NOVEL HYDROGEL FOR 3D TISSUE ENGINEERING

Abstract

It is described a composite hydrogel containing egg white and alginate (EWA) polymers, and a method of producing same, wherein the alginate is cross-linked using frozen calcium chloride disks, creating a scaffold for cells comprising a slow-rate ions diffusion through the matrix, ensuring a homogenous crosslink and smooth surface.

Claims

1. A hydrogel composition comprising egg white and alginate.

2. The composition of claim 1, wherein the egg white and alginate are cross-linked with calcium chloride.

3. The composition of claim 1, comprising 1% to 3% alginate.

4. The composition of claim 1, wherein calcium chloride is a CaCl.sub.2) solution.

5. The composition of claim 4, wherein the CaCl.sub.2) solution is at a temperature of 4° C. to 25° C.

6. The composition of claim 4, wherein the CaCl.sub.2 solution is a frozen CaCl.sub.2 solution.

7. The composition of claim 1, wherein said composition is a scaffold.

8. The composition of claim 7, further comprising seeded cells on said scaffold.

9. The composition of claim 8, wherein the cells are salivary gland cells.

10. The composition of claim 1, further comprising a copolymer.

11. The composition of claim 10, wherein the copolymer is at least one of gelatin, hyaluronic acid, collagen, laminin and carbon nanotubes.

12. A method of producing the hydrogel composition of claim 1 comprising the steps of: a) providing egg whites; b) mixing the egg whites with alginate creating an egg white/alginate (EWA) hydrogel; c) adding the EWA hydrogel on a surface; and d) incorporating a crosslinker into the EWA hydrogel for crosslinking the alginate in said EWA hydrogel.

13. The method of claim 12, wherein the crosslinker is a CaCl.sub.2 solution.

14. The method of claim 13, wherein the CaCl.sub.2) solution is at a temperature of 4° C. to 25° C.

15. The method of claim 12, wherein the EWA hydrogel is crosslinked at a temperature of −20° C. to 25° C.

16. The method of claim 12, wherein the EWA hydrogel is a scaffold.

17. The method of claim 12, wherein the surface is a plate or a 6-well plate.

18. The method of claim 16, further comprising the step e) of seeding cells on top of the scaffold.

19. The method of claim 18, wherein the cells are salivary gland cells.

20. The method of claim 12, wherein the egg whites are mixed with 1% to 3% alginate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Reference will now be made to the accompanying drawings.

[0036] FIG. 1 illustrates a schematic representation of the EWA hydrogel preparation, frozen CaCl.sub.2) disk, and salivary gland organoid formation in accordance to an embodiment.

[0037] FIG. 2 illustrates images of EWA made by two techniques, and the small bubbles in EWA, wherein the EWA images were acquired before (a, c) and after (b, d) the improved crosslinking technique, c) and d) show one well from the 6-well plate where the low quantity of micro and macro bubbles/pit is observed on the improved method (d).

[0038] FIG. 3 illustrates frozen CaCl.sub.2) on aluminum foil pressed against the interior of the wells of a 6-well plate.

[0039] FIG. 4 illustrates optical images of NS-SV-AC cells growing on EWA hydrogels at day 3 after culture wherein cells were seeded on top of the EWA at various concentrations of alginate: 1.0% (a), 1.5% (b), 2.0% (c), 2.5% (d) or 3.0% (e), and magnification ×5 (left panel) and ×10 (right panel) are presented, and scale bar 100 μm. Dotted lines show the area of the magnified images.

[0040] FIG. 5 illustrates surface area and distribution of organoids formed on EWA hydrogels, showing in (a) the surface area (μm.sup.2) of each organoid found in the EWA hydrogels as measured and compared among EWA containing different alginate percentages, data were plotted as boxplot using a box limit of 25.sup.th and 75.sup.th percentiles with a minimum-maximum whisker's range; **** P<0.0001; and in (b) the number of organoids per square millimeter in the EWA scaffolds determined and compared among the different alginate concentration samples, where data are presented as mean±SD, n<3.

