HYDROGEL-BASED ORAL IN VITRO SCREENING SYSTEM WITH MULTILAYERED CELLULAR STRUCTURE

Abstract

The invention relates to a biomimetic in vitro testing system for evaluating the toxicity and / or efficacy of substances to be administered orally or exposed orally.

Description

1 TARIFF HYDROGEL-BASED ORAL INSERTS WITH A MULTI-LAYERED CELLULAR STRUCTURE VITRO SCANNING SYSTEM Technical Field to Which the Invention Relates 5 The invention has applications in pharmaceutical toxicology, bioengineering, and in vitro cell culture modeling. It lies at the intersection of technical fields; especially those to be administered orally. or the toxicity and / or efficacy of substances subject to oral exposure The invention relates to biomimetic in vitro testing systems for evaluation. Specifically, hydrogel-based three-dimensional cell culture structures, with angiogenic factors 10 enriched vascular-like microenvironments and multilayered cells using these organizations, mimicking the oral tissue microenvironment and preclinical a reproducible and scalable in vitro model that can be used in evaluations It is aimed at developing a screening platform. In this context, the invention is used for drug development, 15 in fields such as cosmetic and oral care product testing and biocompatibility analysis This relates to advanced testing systems that can be used. State of the Art Drugs, cosmetics, and oral hygiene products that are to be administered orally or to which one may be exposed orally. Safety and efficacy assessments of personal care products have been largely based on 20 principles for many years. through in vivo animal experiments and two-dimensional (2D) cell culture systems This has been carried out using animal models, allowing for the observation of holistic physiological responses. While it allows for this, due to interspecies biological differences, human oral It cannot fully reflect the texture due to ethical constraints, cost, and time. It has significant disadvantages in this respect. For this reason, in recent years, animal 25 There is an intensive focus on in vitro model systems that could provide an alternative to experiments. There is an orientation involved. However, classical 2D cell cultures; the cells' natural orientation three-dimensional (3D) organization, cell-cell and cell-matrix interactions due to its inability to adequately replicate, especially complex tissue structures and It is insufficient in understanding microenvironmental conditions. This situation is reflected in the 30 obtained. This leads to limited correlation between the data and clinical outcomes. Oral tissue includes epithelial layers, connective tissue components, vascular structures, and various cells. It has a highly complex microenvironment consisting of the dynamic interaction of its types. 2 In this microenvironment; oxygen gradients, nutrient diffusion, growth factors, cytokines Numerous parameters, such as extracellular matrix components, influence cell behavior. It has a direct impact. Current technical solutions mimic this multi-component structure. Different approaches have been developed for this purpose, and especially three-dimensional cell culture systems, organoid structures and biomimetic scaffold materials come to the forefront 5 These include collagen, gelatin derivatives, alginate, and synthetic polymers. cells are studied in a three-dimensional environment using various biocompatible materials such as these. Structures have been created that enable its growth. However, these systems are important. in some cases, tissue-specific microenvironmental signals, and especially vascular-like It appears that the structures (vascularization) are not adequately represented. 10 The absence of vascular structures impairs the long-term viability and differentiation of cells, and This can negatively affect the model's physiological and functional behaviors; This limits its realism. Another approach included in the current technology is the air-liquid interface (15 ALI (Alternative Cell Alternatives) are culture systems that specifically promote the differentiation of epithelial cells and barrier function. It is used to support the examination of its functions. This In these systems, the cells are in contact with the culture medium from the bottom, while the top The surfaces are brought into contact with air, thus providing a more physiological environment. Studies are being conducted. However, the implementation of ALI systems generally involves specific 20 Insert structures and multilayered culture setups are used, which is beneficial for both this increases costs as well as the installation and standardization of the system. This makes things more difficult. Furthermore, in such systems, the differences between cell layers... limited interactions and biochemical processes occurring at tissue depth There are also technical limitations, such as the inadequate representation of processes. 25 Particularly useful in modeling complex cell interactions specific to oral tissue, Existing ALI-based systems do not provide sufficient flexibility and adaptability. It is known. Another 30 encountered in in vitro model systems in the current state of the art. The main problem is in culturing different cell types together in a controlled manner. These are the challenges. In oral tissue, epithelial cells, fibroblasts, endothelial cells, and Different cell types, such as immune cells, are constantly interacting. However, existing In most systems, these cell types are studied either separately or together. 3 When used, microenvironmental control cannot be adequately achieved. This situation, accurate analysis of intercellular signaling and paracrine interactions This makes it difficult. Furthermore, the mechanical properties of the materials used... Porosity and biochemical composition are also decisive factors in cell behavior. and the inability to precisely control these parameters results in model results of 5%. This can negatively affect the repeatability of existing systems. In addition, A significant portion of them are expensive, complex, and have limited accessibility, especially Disadvantage in terms of use in routine screening and high-throughput analysis. This is the case. Some commercially available ready-to-use in vitro test systems provide specific Although suitable for standard tests, their limited flexibility means they are suitable for different 10 their low adaptability to experimental conditions and local production large-scale research and development due to limited alternatives They are unable to offer adequate solutions in their activities. Furthermore, these systems are largely... The majority are imported, and supply times and costs directly affect the research processes. It has an effect. 