Method for preparing three-dimensional culture medium through DNA

Abstract

The present invention relates to a method for preparing a DNA-based three-dimensional culture medium, the method being characterized by comprising: a step for extracting natural DNA from a biological tissue containing double-stranded DNA through a physical or chemical method; a step for generating a first mixed solution by mixing the extracted DNA with a predetermined first solvent which is one of water, PBS, or DMEM; a step for gelling the first mixed solution by generating viscosity by forming a biopolymer network by self-arrangement of the double helix structure of the DNA; and a step for preparing a culture medium for three-dimensional cell culture by molding or casting the formed gelled first mixed solution into a desired structure.

Description

Method for preparing a 3D culture medium using DNA

[0001] The present invention relates to a method for preparing a three-dimensional culture medium using DNA.

[0002] In modern life science and medical research, three-dimensional culture technology is essential for a more precise understanding of cell and tissue characteristics. Three-dimensional cell culture media can reproduce cell-to-cell interactions and tissue characteristics that cannot be provided in conventional two-dimensional culture environments, and are therefore utilized in various fields such as the study of cellular physiological responses, disease modeling, and new drug development. In particular, it is known that the precision and efficiency of research improve as the culture medium provides physical and chemical properties similar to the natural state of the cells.

[0003] Conventional 3D culture media are typically composed of materials such as synthetic polymers, natural protein-based gels, or hydrogels. However, these existing culture media have limitations. Synthetic polymers have low biocompatibility, which can distort physiological responses in cells, while natural protein-based gels possess physical properties unsuitable for specific cell types or limit the diversity of culture environments due to the limited use of gene or protein-based materials. Additionally, hydrogel-based media face difficulties in actual research applications due to their complex composition or the potential for cell damage during the manufacturing process.

[0004] Recently, the development of culture media utilizing bio-derived materials has been attracting attention. Among these, DNA is a biopolymer capable of replicating the natural environment of cells and tissues, offering advantages such as excellent biological stability, biocompatibility, and compositional flexibility. In particular, the double helix structure of DNA provides viscosity and flexibility, enabling the formation of biopolymer networks. However, existing technologies utilizing DNA-based culture media are revealing limitations at the commercialization stage due to issues such as complex DNA purification processes or the inability to efficiently implement gelation processes.

[0005] The present invention aims to solve the aforementioned problems by extracting natural DNA containing a double helix structure from biological tissue and mixing it with a specific solvent to utilize the self-assembly characteristics of DNA, thereby forming a biopolymer network and inducing gelation, and thereby enabling the production of a customized culture medium that provides viscosity, strength, and transparency suitable for cell culture.

[0006] DNA-based culture media provide biological characteristics and physical stability simultaneously, and can provide a culture environment suitable for various cell types and research purposes, and

[0007] By extracting natural DNA and forming a culture medium through a simple gelation process, the manufacturing process can be simplified and time and costs can be reduced, and

[0008] By adjusting DNA concentration and environmental conditions of the solution (pH, ion concentration, etc.), viscosity, strength, transparency, etc. can be designed to produce customized culture media tailored to specific research requirements, and

[0009] The present invention provides a method for manufacturing a three-dimensional culture medium using DNA that can be utilized in various life science and medical research fields, such as organoid culture, neuronal research, and tissue regeneration research, and in particular, can contribute to personalized research and therapeutic development by providing a customized culture environment based on the patient's cells.

[0010] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention comprises: a step of extracting natural DNA from a biological tissue containing double helix DNA through a physical or chemical method; a step of producing a first mixture by mixing the extracted DNA with a first solvent set in which water, PBS, or DMEM is selected; a step of gelling the first mixture by causing the double helix structure of the DNA to self-arrange to form a biopolymer network and generate viscosity; and a step of preparing a culture medium for three-dimensional cell culture by molding or casting the first mixture in a gelled state into a desired structure.

[0011] The step of extracting the natural DNA described above is characterized by comprising: a step of obtaining high-purity DNA by removing proteins and RNA from biological tissue using an enzyme and then precipitating with an ionic solvent and ethanol; and a step of treating the extracted DNA in a low-temperature (4~10°C) environment so that it maintains a high molecular weight of 10kb or more.

[0012] The step of generating the first mixture is characterized by adjusting the concentration of the DNA to a range of 0.1% to 5% so that the first mixture is mixed to convert from a flowing liquid state to a viscous gelled state.

[0013] The gelling step described above is characterized by adjusting the concentration of DNA and the values ​​of multiple environmental condition variables of the solution so that the double helix structure of DNA interacts with a pre-set metal divalent cation to strengthen the biopolymer network.

[0014] The step of forming or casting the first mixture in the gelled state formed above into a desired structure includes the step of designing the thickness, strength, and pore structure of the gel according to the physical properties required in three-dimensional cell culture, and the designing step is characterized by designing a culture medium structure containing a nanostructure for organoid culture and designing a culture medium structure with high transparency for neuronal cell culture.

[0015] The manufactured culture medium contains natural DNA extracted from the patient's biological tissue, and the culture medium provides a customized culture environment that reflects the same genetic and environmental characteristics as the patient's cells. The method is characterized by culturing the first cells of a patient with the first disease, which are induced by the first disease, through the culture medium, and then performing cell responsiveness and drug efficacy evaluations on the colony of the first cells cultured through the culture medium based on this for patient-specific research and therapeutic agent development for the first disease.

[0016] The above patient-specific culture environment includes the step of reproducing the patient's cellular microenvironment by introducing specific signaling molecules, cytokines, or exosomes secreted from the cells of a patient with a first disease into the culture medium, and is characterized in that the specific signaling molecules interact with the physical and chemical properties of the culture medium to mimic the physiological response of the patient's cells in their natural state.

[0017] The step of performing an evaluation of drug efficacy for a first disease using the above culture medium includes adding a pre-established drug related to the first disease to the above culture medium, and then quantitatively analyzing changes in the growth rate, apoptosis rate, inflammatory response, or intracellular metabolic response of patient-derived cells cultured in the above culture medium, and is characterized by evaluating the therapeutic efficacy and potential side effects of the drug based on the analysis results.

[0018] The above culture medium is designed with a structure capable of simultaneously culturing a second patient with a second disease in addition to a first patient with a first disease to form a multi-disease model, and is characterized by the physical structure and chemical properties of the culture medium inducing interaction between the first and second cells to analyze complex disease pathological mechanisms and evaluate the efficacy of co-administration of therapeutic agents based on a multi-disease model.

[0019] The above culture medium is characterized by being utilized for patient-specific gene editing research by culturing a first cell of a patient with a first disease together with a gene editing tool including CRISPR-Cas9 to evaluate in real time the effect of the gene editing on the physiological function, cell signaling pathway, or cell proliferation rate of the first cell, confirming the applicability of the gene editing tool, and developing patient-specific treatment strategies.

[0020] The culture medium containing the above DNA includes a step of adjusting the DNA content and physical properties of the culture medium according to the species of organism, wherein the adjustment step is characterized by adjusting the concentration of the DNA extracted from mammalian tissue for mammalian-derived DNA to 2.5–5% and applying it as a culture medium for cartilage and muscle tissues requiring high viscosity and strength, adjusting the concentration of the DNA extracted from fish tissue for fish-derived DNA to 1.5–3% and applying it as a culture medium emphasizing transparency and flexibility, adjusting the concentration of the DNA extracted from algal tissue for algal-derived DNA to 0.5–2% and applying it as a culture medium for skin regeneration and wound healing research requiring rapid cell migration and proliferation by providing a lightweight substrate environment, and adjusting the concentration of the DNA extracted from insect tissue for insect-derived DNA to 0.1–1% and applying it as a culture medium with a developed microstructure.

[0021] Natural DNA containing a double helix structure is extracted from biological tissue and mixed with a specific solvent to utilize the self-assembly properties of DNA, thereby forming a biopolymer network and inducing gelation. Through this, a customized culture medium can be manufactured that provides viscosity, strength, and transparency suitable for cell culture.