[0041] FIG. 6 illustrates the growth rates of NS-SV-AC seeded on EWA at 1, 1.5, 2, 2.5, or 3% alginate, wherein data are presented as mean±SD, n 3, *P<0.05, ** P<0.01.

DETAILED DESCRIPTION

[0042] In accordance with the present description, it is provided a composite hydrogel containing egg white and alginate (EWA) polymers using a nonconventional method to crosslink the alginate employing frozen calcium chloride disks.

[0043] This crosslinking method provided herewith allows creating a scaffold for cells comprising a slow-rate ions diffusion through the matrix, ensuring a homogenous crosslink and smooth surface compared with the traditional hand drop method. The formulation of the composite provided herewith can be tailored to modify the stiffness of the material, which can be suitable for multiple biomedical applications. This composite hydrogel promotes the formation of salivary organoids controlling their sizes.

[0044] Hydrogels have been used for a variety of biomedical applications; in tissue engineering, it is commonly used as scaffolds to cultivate organoids in a three-dimensional (3D) environment. EWA as described is a novel hydrogel which combines the advantages of both egg white and alginate; the egg white material provides extracellular matrix (ECM)-like proteins that can mimic the ECM microenvironment, while alginate can be tuned mechanically through its ionic crosslinking property to modify the scaffold's porosity, strength, and stiffness.

[0045] It is described a frozen calcium chloride (CaCl.sub.2)) disk technique to homogenously crosslink alginate and egg white hydrogel is presented for 3D culture of human salivary cells. Different EWA formulations were prepared and biologically evaluated as an organoid platform. Although all five EWA hydrogels showed biocompatibility, the EWA with 2.0% alginate presented the highest cell viability, while EWA with 3% alginate promoted the formation of larger size salivary organoids.

[0046] As seen in FIG. 1, the use of frozen CaCl.sub.2) disks allows alginate and EW to crosslink in a more controlled manner through the slow diffusion of Ca.sup.2+ ions. This technique improves the smoothness of the EWA hydrogel surfaces as well as significantly reduces the formation of macro pits and bubbles generated by the addition of the CaCl.sub.2) solution using a slow-dispensing micro-pipetting technique. Furthermore, the use of frozen CaCl.sub.2) disks allow for better cell distribution across the EWA scaffold surface due to the steady homogenous crosslink. Increasing the alginate concentration in the EWA scaffold, SG organoid formation is promoted with high cell viability.

[0047] Alginate is an inert carbohydrate copolymer comprised of 1-4 linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) isolated from brown seaweed and some bacterial species. Divalent ions (e.g. Ca.sup.2+, Ba.sup.2+, Sr.sup.2+) are used as a crosslinker to form an “egg-box” structure by the interaction with blocks of G monomers, producing a stiff and stable 3D structure, thus allowing mechanical modification of the alginate's properties to mimic the ECM stiffness of particular tissues. Alginate is purified by a multi-step extraction procedure yielding high-quality materials that are inert in mammals. It has been shown that no significant inflammatory response is observed when gels formed from commercially available, highly purified alginate are subcutaneously injected into mice.

[0048] Egg white (EW) is a viable scaffold due to its great advantages in cellular attachment, differentiation, and proliferation. EW is comprised of an albumen layer which is mainly composed of ovalbumin (<50%) and other structural proteins that resemble ECM proteins (such as collagen), acting as a substrate for cellular attachment. All these EW proteins make EW a great candidate to be used as complementary material in alginate hydrogels allowing the creation of 3D microenvironments that mimic native mammalian ECM.