15 In recent years, with advancements in the field of bioengineering, it has become more advanced. microfluidic systems (organ-on-a-chip) and multilayer biomimetic models Although improved, these systems generally require advanced technical infrastructure. and their applicability in common laboratory conditions remains limited. Furthermore, these 20 The complexity of the devices and equipment used in such systems increases user dependency. This increases the number of errors and makes it difficult to standardize the results obtained. Therefore, it should be simpler, more accessible, repeatable, and more physiologically meaningful. The need for new model systems capable of producing results continues. All of this... For these reasons, 25 that will be administered orally or to which one may be exposed in the oral environment for use in toxicity and efficacy assessments of substances; cells a system that can better mimic the natural microenvironment, and the interaction of different cell types supporting, biochemical and biophysical parameters can be controlled and the same in vitro model systems that can offer economical and viable solutions in a timely manner The need is clearly evident in the technical field. Existing technical solutions are limited to a certain 30 While it offers advantages in several aspects, due to the limitations mentioned above, studies towards more advanced and realistic modeling approaches The need for its continuation persists. 4 Brief Description and Objectives of the Invention The invention describes the toxicity of substances to be administered orally or subjected to oral exposure. and / or biomimetic in vitro test systems for evaluating their effectiveness. It is explained. The purpose of the invention is to modify substances that are to be administered orally or subjected to oral exposure. toxicity and / or efficacy in a more realistic and reproducible way The aim is to create an in vitro model for evaluation. implementation, preparation of gelatin-methacrylate (GelMA) based hydrogel structures, Cross-linking of these structures using photoinitiators in appropriate ratios and 10 a matrix that allows cells to be cultured in a three-dimensional environment This is made possible by creating a microenvironment in the oral tissue. VEGFA was incorporated into the hydrogel to create a closer biological environment. the inclusion of angiogenic factors such as angiopoietin I and II, thereby enabling the vessel The aim is to create similar microenvironments, and the technical element in question is 15. more physiological processes of cell proliferation, differentiation and signaling It contributes to the realization of these conditions. One aim of the invention is to enable different cell types to interact together in a controlled manner. by creating a multilayered structure that it can enter, cell-to-cell and cell-to-cell interaction in oral tissue. The goal is to model matrix interactions more accurately; to achieve this goal... Development of human vascular endothelial cells (HUVEC) in a hydrogel and then human oral fibroblast (HOF) cells form a separate layer in this structure A layered cell organization is created by adding layers on top of each other. Thus, the biochemical interactions that occur between different cell types are 25. This allows for examination and increases the physiological validity of the model. One aim of the invention is to replace commonly used and cost-effective systems in existing models. by eliminating reliance on insert structures that create implementation difficulties The aim is to develop a more practical and accessible testing system; within this scope, a 30 hydrogel is being considered. its use as an interface and signaling between cell layers this structure Thanks to being provided via this method, the need in classic air-liquid interface (ALI) systems is eliminated. Obtaining similar functions without the need for insert components. This is made possible. This technical approach ensures both the cost effectiveness of the system. it also increases its applicability in broader laboratory conditions. It provides. Within the scope of the invention, the developed model is limited solely to toxicity assessments. not only that, but it also allows for the performance of activity analyses. 5 This is ensured; cytotoxicity on the in vitro system created in this context, different biological analyses such as genotoxicity and reactive oxygen species (ROS) production A testing infrastructure has been designed that makes it possible to implement the test. This allows for testing... The safety and biological activity profiles of the substances obtained were analyzed holistically. It can be evaluated in this way. In addition, the developed system is suitable for 10 different cell types. its adaptability and its ability to contribute to personalized treatment approaches It is also intended to offer a flexible platform; this goal is achieved through hydrogel. composition, biomolecule content and cell types can be modified This is achieved through its design. This technical flexibility allows the model to be used in different research. ensuring that it can be adapted to their needs and biomedical research and product 15 It offers a wide range of applications in development processes. Explanation of the Figures Figure 1. Hydrogel-supported structure designed to facilitate intercellular interaction. Schematic representation of the cultural system. 20 Figure 2. HUVEC cell viability of GelMA hydrogels with different concentrations. its effect on. Figure 3. HUVEC cells in GelMA hydrogel and VEGF on TCPS surfaces. Demonstration of I / II and CD105 gene expression. Figure 4. Percentage of wound closure over time. 25 Figure 5. A) Endothelial tube formation in wells without VEGF addition, B) VEGF Endothelial tube formation in wells enriched with [synthetic material]. Figure 6. Relevant samples from GelMA hydrogels enriched with VEGF, Ang 1 and Ang 2. Time-dependent release of biosignals. Figure 7. 30 GelMA hydrogels enriched with A) VEGF, B) Ang 1 and C) Ang 2. Time-dependent release of biosignals in terms of different kinetic models evaluation. Figure 8. A) HUVEC cells in GelMA hydrogel and on TCPS surfaces. VEGF B) CD105 antibody immunocytochemical staining. 6 Figure 9. SEM microscopy of GelMA hydrogels at 5% (w / v) concentration. images. Figure 10. a) Sodium lauryl sulfate (SLS), b) mercury chloride (HgCl2), c) obidoximine 24 Cell size in 2D (2B) HOF and 3D (3B) ALI models after hourly exposure. Effects on vitality. * Significant differences were found between groups (p<0.05). 5 Figure 11. a) Sodium lauryl sulfate (SLS), b) mercury chloride (HgCl2), c) obidoximine 2 Reactive oxygen species (ROS) in 3D (2D) HOF and 3D (3D) ALI models Effects on production. * Significant differences were found between groups (p<0.05). Figure 12. a) Sodium lauryl sulfate (SLS), b) mercury chloride (HgCl2), c) obidoximine 2 10 studies on genotoxicity in 3D (2B) HOF and 3D (3B) ALI models Effects. * Significant differences were found between groups (p<0.05). Figure 13. a) Sodium lauryl sulfate (SLS), b) mercury chloride (HgCl2), c) obidoximine 2 In the 3D (2B) HOF and 3D (3B) ALI models, a) IL-1β, b) IL-6, c) IL-8, d) TNF- Effects on α levels. * Significant differences were found between groups. (p<0.05), ** Significant difference between groups (p<0.01), *** Significant difference between groups 15 There is a significant difference (p<0.001). Explanation of References in Figures 1. Human oral fibroblast cells 2. Hydrogel 20 3. Culture Medium 4. Human vascular endothelial cells Detailed Description of the Invention The invention relates to the toxicity of substances to be administered orally or exposed orally. and / or biomimetic in vitro test systems for evaluating their effectiveness It is related to the