[0022] DNA-based culture media provide biological characteristics and physical stability simultaneously, and can provide a culture environment suitable for various cell types and research purposes, and

[0023] By extracting natural DNA and forming a culture medium through a simple gelation process, the manufacturing process can be simplified and time and costs can be reduced, and

[0024] By adjusting DNA concentration and environmental conditions of the solution (pH, ion concentration, etc.), viscosity, strength, transparency, etc. can be designed to produce customized culture media tailored to specific research requirements, and

[0025] A method for manufacturing a 3D culture medium using DNA can be provided, which can be utilized in various life science and medical research fields such as organoid culture, neuronal research, and tissue regeneration research, and in particular, can contribute to personalized research and therapeutic development by providing a customized culture environment based on the patient's cells.

[0026] FIG. 1 is a schematic flowchart illustrating a method for preparing a three-dimensional culture medium using DNA according to one embodiment of the present invention.

[0027] FIG. 2 is a diagram illustrating a specific flowchart of a method for preparing a three-dimensional culture medium using DNA according to one embodiment of the present invention.

[0028] FIG. 3 is a diagram illustrating an image of various liquid substances synthesized in the form of a viscous gel in a method for preparing a three-dimensional culture medium using DNA according to one embodiment of the present invention.

[0029] Figure 4 is a graph showing the change in viscosity according to the DNA content of a method for preparing a three-dimensional culture medium using DNA according to one embodiment of the present invention.

[0030] FIG. 5 is a graph showing the change in viscosity according to the DNA content of a method for preparing a three-dimensional culture medium using DNA according to one embodiment of the present invention.

[0031] Figure 6 is a diagram showing the pH measurement results of a culture medium prepared by a method for preparing a three-dimensional culture medium using DNA according to one embodiment of the present invention.

[0032] FIG. 7 is a diagram illustrating storage results to show the storage stability of a culture medium prepared by a method for preparing a three-dimensional culture medium using DNA according to one embodiment of the present invention.

[0033] FIG. 8 is a diagram showing observation results that can be seen in which improved resolution is provided when observing a culture medium prepared by the method for preparing a three-dimensional culture medium through DNA according to one embodiment of the present invention.

[0034] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. In the drawings, identical reference numbers or symbols refer to components that perform substantially the same function, and the size of each component in the drawings may be exaggerated for clarity and convenience of explanation. However, the technical concept of the present invention and its core components and operations are not limited only to the components or operations described in the following embodiments. In describing the present invention, if it is determined that a detailed description of known technologies or components related to the present invention may unnecessarily obscure the essence of the present invention, such detailed description will be omitted.

[0035] In embodiments of the present invention, terms including ordinal numbers, such as first, second, etc., are used solely for the purpose of distinguishing one component from another, and singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, in embodiments of the present invention, terms such as 'composed of,' 'include,' 'have,' etc., should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof. Additionally, in embodiments of the present invention, 'module' or 'part' performs at least one function or operation and may be implemented in hardware or software, or as a combination of hardware and software, or may be integrated into at least one module and implemented as at least one processor. Furthermore, in embodiments of the present invention, 'at least one' among a plurality of elements refers not only to all of the plurality of elements but also to each individual element excluding the remainder or all combinations thereof. Additionally, "configured to" may be used interchangeably with, depending on the context, for example, "suitable for," "having the capacity to," "designed to," "adapted to," "made to," or "capable of." "Configured to" does not necessarily mean that it is "specifically designed to" in hardware. Instead, in some situations, the expression "device configured to" may mean that the device is "capable of" doing so in conjunction with other devices or components.For example, the phrase “a processor configured (or set) to perform A, B, and C” may mean a dedicated processor for performing said operations (e.g., an embedded processor), or a generic-purpose processor (e.g., a CPU or an application processor) capable of performing said operations by executing one or more software programs stored in a memory device.

[0036] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. This description is intended to be detailed enough for a person skilled in the art to easily practice the invention, and it should be noted that the technical scope and concept of the present invention are not limited thereby.

[0037] FIG. 1 is a schematic flowchart illustrating a method for preparing a three-dimensional culture medium using DNA according to an embodiment of the present invention; FIG. 2 is a detailed flowchart illustrating a method for preparing a three-dimensional culture medium using DNA according to an embodiment of the present invention; FIG. 3 is an image illustrating various liquid substances synthesized in the form of a viscous gel in a method for preparing a three-dimensional culture medium using DNA according to an embodiment of the present invention; FIG. 4 is a graph illustrating the change in viscosity according to the DNA content in a method for preparing a three-dimensional culture medium using DNA according to an embodiment of the present invention; FIG. 5 is a graph illustrating the change in viscosity according to the DNA content in a method for preparing a three-dimensional culture medium using DNA according to an embodiment of the present invention; FIG. 6 is a diagram illustrating the pH measurement results of a culture medium prepared by a method for preparing a three-dimensional culture medium using DNA according to an embodiment of the present invention; FIG. 7 is a diagram illustrating storage results to indicate the shelf life of a culture medium prepared by a method for preparing a three-dimensional culture medium using DNA according to an embodiment of the present invention; FIG. 8 is a diagram of the present invention This is a drawing showing observation results that confirm that the culture medium prepared by the method for preparing a three-dimensional culture medium using DNA according to one embodiment provides improved resolution when observed under a microscope.

[0038] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention includes the step of extracting natural DNA from a biological tissue containing double helix DNA through a physical or chemical method.

[0039] In a method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention, the step of extracting natural DNA from a biological tissue containing double helix DNA through a physical or chemical method is a key process for obtaining high-purity DNA, which is the basic material of the culture medium. This step aims to efficiently extract high molecular weight DNA from tissues derived from various biological tissues, such as mammals, fish, birds, insects, etc.

[0040] Specifically, physical methods include procedures that crush or homogenize tissue to destroy cells and cell nuclei and release the DNA present inside. Techniques such as sonication, mechanical compression, or high-speed centrifugation may be utilized in this process. For example, in the case of animal tissue, a tissue sample is added to a buffer solution, and then a homogenization device is used to destroy the cells, followed by centrifugation to remove cell debris.

[0041] Chemical methods include a process of purifying DNA by selectively removing non-nucleic acid materials such as proteins and RNA. Specifically, a protease (e.g., proteinase K) is used to remove proteins, and RNase is added to induce RNA degradation to remove RNA. Subsequently, residual impurities are separated using an ionic solvent (e.g., a phenol / chloroform mixed solvent) to increase the purity of the DNA, and finally, the DNA is recovered through ethanol or isopropanol precipitation.

[0042] The extracted DNA is processed at low temperatures (4–10°C) or below 40°C to maintain its high molecular weight (10 kb or more). This is to preserve the structural stability of the DNA and protect its double helix structure from damage. These low-temperature conditions also reduce the risk of DNA hydrolysis or denaturation and ultimately allow the physical and chemical properties of the culture medium to be maintained uniformly.

[0043] In an realized embodiment, fish cartilage tissue is selected as the biological tissue, homogenized, and then treated sequentially with proteinase K and RNase to remove proteins and RNA. Subsequently, impurities are removed through phenol / chloroform extraction, and high-purity DNA is recovered through ethanol precipitation. The recovered DNA is stored under low-temperature conditions and used as a component of the culture medium in the next step.

[0044] This process optimizes the macromolecular properties of natural DNA, contributing to the promotion of biopolymer network formation utilizing the double helix structure of DNA during the subsequent preparation of culture media. The culture media prepared in this way provide unique viscosity and stability in a cell culture environment and serve as the basis for three-dimensional culture media applicable to various cell types and tissue models.

[0045] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention includes the step of producing a first mixture by mixing the extracted DNA with a first solvent that is a predetermined solvent, which is any one of water, PBS, or DMEM.

[0046] In a method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention, the step of mixing the extracted DNA with a predetermined first solvent, which is one of water, PBS (Phosphate Buffered Saline), or DMEM (Dulbecco's Modified Eagle Medium), to produce a first mixture is an important process for preparing a mixture that forms the basis for the formation of the culture medium. This step serves as a pretreatment step to control the physical and chemical properties of the culture medium through an appropriate combination of DNA and solvent, and to finally ensure gelation and structural stability.