[0049] EW-based biomaterials have been proven advantageous in tissue engineering: (i) it can provide sufficient nutrition for chicken gametes to grow into a baby chick; (ii) its transparency makes it convenient to monitor and record the changes in cell morphology; and (iii) it is inexpensive and widely available. A study reported that epithelial breast tumor cell lines grown on EW have comparable phenotypes as those grown on Matrigel (Kaipparettu et al., 2008, Biotechniques, 45: 165-8, 170-1). Similar observations were found in a study comparing human umbilical vein endothelial cells grown on EW and Matrigel (Mousseau et al., 2014, Lab Invest, 94: 340-349). These studies highlight the feasibility of using egg white as an alternative biomaterial to Matrigel.

[0050] One challenge for EWA hydrogels is the instantaneous formation of gels when cross-linked via divalent ion solutions at room temperature, which produces gels that have rough surfaces and is also highly heterogeneous, in addition to their properties being difficult to control. The use of frozen alginate or calcium/solvent solution is proposed as a methodology to create more homogeneous cross-linked hydrogels. The fluidity of EWA decreases with decreasing temperature, thus, a more homogeneous hydrogel can be maintained with the 3D structure that was originally intended.

[0051] It is also encompassed that the described EWA can further comprise in combination a copolymer such as for example, and not limited to, gelatin, hyaluronic acid, collagen, laminin, and carbon nanotubes.

[0052] FIG. 1 shows the general protocol followed to create EWA scaffolds using frozen CaCl.sub.2) disks as the crosslinker, and the formation of salivary gland organoids. Egg white material is extracted from eggs and mixed with different concentrations of alginate to create various EWA hydrogels. For crosslinking, wells from a 6-well plate are covered with sterile aluminum foil, CaCl.sub.2 solution is added and incubated at −20° C. overnight. In another 6-well plate, EWA is added, and then frozen disks are placed on the top to crosslink it. Next, salivary gland cells are seeded on the top of the EWA scaffolds and incubated for 7 to 10 days allowing the formation of organoids.

[0053] Calcium chloride is one of the most common crosslinkers used in alginate gelation, which has less cytotoxicity compared to other divalent ionic crosslinkers such as Ba.sup.2+. These divalent cations cooperatively interact with blocks of G monomers to generate ionic bridges between different polymer chains of alginate that entraps water eventually forming a hydrogel. It has been shown that the gelation rate of alginate increased with temperature, while low temperatures reduce the diffusion rate of Ca.sup.2+ ions, leading to a slower crosslinking process which generates a more ordered network structure, and hence enhanced mechanical properties. This is crucial because for tissue engineering scaffolds, structural uniformity is important not only for well-controlled material properties but also for uniform cell distribution.

[0054] At first, CaCl.sub.2 was added to the EWA solution by slowly pipetting the crosslinking solution directly onto the EWA. Despite dropping it carefully and slowly, pits and bubbles were inevitably produced on the surface of the EWA (see FIGS. 2a and c). The size and depth of those pits varied and was extremely challenging to make uniform. This heterogeneity in the material macro-porosity often resulted in higher cell density at the bottom of the pits where they clumped together while the flat surfaces had sparse amounts of cells. This could ultimately be the result of gravity, causing cells to slide down along the sidewalls of pits before they firmly attach to the scaffold.

[0055] EWA was fabricated with smoother surfaces by placing frozen CaCl.sub.2) disks (see FIG. 3) on the surface of the hydrogel, allowing a slow-rate crosslinking process as the CaCl.sub.2) disk thawed. This method allows the formation of a smoother, uniform EWA surface (FIG. 2C and d) with a visibly lower amount of bubbles within the scaffold (FIG. 2d). It is believed that the two main reasons leading to this improved outcome are: (a) the low temperature of crosslinking solution, which ensures the homogenous and slow-rate crosslink due to the slow release of CaCl.sub.2) molecules as the disk thaws; (b) the smooth surface created in the bottom surface of the frozen CaCl.sub.2) disks that is in direct contact with the EWA surface. After the improvement in crosslinking technique, it was observed that cells and cell clumps were homogeneously distributed across the hydrogel. This improvement on EWA surface smoothness could provide more reliable results, driven by better cell distribution and cellular attachment to the scaffold.