system. The system is based on gelatin-methacrylate (GelMA) hydrogel (2) structures. three-dimensional matrix obtained by cross-linking with photoinitiators It is based on the culturing of cells within it; this matrix with VEGFA By enriching the vessel with angiogenic factors such as angiopoietin I and II, 30 This allows for the creation of similar microenvironments. This is the preferred approach. VEGFA, angiopoietin-I and angiopoietin-II biomolecules in the form of GelMA hydrogel (2) is integrated into the system by premixing method; the growth in question 7 factors are added to the hydrogel (2) precursor solution before gelation, and the hydrogel (2) They are ensured to show a homogeneous distribution within the matrix. The invention describes a multilayered in vitro cell culture system, GelMA (gelatin methacrylate). a hydrogel (2) matrix based on the said hydrogel (2) enables cross-linking of the said hydrogel (2) 5 At least one photocrossing human located in a hydrogel (2) matrix vascular endothelial cells (4) and at least one located on the upper surface of the hydrogel (2) matrix second cell layer, preferably human oral fibroblast cells (1), located within the system The system contains a culture medium (3) to maintain the viability of the cells in the area. without direct contact between the said cell layers, hydrogel (2) 10 diffusion-based biochemical interactions occurring across the matrix It is structured to withstand this. Thanks to this structure, intercellular signal transmission is possible. is provided in a controlled manner and mimics physiological microenvironment conditions. This is possible. In the preferred application modes of the invention, system performance is improved. to increase vascular endothelial growth factor A (VEGFA), Angiopoietin-1 15 Biomolecules such as (Ang-1) and / or Angiopoietin-2 (Ang-2) hydrogel (2) matrix These components can be included, although they are not mandatory. It contributes to the enhancement of the angiogenic response. The aforementioned biomolecules, hydrogel (2) precursor before gelation in preferred application It is included in the solution by premixing method and 20 in the hydrogel (2) matrix. It is applied in a way that ensures a homogeneous distribution. In this application, VEGFA, Angiopoietin-1 and Angiopoietin-2 biomolecules at a final concentration of 0.3 µg / mL. It can be used. In addition, the cell types that can be used in the system are only human vascular cells. not limited to endothelial cells (4) and human oral fibroblast cells (1), epithelium Different cell types, such as 25 cells and / or immune cells, are also integrated into the system. However, the mechanical and structural properties of the hydrogel (2) matrix are determined; in terms of parameters such as hardness, porosity and biomolecule loading capacity It can be modified in an adjustable manner, thus enabling it to target specific biological agents. precise optimization of application-specific microenvironmental conditions It is made possible. 30 The method for preparing the biomimetic in vitro test system, which is the subject of this invention, is as follows: i. Preparation of GelMA based hydrogel (2) precursor solution, 8 ii. cross-linking of the hydrogel (2) to the hydrogel (2) precursor solution. the addition of at least one photocrossover to enable coupling, iii. VEGFA, gelation of angiopoietin-I and angiopoietin-II biomolecules beforehand, hydrogel (2) precursor solution is added by premixing method done, 5 iv. the aforementioned VEGFA, angiopoietin-I and angiopoietin-II biomolecules homogeneous distribution within the hydrogel (2) matrix ensuring, v. hydrogel (2) of human vascular endothelial cells (4) before gelation Adding to the precursor solution, 10 vi. hydrogel (2) precursor solution of human vascular endothelial cells (4). homogeneous distribution within it, vii. Hydrogel (2) precursor containing human vascular endothelial cells (4) transferring the solution into a multi-well plate, viii. In the preferred practice, human vascular endothelium 15 in a 96-well plate Adjustment of cell density of cell (4), ix. Hydrogel (2) precursor containing human vascular endothelial cells (4). photocrosslinking of the solution under UV light Formation of a three-dimensional hydrogel (2) matrix by means of, x. photocrosslinking after human vascular endothelial cells (4) 20 by positioning it within a hydrogel (2) matrix, it resembles a vessel. Obtaining a substrate / intermediate layer suitable for microenvironment formation, xi. Cross-linking of the hydrogel (2) system with UV followed by human oral Addition of fibroblast cells (1) to the system, xii. 25 human oral fibroblast cells (1) on the upper surface of the hydrogel (2) matrix placement in the system in such a way that it will be positioned xiii. In the preferred practice, human oral fibroblasts are placed in a 96-well plate. planting of cell (1), xiv. Human oral fibroblast cells (1) in low serum culture medium containing FBS (3) incubation under these conditions, 30 xv. human vascular endothelial cells (4), endothelial proliferation and tubule standard endothelial culture medium containing FBS to support its formation (3) to be maintained under the conditions 9 xvi. human oral fibroblast cells (1) and human vascular endothelial cells (4), Diffusion-based biochemical activity through hydrogel (2) matrix acculturation in a way that allows for interactions, xvii. Culturing of cells within the system, xviii. Hydrogel (2) system, scratch test or similar stability evaluations 5 incubating it in a way that maintains its structural integrity throughout the period, xix. The system, substances to be administered orally or subjected to oral exposure Ready for use for the evaluation of toxicity and / or efficacy. to be made It includes the steps of the process. 10 In one application of the invention, the biomimetic in vitro test system that is the subject of the invention Preparation method; i. Preparation of GelMA based hydrogel (2) precursor solution (%5 w / v), ii. cross-section of the hydrogel (2) into the precursor solution (2) of the hydrogel (2) 15 additional photocrossover (0.5%) to enable binding being done, iii. VEGFA, gelation of angiopoietin-I and angiopoietin-II biomolecules beforehand, hydrogel (2) precursor solution is added by premixing method done, 20 iv. the aforementioned VEGFA, angiopoietin-I and angiopoietin-II biomolecules homogeneous distribution within the hydrogel (2) matrix ensuring, v. VEGFA, hydrogel of angiopoietin-I and angiopoietin-II biomolecules (2) Adjustment to a final concentration of 0.3 µg / mL, 25 vi. hydrogel (2) of human vascular endothelial cells (4) before gelation Adding to the precursor solution, vii. hydrogel (2) precursor solution of human vascular endothelial cells (4). homogeneous distribution within it, viii. Hydrogel (2) precursor 30 containing human vascular endothelial cells (4) transferring the solution into a multi-well plate, ix. In the preferred application, 1.5 × 10⁴ human cells per 96-well plate. cell density of vascular endothelial cell (4) to be used adjustment, x. hydrogel (2) precursor containing human vascular endothelial cells (4) photocrosslinking of the solution under UV light Formation of a three-dimensional hydrogel (2) matrix by means of, xi. photocrosslinking