Method for manufacturing hydrogel for three-dimensional cell culture, capable of controlling oxygen concentration, and method for constructing disease model

Abstract

In the present invention, an atopic dermatitis model capable of controlling oxygen concentration was developed on the basis of a hydrogel, and is characterized in that a precise disease environment can be simulated through three-dimensional cell culture and oxygen concentration control in the hydrogel. In addition, it is possible to easily control the phase transition time and mechanical properties by changing various components of the polymer, and the stability of the hydrogel was also confirmed through excellent cell compatibility. It was confirmed that factors related to atopic dermatitis were expressed through hydrogel-based three-dimensional cell culture and oxygen concentration control, and expression changes according to treatment drugs were also confirmed. In addition, the present invention can overcome the limitations of existing skin models through three-dimensional cell culture, can provide a platform for drug screening, and furthermore can be applied to various disease models.

Description

Method for manufacturing a hydrogel for 3D cell culture with adjustable oxygen concentration and method for manufacturing a disease model

[0001] <Technology>

[0002] The present invention relates to a three-dimensional cell culture hydrogel capable of controlling oxygen concentration and a method for manufacturing an artificial disease model, and more particularly, to a method for manufacturing an in situ cross-linked hydrogel based on a hydrogel and the development of a three-dimensional artificial atopic skin model / platform with controlled oxygen concentration using the same. More particularly, the present invention relates to the development of a three-dimensional artificial skin model capable of simulating an atopic dermatitis environment through controlled oxygen concentration based on a hydrogel manufactured through disulfide bonds (-SS-) and a thiol-ene reaction between a gelatin derivative (GtnSH) to which a thiol group has been introduced and a gelatin derivative (GtnMI) to which a maleimide group has been introduced.

[0003] Furthermore, compared to existing artificial disease models, the present invention enables simple and rapid hydrogel production through polymer crosslinking without the use of a crosslinking agent. Furthermore, the physical, chemical, and biological properties of the hydrogel can be easily controlled depending on the manufacturing conditions. Furthermore, the present invention can be utilized as a 3D cell culture model based on its excellent cell compatibility, and it has the advantage of being able to simulate the atopic microenvironment by controlling oxygen concentration.

[0004] <National Research and Development Project Supporting This Invention>

[0005] This patent application is the result of a basic research support project supported by the National Research Foundation of Korea with funding from the Ministry of Science and ICT (Project No. 2021R1A4A5032185).

[0006] This patent application is the result of a mid-career researcher support project supported by the National Research Foundation of Korea and funded by the Ministry of Science and ICT (Project No. 2022R1A2C1010024).

[0007] Polymeric hydrogels are three-dimensional networks formed through various physicochemical cross-linking reactions of hydrophilic polymers. They feature excellent biocompatibility, mechanical compliance, high permeability through their porous structure, excellent cell adhesion, and easy loading of therapeutic agents. Due to these characteristics, in situ cross-linked hydrogels have been utilized in fields such as tissue engineering scaffolds, drug / cell delivery systems, and artificial tissue models. In particular, in situ cross-linked hydrogels can mimic the properties of the extracellular matrix (ECM) by controlling various physical and chemical properties, such as phase transition time and mechanical properties. Because they can support cells based on their excellent cytocompatibility, they have been used in the development of various tissue models, including artificial skin models. These in situ cross-linked hydrogels can be manufactured using natural and synthetic polymers, and hydrogel formation is possible through various chemical and physical cross-linking processes.

[0008] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by severe itching, recurrent eczematous lesions, swelling, and rash. A hypoxic environment (<5% pO2) and the release of inflammatory cytokines are known to occur in various allergic diseases, including atopic dermatitis. Atopic dermatitis presents with complex physiological conditions and has diverse etiologies. Furthermore, the clinical phenotype varies across patients, making accurate predictions difficult. Currently, there is no clear treatment, placing a significant burden on patients and their families and leading to high medical costs. Therefore, the development of novel treatments for atopic dermatitis is essential, as is the analysis of its basic pathogenesis and phenotype, as well as the development of personalized treatment strategies.

[0009] To study atopic dermatitis, various in vivo and in vitro skin models are being developed and utilized for toxicity assessments of cosmetics and materials, drug screening, and other applications. Currently, animal models and 2D cell culture models are widely used. Animal models inherently have the limitation of being species-specific, and 2D cell culture models struggle to replicate the sophisticated 3D microenvironment of actual skin. Developing an ideal atopic dermatitis model requires replicating complex physiological conditions such as epidermal barrier defects, epidermal hyperproliferation, and overexpression of relevant cytokines and chemokines. However, currently commercialized atopic dermatitis models are limited to mere anatomical simulations of human skin tissue and fail to accurately replicate complex factors such as the various biological mechanisms occurring in skin lesions, cell-to-cell interactions, and physicochemical properties such as oxygen and reactive oxygen species.

[0010] Accordingly, the need for developing a hydrogel-based artificial atopic dermatitis model for drug screening by more precisely simulating the three-dimensional skin microenvironment by utilizing the advantages of in situ cross-linked hydrogels is being highlighted.

[0011] The present invention aims to address the aforementioned needs by developing an in situ cross-linked polymer hydrogel-based, oxygen-concentration-controlled, three-dimensional artificial atopic dermatitis model. The technical objectives of the present invention are to provide a method for manufacturing an in situ cross-linked polymer hydrogel with easily controllable physical and chemical properties, and to use the same to provide a three-dimensional cell culture system and a platform capable of simulating the disease microenvironment through oxygen concentration control.