[0056] Another challenge with using EWA is that EWA scaffolds shrink slightly during the crosslinking process due to the reorganization of the alginate chains to form a stiffer structure. An average of 15% decrease in size of the EWA scaffolds post-crosslinking was noted, creating spaces between the hydrogel and the walls of the 6-well plate. The smaller scaffold size allows cells to be displaced off of the scaffold and instead attach to the bottom of the wells during the initial cell-seeding phase. As a result, for all cell experiments, wells that contained EWA without gaps were only included.

[0057] Several studies have demonstrated that some mammalian cells are able to form organoids or spheroids in 3D culture, whether the cells are embedded in the hydrogel or seeded on the scaffold. The ability of five different formulations of EWA on the SG organoid formation was evaluated. FIG. 4 shows the organoid formation of the NS-SV-AC cell line seeded on EWA at day 3. Using 1% alginate, most of the cells were attached to the EWA surface and spread as a monolayer, just a few organoids were observed on the cultures (FIG. 4a). In the case of EWA with 1.5% alginate, more organoid-like structures were visible (FIG. 4b). This observable trend appears to continue with the increase of alginate percentage in the final EWA biomaterial, with 3% alginate showing the most numbers of organoids (see FIGS. 4c, d and e). These results suggest that alginate concentration promotes the NS-SV-AC organoid formation. As observed, scaffolds with a higher concentration of alginate formed larger organoids and their sizes increased with increasing alginate concentration.

[0058] Following the characterization of the organoid formation in EWA, the surface area of the organoids in each hydrogel was measured. The measure of surface area is used as an additional parameter to determine the efficiency of the hydrogels in promoting organoid formation in 3D structures. FIG. 5 shows the organoid sizes and distribution in different EWA blends. EWA 1% produces organoid no larger than 5×10.sup.3 μm.sup.2; increasing the concentration of alginate in the EWA material resulted in larger organoid formation (FIG. 5a). No significant differences among EWA 1% to 2.5% in mean surface area was observed; however, the EWA 3% hydrogel showed significant differences when compared with the rest of the EWA material, where organoid sizes ranged from 5×10.sup.3 μm.sup.2 to 4×10.sup.4 μm.sup.2 (FIG. 5a).

[0059] Organoid/spheroid sizes were classified based on surface area: small (500-10,000 μm.sup.2), medium (10,000-20,000 μm.sup.2), and large (>20,000 μm.sup.2). The formation of a few medium size organoids was seen (between 1 and 3) in 6-well plates containing EWA 1.5%, 2.0%, and 2.5% samples, but found higher amounts of medium and large-sized organoids in the wells containing EWA 3.0%. These results suggest that higher concentrations of alginate in EWA hydrogels promotes the formation of larger organoids.

[0060] The number of organoids per square millimeter was quantified. No significant differences among the five EWA samples was found, however, the number of organoids/mm.sup.2 in EWA 2.0% was slightly larger than the others (FIG. 5b). These differences in organoid sizes and quantities could be related to the mechanical properties provided by the alginate concentration in the scaffolds, where the stiffness of the scaffold plays an important role in cell growth, migration, and survivability.