of human vascular endothelial cells (4) by positioning it within the hydrogel (2) matrix, it forms a vessel-like 5 Obtaining a substrate / intermediate layer suitable for microenvironment formation, xii. human body 1 hour after UV crosslinking of the hydrogel (2) system. Addition of oral fibroblast cells (1) to the system, xiii. human oral fibroblast cells (1), on the upper surface of the hydrogel (2) matrix placement into the system in such a way as to be positioned, 10 In the preferred application (xiv), 1.5 × 10⁴ wells per well in a 96-well plate. Inoculation of human oral fibroblast cells (1), xv. human oral fibroblast cells (1) in low serum solution containing 2% FBS incubation under culture medium (3) conditions, xvi. human vascular endothelial cells (4), endothelial proliferation and tube 15 Standard endothelial containing 10% FBS to support its formation continuation under culture medium (3) conditions, xvii. human oral fibroblast cells (1) and human vascular endothelial cells (4), Diffusion-based biochemical activity through hydrogel (2) matrix acculturation in a way that allows for interactions, 20 xviii. Culturing the cells within the system for 3 days, xix. Scratch test or similar stability assessments of the hydrogel (2) system maintaining its structural integrity for at least 5 days during this period incubation, xx. system, substances to be administered orally or subjected to oral exposure 25 Ready for use for the evaluation of toxicity and / or efficacy. to be made It includes the steps involved in the process. In the system described in the invention, VEGFA, angiopoietin-I and angiopoietin-II are 0.3 µg / mL for the last 30 days. It is used in high concentrations. This structure is involved in cell proliferation, differentiation, and allows intercellular signaling to occur under more physiological conditions. It recognizes human vascular endothelial cells (4) and human oral By presenting a structure in which fibroblast cells (1) are organized in a layered manner, the cell- 11 This enables more accurate modeling of cell and cell-matrix interactions. Human vascular endothelial cells (4) make hydrogel (2) before gelation. It is homogeneously distributed into the precursor solution, which is the preferred application. The system contains 1.5 × 10⁴ human vascular endothelial cells per 96-well plate. (4) is used. Human oral fibroblast cells (1) are used in human vascular endothelium 5 UV cross-linking of GelMA hydrogel (2) system containing cells (4) It is added to the system in approximately 1 hour; the preferred application has 96 wells. 1.5 × 10⁴ human oral fibroblast cells (1) are seeded per well of the plate and 2% FBS The cells are incubated under low serum culture conditions. They are cultured inside for 3 days; at the end of this period, cell viability is 10 Migration / wound closure and related biological responses are being evaluated. The hydrogel... (2) thanks to its use as an interface, in classic air-liquid interface systems Similar functionality can be achieved without the need for insert components. This ensures that it is done; this is advantageous both in terms of cost and ease of implementation. It offers advantages. The platform described in the invention exhibits cytotoxicity, genotoxicity and reactive properties. high-performance facilities capable of carrying out multiple biological analyses, such as oxygen species production. It offers a test infrastructure with repeatability and scalability; hydrogel (2) the composition, biomolecule content and cell types can be modified thanks to different research needs and personalized applications It presents an adaptable structure. 20 In current three-dimensional (3D) cell culture systems, hydrogel (2) structures are mostly as passive components that provide only mechanical and structural support In contrast, the hydrogel (2) matrix used in the invention is cell a functional 25 that enables biochemical signal transmission between its layers It is structured as an interface. In this context, the hydrogel (2) is only for the cells. It is not a carrier medium that allows adhesion, but rather an intercellular medium. It functions as an active microenvironmental component regulating communication. The invention Furthermore, the air-liquid interface (Air-) commonly used in the known state of the art. It differs from Liquid Interface (ALI) systems. ALI systems have 30 creating a physical air phase to ensure biological functions However, in the invention, a hydrogel (2) matrix is ​​created without the need for such an air phase. diffusion mechanisms and biochemical signal gradients created within it Similar biofunctional effects are achieved through this method. In this respect, the invention is more... 12 It offers a compact, controlled and practical microenvironment. On the other hand, The invention eliminates the dependence on the use of inserts. This situation allows for the use of hydrogels. (2) the matrix will be semi-permeable and allow biochemical signal transmission This is achieved through its specially structured design. The structure in question, without requiring direct physical contact between cell layers, controlled and 5 It enables selective molecular transport, thus facilitating cellular interactions more effectively. It allows for precise and predictable regulation. Within the scope of the invention, the formation of vessel-like structures in hydrogel (2) is multiple This can be evaluated using validation approaches. First, human vascular endothelium 10 capillary-like organization of the cells (4) within the GelMA hydrogel (2). The capacity to form endothelial tubes can be analyzed with the VEGF test; under supported conditions, human vascular endothelial cells (4) within 24 hours It can be microscopically shown that it forms tubular structures. Furthermore... Immunofluorescence staining for confirmation of endothelial phenotype 15 This can be done and CD105 and VEGF expressions can be evaluated. Human vascular endothelial cells (4) cultured in GelMA hydrogel (2), Stronger CD105 and VEGF fluorescence signals compared to the TCPS control surface. observation of the endothelial activity and angiogenic behavior of the hydrogel (2) medium This shows that it supports it. In addition, VEGFR I / II and 20, which are associated with vascularization. CD105 gene expression was analyzed using droplet digital PCR (ddPCR). Human vascular endothelium cultured in GelMA hydrogel (2) can be done; In cells (4) VEGFR2 expression increased approximately 2.92-fold, CD105 An increase of approximately 5.65 times in expression can be detected. In addition, low In scratch / migration analyses performed under serum conditions, human blood vessels 25 endothelial cells (4) are faster on GelMA hydrogel (2) than on TCPS surface exhibiting migration and closing the wound area in a shorter time, hydrogel (2) its structure provides a microenvironment that supports vascular organization It supports. Different cell type-specific culture media (3) conditions can be used in the system. Human Serum optimization is performed for oral fibroblast cells (1) and low in serum conditions, preferably in culture medium (3) containing 2% FBS, cells The preservation of viability is ensured. In contrast, human vascular endothelial cells 13 (4), preferably 