[0012] In order to achieve the above-mentioned technical task, the present invention provides an in situ cross-linked polymer hydrogel by inducing a thiol-ene reaction or formation of a disulfide bond (-SS-) in a natural / synthetic polymer having a thiol (-SH) reactive group and a maleimide reactive group in an aqueous solution.

[0013] 1) In the present invention, the hydrogel is manufactured through a crosslinking reaction between a gelatin derivative having a thiol group introduced and a gelatin derivative having a maleimide group introduced without a crosslinking agent.

[0014] 2) By controlling the amount of initial cystamine dihydrochloride / MHA / EDC / NHS used in polymer synthesis, the content of thiol reactive groups and maleimide reactive groups introduced into the polymer main chain can be controlled.

[0015] 3) Provided is an in situ hydrogel manufacturing method that can easily control physical / chemical properties such as phase transition time and mechanical strength of hydrogel by controlling the concentration of gelatin, the concentration ratio of polymer, and the equivalent ratio of reactor (thiol, maleimide).

[0016] In addition, the present invention provides a three-dimensional cell culture environment based on the excellent cell compatibility of the hydrogel, and provides an environment similar to atopic dermatitis by controlling the oxygen concentration inside the hydrogel.

[0017] Additionally, the present invention provides a hydrogel-based three-dimensional artificial disease model and a platform for drug screening.

[0018] The in situ cross-linked hydrogel according to the present invention can be manufactured simply and rapidly through a cross-linking reaction between polymers without a cross-linking agent, and its physical and chemical properties can be controlled according to manufacturing conditions. Furthermore, it allows for three-dimensional cell culture within the hydrogel, and exhibits excellent cytocompatibility and long-term culture. Furthermore, it is possible to control the oxygen concentration within the hydrogel, thereby inducing the expression of factors associated with atopic dermatitis.

[0019] In other words, the present invention overcomes the limitations of existing artificial skin models and, through three-dimensional culture and controlled oxygen concentration, allows for a more precise simulation of the atopic microenvironment. Furthermore, based on controlled physical and chemical properties and excellent biocompatibility, it can be applied to various disease models, artificial tissue manufacturing, drug delivery systems, and drug screening for new therapeutics.

[0020] Figure 1 is a schematic diagram of the synthesis of thiolated gelatin (GtnSH).

[0021] Figure 2 is a schematic diagram of the synthesis of maleimide-conjugated gelatin (GtnMI).

[0022] Figure 3 is a schematic diagram of the preparation of GtnSH / GtnMI hydrogel.

[0023] Figure 4 is a graph showing the phase transition time of a hydrogel according to the concentration of gelatin, the concentration ratio of polymers, and the equivalent ratio of the reactor (thiol, maleimide).

[0024] Figure 5 is a graph showing the mechanical strength of hydrogel according to the concentration of gelatin, the concentration ratio of polymer, and the equivalent ratio of the reactor (thiol, maleimide).

[0025] Figure 6 is a schematic diagram of a hydrogel-based 3D cell culture.

[0026] Figure 7 is a drawing and graph showing the cell compatibility of the hydrogel.

[0027] Figure 8 is a schematic diagram of a hydrogel in situ oxygen measurement system and a graph showing the oxygen measurement results.

[0028] Figure 9 is a graph showing the expression of the HIF-1a gene according to oxygen concentration.

[0029] Figure 10 is a diagram and graph showing the analysis of the expression of atopic dermatitis-related factors (Periostin) according to oxygen concentration.

[0030] Figure 11 is a graph showing the gene expression of additional atopic dermatitis-related factors (Tenascin-C, WNT5A, COL6A5).

[0031] Figure 12 is a schematic diagram of drug resistance evaluation and a diagram and graph analyzing changes in cell compatibility and atopic dermatitis-related factor (Periostin) expression according to drug concentration.

[0032] Figure 13 is a schematic diagram showing the method for manufacturing a hydrogel for cell culture and its use, etc. of the present invention.

[0033] Hereinafter, the present invention will be described in more detail.

[0034]

[0035] Through extensive research, the inventors have confirmed that in situ-forming hydrogels can be fabricated while simultaneously simulating an atopic dermatitis environment through 3D cell culture and controlled oxygen concentration. Furthermore, they have demonstrated that polymer hydrogels can be simply prepared by mixing polymer solutions, while easily controlling their physical and chemical properties by adjusting the manufacturing conditions. Furthermore, they have demonstrated that the expression of atopic dermatitis-related factors can be induced in a hydrogel-based 3D cell culture system.

[0036] The present invention mixes a natural polymer having a maleimide reactive group with a natural polymer having a thiol reactive group to induce a thiol-ene reaction and a disulfide bond, thereby producing a hydrogel for in situ cell culture.

[0037] The above cell culture hydrogel may include, but is not limited to, a step of crosslinking a polymer containing a thiol functional group and a polymer containing a maleimide functional group.

[0038] According to another embodiment of the present invention, a method for preparing a hydrogel is provided, comprising a step of mixing and crosslinking a polymer including a thiol functional group and a polymer including a maleimide functional group.

[0039] The above hydrogel can be manufactured by the above manufacturing method, and the above hydrogel can be used for cell culture.

[0040] The characteristics of the hydrogel described below will also be applied to the manufacturing method.

[0041] Specifically, the above step is performed by mixing a solution in which each polymer is dissolved in a solvent, and the solvent for dissolving the polymer may be any one selected from the group consisting of PBS (Phosphate Buffered Saline), Hanks buffer, HEPES buffer, DPBS (Dulbecco's phosphate buffered saline) buffer, and MOPS buffer, but is not limited thereto.

[0042] The above polymer is characterized by containing a carboxyl group and an amine group.

