PBS was then replaced with stiffening solution (250 pL, Eosin Y and PEG-4-SH, 10 kDa) overnight (~12 hr) at room temperature for final concentrations of 0.5 mM and 6.6 mM in the hydrogels. Modulus was measured. Gel was then placed into a well plate with 250 pL PBS and exposed to 530 nm, 4 mW/cm² for a reported amount of time. Modulus of gel was remeasured. Gels were placed back in 250 pL PBS during the illumination of the rest of the gels (10 min - 6 hr) and then additionally washed for 2 times 1 h wash. Stiffening was repeated up to 3 times each time assuming 0 mM of PEG-4-SH or Eosin Y in gel when swelling in additional components. After stiffening was completed and the final washes were done the gels washed in last PBS wash overnight and the full extent of stiffening was measured.

9.5. Stiffening in Cell Culture

Hydrogel stiffening for cell culture applications is detailed in the following. A hydrogel solution was made as descripted previously. 15 pL was used for each gel and was deposited on the Transwell surface by adding the first drop to the center of the well, spreading the rest around the gel, and adding the last drop to the middle of the well. Transwells were then incubated overnight. PBS was added to the top and bottom of the well the following day and swelled for 24 hours. Cells were then seeded onto the gel with media replacing the PBS. The cell growth was monitored and moved to ALI at 90% confluency. After they had been in ALI conditions for 7 days the media was replaced with the stiffening solutions of PEG-4-SH, Eosin Y, and media at concentrations with made the hydrogel have 6.6 mM thiol from PEG-4-SH and 0.5 mM Eosin Y. The next day, gels were stiffened one by replacing the stiffening solution with 700 pL of media and then illuminating with 530 nm, 4 mW/cm², 30 min. Each gel was then washed 3 times for 30 min each with media. For repeated stiffening, a fresh stiffening solution was added again, assuming that there are 0 mM of thiol groups or Eosin Y, stiffening done the following day, and the process repeated up to 3 total times. Gels were fixed for immunostaining along the way.

All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and/or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED:

1. A hydrogel comprising a first macromolecule, a second macromolecule, and a photoinitiator, wherein the first macromolecule has three or more functional groups and the second macromolecule has two or more functional groups, wherein either the first macromolecule or the second macromolecule has a cleavage site, and wherein the hydrogel degrades at the cleavage site upon irradiation with a first light at a first wavelength and crosslinks upon irradiation with a second light at a second wavelength.

2. The hydrogel of claim 1, wherein the first macromolecule is functionalized with a cyclooctyne, wherein the second macromolecule is functionalized with a coumarinazide, and wherein the first wavelength is 365-450 nm.

3. The hydrogel of claim 1, wherein the first macromolecule is functionalized with a bicyclooctyne (BCN), wherein the second macromolecule is functionalized with an azide, and wherein the second wavelength is 450-550 nm.

4. The hydrogel of claim 1, wherein the first macromolecule is functionalized with a bicyclooctyne (BCN), the second macromolecule is functionalized with a coumarinazide, the photoinitiator is Eosin Y, the first wavelength is 365-450 nm, and the second wavelength is 450-550 nm.

5. The hydrogel of claim 4, wherein the first macromolecule is 4-arm polyethylene glycol (PEG)-bicyclononyne (exo) (BCN-exo) (PEG-4-BCN) or 8-arm PEG- bicyclononyne (exo) (PEG-8-BCN), and wherein the second macromolecule is PEG-di- coumarin-azide.

6. The hydrogel of any one of claims 1-5, wherein the hydrogel has a Young’s modulus of 0.5-5kPa.

7. The hydrogel of any one of claims 1-5, wherein the hydrogel has a Young’s modulus of 5-20 kPa.

8. The hydrogel of any one of claims 1-7, further comprising an extracellular matrix molecule.

9. A cell culture comprising cells, the hydrogel of any one of claims 1-8, and a culture medium, wherein the cells are seeded onto the hydrogel and grown in the culture medium.

10. The cell culture of claim 9, wherein the cells are epithelial cells and form an epithelium on the hydrogel.

11. The cell culture of claim 9 or 10, wherein the epithelium is formed under a submerge culture condition.

12. The cell culture of claim 9 or 10, wherein the epithelium is formed at an airliquid interface (ALI).

13. A method for monitoring a cell response in an in vitro injury model, comprising

(a) growing the cells in the cell culture of any one of claims 9-12;

(b) irradiating a light to a predetermined area in the hydrogel, whereby the stiffness of the hydrogel in the predetermined area is changed; and

(c) measuring a response of the cells in the predetermined area after step (b).

14. The method of claim 13, wherein the cells are epithelial cells, and wherein step (a) comprises forming an epithelium on the hydrogel.

15. The method of claim 14, wherein the epithelium is formed under a submerge culture condition.

16. The method of claim 14, wherein the epithelium is formed at an air-liquid interface (ALI).

17. The method of any one of claims 13-16, wherein step (c) comprises quantifying proliferation of the cells.

18. The method of any one of claims 13-16, wherein step (c) comprises quantifying expression of a biomarker by the cells.

19. The method of any one of claims 13-18, wherein step (b) comprises irradiating to a first predetermined area in the hydrogel with the first light at the first wavelength, whereby the first predetermined area in the hydrogel degrades at the cleavage site.

20. The method of any one of claims 13-18, wherein step (b) comprises irradiating to a second predetermined area in the hydrogel with the second light at the second wavelength, whereby the second predetermined area of the hydrogel crosslinks.

21. The method of any one of claims 13-18, wherein step (b) comprises:

(i) irradiating to a first predetermined area in the hydrogel with the first light at the first wavelength, whereby the first predetermined area degrades at the cleavage site; and

(ii) irradiating to a second predetermined area in the hydrogel with the second light at the second wavelength, whereby the second predetermined area crosslinks.