[0047] Specifically, the mixing process of DNA and the first solvent is designed to ensure optimal interaction between the physical properties of DNA and the chemical properties of the solvent. For example, water serves as the primary solvent, facilitating DNA dissolution and providing an inert environment to maintain the DNA's double helix structure. PBS provides a physiological pH and supports DNA self-assembly and network formation during the gelation process by appropriately regulating ion concentrations while maintaining the structural stability of DNA. DMEM is a solvent optimized for cell culture; containing additional nutrients and ions, it provides an environment in which the culture medium supports cell growth and proliferation.

[0048] In the mixing process, the ratio of DNA concentration to solvent acts as an important factor in determining the viscosity and gelation characteristics of the culture medium. For example, when extracted DNA is mixed at a concentration ranging from 0.1% to 30%, preferably from 0.1% to 5%, the mixture can transition from a low-viscosity liquid state to a high-viscosity gel state. The type of mixer used and the mixing speed also play an important role in improving the uniform dispersion and solubility of the DNA. Mixing slowly at a low speed can produce a homogeneous mixture while minimizing structural denaturation of the DNA.

[0049] One realized embodiment involves a process of preparing a first mixture by mixing DNA extracted from fish tissue with water at a concentration of 2%. In this case, the water maximizes the solubility of the DNA, and after mixing, an appropriate temperature (25°C) and time (30 minutes) are maintained to ensure that the DNA is completely dissolved and a homogeneous mixture is formed. Another embodiment involves a process of preparing a first mixture at a concentration of 3% by mixing DNA derived from mammals with PBS. PBS maintains a physiological pH (7.4) to support the DNA in maintaining a stable double helix structure, and the mixture provides the initial viscosity of the culture medium.

[0050] This process serves as a preparatory step for DNA to interact with the solvent and form a biopolymer network, providing a foundation for maximizing the physical properties of DNA during the subsequent gelation and molding stages. The resulting first mixture offers high stability and flexibility during cell culture, contributing to an environment suitable for various cell models and tissue engineering applications.

[0051] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention includes the step of gelling the first mixture by self-arranging the double helix structure of DNA to form a biopolymer network and generating viscosity.

[0052] In a method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention, the step in which the double helix structure of DNA self-arranges to form a biopolymer network, thereby generating viscosity and gelling the first mixture, is a key process that determines the physical properties of the culture medium. This step is a process of converting the mixture from a liquid state to a gelled state by utilizing the unique structural properties of DNA and the environmental conditions of the solvent, and is designed so that the DNA performs its role as a biopolymer.

[0053] Specifically, the double helix structure of DNA forms a network through self-assembly under specific environmental conditions. This process is stably maintained by the phosphate backbone and hydrogen bonds of DNA molecules, and is facilitated by ion concentration and pH in the solvent. For example, metal divalent cations (e.g., Mg²⁻¹, Ca²⁻¹) enhance interactions between DNA molecules, leading to the formation of a stronger network. This network formation increases the viscosity of the mixture and plays a crucial role in the mixture's transition into a gel state.

[0054] One realized embodiment is a case in which Mg²⁺ ions are added at a concentration of 10 mM to a first mixture of 2% DNA and PBS to induce a gelation process. In this case, the Mg²⁺ ions strengthen the cross-linking between DNA molecules to form a robust biopolymer network, causing the mixture to convert into a viscous gel state. Another embodiment is a case in which 3% DNA and DMEM are mixed, and then gelation is carried out by reacting at 25°C for 1 hour while maintaining the pH at 7.0. In this process, the double helix structure of DNA is stably arranged by the ion concentration in the solvent and appropriate pH conditions to form a uniform gel structure.

[0055] The viscosity generated during the gelation process acts as an important factor in determining the physical properties of the culture medium. Highly viscous culture media promote cell adhesion and growth, and provide a structurally stable environment. For example, gelled culture media for neuronal culture support intercellular signaling and network formation, while gelled culture media for organoid culture provide an environment where cell clusters can grow while maintaining a three-dimensional structure.

[0056] The gel generated at this stage possesses high biocompatibility in itself and remains stable even under external stimuli or environmental changes during cell culture. This gelation process simultaneously imparts mechanical strength and flexibility to the culture medium, creating an environment suitable for cell model research and tissue engineering applications. Furthermore, by controlling DNA concentration and solvent conditions during the gelation process, the viscosity of the culture medium can be finely adjusted, enabling customized designs tailored to the culture requirements of various cells and tissues.

[0057] The step of extracting natural DNA in a method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention includes: a step of obtaining high-purity DNA by removing proteins and RNA from biological tissues using an enzyme and then precipitating with an ionic solvent and ethanol; and a step of treating the extracted DNA in a low-temperature environment (4 to 10°C) so that the extracted DNA maintains a high molecular weight of 10kb or more.

[0058] In the method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention, the step of extracting natural DNA is designed as an essential process to secure pure DNA by removing various impurities present in biological tissue and to maintain it at a high molecular weight. This step starts from the raw state of the biological tissue and ultimately provides high-purity DNA that can be used as a culture medium.

[0059] First, proteins and RNA present in biological tissues are selectively removed through enzymatic treatment. In this process, proteolytic enzymes such as proteases degrade and lyse proteins, while RNAolytic enzymes such as RNases remove RNA, leaving only DNA. For example, after pulverizing biological tissues, proteolytic and RNAolytic enzymes are added to a lysis buffer and reacted at 37°C for one hour, resulting in the degradation of most proteins and RNA. This enzymatic treatment serves as an initial step to ensure DNA purity and stability, playing a crucial role in enhancing the efficiency of subsequent processes.

[0060] After enzymatic treatment is completed, pure DNA is separated and purified through ionic solvent and ethanol precipitation. Ionic solvents facilitate the separation of DNA from residual impurities; for example, when using a salt such as NaCl, the DNA remains in a soluble state while other impurities precipitate. Subsequently, the DNA is precipitated and concentrated via ethanol precipitation. An implemented embodiment involves adding 70% ethanol and reacting at -20°C for 30 minutes to precipitate high-purity DNA. The precipitated DNA is collected by centrifugation and redissolved in distilled water or TE buffer to prepare it for use.

[0061] In addition, a process of treating the extracted DNA in a low-temperature environment is included to maintain its high molecular weight (10 kb or more). Since DNA can degrade or suffer structural damage at high temperatures, stability is ensured by maintaining a low temperature (4–10°C). For example, the extracted DNA is stored at 4°C, and an ice box or cooling device is used during the experiment to prevent the DNA from being exposed to high temperatures. This treatment maintains the double helix structure of the DNA, contributing to the preservation of the ability to form a biopolymer network required during the subsequent gelation process.

[0062] This step contributes to improving the quality of DNA used in the preparation of culture media by stably securing high-purity and high-molecular-weight DNA. Consequently, the culture media produced provides high biological stability during cell culture and can create an environment suitable for various cell and tissue studies. In particular, by appropriately combining enzymatic treatment and physical environmental conditions during the extraction process, the purity and structural stability of DNA are maximized, laying the foundation for precisely adjusting the physical and chemical properties of the culture media.

[0063] The step of producing a first mixture of a DNA-based three-dimensional culture medium according to one embodiment of the present invention is characterized by adjusting the concentration of the DNA to a range of 0.1% to 5% and mixing so that the first mixture is converted from a flowing liquid state to a viscous gelled state.

[0064] In a method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention, the step of generating a first mixture is a process of adjusting the physical properties required as a culture medium using extracted DNA, and the key is to adjust the DNA concentration to a range of 0.1% to 5%. This process contributes to creating an environment suitable for cell culture by controlling the viscosity and gelation state of the culture medium.

[0065] First, the extracted DNA is mixed with one of the following solvents: water, PBS (Phosphate-Buffered Saline), or DMEM (Dulbecco's Modified Eagle Medium). During the mixing process, the DNA concentration is adjusted to a range of 0.1% to 5%, which acts as a key variable determining the gelation characteristics of the culture medium. For example, setting the DNA concentration to 0.1% results in a mixture with relatively low viscosity and maintains high fluidity. This is suitable for experiments requiring specific cell motility or where cells need to freely explore the culture environment. Conversely, adjusting the DNA concentration to a level close to 5% transforms the mixture into a gel state with very high viscosity. This high viscosity helps cells grow stably within a fixed structure and is suitable for forming or maintaining three-dimensional tissue structures.