[0043] Therefore, the polymer including the thiol functional group can be manufactured by a method including a step of binding cystamine dihydrochloride to a carboxyl group of the polymer and a step of breaking a disulfide bond (-SS-), and the polymer including the maleimide functional group can be manufactured by a method including a step of binding 6-maleimidohexanoic acid (MHA) to an amine group of the polymer.

[0044] Referring to FIG. 1, a gelatin derivative having a thiol reactive group is prepared by bonding cystamine dihydrochloride to a carboxyl group of gelatin through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) reaction, and then adding DL-dithiothreitol (DTT) to break the disulfide bond (-SS-) to produce a polymer main chain having a thiol group. The polymer main chain may be any one selected from the group consisting of gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, hyaluronic acid, fibrinogen, and multi-polymers, but is not necessarily limited thereto.

[0045] Referring to FIG. 2, a gelatin derivative (GtnMI) having a maleimide reactive group is prepared by bonding 6-maleimidohexanoic acid (MHA) to an amine group of gelatin through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) reaction to produce a polymer main chain having a maleimide group. The polymer main chain may be any one selected from the group consisting of gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, hyaluronic acid, fibrinogen, and multi-polymers, but is not necessarily limited thereto.

[0046] The above multi-polymer may be one or more polymers selected from the group consisting of one or more multi-polyethylene glycols selected from 3-arm-polyethylene glycol (3armPEG), 4-arm-polyethylene glycol (4armPEG), 6-arm-polyethylene glycol (6armPEG), and 8-arm-polyethylene glycol (8armPEG); and Tetronic series (4arm-PPO-PEO), but is not limited thereto.

[0047] Referring to Figure 3, an in situ cross-linked hydrogel can be manufactured by mixing a polymer main chain having a thiol group and a polymer main chain having a maleimide group to induce a thiol-ene reaction and a disulfide bond.

[0048] By the above thiol-ene reaction Such combinations can be induced.

[0049] The above in situ hydrogel has the advantage of easily controlling physical and chemical properties, such as phase transition time and mechanical strength, by adjusting gelatin concentration, polymer concentration ratio, and reactor equivalence ratio. Furthermore, it has the advantage of enabling long-term 3D cell culture based on excellent cytocompatibility. Furthermore, it has the advantage of being able to mimic the microenvironment of atopic dermatitis by controlling the oxygen concentration within the hydrogel.

[0050] In addition, it is characterized in that the phase transition time decreases as the ratio of the polymer containing a maleimide functional group to the polymer containing a thiol functional group increases.

[0051] In addition, the equivalence ratio of the thiol functional group is characterized by being smaller than the equivalence ratio of the maleimide functional group, and the smaller the equivalence ratio of the thiol functional group, the shorter the phase transition time.

[0052] The above hydrogel is characterized by the ability to control the oxygen concentration inside.

[0053] According to another embodiment of the present invention, a method for producing a disease model is provided, comprising the steps of mixing cells with either a polymer solution containing a thiol functional group or a polymer solution containing a maleimide functional group, and adding the other of the polymer solutions to crosslink the polymer. The disease model may be an artificial disease model, and the cells may be cells having disease characteristics.

[0054] The above disease model manufacturing method may further include a step of culturing the cross-linked polymer in a growth medium.

[0055] Additionally, the present invention provides a hydrogel-based three-dimensional artificial disease model and a platform for drug screening.

[0056] The above artificial disease model may be any one selected from the group consisting of an immune disease model (atopic dermatitis, allergy, asthma, rhinitis), a skin inflammation model, a cardiovascular disease model, a nervous system disease model, a musculoskeletal disease model, a tumor model, and an infectious disease model, but is not necessarily limited thereto.

[0057] The above drug screening may be one or more selected from the group consisting of peptide or protein drugs, antibacterial agents, anticancer agents and anti-inflammatory agents, but is not necessarily limited thereto.

[0058] The above peptide or protein drug product is selected from the group consisting of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), bone morphogenetic protein (BMP), human growth hormone (hGH), porcine growth hormone (pGH), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interferon-α,β,γ, It may be any one selected from the group consisting of interleukin-2 (IL-2), calcitonin, nerve growth factor (NGF), growth hormone-releasing factor, angiotensin, luteinizing hormone-releasing hormone (LHRH), LHRH agonist, insulin, thyrotropin-releasing hormone (TRH), angiostatin, endostatin, somatostatin, glucagon, endorphin, bacitracin, mergain, colistin, bacitracin, monoclonal antibodies, vaccines and mixtures thereof, but is not necessarily limited thereto.

[0059] The above antibacterial agent may be any one selected from the group consisting of minocycline, tetracycline, ofloxacin, fosfomycin, mergain, profloxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, norcadicin, gentamicin, neomycin, kanamycin, paromomycin, micronomycin, amikacin, tobramycin, dibekacin, cefotaxime, cefaclor, erythromycin, ciprofloxacin, levofloxacin, enoxacin, vancomycin, imipenem, fusidic acid, and mixtures thereof, but is not necessarily limited thereto.

[0060] The above anticancer agent may be any one selected from the group consisting of paclitaxel, taxotir, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carboplatin, doxorubicin, daunorubicin, idarubicin, 5-fluorouracil, methotrexate, actinomycin-D, and mixtures thereof, but is not necessarily limited thereto.

[0061] The above anti-inflammatory agent may be any one selected from the group consisting of acetaminophen, aspirin, ibuprofen, diclofenac, indomethacin, piroxicam, fenoprofen, flurbiprofen, ketoprofen, naproxen, suprofen, loxoprofen, cinoxicam, tenoxicam and mixtures thereof, but is not necessarily limited thereto.