10% to support endothelial proliferation and tube formation. It can be maintained under standard endothelial medium containing FBS (3). During the scratch test, the hydrogel (2) system maintained its structural integrity for 5 days. It is able to preserve and does not show significant degradation. Therefore, the system It is considered to be able to function stably for at least 5 days; cell viability, 5 Optimal incubation in terms of migration and tube formation assessments. Although the duration depends on the experimental parameter, a suitable range is 3–5 days. It is located. The reproducibility of the system described in the invention; cell viability, ROS analysis, genotoxicity, 10 Similar results were obtained in different replicate tests in ddPCR, ELISA, and wound closure experiments. This is supported by the results obtained. These analyses were conducted by at least three independent researchers. This can be repeated and the results are statistically significant. It can be evaluated. In addition, the hydrogel (2) structure during the wound closure test. Maintaining its structural integrity for at least 5 days, ensuring the system operates stably. This shows that the scalability of the system is based on GelMA hydrogel (2) The fact that the formulation can be prepared under standard laboratory conditions, multi-well can be applied to plate systems and cells within the hydrogel (2) matrix This is supported by its ability to be distributed homogeneously. This structure allows for different experiments. It is adaptable to their designs and cell densities. 20 Cell viability / cytotoxicity analysis of the system described in the invention was performed using the MTT test. This can be achieved. Reactive oxygen species (ROS) levels, 2',7'- Fluorescence measurements using the dichlorodihydrofluorescein diacetate (H2DCFDA) method. This can be determined by measurements using 485 nm excitation and 520 nm emission wavelengths. This can be done in their respective lengths. Genotoxicity assessments are performed using alkaline mononucleosis. Gene expression can be determined using cell gel electrophoresis (Comet Assay) method. The analyses can be performed using the droplet digital PCR (ddPCR) method, and protein ELISA method can be used in the analysis of inflammation biomarkers. In addition, cell migration and hydrogel (2) stability wound closure 30 This can be examined with a (scratch / wound healing) test. The results obtained... For verification purposes, the system was used as a standard two-dimensional cell culture platform. Human oral fibroblasts cultured on tissue culture polystyrene (TCPS) cells (1) can be compared. In comparative analyses, cell viability, 14 Genotoxicity, reactive oxygen species (ROS) levels, PCR analyses, ELISA analyses and wound closure experiments can be carried out; GelMA hydrogel (2) and human Results obtained from hydrogel (2) system containing vascular endothelial cells (4) TCPS can be evaluated by comparing it with control groups. Especially cell 5 observed in terms of behavior, migration capacity and endothelial activity These differences support the biomimetic and functional nature of the system. Comparative analyses were conducted with TCPS control groups within the scope of the study. It provides in vitro validation of the system. 10 GelMA hydrogels prepared with cells at different concentrations The MTT test was used to evaluate their interactions. On a 96-well plate... HUVEC cells were seeded in each well at a rate of 2 x 10³ cells, and In order to allow the cells to adhere to the surface, a mixture of 37°C and 5% CO2 was used for 24 hours. The cells were incubated in the medium. After incubation, the cells adhered to the surface of the culture medium. GelMA 15 prepared in different concentrations (%5-%7 (w / v)) by removing the gel The hydrogels were added to each well at a thickness not exceeding 0.5 mm, in amounts of 30 µL per well. Plates for cross-linking hydrogels were added, at a wavelength of 365 nm. J / cm2 was exposed to UV light. Following cross-linking, 100 µL was added to each well. Culture medium was added and the plates were incubated for 24 hours. Incubation Subsequently, the MTT test was performed to evaluate cell viability analysis. 20 The results showed a decrease in cell viability for both GelMA concentrations. However, loss of viability was shown in cells treated with 5% (w / v) GelMA. It was observed to be 15% lower compared to the 7% (w / h) GelMA group, and this The difference was not found to be statistically significant. HUVEC cells in GelMA hydrogels doped with growth factor Their behavior is also influenced at the gene level by the VEGFR I / II and CD105 genes. This was analyzed through the proliferation level. For this, first, the primers were examined. The optimization was performed and it was found that all three genes could be studied at an average temperature of 52.5°C. It has been understood. However, in droplet PCR analysis, droplets are formed and 30 It has also been tested that it cannot be created. During these preliminary verifications, directly HUVEC cells were used. HUVEC cells and growth factors... When analyses were performed on HUVEC cells embedded in enriched GelMA droplet reactions occur; on average 6000- directly for HUVEC cells. The analysis of 8000 droplets was carried out, while in the gel formulation this number was 10000. It has been understood that it can reach that level. In this environment, the signal multiplies, resulting in higher outcomes. The presence of gene populations that will give rise to β- is indicated after gel addition. When normalization is performed according to actin groups, the results obtained from growth on GelMA... The cells were 2.92 and 5.65 times more concentrated, respectively, in terms of VEGFR2 and CD105. It appears to be too expressive. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium is used in determining cell viability. The bromide (MTT) method was used. The effect of the substances on cell viability was investigated. For evaluation, cells were prepared with culture medium at different concentrations. The substance was exposed to the solutions for 24 hours. The exposure time... Finally, the inserts were removed and each insert was washed with PBS. Then the cells The cells were removed using gentle cell dissociation reagent (GCDR) and then placed in 200 μL Mixed with freshly prepared MTT (0.5 mg / mL) solution, in 96-well plates This mixture was added and incubated at 37 °C for 4 hours. At the end of the incubation period, 15 The plates were centrifuged at 500 rpm for 18 minutes, and the solutions in the plates were discarded. 100 µL of DMSO was added to each well. Then, the absorbance of the solutions was measured. Measurements were taken at 450 nm. The results obtained in the ALI model are similar to those in 2D cells. For comparison with the results, HOF cells were sampled in 2D with 96 wells. The cells were cultured in plates with 15,000 cells in each well, and 20 The cells were placed in SLS, HgCl2, and Obi solutions at the same concentrations as in the ALI model. They were exposed for 24, 48, and 72 hours. Incubation with substance solutions. Then the drug solutions were removed from the wells, and 20 μL of fresh solution was added to each well. Prepared MTT (0.5 mg / mL) solution was added and 96-well plates were incubated at 37 °C for 4 months. They were incubated for 25 hours. At the end of the incubation, the solutions in the plates were discarded and each plate was prepared for 25 minutes. 