[0066] An realized embodiment is a case in which extracted DNA is mixed with PBS to set the DNA concentration to 2%. In this case, the mixture achieves a balance between viscosity and fluidity, ensuring both cell adhesion and motility during cell culture. The mixing process is generally carried out using a magnetic stirrer or ultrasonic treatment to ensure that the DNA is uniformly dispersed in the solvent. After mixing is complete, the mixture gradually becomes viscous, and the double helix structure of the DNA self-assembles to form a biopolymer network.

[0067] The reason for adjusting the DNA concentration to a range of 0.1% to 5% is to secure physical properties suitable for cell culture. Low concentrations promote cell motility and environmental exploration, while high concentrations help stably fix cells and maintain tissue structure. Furthermore, by optimizing the concentration range, the lysis stability of DNA can be maintained, and uneven viscosity distribution during the gelation process can be prevented. As a result, the prepared mixture can provide a culture environment suitable for various cell types and culture requirements.

[0068] This step enables fine-tuning of the physical environment for cell culture and allows the culture medium to be designed to meet various physiological requirements. For example, low concentrations provide flexibility for cell migration and differentiation in stem cell culture, while high concentrations are suitable for research requiring fixed structures, such as organoid formation. Through this, the present invention can provide customized culture media applicable to various biological research and medical applications.

[0069] The gelling step of the method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention is characterized by adjusting the concentration of DNA and the values ​​of a plurality of environmental condition variables of the solution so that the double helix structure of DNA interacts with a pre-set metal divalent cation to strengthen the biopolymer network.

[0070] In a method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention, the gelation step is a process that allows the double helix structure of DNA to form a stable and strong biopolymer network through the adjustment of environmental conditions in the solution and specific chemical interactions. In this step, the concentration of DNA is adjusted to a range of 0.1% to 5%, and the environmental conditions of the solution include pH, ion concentration, and the concentration of specific metal divalent cations. These variables play an important role in the double helix structure of DNA forming a network through self-assembly.

[0071] First, DNA concentration is a key variable determining the density of the biopolymer network. Higher concentrations strengthen the interactions between DNA double helix structures, forming a more viscous gel state. Conversely, lower concentrations result in a looser network structure, characterized by increased fluidity. This allows the culture medium to be adjusted to possess various physical properties depending on the purpose of cell culture.

[0072] Among the environmental conditions of a solution, pH affects the charge state of DNA, thereby regulating the stability of the double helix structure. For example, DNA exhibits maximum stability when the pH is in the neutral range (6.8–7.4), allowing the double helix structure to efficiently form a network through self-assembly processes. Ion concentration influences the electrical interactions of DNA molecules, and the binding strength between DNA molecules can be adjusted by adding ions such as sodium chloride (NaCl) or calcium chloride (CaCl₂) at specific concentrations.

[0073] In particular, metal divalent cations play a crucial role in enhancing the mechanical strength of the network by forming strong bonds with the DNA phosphate backbone. For example, when calcium chloride (CaCl₂) is added to the solution, Ca²⁺ ions bind to the DNA phosphate backbone, promoting interactions between DNA molecules; consequently, the resulting biopolymer network possesses more stable and robust physical properties. An realized embodiment involves adjusting the DNA concentration to 3% and performing a gelation process by adding 2 mM Ca²⁺ to a PBS solution maintained at pH 7.0. In this case, the gelled mixture exhibits strong viscosity and mechanical stability, providing a suitable culture environment for three-dimensional cell culture.

[0074] The effect of the gelation step lies in maximizing the stability and efficiency of cell culture. By reinforcing the DNA network, structural stability is ensured while cells migrate or attach within the culture medium, which has a significant impact on research into cell proliferation and differentiation. Furthermore, by controlling the concentration of divalent metal cations, the culture medium can be designed to meet the physical requirements of various tissue types. For example, the concentration can be adjusted by increasing it for cartilage tissue culture, which requires high strength, and by lowering it for neural tissue culture to maintain transparency.

[0075] In conclusion, the gelation step of the present invention is a key process for manufacturing customized culture media by utilizing the interaction between the double helix structure of DNA and environmental conditions, providing the advantage of being widely applicable to various cell and tissue culture studies.

[0076] The step of forming or casting a first mixture in a gelled state formed in a method for manufacturing a DNA-based three-dimensional culture medium according to one embodiment of the present invention into a desired structure includes the step of designing the thickness, strength, and pore structure of the gel according to the physical properties required in three-dimensional cell culture, wherein the designing step is characterized by designing a culture medium structure containing a nanostructure when culturing organoids and designing a culture medium structure with high transparency when culturing nerve cells.

[0077] The step of molding or casting a first mixture in a gelled state formed in a method for manufacturing a DNA-based three-dimensional culture medium according to one embodiment of the present invention into a desired structure is a process of designing to optimize the physical properties of the culture medium according to the purpose and need of cell culture. This step mainly involves adjusting physical factors that have an important influence on the culture environment, such as the thickness, strength, and pore structure of the gel.

[0078] First, the thickness of the gel is closely related to the diffusion characteristics of oxygen and nutrients required for cell culture. For example, while a thick gel provides stability in long-term culture environments, it may impair the delivery of oxygen and nutrients within the cells. To address this, the present invention is designed so that the gel thickness can be adjusted within a range of 0.5 mm to 5 mm depending on the cell type and culture conditions. A thin gel is suitable for culturing highly mobile cells such as neurons, while a thick gel is suitable for culturing cells that form complex structures, such as hepatocytes or organoids.

[0079] The strength is designed to respond to physical stress that may occur during culture. In the present invention, the strength of the gel can be varied from 10 kPa to 100 kPa by adjusting the DNA concentration and gelation conditions. For example, in cell cultures requiring a high-strength support structure such as cartilage tissue, the strength can be increased by increasing the DNA concentration and adjusting the metal cation concentration, whereas, conversely, in cases where a delicate structure such as nerve tissue is required, the strength of the gel can be adjusted through a low DNA concentration.

[0080] Furthermore, the design of the pore structure plays an important role in promoting cell motility, differentiation, and cell-cell interactions. In the present invention, pneumatic molding, freeze-drying, or 3D printing technologies may be used to control pore size and distribution during the casting process. For example, during organoid culture, a nanostructure containing pores with a diameter of 50 to 200 μm is formed to allow cells to grow three-dimensionally, which provides conditions for cells to move freely and interact with the internal environment.

[0081] Highly transparent culture media are required for neuronal cell culture. To this end, the present invention improves optical transparency by adjusting the DNA concentration and the refractive index of the solvent. Transparent culture media offer the advantage of allowing real-time observation of neuronal growth and connectivity patterns, and are particularly suitable for research utilizing fluorescence microscopy or imaging techniques. For example, highly transparent culture media are advantageous for monitoring neuronal network formation or observing responses after drug treatment.

[0082] One realized embodiment involves a culture environment in which a three-dimensional culture medium containing a nanostructure was prepared using DNA at a concentration of 2.5% for organoid culture, thereby creating uniform pores with a diameter of 150 μm. This culture medium provides an optimal environment for hepatocyte organoid formation and significantly improved cell viability and functionality. As another example, a culture medium with increased transparency was prepared using DNA at a concentration of 1.5% for neuronal culture, which allowed for the clear observation of neural network formation and synaptic activity.

[0083] In conclusion, the culture medium of the present invention allows for the adjustment of various physical properties according to the intended use, thereby maximizing the efficiency of cell culture and providing an optimized environment for cell growth and interaction. This provides a platform that can substantially contribute to various biological studies and drug development.

[0084] The culture medium prepared by the method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention comprises natural DNA extracted from a patient's biological tissue, and the culture medium provides a customized culture environment that reflects the same genetic and environmental characteristics as the patient's cells.

[0085] A DNA-based three-dimensional culture medium according to one embodiment of the present invention includes natural DNA extracted from a patient's biological tissue, thereby providing a customized culture environment capable of mimicking the same genetic characteristics and environmental conditions as the patient's cells. Since this culture medium utilizes DNA directly extracted from the patient's tissue, it has the characteristic of being able to precisely observe cellular responsiveness to specific diseases by reflecting the patient's genetic variations.