[0062]

[0063] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples are intended only to aid understanding of the present invention and are not intended to limit the scope of the present invention in any way.

[0064]

[0065] <Example 1> Preparation of materials

[0066] Gelatin (type A from porcine skin, >300 bloom), cystamine dihydrochloride, 6-maleimidohexanoic acid (MHA), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-Hydroxysuccinimide (NHS), and DL-dithiothreitol (DTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). Hydrogen chloride (1 N hydrochloric acid) solution was purchased from Daejung (Siheung, Gyeonggi), and dialysis membrane bags (molecular weight cutoff = 3500 Da) were purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). In addition, phosphate-buffered saline (Dulbecco's phosphate-buffered saline; DPBS) was purchased from Gibco (Grand Island, NY, USA).

[0067] Deuterium oxide (D2O) was purchased from Sigma Aldrich (St. Louis, MO, USA), and 5,5′-thio-bis(2-nitrobenzoic acid) (Ellman's reagent) was purchased from Thermo Scientific (Rockford, IL, USA).

[0068] Human dermal fibroblasts (HDFs) were purchased from Lonza (Walkersville, MD, USA). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin / streptomycin (P / S), and 0.25% trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA).

[0069] Live / Dead Viability / Cytotoxicity Kit, Periostin Polyclonal Antibody, and Human Periostin ELISA kit were purchased from Invitrogen (Grand Island, NY, USA). Recombinant human IL-4 was purchased from PeproTech (Cranbury, NJ), and primers (GAPDH, HIF-1a, Periostin, Tenascin-C, WNT5A, COL6A5) were purchased from Cosmogenetech (Seongdong-gu, Seoul). Donkey Anti-Rabbit IgG H&L Alexa Flour 488 was purchased from Abcam (Cambridge, UK), and 4',6-diamidino-2-phenylindole (DAPI) solution was purchased from Molecular Probes (Eugene, OR). Additionally, dexamethasone was purchased from Sigma Aldrich (St. Louis, MO, USA).

[0070] Other chemicals and solvents were used without further purification.

[0071]

[0072] <Example 2> Synthesis and structural analysis of thiol-bound gelatin derivatives (Figure 1)

[0073] 250 mg of gelatin was dissolved in 125 mL of deionized water (DIW), and the solution was mixed with a solution of cystamine dihydrochloride dissolved in 2.5 mL of DIW for 10 minutes. Then, EDC and NHS were dissolved in 2.5 mL of DIW and mixed. After reaction for 2 hours, a solution of DTT dissolved in 5 mL of DIW was added to the mixture and reacted. After reaction for 24 hours, the reaction mixture was placed in a dialysis membrane and replaced with a 5 mM HCl solution and a 1 mM HCl solution daily for 2.5 days. The solution obtained through vacuum filtration was freeze-dried to obtain a thiolated gelatin (GtnSH) polymer.

[0074] The amount of thiol introduced into GtnSH is NMR ( 1 It was measured qualitatively and quantitatively using H-NMR and Ellman's assay.

[0075] For qualitative analysis, 6 mg of GtnSH was dissolved in 600 μL of D2O and placed in an NMR tube for measurement. The measurement results confirmed the introduction of a thiol group into gelatin through the formation of cystamine peaks at 2.8 ppm and 3.5 ppm.

[0076] For quantitative analysis, cysteine ​​(L-cysteine) was used as a standard curve. 100 μL of cysteine ​​solutions at various concentrations and 1 mg / mL GtnSH solution were mixed with 100 μL of Ellman's reagent solution, blocked from light, and allowed to react at room temperature for 20 minutes. The absorbance was measured at 405 nm using an absorbance detector, and the thiol content in GtnSH was calculated using the cysteine ​​standard curve. By adjusting the amounts of cystamine dihydrochloride / EDC / NHS / DTT introduced, the thiol content could be controlled from an average of 86.89 to 165.83 μmol / g (polymer).

[0077]

[0078] <Example 3> Synthesis and structural analysis of gelatin derivatives containing maleimide (Fig. 2)

[0079] Maleimide-conjugated gelatin derivatives were prepared by dissolving 250 mg of gelatin in 50 mL of DPBS, and mixing the solution with a solution of MHA dissolved in 120 mL of DPBS for 10 minutes under protection from light. EDC and NHS were then dissolved in 5 mL of DPBS, mixed, and the reaction was carried out under protection from light for 24 hours. The reacted mixture was placed in a dialysis membrane, and the DI water was replaced three times over three days. The solution obtained through vacuum filtration was freeze-dried to obtain a maleimide-conjugated gelatin (GtnMI) polymer.

[0080] The amount of maleimide introduced into GtnMI is NMR ( 1 It was measured qualitatively and quantitatively using H-NMR and Ellman's assay.

[0081] For qualitative analysis, 6 mg of GtnMI was dissolved in 600 μL of D2O and placed in an NMR tube for measurement. The measurement results confirmed that a maleimide group was introduced into gelatin through a decrease in the primary amine peak of gelatin at 3.0 ppm and the formation of a maleimide peak at 6.8 ppm.

[0082] For quantitative analysis, 6-maleimidohexanoic acid (MHA) was used as a standard and cysteine ​​(L-cysteine) was used as a thiol-ene reaction substrate. 50 μL of each 0.04 mg / mL cysteine ​​solution was mixed with 50 μL of MHA solutions at various concentrations and 1 mg / mL GtnMI solution, and the thiol-ene reaction was induced at room temperature under light protection for 10 minutes. After the reaction, the mixture was mixed with 50 μL of Ellman's reagent solution and reacted at room temperature under light protection for 20 minutes. The absorbance was measured at 405 nm using an absorbance detector, and the maleimide content in GtnMI was calculated using a maleimide standard curve. By adjusting the amount of MHA / EDC / NHS introduced, the maleimide content could be controlled from an average of 47.54 to 102.57 μmol / g (polymer).