100 µL of DMSO was added to the well. Then the absorbance of the solutions was 570. Measured at nm. Based on absorbance values ​​measured using the MTT method. Different values ​​were calculated by determining %cell viability and 50% inhibitory concentration (IC50). incubation times and exposure to different concentrations of substance solutions The effects on cells were evaluated. 30 drug-free cells were used as a negative control. Cells treated with culture medium were used. 16 Determining cell viability; The viability of the tested cells was compared to the negative control according to the equation below. Expressed as a percentage: Reactive oxygen species (ROS) levels in cells were measured with 2,7-dichlorodihydrofluorescein diacetate. Determined via the (H2DCFDA) method. Cell removal with GCDR. The mixture was prepared and added to 96-well plates, and the plates were spun at 500 rpm for 18 minutes. They were centrifuged. Both the cells in the ALI model and those in 96-well plates were centrifuged. For HOF cells cultured in 2B, PBS was used as a blind spot and culture medium as a negative control. 100 µM H2O2 was used as the site and positive control, and the cells were incubated for 1 hour. Drug solutions at concentrations used in cytotoxicity studies are stored at 37°C. and exposed to 5% CO2 in an incubator. After incubation, the drug... The solutions were discarded, and each well was washed with 100 µL of PBS. The following 15... The studies in these stages were conducted in the dark because H2DCFDA is affected by light. This was carried out. The plate was placed on ice, and 100 μL of H2DCFDA was added to each well. The plates were added and incubated for 1 hour in an incubator at 37°C and 5% CO2. The solutions in each well were discarded at the end of the incubation period. Each well was washed with 100 µL of PBS and 100 µL of PBS was added to each well 20 times. added. Then, the absorbance given by each well (absorption: 485 nm, Emission (520 nm) was measured. Alkaline substances are used to determine their genotoxic effects on cells. The single-cell gel electrophoresis (Comet) method was used. 25 cells were used in this method. IC50 values ​​obtained from cytotoxicity determination at three different concentration values Selected from among low concentrations. Single-cell gel electrophoresis (Comet) For genotoxicity determination using this method, cells are placed in a culture medium containing the prepared substance. They were incubated with the solutions for 3 hours. After incubation... The culture medium in which the cells were located was removed, and each well was filled with PBS for 30 minutes. The cells in the ALI model were washed with GCDR and cultured in 2B HOF. 17 The cells were removed with trypsin-EDTA solution and placed on a counting slide. The cells were applied and the live cells were counted using a counting slide, resulting in 1 mL of cells. The number of cells in the suspension has been determined. Each slide contains 10,000-20,000 cells. Dilute with culture medium as follows: 100 µL melted at 37°C ± 0.5°C. 1% low melting point agar (LMA) solution mixed with 50 µL of cell suspension. This mixture was then mixed. Next, this mixture was added to a pre-prepared 1% standard melting point solution. Dipped in dotted agar (NMA) solution and spread onto agar-coated slides, and The slides were covered with coverslips. The slides were placed on an ice surface for about 5 minutes. After being left to stand and allowing the agar to solidify, the coverslip is placed on the slide. They were carefully removed, placed in the chalets, and then 10 pre-prepared items were placed on top of them. A cold lysis solution kept at +4ºC was added and left at +4°C for 1 hour. For the electrophoresis process, the tank is filled with cold electrophoresis solution. Lysis After the procedure, the slides were placed with the agar-spreading sides facing upwards for electrophoresis. 20 placed in the bathtub without leaving any gaps and without applying any current It was kept in the electrophoresis cuvette for 15 minutes. Then, a current of 25 V and 300 mA was applied. Electrophoresis was applied for 20 minutes using this method. After the electrophoresis process was completed... Then, the carefully removed slides were placed in trays and immersed in PBS solution for 15 minutes. They were left to stand. Then the slides were immersed in 50%, 75% and 99% ethanol, respectively. They were left to stand in the solution for 5 minutes each. During the evaluation of genotoxic effect... Ethidium bromide solution was added to the slides. 100 cells per slide (20°C) DNA damage level determined using a computer program under a fluorescence microscope. It was evaluated using the moment parameter. The invention includes VEGF, Ang 1 and Ang 2 GelMA at a concentration of 16 ng / cm2. They are incorporated into hydrogels. The aforementioned biosials are contained within the gels and are suitable for UV application. 25 determining whether it remains active afterwards and biosignals from these gels Analyses regarding emission levels were performed using the ELISA method. Analyses showed that 23.82 ± 0.23% of VEGF was released at the end of 120 hours. This has been shown. This value was determined as 9.34 ± 0.22% for Ang 1, while for Ang 2 it was % It was determined as 27.00 ± 0.06. Consistent with 120-hour release inflation study of 30. Since gel integrity is maintained, biosignal release from the gels remains limited. It has been observed that the released proteins retain their activity. It has been understood that data obtained from emission studies can be used on kinetic models. 18 When studied, all three biosignals showed an emission curve consistent with the Higuchi model. It has been understood that it was created. After HUVEC cells were placed in the gel, CD105 and VEGF markers were used. Staining was performed in this regard. The cells were stained alive using CellBrite. The form was dyed green and its condition within the hydrogel was observed. The findings indicate that the presence of hydrogel signals in both markers were higher compared to the TCPS group. It has been shown to increase significantly. The system described in the invention is capable of mimicking the oral tissue microenvironment and producing 10 microscopic microorganisms in vitro. a multi-layered system that enables toxicity / efficacy analyses to be carried out It is a cell culture system; • Human oral fibroblast cells (1); represent the connective tissue component of oral tissue. It will be positioned at the top layer of the system in such a way as to be tested. cellular response to direct contact with substances (proliferation, 15 It enables the evaluation of cytotoxicity, genotoxicity, etc. • Hydrogel (2); forming a three-dimensional extracellular matrix (ECM)-like structure It enables both cell attachment and multiplication, as well as different cells. an interface that allows biochemical signal transmission between its layers performing its duty, 20 • Culture medium (3); to maintain the viability of the cells