[0086] For example, a culture medium prepared using DNA extracted from the tissues of cancer patients provides optimal conditions to simulate the growth and metastasis of cancer cells. This culture environment can reproduce the microenvironment experienced by the patient's cancer cells in the body, thereby enabling the effective analysis of cellular responsiveness to specific drugs and drug resistance. In an realized embodiment, when patient-derived cancer cells were cultured in a medium prepared using DNA extracted from the tissues of breast cancer patients, the cell death rate and proliferation inhibitory effect following drug treatment could be reproduced in a manner similar to the in vivo environment.

[0087] Furthermore, the culture medium of the present invention is prepared based on DNA extracted from the tissues of patients with immune diseases and can be utilized to study the activation and responsiveness of immune cells. For example, a culture medium prepared from the DNA of a patient with an autoimmune disease can accurately reproduce the antigen and cytokine environment experienced by immune cells within the body, thereby simulating the abnormal activation process of immune cells. Through this, it is possible to develop patient-specific immunosuppressants and verify therapeutic efficacy.

[0088] In addition, to accurately reflect the cellular microenvironment of the patient, the culture medium of the present invention may additionally include biosignal molecules such as cytokines and exosomes secreted from the patient's tissue. These components interact with DNA within the culture medium to contribute to maintaining the natural state of the cell or enhancing a specific environment. In an realized embodiment, when neurons were cultured in a culture medium based on DNA derived from the brain tissue of a patient with a neurodegenerative disease, the growth and synapse formation processes of the neurons appeared similar to those in the body, providing useful data for the development of therapeutic agents and efficacy evaluation.

[0089] In conclusion, the culture medium of the present invention, by including patient-derived DNA, provides a level of accuracy and a patient-specific research environment that conventional synthetic or non-specific culture media cannot offer. Through this, it can be utilized as an innovative platform capable of conducting in-depth research on the pathological mechanisms of diseases and contributing to the development of personalized therapeutic agents.

[0090] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention is implemented such that, after culturing the first cells of a patient with a first disease that have been induced with the first disease through the culture medium, cell responsiveness and drug efficacy evaluations are performed on the cluster of the first cells cultured through the culture medium when conducting patient-specific research and developing a therapeutic agent for the first disease based on the above.

[0091] A method for manufacturing a DNA-based three-dimensional culture medium according to one embodiment of the present invention is designed to reproduce a cellular environment that reflects the disease characteristics of a patient by utilizing cells derived from a patient with a first disease. The culture medium produced using DNA extracted from the tissue of a patient with the first disease provides a unique culture platform capable of reflecting the patient's unique genetic variations and cellular microenvironment. Through this, substantial and meaningful data can be obtained in the analysis of disease pathology and the development of therapeutic agents.

[0092] In an realized embodiment, a culture medium was prepared using DNA extracted from the tissue of a patient with a specific cancer type (e.g., liver cancer), and patient-derived liver cancer cells were cultured in the medium. The cultured liver cancer cells replicated the growth environment within the patient's body, providing an environment for studying the proliferation rate of cancer cells, intercellular signaling, and resistance and sensitivity to drugs. For example, when an anticancer drug was applied to a colony of liver cancer cells cultured in the medium, the cytotoxic effects of the drug and the activation of autophagy in the cancer cells appeared in a manner similar to that observed in the body. This can be utilized as evidence to accurately predict the efficacy and side effects of the drug.

[0093] In another embodiment, patient-derived neurons were cultured using a culture medium prepared with DNA extracted from the brain tissue of patients with Alzheimer's disease. During the culture process, the neurons reproduced pathological features observed in Alzheimer's disease, including amyloid beta aggregation and modification of tau proteins. Under these conditions, specific drug candidates were treated to evaluate neuronal viability, neuroinflammatory response, and synaptic connectivity. This allowed for the quantitative analysis of the drugs' neuroprotective effects and potential therapeutic potential.

[0094] The aforementioned culture medium is also utilized as a platform for developing patient-specific therapeutic agents. For example, cells derived from the tissues of patients with autoimmune diseases were cultured in the medium, and their responsiveness was monitored after administering immunosuppressants. By observing the reduction of cellular inflammatory responses and the inhibition of activation in specific immune signaling pathways, the efficacy and safety of the immunosuppressants could be evaluated.

[0095] In conclusion, the culture medium of the present invention provides an environment for precisely evaluating cellular reactivity based on cells from patients with the first disease, and enables the acquisition of personalized data during the drug efficacy verification process. This offers a technological advantage differentiated from existing non-specific culture systems and can play a key role in the development of new therapeutic agents and the establishment of patient-specific treatment strategies.

[0096] A patient-specific culture environment according to a method for manufacturing a DNA-based three-dimensional culture medium according to one embodiment of the present invention comprises the step of introducing a specific signal molecule, cytokine, or exosome secreted from the cells of a patient with a first disease into the culture medium to reproduce the patient's cellular microenvironment, wherein the specific signal molecule interacts with the physical and chemical properties of the culture medium to mimic the physiological response of the patient's cells in their natural state.

[0097] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention includes the step of introducing physiological elements, such as specific signaling molecules, cytokines, or exosomes secreted from cells of a patient with a first disease, into the culture medium to realize a patient-specific culture environment. This provides a feature that can induce cellular responsiveness that could not be observed in conventional standard culture environments by more precisely reproducing the cellular microenvironment through the integration of molecules actually secreted from patient cells into a culture medium composed of patient-derived DNA.

[0098] Specifically, TGF-β cytokines secreted by cancer cells were added to a culture medium prepared based on DNA extracted from the tissues of patients with specific cancer types (e.g., pancreatic cancer). TGF-β is known as a key signaling molecule that regulates cancer cell growth and metastasis, and when incorporated into the culture medium, it contributed to replicating the fibrotic response and metastatic characteristics exhibited by cancer cells in actual patient bodies. It was confirmed that the growth rate, substrate reorganization, and signaling pathways of cancer cells were activated in a manner similar to in vivo conditions under such a culture environment.

[0099] In addition, exosomes derived from the cells of Alzheimer's disease patients were introduced into the culture medium to culture neurons. Exosomes are vesicles that play a crucial role in intercellular signaling and act as major mediators transmitting pathological signals in neurodegenerative diseases such as Alzheimer's disease. Neurons cultured in the culture medium containing exosomes mimicked pathological responses, including amyloid beta aggregation and tau protein modification, and exhibited characteristic disease responses such as inflammatory reactions and reduced synaptic connections between cells. This provided an experimental environment capable of monitoring the progression of Alzheimer's disease and evaluating the efficacy of exosome-targeted therapies.

[0100] In another example, to culture immune cells from rheumatoid arthritis patients, inflammatory cytokines such as IL-6 secreted from patient cells were added to the culture medium. IL-6 is a major factor that amplifies inflammatory responses; as a result of culturing immune cells in a culture medium containing it, excessive activation of inflammatory response pathways and an increase in the rate of cell proliferation were observed. This culture environment was utilized to evaluate the efficacy and potential side effects of anti-inflammatory drugs.

[0101] The culture medium of the present invention exhibits excellent effects in reproducing cellular responses occurring in a patient's body by allowing physiological elements such as specific signaling molecules, cytokines, or exosomes to interact with the physical and chemical properties of the culture medium. This provides an environment similar to the natural state of cells that could not be observed in existing standardized culture systems, and can contribute to increasing the accuracy of disease research and the development of therapeutic agents.

[0102] The step of performing a drug efficacy evaluation for a first disease using a culture medium of a method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention includes adding a pre-set drug related to the first disease to the culture medium, and then quantitatively analyzing changes in the growth rate, cell death rate, inflammatory response, or intracellular metabolic response of patient-derived cells cultured in the culture medium, and is characterized by evaluating the therapeutic efficacy and potential side effects of the drug based on the analysis results.

[0103] The step of performing a drug efficacy evaluation using a culture medium according to a method for manufacturing a DNA-based three-dimensional culture medium according to one embodiment of the present invention consists of a series of analysis procedures to objectively verify the therapeutic effect and safety of a drug related to a first disease. Specifically, it includes the process of adding a pre-established drug associated with the first disease to the culture medium, and then measuring and analyzing various physiological and metabolic characteristics of patient-derived cells cultured in the culture medium.