[0083]

[0084] <Example 4> Formation of GtnSH / GtnMI hydrogel (Fig. 3)

[0085] Hydrogels were prepared by simply mixing solutions of GtnSH and GtnMI polymers dissolved in DPBS without any additional cross-linking agent.

[0086]

[0087] <Experimental Example 1> Measurement of the phase transition time of hydrogel (Fig. 4)

[0088] The phase transition time of the hydrogel was evaluated using the vial tilting method. 1) The measurement was performed by fixing the ratio of GtnSH and GtnMI to 1:1 and changing the final concentration of gelatin to 6, 10, and 14 wt%. 2) The measurement was performed by fixing the final concentration of gelatin to 10 wt% and changing the concentration ratio of the polymers (GtnSH:GtnMI) to 3:7, 5:5, and 7:3. 3) The measurement was performed by fixing the final concentration of gelatin to 10 wt%, the ratio of GtnSH:GtnMI to 5:5, and adjusting the equivalent ratio of the reactive groups (thiol group, maleimide group) to 1 / 2 and 1. More detailed hydrogel manufacturing conditions are shown in Tables 1 to 3.

[0089] Hydrogel GtnSH Concentration (wt%) GtnMI Concentration (wt%) Final Concentration (wt%) G3MI3336 G5MI55510 G7MI77714 G3MI73710 G7MI37310

[0090] GtnSH equivalent ratioCystamine dihydrochloride(mg)EDC(mg)NHS(mg)DTT(mg)GtnSH 1 / 2225.39658308.5GtnSH 1450.5192116617

[0091] GtnMI Equivalence RatioMHA(mg)EDC(mg)NHS(mg)GtnMI 1 / 2105.6115.180.6GtnMI 1211.2230.1161.1

[0092]

[0093] Experimental results showed that the phase transition time could be controlled from 10 to 130 seconds by adjusting various polymer conditions. 1) As the final gelatin concentration increased, the phase transition time decreased. This is because the thiol-ene reaction and disulfide bond formation were promoted as the amount of polymer increased, shortening the hydrogel formation time.

[0094] 2) As the ratio of GtnMI increased, the phase transition time decreased. This can be explained by the fact that the hydrogel formation time shortened because the thiol-ene reaction rate increased as the ratio of maleimide groups increased.

[0095] 3) As the equivalence ratio of the reactor increased, the phase transition time decreased. This is because the thiol-ene reaction and disulfide bond formation are accelerated as the amount of polymer reactor increases, shortening the hydrogel formation time. Furthermore, the hydrogel formation time can be shortened because the thiol-ene reaction rate increases as the amount of maleimide reactor increases.

[0096]

[0097] <Experimental Example 2> Analysis of mechanical strength of hydrogel (Fig. 5)

[0098] The mechanical strength of the hydrogel was analyzed using a rheometer. 1) The ratio of GtnSH and GtnMI was fixed at 1:1, and the final concentration of gelatin was varied to 6, 10, and 14 wt%, and the measurement was performed. 2) The final concentration of gelatin was fixed at 10 wt%, and the polymer concentration ratios of GtnSH:GtnMI were varied to 3:7, 5:5, and 7:3, and the measurement was performed. 3) The final concentration of gelatin was fixed at 10 wt%, the ratio of GtnSH:GtnMI was fixed at 5:5, and the measurement was performed by adjusting the equivalent ratio of the reactive groups (thiol group, maleimide group) to 1 / 2 and 1. More detailed hydrogel manufacturing conditions are shown in Tables 1 to 3.

[0099] Experimental results showed that the mechanical strength could be controlled from 90 to 1500 Pa by adjusting various polymer conditions. 1) As the final gelatin concentration increased, the mechanical strength increased. This is because as the amount of polymer increased, the thiol-ene reaction and disulfide bond formation were promoted, increasing the degree of crosslinking.

[0100] 2) When the ratio of GtnSH and GtnMI was 5:5, the mechanical strength increased. This can be explained by the fact that when GtnSH and GtnMI were reacted at a ratio of 1:1, the thiol-ene reaction between the polymer chain with a group (GtnSH) and the polymer chain with a maleimide group (GtnMI) was promoted, thereby increasing the degree of cross-linking.

[0101] 3) When the content of thiol and maleimide groups was quantitatively analyzed using the previously performed Ellman analysis, the average reactive group contents were measured to be 86.89 μmol / g (polymer) and 102.57 μmol / g (polymer) in GtnSH (1 / 2) and GtnMI (1), respectively. This confirmed that the amount of thiol groups and the amount of maleimide groups reacted in a 1:1 ratio. As a result of the experiment, the highest mechanical strength was measured when the amount of reactive groups (thiol:maleimide) contained in the polymer reacted in a 1:1 ratio. This can be explained by the fact that when the ratio of thiol groups and maleimide groups was 1:1, the thiol-ene reaction was promoted, thereby increasing the degree of crosslinking.

[0102]

[0103] Hydrogel conditions suitable for 3D cell culture were selected through hydrogel phase transition time analysis and mechanical strength measurement. The hydrogel conditions were fixed at GtnSH(1 / 2) (5 wt%) and GtnMI(1) (5 wt%) and the experiment was conducted.

[0104]

[0105] <Experimental Example 3> 3D cell culture (3D encapsulation) using hydrogel (Fig. 6)

[0106] To evaluate the 3D cell-carrying ability of hydrogels and to optimize culture conditions, 3D cell culture (3D encapsulation) of human dermal fibroblasts (HDFs) was performed.