[0061] To determine the viability of the cells and organoids cultured in the EWA material, a cell viability test was performed using an AlamarBlue assay. AlamarBlue monitors the reducing environment of the living cell. The active ingredient is resazurin (IUPAC name: 7-hydroxy-10-oxidophenoxazin-10-ium-3-one); it is non-toxic which allows for continuous monitoring of cells in culture over multiple time points. As the indicator dye accepts electrons, it can be reduced by NADPH, FADH, FMNH, NADH, and cellular cytochromes, thus changing from the oxidized, non-fluorescent, blue state to the reduced, fluorescent, pink state. Therefore, the change from oxidized to reduced state, a measure of cellular activity, can be quantitatively measured as colorimetric and/or fluorometric readings, where more detection of reduction reflects higher cell viability. First, NS-SV-AC cells were seeded on EWA containing either 1%, 1.5%, 2%, 2.5%, or 3% alginate. Then the samples were cultured for 10 days, taking samples at 1, 3, 5, 7, and 10 days after the initial seeding period (day 0). FIG. 6 shows the growth rates of NS-SV-AC cells seeded on EWA. The viability and proliferation increased over time in all samples from day 1 to day 7, slowing down thereafter, likely contributable to either the formation of large organoids or the number of cells/organoids nearing the maximum growth capacity of the scaffold volume. On day 5, the cell viability of EWA 2.0% and 2.5% alginate showed significantly higher values than the EWA 1.5% and 3.0%. Additionally, on day 7, only the EWA 2% hydrogel showed higher cell viability than the other samples. This result suggests that EWA hydrogels promoted the proliferation of NS-SV-AC as well as their viability, but the EWA with 2% alginate showed the greatest cell viability and proliferation.

[0062] Accordingly, it is provided that a smoother surface in the EWA hydrogel can be generated by decreasing the cross-linking rate using a frozen CaCl.sub.2 solution. In addition, salivary gland organoid formation can be controlled by modifying the concentration of alginate in the EWA material. Visual differences in organoid formation across five different EWA groups are evident, showing that the 2%, 2.5%, and 3% alginate groups of the EWA material are the better hydrogels for promoting cell self-assembly, with high cell proliferation and cell attachment.

[0063] As further encompassed, the provided scaffold can be used as an alternate 3D culture scaffold for studies on drug-screening, cell-migration, or as an in vitro disease model. In addition, the provided EWA can be used as a potential source for cell transplantation. The low cost of producing EWA is an added advantage. Using egg white-based hydrogels as described herewith promotes cells reorganization as spheroids, suggesting that using primary cultures of cells or stem cells, it is possible to promote salivary organoids in EWA. Spheroid and organoid structures can be injured (e.g. irradiation-injury) in order to create an in vitro disease model that mimics the in vivo injury-damaged salivary glands after irradiation (e.g. radiotherapy). By having this in vitro irradiation-injury model, the efficacy of newly developed or current drugs/therapies (for example, medicines for xerostomia, dry mouth) can be tested directly on these functional salivary spheroid structures to determine cell response (e.g. viability) and function. Furthermore, this in vitro model can be used in the study of cell migration because of its transparent property, which supports the use of optical microscopes, as cells live and move within the gel just as they do in vivo. In addition, EWA can support organoid generation, is biodegradable and is not immunogenic, and it can then be used as a platform to culture and expand salivary cells for cell transplantation. For example, it is encompassed to transplant salivary organoids grown in EWA into the site of the damaged salivary glands.

Example I

Fabrication Procedure and Testing of EWA

Egg White Isolation and Heat Treatment

[0064] Fresh eggs (Large White Eggs Omega-3) were purchased from a local retail store (Montreal, QC, Canada). Eggs were sprayed with 70% ethanol then were decontaminated under a biological safety cabinet (BSC). The apex of the shells was cracked and removed (an approximately 1 cm diameter hole was created). Then, the EW material was poured into a 50 ml conical centrifuge tube using forceps to pull the EW out, ensuring no visible contamination from the egg yolk; all other contents (chalaza, yolk, and watery content) were discarded. Each egg harvested provided approximately 25 ml of EW. Next, the tubes were placed in an incubator at 58° C. for 1 hour to sterilize the EW material (pasteurize).