[0104] First, the growth rate of patient-derived cells cultured in a culture medium is quantitatively measured. For example, when tumor cells derived from a cancer patient are cultured in the culture medium of the present invention and an anticancer drug is added, the change in the cell proliferation rate can be evaluated. This evaluation is generally performed through MTT analysis, cell viability measurement, or DNA synthesis measurement using BrdU. Through this, the inhibitory effect of the drug on cell proliferation can be precisely confirmed.

[0105] Secondly, drug toxicity is evaluated by analyzing the cell death rate following drug treatment. Techniques such as Annexin V / PI staining are utilized to determine the extent of drug-induced apoptosis or necrosis in cultured patient-derived cells. This analysis clarifies the mechanism by which the drug acts at the cellular level and contributes to the simultaneous evaluation of therapeutic efficacy as well as potential toxicity.

[0106] Third, the effect of the drug on the inflammatory response is quantitatively analyzed. When immune cells from patients with inflammatory diseases are cultured in the culture medium of the present invention and then treated with an anti-inflammatory agent, the anti-inflammatory effect of the drug can be confirmed by measuring changes in the secretion of inflammatory cytokines (e.g., IL-6, TNF-α). This evaluation is performed using quantitative techniques such as ELISA analysis or qPCR, and the effect of the drug on suppressing the inflammatory response is systematically analyzed.

[0107] Fourth, the effect of the drug on intracellular metabolic reactions is evaluated. By quantitatively measuring changes in intracellular ATP production, glucose consumption, or lactic acid production after drug treatment, the effect of the drug on cellular energy metabolism can be analyzed. This allows for a clearer understanding of the drug's mechanism of action and verification of metabolic effects at the cellular level.

[0108] All analysis results obtained at this stage are used to evaluate the therapeutic efficacy of the drug while simultaneously identifying potential side effects. For example, if a drug inhibits cell growth without causing intracellular toxicity, it may be deemed a drug with high therapeutic potential. Conversely, if the rate of cell death is abnormally high, there are concerns regarding drug toxicity, which may necessitate further verification or dosage adjustment.

[0109] Therefore, the evaluation of drug efficacy using the three-dimensional culture medium of the present invention provides a useful method for comprehensively evaluating the therapeutic effect and safety of a drug based on quantitative data, and thereby can provide more efficient and accurate information for the development of patient-specific treatment strategies and drug design.

[0110] The culture medium of the method for manufacturing a DNA-based three-dimensional culture medium according to one embodiment of the present invention is designed to have a structure capable of simultaneously culturing a second cell of a patient with a second disease in addition to a first cell of a patient with a first disease to form a multi-disease model, and the physical structure and chemical properties of the culture medium induce interaction between the first cell and the second cell, thereby analyzing the complex disease pathological mechanism and evaluating the efficacy of co-administration of a therapeutic agent based on a multi-disease model.

[0111] The culture medium of the method for manufacturing a DNA-based three-dimensional culture medium according to one embodiment of the present invention is designed to form a multi-disease model, thereby providing an innovative environment for simultaneously studying complex pathological mechanisms. Specifically, it is designed to allow the simultaneous culture of a first cell from a patient with a first disease and a second cell from a patient with a second disease in the same culture medium. This multi-disease model overcomes the limitations of existing single-disease models and supports the precise analysis of inter-cell interactions and pathological connectivity in different disease states.

[0112] The above culture medium is optimized in terms of its physical structure and chemical properties to induce interactions between cells. For example, a DNA-based biopolymer network contained within the culture medium provides a stable three-dimensional structure that allows cells to interact in physically close proximity, and this network facilitates intercellular signaling and secretion exchange. Additionally, the chemical properties of the culture medium are adjusted to reflect the physiological characteristics of the first and second cells, respectively. For example, the pH, ion concentration, and concentration of specific chemical additives of the culture medium are controlled so that the first cells activate signaling pathways associated with inflammatory diseases, while the second cells reflect metabolic processes associated with cancer cells.

[0113] This design serves two main purposes. First, it is utilized to analyze complex pathological mechanisms through the interactions between first and second cells. For example, in cases where inflammatory diseases and cancer coexist, it allows for the study of how inflammatory cytokines promote the growth and motility of cancer cells. Through this, it enables a systematic understanding of interaction mechanisms in complex disease states and provides foundational data necessary for establishing treatment strategies.

[0114] Secondly, the multi-disease model enables the evaluation of the efficacy of combination drugs. After introducing combination drugs acting on both the first and second diseases into a culture medium, the effects of the drugs on first and second cells can be analyzed independently. For example, by simultaneously introducing an anti-inflammatory agent and an anticancer agent, the impact of each drug on the corresponding cells can be quantitatively evaluated, and potential side effects caused by drug interactions can be identified in advance.

[0115] As a realized embodiment, there is a case in which intestinal epithelial cells from a patient with inflammatory colitis as the first disease and cancer cells from a patient with colorectal cancer as the second disease were cultured in the same culture medium. In this case, the culture medium possesses physical strength and chemical properties capable of appropriately supporting each of the two types of cells, and provides transparency that allows for the observation of intercellular interactions. Through this, it was possible to analyze the mechanism by which an inflammatory environment promotes the growth and motility of cancer cells, and to confirm the effects of combination-administered anti-inflammatory and anticancer agents based on this multi-disease model.

[0116] Therefore, the multi-disease model culture medium of the present invention reproduces complex disease environments to deepen pathological understanding and contributes to significantly improving efficiency and accuracy in the therapeutic agent development process.

[0117] The culture medium according to one embodiment of the present invention is characterized by being utilized for patient-specific gene editing research by culturing a first cell of a patient with a first disease together with a gene editing tool comprising CRISPR-Cas9 to evaluate in real time the effect of the gene editing on the physiological function, cell signaling pathway, or cell proliferation rate of the first cell, confirming the applicability of the gene editing tool, and developing a patient-specific treatment strategy.

[0118] The culture medium according to one embodiment of the present invention is designed to be utilized in conjunction with a gene editing tool comprising CRISPR-Cas9 to effectively perform patient-specific gene editing research. The culture medium comprises a first cell derived from a patient with a first disease, and when the said cell is cultured in the culture medium of the present invention, it mimics the patient's cellular environment, thereby providing optimal conditions for substantially evaluating the applicability of the gene editing tool.

[0119] Specifically, the CRISPR-Cas9 gene editing tool targets specific genes to induce desired genetic modifications within cells. The culture medium of the present invention provides an environment in which the effects of gene editing on key cellular activities, such as physiological functions, signaling pathways, or cell proliferation rates, can be monitored in real time. For example, when the expression of a specific gene in a first cell is suppressed or activated by the gene editing tool, the effect of that gene on the cell's metabolic activity can be directly observed and analyzed within this culture medium. Through this, not only the accuracy and efficacy of the gene editing tool but also potential side effects or unexpected reactions that may occur in the patient's cells can be evaluated in advance.

[0120] One realized embodiment involves a study in which a CRISPR-Cas9 tool is applied to cancer cells with specific genetic mutations of patients with the first disease to activate tumor suppressor genes or inactivate tumor-promoting genes. In this case, the culture medium of the present invention provided sufficient transparency and cellular responsiveness to enable real-time analysis of the gene editing effects on the proliferation rate and apoptosis pathways of cancer cells. By monitoring the signaling molecules and metabolites secreted by the cells during the culture process, it was possible to quantitatively evaluate the impact of gene editing on intercellular signal transduction.

[0121] Furthermore, this culture medium can be utilized to develop patient-specific gene editing strategies. For example, customized CRISPR RNA was designed based on specific patient genetic mutations and introduced into patient cells within this culture medium to evaluate accuracy against target genes. This demonstrated the potential for developing customized gene editing tools applicable to other patients with the same disease.