[0107] For 3D cell culture, HDFs cell suspension was first mixed with GtnMI solution (5 wt%), and then mixed with GtnSH solution (5 wt%) to prepare cell-loaded hydrogels before gel formation. After 20 minutes of hydrogel stabilization, DMEM growth medium was added to culture the cells. The concentration of cells used in the experiment was 2.0 X 10 6 cells / mL and cultured under conditions of 37°C and 5% CO2. The cell-loaded hydrogel was produced using the direct loading method and the disk type method.

[0108] The direct loading method was performed by injecting 120 μL of a mixture of HDFs / GtnSH / GtnMI into a 48-well plate. For the disk-type method, 60 μL of a mixture of HDFs / GtnSH / GtnMI was injected into a 1-mL syringe to create disk-shaped hydrogels. After a 20-min stabilization period, the hydrogels were floated on a 48-well plate and cultured. More detailed 3D cell culture conditions are shown in Table 4.

[0109] 3D culture conditions Manufacturing method DMEM medium volume (μl) GtnSH concentration (wt%) GtnMI concentration (wt%) Solvent Hydrogel volume (μl) Cell density (cells / mL) Direct loading 500 55 DPBS 120 2.0 × 10 6 Disk type50055DPBS602.0 × 10 6

[0110] Experimental results showed that the cell-loaded hydrogel maintained its morphology without degradation for up to 14 days. This confirmed that the hydrogel can provide an environment suitable for 3D cell culture and that long-term culture is possible.

[0111]

[0112] <Experimental Example 4> Evaluation of cell compatibility of hydrogel (Fig. 7)

[0113] To evaluate the cytocompatibility of hydrogels, 3D cell-bearing hydrogels were fabricated using a disk-type method, and then cytotoxicity was evaluated. 3D cell culture was performed at 37°C and 5% CO 2, The cytotoxicity of the hydrogel was evaluated using a live / dead assay after culturing for 1 and 3 days, respectively, under 5% / 21% O2 conditions. The live / dead assay is a method for evaluating cytotoxicity by staining dead cells red and living cells green.

[0114] Experimental results showed that most cells were viable, as demonstrated by optical microscopy and live / dead images. Quantitative analysis confirmed that cell viability exceeded 80% in all oxygen groups. This confirmed the excellent cytocompatibility of the hydrogel.

[0115]

[0116] <Experimental Example 5> In situ oxygen measurement system of hydrogel and real-time oxygen measurement results according to oxygen concentration (Fig. 8)

[0117] To verify whether the oxygen concentration inside the hydrogel could be controlled, the oxygen concentration was measured in real time using an in situ oxygen measurement system.

[0118] Real-time oxygen measurement was performed by attaching an oxygen sensor patch to the center bottom of a 48-well plate using vacuum grease, and fabricating a 3D cell-supported hydrogel using a direct loading method. Oxygen concentration was measured in real time for 3 days using a noninvasive oxygen sensor and an oxygen-controlled incubator (0%–21% pO2).

[0119] Experimental results showed that under 21% conditions, the oxygen concentration within the hydrogel was maintained at 11% to 13% pO2. Conversely, under 5% conditions, the oxygen concentration remained below 5% pO2 after 8 hours of incubation. This confirmed that the oxygen concentration within the hydrogel can be controlled using an oxygen-controlled incubator.

[0120]

[0121] <Experimental Example 6> Analysis of HIF-1a gene expression according to oxygen concentration (Fig. 9)

[0122] To determine whether a hypoxic (<5% pO2) environment was induced inside the hydrogel, an analysis of hypoxia-inducible factor 1 alpha (HIF-1a) expression according to oxygen concentration was performed.

[0123] HIF-1a expression analysis was performed by producing hydrogels using the direct loading method and culturing them in 48-well plates. 3D cell culture was performed at 37°C and 5% CO 2, RNA extraction was performed after culturing for 6 and 24 hours, respectively, under 5% and 21% O2 conditions. RNA extraction was performed by homogenizing the cultured hydrogels and isolating RNA using Tri-RNA reagent. cDNA was then synthesized via reverse transcription, and HIF-1a was quantitatively analyzed using qRT-PCR. GAPDH was used as a control to normalize the amount of cDNA, and the relative fold change in gene expression was calculated using the comparative Ct method. The sequences of the primers used are shown in Table 5.

[0124] GAPDHFwGGG AGC CAA AAG GGT CAT CAT CTCRvGAG GGG CCA TCC ACA GTC TTCHIF-1aFwGTT TAC TAA AGG ACA AGT CAC CRvTTC TGT TTG AAG GGA GPeriostinFwCAG AGA AAT CCC TCC ATG AAARvCAG GAG CTC TTT CAA GTC TGCTenascin-CFwTCC CAG TGT TCG GTG GAT CTRvTTG ATG CGA TGT GTG AAG ACAWNT5AFwTAC GAG AGT GCT CGC ATC CTC ARvTGT CTT CAG GCT ACA TGA GCC GCOL6A5FwCAG TAC AGC GAC GAG TTT CACRvAGT AGG TCC TAT GAG CCT CCT

[0125] As a result of the experiment, the expression of HIF-1a increased under the condition of culturing for 6 hours at 5%. On the other hand, it was confirmed that the expression of HIF-1a decreased under the condition of culturing for 24 hours at 5%. This is because HIF-1a is a gene that is initially overexpressed in a hypoxic environment, and its initial expression increases for 6 hours and decreases after adaptation to the hypoxic environment. This confirmed that the 3D cell-cultured hydrogel can simulate the hypoxic environment of atopic skin tissue, and that the initial expression of HIF-1a induced a hypoxic environment inside the hydrogel.