within the system by providing the necessary nutrients, growth factors and a suitable chemical environment enabling the continuation of cellular metabolic activities, • Human vascular endothelial cells (4); in hydrogel (2) matrix by being positioned, it enables the formation of vascular-like microstructures and 25 Thus, by mimicking angiogenesis-like processes, a more realistic result can be obtained. It contributes to the creation of an in vitro microenvironment. Thanks to the combined action of these elements, oral exposure or The toxicity and / or efficacy of the substances to be applied should be closer to the actual tissue. under these conditions, it is possible to analyze in a repeatable and scalable manner. It is being done. 19 The effect of substances with known oral toxic effects on the system covered by the invention research into its effects 2D (2-dimensional) HOF cells and SLS, HgCl2, and Obi in the generated 3D ALI model. The cytotoxic effects were investigated after 24 hours of exposure to SLS, HgCl2, and 5. In both 2B HOF cells exposed to obidoxime for 24 hours In the 3D model created, cell viability depends on the increase in concentration. It has been observed that there is a decrease. However, especially at high concentrations As the view progresses, cell viability decreases more significantly in the 3D model. This indicates that a response similar to that of the organism is beginning to be obtained. Toxic 10 IC50 in 3D cells treated with SLS and obidoxime, which are known to have significant effects. The fact that its value is lower compared to 2B cell culture also indicates that this culture is toxic. This shows that it is more sensitive to the effects. Viability studies, 2D and 3D cultures. There are significant differences between the systems; these differences are partly due to 2D. The physical transition to the 3D environment causes changes and the cellular membrane 15 It is also claimed that this stems from its effect on cellular function. 3D culture The use of matrices, compared to cells cultured in traditional 2D systems increased viability or decreased toxicity or cellular resistance resulting This is how it is interpreted. The results obtained in this study also indicate cell viability. It was found that it did not change with culturing in 3D environments but showed significant toxic effects. 20 It shows. 2D (2-dimensional) HOF cells and SLS, HgCl2, and Obi in the generated 3D ALI model. The effects of SLS, HgCl2, and obidoxime on ROT production have been investigated. ROT production increased in 3D cells as expected, with a concentration increase of 25. and the cells' response to ROT production increased together and the response of 2B cells It was determined to be more pronounced according to the answer given. Observed in the 3D ALI model. A more pronounced ROT response indicates that this system monitors the cellular microenvironment and metabolic activity. This can be associated with better reflection. The polarization of cells, cell-to-cell and With increased cell-matrix interactions, oxidative metabolism becomes more active. 30 This leads to an amplification of ROS production in response to toxic agents. This can lead to... Also, under ALI conditions, direct contact of cells with air, By increasing basal oxidative stress levels, it leads to a more sensitive response to external stimuli. It can contribute to its formation. In addition, diffusion occurring in the 3D structure. gradients cause local toxic substance accumulation (ROS) in some areas. This can lead to a more precise measurement of production. In contrast, in 2D cell cultures... cells that are less differentiated and have lower metabolic activity This may have resulted in a more limited oxidative stress response. 2D (2-dimensional) HOF cells and SLS, HgCl2, and Obi in the generated 3D ALI model. It has been found that increasing concentration leads to an increase in genotoxic effects. However, the lack of a clear difference between the two cultural models suggests that the methods used... This may be related to the agents' potential to create strong and direct cellular stress. In particular, small and reactive molecules such as HgCl₂ can disrupt membrane integrity. SLS may have limited the diffusion and barrier advantages that 3D modeling could provide. Additionally, the high concentrations applied may have a preservative effect on 3D culture. masking the effect, similar level of DNA damage in 2D and 3D systems. This may have led to its formation. This finding suggests that the 3D ALI model is resistant to toxic agents. It does not provide absolute resistance, but in a more physiological microenvironment, genotoxic 15 This indicates that it allows for the evaluation of the response. IL-1β, an inflammation biomarker, was found to be correlated between 2D HOF cells and a 3D model. IL-6, IL-8, and TNF-α levels were compared. All panels were analyzed together. When evaluated, cytokine levels in 2D cell culture were 20% higher compared to the 3D model. It is observed that it is significantly higher. This situation indicates that 2B cell culture that it showed a higher inflammatory response to the substances applied. This shows that, in contrast, TNF-α and interleukin levels are higher in the 3D model. The fact that it remains low and limited allows this model to better mimic the cellular microenvironment. as a result, the inflammatory response occurs at a more controlled and physiological level. 25 This suggests that the findings indicate an inflammatory response in 2D cell cultures. It can reflect a higher level than it actually is, whereas 3D models are more in vivo This indicates that it presents a cytokine response that is close to and balanced with the conditions. Therefore, 3D cell models are more physiological and reliable for inflammation-based studies. It is considered that it can yield results. 30 21 Industrial Applicability of the Invention The invention relates to the toxicity of substances to be administered orally or exposed orally. and / or biomimetic in vitro test systems for evaluating their effectiveness. It is related to and applicable to industry. The invention is not limited to the above descriptions, and a person skilled in the field can easily make further discoveries. It can demonstrate different applications of the invention. These are the claims and demands of the invention. It should be evaluated within the scope of the protection granted. 15 25

Claims

22 REQUESTS 1. It is a multilayered biomimetic in vitro cell culture system; its characteristic is GelMA. (gelatin methacrylate) based hydrogel (2) matrix, the hydrogel (2) At least one photocrossover agent that enables cross-linking, hydrogel (2) matrix Human vascular endothelial cells (4) and hydrogel (2) positioned within it 5 at least one second layer of cells located on the top surface of the matrix, system a culture medium (3) in order to maintain the viability of the cells contained within it It includes.

2. A cell culture system according to claim 1, characterized by its second cell layer. It is human oral fibroblast cells (1). 10 3. A cell culture system according to claim 1, characterized by its vascular endothelial properties. Growth factor A (VEGFA), Angiopoietin-1 (Ang-1) and / or Angiopoietin-2 It contains (Ang-2).