[0122] The culture medium of the present invention reproduces the natural physiological environment of patient cells, thereby overcoming problems such as unrealistic cell responses or reduced editing efficiency that occurred in existing two-dimensional culture systems. In particular, it can evaluate the applicability of CRISPR-Cas9 tools with high precision, thereby improving accuracy in the development of patient-specific treatment strategies and simultaneously ensuring the efficacy and safety of therapeutic agents. These characteristics can make a significant contribution to research on patient-specific gene therapies and the development of personalized medical strategies.

[0123] A culture medium containing DNA according to one embodiment of the present invention includes a step of adjusting the DNA content and physical properties of the culture medium according to the species of organism, wherein the adjustment step is characterized by adjusting the concentration of the DNA extracted from mammalian tissue to 2.5 to 5% for mammalian-derived DNA to be applied as a culture medium for cartilage and muscle tissues requiring high viscosity and strength, adjusting the concentration of the DNA extracted from fish tissue to 1.5 to 3% for fish-derived DNA to be applied as a culture medium emphasizing transparency and flexibility, adjusting the concentration of the DNA extracted from algal tissue to 0.5 to 2% for algal-derived DNA to be applied as a culture medium for skin regeneration and wound healing research requiring rapid cell migration and proliferation by providing a lightweight substrate environment, and adjusting the concentration of the DNA extracted from insect tissue to 0.1 to 1% for insect-derived DNA to be applied as a culture medium with a developed microstructure.

[0124] A culture medium containing DNA according to one embodiment of the present invention is designed to provide a culture environment suitable for various biological applications by adjusting the DNA content and the physical characteristics of the culture medium according to the species of organism. This adjustment process reflects the content and structural characteristics optimized to match the physical and chemical characteristics of the DNA of each species and the corresponding culture purpose.

[0125] Specifically, mammalian-derived DNA forms a culture medium suitable for culturing cartilage and muscle tissues that require high viscosity and strength by adjusting high-purity DNA extracted from mammalian tissues to a concentration of 2.5–5%. For example, cartilage cells or muscle fiber tissues require a high-strength substrate environment during the culture process, and these requirements are met by adjusting the concentration of mammalian DNA to the above range. This culture medium enables effective tissue culture by allowing cells to stably attach and proliferate and to reproduce tissue-specific mechanical stimuli.

[0126] Fish-derived DNA is formed by adjusting DNA extracted from fish tissues to a concentration of 1.5–3% to create a culture medium that emphasizes transparency and flexibility. This is because fish DNA possesses inherently low viscosity and high transparency, making the medium suitable for culturing vascular tissues or neural tissues where transparency is critical. For instance, when real-time observation of intercellular interactions is required during neural network formation, a culture medium composed of fish DNA is efficient due to the high visual transparency it provides. Furthermore, this medium offers minimal resistance to cell growth and migration, making it suitable for cell dynamics research.

[0127] Algae-derived DNA provides a lightweight substrate environment by adjusting DNA extracted from algae tissues to a concentration of 0.5–2%. This is ideal for applications requiring rapid cell migration and proliferation in skin regeneration and wound healing studies. Algae DNA possesses a relatively low molecular weight and light physical properties; applying this to culture media can maximize cell motility and enhance the rate of cell proliferation. In particular, algae DNA-based culture media can yield effective results in the evaluation of wound healing agents or skin tissue restoration research.

[0128] Insect-derived DNA forms a culture medium with a developed microstructure by adjusting DNA extracted from insect tissues to a concentration of 0.1–1%. Insect DNA possesses a unique nanostructure, and this characteristic contributes to providing a sophisticated cell culture environment. For example, it can be utilized in studies requiring intricate microstructures, such as vascular mimicry or research on the connectivity between complex tissues. The low concentration of insect DNA reduces unnecessary mechanical resistance during cell culture while providing an appropriate support structure, thereby maintaining the natural morphology and function of the cells.

[0129] Therefore, the culture medium of the present invention provides an optimal culture environment tailored to the characteristics and culture objectives of each biological species by adjusting DNA content and physical properties according to the species. This adjustment process can maximize efficiency and effectiveness in various cell culture studies and tissue engineering applications, and enables customized culture environments that conventional standardized culture media could not provide.

[0130] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention includes the feature of adjusting the ratio of DNA to reflect DNA species differentiation between a species used for research purposes and a species to be cultured. This adjustment is designed so that the culture medium provides optimal cell viability, growth rate, and functionality by considering the biological characteristics, genetic similarity, and responsiveness according to the cell environment of each species.

[0131] Specifically, culture media for culturing human cells are designed to contain human-derived DNA at a concentration of 3–5%. Human cells generally possess complex physiological characteristics, and high-concentration DNA-based culture media provide a support structure that allows cells to grow and differentiate stably, just as they would in their natural microenvironment. For example, when culturing human skin cells, a culture medium containing 4% human DNA can accelerate the regeneration rate of skin tissue and improve the adhesion of epithelial cells. Additionally, when culturing human immune cells, high concentrations are advantageous for simulating immune responses by activating intercellular signaling and interactions.

[0132] On the other hand, culture media for culturing cells of canids are designed to contain canine-derived DNA at a concentration of 2–4%. This adjustment of DNA concentration is intended to support the relatively high motility and proliferative characteristics of canine cells. For example, when culturing canine chondrocytes, a culture medium containing 3% canine DNA enhances the specific secretory activity (e.g., collagen synthesis) of chondrocytes and promotes the formation of extracellular matrix, providing an optimal environment for cartilage regeneration research. Furthermore, in arthritis models commonly studied in canine tissues, such concentrations maintain the physiological responsiveness of the cells and are advantageous for pathological analysis of the disease.

[0133] When culturing cells from small mammals such as cats, the culture medium is designed to contain cat-derived DNA at a concentration of 1.5–3%. Cat cells possess sensitive metabolic characteristics, and low-concentration DNA-based culture media provide a stable growth environment while preventing metabolic overload on the cells. In particular, when culturing cat neurons, a culture medium containing 2% cat DNA promotes the formation of neurites and enhances neural network connectivity, making it suitable for research on neurological diseases and drug development.

[0134] In addition, when culturing experimental rodent cells such as mice, DNA-based culture media are designed by adjusting mouse-derived DNA to a concentration of 1–2.5%. Mouse cells are generally characterized by rapid proliferation and differentiation, and relatively low concentrations optimally support these growth characteristics. For example, when culturing mouse embryonic stem cells, a culture medium containing 1.5% mouse DNA improves the reproducibility of the experiment by providing proper cell adhesion while preventing excessive mechanical resistance during the differentiation induction process.

[0135] Therefore, the manufacturing method of the present invention reflects the biological differences between the research target species and the culture target species in DNA concentration and physical characteristics, thereby providing a species-specific culture environment. This method overcomes the limitations of existing uniform culture media and provides high efficiency and reliability in various cell culture experiments and tissue engineering research.

[0136] The method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention can be confirmed through the relationship between the ratio of DNA and viscosity using the viscosity graph presented in FIG. 4-5. The graph shows a tendency for the viscosity of the culture medium to increase as the concentration of DNA increases. This indicates that the culture medium of the present invention can predict and control changes in physical properties simply according to changes in concentration. In particular, this increase in viscosity plays an important role in creating an environment for the stable attachment and proliferation of cells.

[0137] In addition, as shown in FIG. 6, the culture medium of the present invention maintains a stable gel state at pH 7, which demonstrates the durability and chemical stability of the biopolymer network formed at neutral pH. Through this, the culture medium does not degrade in acidic or alkaline environments and is designed to be suitable for a wide range of biological research conditions.

[0138] Furthermore, the data in Fig. 7 confirms that the culture medium of the present invention has stability against temperature changes. The culture medium maintains its physical properties in a temperature range from -20°C to 37°C, and this thermal stability contributes to increased usability in various storage and research environments. For example, the culture medium can support cell culture without deformation even during long-term storage or thawing after freezing.

[0139] The results presented in Fig. 8 demonstrate that the culture medium is also excellent in optical properties. The culture medium of the present invention has excellent transparency and homogeneity, which minimizes light diffraction and refraction during optical microscopy observation, thereby improving resolution. This provides a significant advantage in precisely observing the microstructure of cells and intercellular interactions. In particular, high analytical accuracy can be secured in studies dealing with complex cell morphologies, such as neurons.