[0126]

[0127] <Experimental Example 7> Analysis of expression of atopic dermatitis-related factor (Periostin) according to oxygen concentration (Figure 10)

[0128] To evaluate whether 3D cell culture hydrogels could mimic the microenvironment of atopic dermatitis, we analyzed the expression of periostin, a factor associated with atopic dermatitis. It is known that in atopic dermatitis, T cells stimulated by external allergens express cytokines such as IL-4 / IL-13, and periostin expression is induced in dermal fibroblasts. Therefore, after 3D cell culture, we treated cells with IL-4 at various concentrations (0, 25, and 50 ng / mL) under 5% and 21% oxygen conditions to analyze periostin expression. Periostin expression was qualitatively and quantitatively analyzed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). More detailed experimental conditions are shown in Table 6.

[0129] Group Oxygen Concentration (%) IL-4 Concentration (ng / mL) 21 %_021021 %_25212521 %_5021505 %_0505 %_255255 %_50550

[0130] (1) Analysis of periostin gene expression by qRT-PCR

[0131] Periostin gene expression analysis was performed by fabricating hydrogels using the direct loading method and culturing them in 48-well plates for 24 hours. IL-4 was then treated at various concentrations (0, 25, and 50 ng / mL) and cultured for 24 hours before RNA extraction. After RNA extraction using the same method as above, periostin expression was quantitatively analyzed using qRT-PCR. Three-dimensional cell culture was performed at 37°C and 5% CO2. 2, Cultured under 5% / 21% O2 conditions. The sequences of the primers used are shown in Table 5.

[0132] The experimental results showed that periostin expression increased in the IL-4 treatment groups (25, 50 ng / mL) compared to the 0 ng / mL group at both 5% and 21% oxygen concentrations. However, no significant difference was observed depending on the concentration of IL-4 treated. Therefore, in the subsequent periostin expression analysis experiment, the IL-4 treatment concentration was fixed at 25 ng / mL and the experiment was conducted. The fact that periostin expression increased only in the IL-4 treated group confirmed that IL-4 stimulated fibroblasts to induce periostin expression.

[0133]

[0134] (2) Analysis of periostin gene expression by immunofluorescence staining

[0135] Periostin immunostaining was performed by fabricating hydrogels using the disk type method and culturing them in a 48-well plate for 24 hours. After treatment with IL-4 at various concentrations (0 and 25 ng / mL), the cells were fixed with paraformaldehyde solution after 24 hours of incubation. After permeabilization with 200 μL of 0.1% Triton X-100 solution for 20 minutes, 200 μL of 1% BSA solution was treated for protein blocking, and incubated for 1 hour at 4°C. Periostin polyclonal antibody (1:100 dilution) was treated as the primary antibody and incubated overnight at 4°C. After washing, Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:500 dilution) was treated and incubated for 4 hours at 25°C. Cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) solution for 15 minutes and immunofluorescence staining was performed. Periostin expression was confirmed by staining periostin in green and nuclei in blue. The expression of periostin was qualitatively analyzed in three dimensions using a confocal laser scanning microscope. The three-dimensional cell culture was performed at 37°C and 5% CO 2, Cultured under 5% / 21% O2 conditions.

[0136] As a result of the experiment, periostin staining around the nucleus was observed in the IL-4-treated group (25 ng / mL). This qualitatively confirmed the expression of periostin.

[0137]

[0138] (3) Analysis of periostin gene expression by ELISA

[0139] Periostin protein expression analysis was performed by fabricating hydrogels using the direct loading method and culturing them in 48-well plates for 24 hours. IL-4 was then treated at various concentrations (0 and 25 ng / mL) and cultured for 24 hours. After incubation, the hydrogels were degraded by collagenase treatment for 2 hours, and cell lysates were extracted using protein extraction buffer. Periostin protein expression was quantitatively analyzed using a human periostin ELISA kit. The 3D cell culture was performed at 37°C and 5% CO2. 2, Cultured under 5% / 21% O2 conditions.

[0140] Experimental results showed that periostin protein expression increased only in the IL-4-treated group (25 ng / mL). The 0 ng / mL group was measured below the minimum detection limit, confirming that no protein was detected. Furthermore, we confirmed that periostin protein expression significantly increased in a hypoxic environment (<5% pO2).

[0141]

[0142] In summary, we confirmed that periostin expression increased in a hypoxic (<5% pO2) environment and in the IL-4-treated group (25 ng / mL). This confirms that the 3D cell culture hydrogel conditions can mimic the atopic dermatitis microenvironment through IL-4 treatment and oxygen concentration control. This suggests that the hydrogel-based 3D cell culture system has potential as an artificial atopic dermatitis model.

[0143]

[0144] <Experimental Example 8> Analysis of additional atopic dermatitis-related factors (Tenascin-C, WNT5A, COL6A5) (Figure 11)

[0145] To analyze additional atopic dermatitis-related factors, we performed expression analysis of Tenascin-C, WNT5A, and COL6A5. After 3D cell culture, the cells were treated with IL-4 at various concentrations (0, 25, and 50 ng / mL) under 5% and 21% oxygen conditions, and the expression of Tenascin-C, WNT5A, and COL6A5 was analyzed.

[0146] Gene expression analysis was performed using the same method as above, and quantitative analysis was performed using qRT-PCR after RNA extraction. 3D cell culture was performed at 37°C and 5% CO 2, Cultured under 5% / 21% O2 conditions. The sequences of the primers used are shown in Table 5.