4. A cell culture system according to any of claims 1-3, characterized by: VEGFA, Angiopoietin-1 and Angiopoietin-2 biomolecules at 0.3 µg / mL for the last 15 It contains it in a specific concentration.

5. A cell culture system according to any of claims 1-4, characterized by its human component. human oral vascular endothelial cells (4) in hydrogel (2) matrix fibroblast cells (1) are on the upper surface of the hydrogel (2) matrix It is its location. 20 6. Is it a cell culture system according to any of the previous requirements? Its feature is that it is structured to mimic the oral tissue microenvironment. It is the fact that.

7. Cytotoxicity of a cell culture system according to any of claims 1-6, 25 in performing genotoxicity and / or reactive oxygen species analyses usage.

8. Preparation of a multilayer biomimetic in vitro cell culture system. Its method and characteristic is; i. Preparation of GelMA based hydrogel (2) precursor solution, ii. cross-section of the hydrogel (2) into the precursor solution (2) of the hydrogel (2) 30 the addition of at least one photocrossover to enable coupling, iii. VEGFA, gelation of angiopoietin-I and angiopoietin-II biomolecules beforehand, hydrogel (2) precursor solution is added by premixing method being done, 23 iv. VEGFA, angiopoietin-I and angiopoietin-II in question. homogeneous distribution of biomolecules within the hydrogel (2) matrix to ensure that it is shown, v. hydrogel (2) of human vascular endothelial cells (4) before gelation Adding to the precursor solution, 5 vi. hydrogel (2) precursor solution of human vascular endothelial cells (4). homogeneous distribution within it, vii. Hydrogel (2) precursor containing human vascular endothelial cells (4) transferring the solution into a multi-well plate, viii. In the preferred practice, human vascular endothelium 10 in a 96-well plate Adjustment of cell density of cell (4), ix. Hydrogel (2) precursor containing human vascular endothelial cells (4). photocrosslinking of the solution under UV light Formation of a three-dimensional hydrogel (2) matrix by means of, x. After photocrosslinking, human vascular endothelial cells (4) 15 by positioning the vessel within the hydrogel (2) matrix Obtaining a substrate / intermediate layer suitable for creating a similar microenvironment, xi. Cross-linking of the hydrogel (2) system with UV followed by human oral Addition of fibroblast cells (1) to the system, xii. The top 20 of the hydrogel (2) matrix of human oral fibroblast cells (1). placement of the system in such a way that it will be positioned on the surface xiii. In the preferred practice, human oral fibroblasts are placed in a 96-well plate. planting of cell (1), xiv. human oral fibroblast cells (1) low serum containing FBS incubation under culture medium (3) conditions, 25 xv. human vascular endothelial cells (4), endothelial proliferation and tubule standard endothelial culture medium containing FBS to support its formation (3) to be maintained under the conditions xvi. human oral fibroblast cells (1) and human vascular endothelial cells (4), diffusion based 30 through hydrogel (2) matrix culturing in a way that allows for biochemical interactions, xvii. Culturing of cells within the system, 24 xviii. hydrogel (2) system, scratch test or similar stability to maintain its structural integrity during the evaluation process incubation, 19. The system, to be administered orally or subjected to oral exposure. 5 for the purpose of evaluating the toxicity and / or efficacy of substances made ready for use It includes the steps of the process.

9. This is a method for preparing a cell culture system according to claim 8, and its characteristics are: i. Preparation of GelMA based hydrogel (2) precursor solution (%5 w / v), ii. cross-section of the hydrogel (2) into the precursor solution (2) of the hydrogel (2) 10 additional photocrossover (0.5%) to enable binding being done, iii. VEGFA, gelation of angiopoietin-I and angiopoietin-II biomolecules beforehand, hydrogel (2) precursor solution is added by premixing method done, 15 iv. VEGFA, angiopoietin-I and angiopoietin-II in question. homogeneous distribution of biomolecules within the hydrogel (2) matrix to ensure that it is shown, v. VEGFA, hydrogel of angiopoietin-I and angiopoietin-II biomolecules (2) Adjustment to a final concentration of 0.3 µg / mL, 20 vi. hydrogel (2) of human vascular endothelial cells (4) before gelation Adding to the precursor solution, vii. hydrogel (2) precursor solution of human vascular endothelial cells (4). homogeneous distribution within it, viii. Hydrogel (2) precursor containing human vascular endothelial cells (4) 25 transferring the solution into a multi-well plate, ix. In the preferred application, 1.5 × 10⁴ human cells per 96-well plate. cell density of vascular endothelial cell (4) to be used adjustment, x. hydrogel (2) precursor containing human vascular endothelial cells (4) 30 photocrosslinking of the solution under UV light Formation of a three-dimensional hydrogel (2) matrix by means of, xi. photocrosslinking of human vascular endothelial cells (4) by positioning the vessel within the hydrogel (2) matrix Obtaining a substrate / intermediate layer suitable for creating a similar microenvironment, xii. 1 hour after UV crosslinking of the hydrogel (2) system Addition of human oral fibroblast cells (1) to the system, 5 xiii. human oral fibroblast cells (1), on top of the hydrogel (2) matrix placement of the system in such a way that it will be positioned on the surface In the preferred application (xiv), 1.5 × 10⁴ wells per well in a 96-well plate. Inoculation of human oral fibroblast cells (1), xv. human oral fibroblast cells (1) in low serum 10% FBS incubation under culture medium (3) conditions, xvi. human vascular endothelial cells (4), endothelial proliferation and tubule Standard endothelial containing 10% FBS to support its formation continuation under culture medium (3) conditions, xvii. Human oral fibroblast cells (1) and human vascular endothelial cells 15 (4), diffusion based on hydrogel (2) matrix culturing in a way that allows for biochemical interactions, xviii. Culturing the cells within the system for 3 days, xix. hydrogel (2) system, scratch test or similar stability During the evaluation process, its structural integrity will be assessed for at least 5 days. incubating in a way that will protect it, xx. system, to be administered orally or subjected to oral exposure for the purpose of evaluating the toxicity and / or efficacy of substances made ready for use It includes the steps of the process. 25 10. Multilayer biomimetic in prepared by a method according to claim 8 or 9. in vitro cell culture system.

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