Egg White-Alginate Hydrogel Preparation

[0065] Sodium alginate (Protanal LF 5/60, FMC BioPolymer) solutions (1%, 1.5%, 2%, 2.5%, and 3%) were prepared by dissolving the alginate into 1:3 Hank's Balanced Salt Solution (HBSS) (GIBCO, 14025076)/Epi Max, following by shaking manually ten times. Then, the tube was placed on a Speci-Mix Aliquot Mixer (Thermolyne, M71015) in a 37° C. incubator for approximately 30 min for further dissolution. The samples were stored at 4° C. until use. A crosslinking solution was prepared by dissolving 90 mM calcium chloride (CaCl.sub.2)) (Fisher Scientific, C77-500) in sterile double distilled water (ddH.sub.2O) under a BSC.

[0066] To create the EWA, EW was poured together with sodium alginate (1%, 1.5%, 2%, 2.5%, or 3% w/v) solutions (2:1) into a 50 mL conical tube. The mixture was homogenized by pipetting. Once homogenous, the EWA mixture was centrifuged at 300 g×3 min at 4° C. to eliminate bubbles from the solution; the bubble foam produced on the surface was discarded. 2 ml of the EWA solution was placed into each well of a 6-well plate. Each well containing EWA was cross-linked with 90 mM CaCl.sub.2 solution as follows: sterile aluminum foil sheets were placed to line the bottom of each well. Next, 3 ml of CaCl.sub.2) solution was added into each aluminum-coated well. The plate was then placed in a freezer at −20° C. for 6 h to freeze the CaCl.sub.2 solution. Once frozen, the frozen CaCl.sub.2 disks were removed from the plate and all aluminum foils were peeled away. Finally, the frozen CaCl.sub.2) disks were gently placed on the top of the EWA-coated 6-well plate, allowing the melting process to occur at 37° C. and crosslink over 12 h to create the 3D EWA scaffold. Excess CaCl.sub.2) solution was aspirated and the EWA scaffolds were rinsed with PBS. All procedures were performed under sterile conditions.

Biological Testing

[0067] The normal salivary simian virus 40-immortalized acinar cells (NS-SV-AC) were cultured at 5% CO.sub.2, 37° C. in Epi Max culture medium (Wisent Bio Products, 002-010-CL) supplemented with antibiotic-antimycotic (100 μg/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B) (Thermo Fisher, 15240062) in a culture petri dish (Sarstedt). Then, the NS-SV-AC culture was rinsed twice with sterilized PBS and the cells were detached with 0.05% Trypsin (GIBCO, 25200-056) when confluency reached 90%. All biological testing with cells were performed between the 3.sup.rd-6.sup.th cell passage. 50,000 cells/well were seeded into EWA-coated plates at 5 different concentrations of alginate (1%, 1.5%, 2%, 2.5%, and 3% w/v). Then, they were cultured with Epi Max growth medium at 37° C., 5% CO.sub.2 for 7 days; the culture medium was replaced with fresh medium every 2 days. Samples were taken on day 1, 3, 5, 7 and 10, where cell viability was measured using the AlamarBlue Cell Viability Reagent (Invitrogen, DAL1025). For each well, 1.5 ml culture medium was replaced with 1.5 ml of the AlamarBlue solution. EWA-coated wells without cells were used as a control. Samples were protected from light and incubated at 37° C. and 5% CO.sub.2 for 8 hours. The oxidation-reduction of the AlamarBlue reagent was analyzed by absorbance measurements at 562 nm and 595 nm wavelengths using 100 μl of the solution from each well and a microplate reader (Bio-Tek Instruments, EL800). All experiments were performed in triplicate for every time point. NS-SV-AC organoid formation was tracked by optical microscopy using a Leica DM IL microscope at ×5 and ×10 magnifications.

Statistical Analysis

[0068] All test samples were performed in triplicate. Data are presented as mean±SD. One-way ANOVA was performed with a Tukey's post hoc test with a P value <0.05. Data from the surface are were plotted as Boxplot graphs using the Origin Pro 9 software, with a box limit of 25.sup.th and 75.sup.th percentiles and a minimum-maximum whisker's range.

[0069] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.