[0140] A method for preparing a DNA-based three-dimensional culture medium according to one embodiment of the present invention is based on the characteristic that the relationship between DNA concentration and the increase in viscosity is exponentially proportional. In particular, when the DNA concentration increases from 1% to 3%, the viscosity increases to more than approximately twice the initial value, and when it is further increased from 3% to 5%, the viscosity rises steeply in a non-linear manner, and it was confirmed that it improves by up to about 5 times depending on the experimental conditions. This non-linear increase in viscosity is analyzed to be due to the double helix structure of DNA forming a more dense biopolymer network as the concentration increases.

[0141] In addition, this embodiment further considered the effect of metal cation concentration. Experimental results showed that as the concentration of divalent metal cations in the solution increased from 10 mM to 50 mM, the strength of DNA network formation increased exponentially, and the viscosity of the culture medium formed as a result tended to improve by approximately three times. This is because the interaction between the phosphate groups of DNA and metal ions strengthens the network structure, contributing to the increase in viscosity. In particular, this increase in viscosity directly affects cell adhesion efficiency and cell proliferation rate, enabling efficient research under various culture conditions.

[0142] In addition, the present invention added physical stirring speed as a variable along with concentration to control the pore size and permeability of the DNA mixture. When the stirring speed was increased from 100 rpm to 300 rpm at a DNA concentration of 2%, it was confirmed that the pore structure in the culture medium was formed more uniformly, and cell migration and material exchange increased by up to 20%. Adjusting these physical variables is effective in providing an essential microenvironment for organoid culture or tissue regeneration research.

[0143] Meanwhile, the culture medium of the present invention has the characteristic of adaptability to temperature changes. In this embodiment, as a result of testing a temperature range of -20°C to 37°C while varying the DNA concentration from 1% to 5%, it was confirmed that the high-concentration DNA culture medium maintains a stable gel structure by minimizing shrinkage at low temperatures and expansion at high temperatures. This thermal stability enables the medium to be used without structural deformation during long-term storage and transportation after cell culture.

[0144] Additionally, in this embodiment, changes in optical properties according to DNA concentration were analyzed. As DNA concentration increased, the refractive index of the culture medium rose linearly from approximately 1.33 to 1.37, while transparency was maintained at over 98%. These optical properties reduce light scattering when observing the detailed structure of cells, significantly improving the accuracy of observation for researchers. In particular, in neuronal cell culture studies or cell microstructure analysis where transparency is important, this culture medium was found to provide optical resolution improved by more than 30% compared to conventional culture media.

[0145] Accordingly, the present embodiment provides a platform that can be flexibly applied according to various cell culture purposes and research environments by comprehensively optimizing the physical and chemical performance of the culture medium through a combination of DNA concentration, metal ion concentration, physical stirring speed, temperature stability, and optical properties. This is a major advantage of the present invention that differentiates it from existing technologies and is expected to play an important role in biological research and the development of personalized therapeutic agents.

Claims

1. A method for preparing a DNA-based three-dimensional culture medium, A step of extracting natural DNA from biological tissue containing double helix DNA through physical or chemical methods; A step of producing a first mixture by mixing the extracted DNA with a first solvent that is a predetermined first solvent, which is any one of water, PBS, or DMEM; A step of gelling the first mixture by self-assembling the double helix structure of the DNA to form a biopolymer network and generating viscosity; and A method for manufacturing a DNA-based three-dimensional culture medium, characterized by including the step of molding or casting the first mixture in a formed gelled state into a desired structure to produce a culture medium for three-dimensional cell culture.

2. In Paragraph 1, The step of extracting the above natural DNA is, A step of obtaining high-purity DNA by removing proteins and RNA from biological tissues using enzymes, followed by precipitation with an ionic solvent and ethanol; and A method for preparing a DNA-based three-dimensional culture medium characterized by including the step of treating the extracted DNA to maintain a high molecular weight of 10kb or more.

3. In Paragraph 2, The step of generating the first mixture above A method for preparing a DNA-based three-dimensional culture medium, characterized by adjusting the concentration of the DNA to a range of 0.1% to 5% so that the first mixture is mixed to convert from a flowing liquid state to a viscous gelled state.

4. In Paragraph 3, The above gelling step is, A method for preparing a DNA-based three-dimensional culture medium characterized by adjusting the concentration of DNA and multiple environmental condition variable values ​​of the solution so that the double helix structure of DNA interacts with a preset metal divalent cation to strengthen the biopolymer network.

5. In Paragraph 4, The step of molding or casting the first mixture in the gelled state formed above into a desired structure is It includes a step of designing the thickness, strength, and pore structure of the gel according to the physical properties required for three-dimensional cell culture, and The above-mentioned design step Designing a culture medium structure containing nanostructures for organoid culture, A method for manufacturing a DNA-based three-dimensional culture medium characterized by designing a culture medium structure with high transparency during neuronal cell culture.

6. In Paragraph 5, The above-mentioned culture medium contains natural DNA extracted from the patient's biological tissue, and The above culture medium provides a customized culture environment that reflects the same genetic and environmental characteristics as the patient's cells, and A method for manufacturing a DNA-based three-dimensional culture medium characterized by culturing first cells in which the first disease is induced in a patient with a first disease through the culture medium, and then evaluating cell responsiveness and drug efficacy in the colony of first cells cultured through the culture medium based on the first cells when conducting patient-specific research and developing a therapeutic agent for the first disease.

7. In Paragraph 6, The above patient-specific culture environment includes the step of reproducing the patient's cellular microenvironment by introducing specific signaling molecules, cytokines, or exosomes secreted from the cells of a patient with a first disease into the culture medium. A method for preparing a DNA-based three-dimensional culture medium characterized by the above-mentioned specific signal molecule interacting with the physical and chemical properties of the culture medium to mimic the physiological response of patient cells in their natural state.

8. In Paragraph 7, The step of performing a drug efficacy evaluation for the first disease using the above culture medium is, After adding a pre-established drug related to the first disease to the culture medium, The method includes the step of quantitatively analyzing changes in the growth rate, apoptosis rate, inflammatory response, or intracellular metabolic response of patient-derived cells cultured in the above culture medium, A method for preparing a DNA-based three-dimensional culture medium characterized by evaluating the therapeutic efficacy and potential side effects of the above drug based on the above analysis results.

9. In Paragraph 8, The above culture medium is designed with a structure that allows for the simultaneous culture of second cells from a patient with a second disease, in addition to first cells from a patient with a first disease, in order to form a multiple disease model. A method for preparing a DNA-based three-dimensional culture medium characterized by the physical structure and chemical properties of the culture medium inducing interaction between the first cell and the second cell, thereby analyzing complex disease pathological mechanisms and evaluating the efficacy of combined administration of therapeutic agents based on a multi-disease model.

10. In Paragraph 9, The above culture medium is for patient-specific gene editing research, By culturing the first cells of a patient with the first disease together with a gene editing tool containing CRISPR-Cas9, The effects of the above gene editing on the physiological function, cell signaling pathway, or cell proliferation rate of the above first cell are evaluated in real time, and A method for preparing a DNA-based three-dimensional culture medium characterized by verifying the applicability of the above-mentioned gene editing tool and utilizing it to develop patient-specific treatment strategies.

11. In any one of paragraphs 1 through 10, The culture medium containing the above DNA includes a step of adjusting the DNA content and the physical properties of the culture medium according to the species of organism, and The above adjustment step Mammalian-derived DNA is obtained by adjusting DNA extracted from mammalian tissues to a concentration of 2.5–5% and is applied as a culture medium for cartilage and muscle tissues requiring high viscosity and strength. Fish-derived DNA is obtained by adjusting DNA extracted from fish tissue to a concentration of 1.5–3% and applying it as a culture medium that emphasizes transparency and flexibility, and Algae-derived DNA is prepared by adjusting DNA extracted from algae tissues to a concentration of 0.5–2% to provide a lightweight substrate environment, and is applied as a culture medium for skin regeneration and wound healing research requiring rapid cell migration and proliferation. A method for preparing a DNA-based three-dimensional culture medium characterized by adjusting insect-derived DNA extracted from insect tissue to a concentration of 0.1 to 1% and applying it to a culture medium with a developed microstructure.

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