[0147] As a result of the experiment, the expression of Tenascin-C increased in the IL-4 treatment group (25, 50 ng / mL) compared to the 0 ng / mL group at both 5% and 21% oxygen concentrations. However, there was no significant difference depending on the concentration of IL-4 treated. WNT5A and COL6A5 did not show a significant difference in gene expression depending on the concentration of IL-4 treatment. We further analyzed factors related to atopic dermatitis and confirmed that the hydrogel-based 3D cell culture system could simulate the atopic dermatitis environment.

[0148]

[0149] <Experimental Example 9> Drug resistance evaluation (Fig. 12)

[0150] To evaluate the potential of a hydrogel-based atopic dermatitis model as a drug screening model, a drug resistance assessment was performed. The drug used in the experiment was the steroid dexamethasone, which, along with moisturizers, is currently the most widely used drug for the treatment of atopic dermatitis. Therefore, after 3D cell culture, we treated the cells with IL-4 and dexamethasone to analyze changes in periostin expression.

[0151]

[0152] To assess cell compatibility according to drug concentration, the cytotoxicity of the drug was assessed using a live / dead assay. The live / dead assay was performed using the same disk-type method as the previously performed cell compatibility assessment. Dexamethasone was treated at various concentrations (0 and 100 μM) and cell viability was analyzed after 3 days of culture.

[0153] As a result of the experiment, all groups showed a cell viability rate of over 80% through live / dead photographs and quantitative results, and it was confirmed that there was no cytotoxicity according to drug concentration.

[0154]

[0155] Drug resistance was assessed by direct loading. After 3D cell culture, periostin expression was induced by treatment with 25 ng / mL IL-4 under 5% oxygen conditions. Then, dexamethasone was treated at various concentrations (0 and 100 μM) and changes in periostin expression were analyzed after 3 days of culture.

[0156] Gene expression analysis was performed using the same method as above, and after RNA extraction, quantitative analysis was performed using qRT-PCR. 3D cell culture was performed at 37°C and 5% CO 2, Cultured under 5% O2 conditions. The sequences of the primers used are shown in Table 5.

[0157] Experimental results showed that periostin expression was significantly reduced only in the dexamethasone-treated group (100 μM). This indicates that periostin expression was suppressed by drug treatment, confirming the potential of the hydrogel-based 3D cell culture system as a drug screening model.

[0158]

[0159] As described above, although the present invention has been described by limited embodiments and drawings, the present invention is not limited thereto, and various modifications and variations are possible within the scope of the technical idea of ​​the present invention and the equivalent scope of the claims to be described below by a person having ordinary skill in the art to which the present invention pertains.

Claims

1. A method for producing a hydrogel for cell culture, comprising a step of mixing and crosslinking a polymer containing a thiol functional group and a polymer containing a maleimide functional group.

2. In paragraph 1, The above step is a method for producing a hydrogel for cell culture, characterized in that the solution in which each polymer is dissolved in a solvent is mixed.

3. In paragraph 2, A method for producing a hydrogel for cell culture, characterized in that the solvent is any one selected from the group consisting of PBS (Phosphate Buffered Saline), Hanks buffer, HEPES buffer, DPBS (Dulbecco's phosphate buffered saline) buffer, and MOPS buffer.

4. In paragraph 1, A method for producing a hydrogel for cell culture, characterized in that the polymer comprises a carboxyl group and an amine group.

5. In paragraph 1, A method for producing a hydrogel for cell culture, characterized in that the polymer is any one selected from the group consisting of gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, hyaluronic acid, fibrinogen, and multi-polymers.

6. In paragraph 5, A method for producing a hydrogel for cell culture, characterized in that the multi-polymer is one or more multi-polyethylene glycols selected from 3-arm-polyethylene glycol (3armPEG), 4-arm-polyethylene glycol (4armPEG), 6-arm-polyethylene glycol (6armPEG), and 8-arm-polyethylene glycol (8armPEG); and Tetronic series (4arm-PPO-PEO).

7. In paragraph 1, The polymer containing the above thiol functional group A step of binding cystamine dihydrochloride to the carboxyl group of the polymer; and A method for producing a hydrogel for cell culture, characterized in that it is produced by a method including a step of breaking a disulfide bond (-SS-).

8. In paragraph 1, The polymer containing the above maleimide functional group A method for producing a hydrogel for cell culture, characterized in that it is produced by a method including a step of binding 6-maleimidohexanoic acid (MHA) to an amine group of the polymer.

9. In paragraph 1, A method for producing a hydrogel for cell culture, wherein the hydrogel is characterized in that the polymer is crosslinked by a thiol-ene reaction and a disulfide bond.

10. In paragraph 1, A method for producing a hydrogel for cell culture, characterized in that the phase transition time decreases as the ratio of a polymer containing a maleimide functional group to a polymer containing a thiol functional group increases.

11. In paragraph 1, A method for producing a hydrogel for cell culture, characterized in that the equivalent ratio of the thiol functional group is smaller than the equivalent ratio of the maleimide functional group.

12. In paragraph 1, A method for producing a hydrogel for cell culture, characterized in that the oxygen concentration inside the hydrogel can be controlled.

13. A hydrogel for cell culture manufactured by the manufacturing method of any one of claims 1 to 12.

14. A step of mixing cells with either a solution of a polymer solution containing a thiol functional group or a solution of a polymer solution containing a maleimide functional group, and A method for producing a disease model, comprising a step of crosslinking a polymer by adding another solution among the above polymer solutions.

15. In paragraph 14, A method for producing a disease model, characterized in that it further comprises a step of culturing a cross-linked polymer in a growth medium.

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