KR-20260062812-A - METHOD FOR PRODUCING A TISSUE FREEZE GRINDING COMPOSITION

Abstract

According to one embodiment of the present invention, a method for preparing a tissue freeze-ground composition can be provided, comprising: a cutting step of cutting a biological tissue into pieces of a predetermined size; a mixing step of mixing the cut biological tissue with a solid crystalline granular body in a predetermined content ratio; a freeze-grounding step of freeze-grounding the mixed composition; a washing step of washing the freeze-ground composition; and a drying step of freeze-drying the washed composition by dissolving it in distilled water.

Inventors

• 권성근
• 김현성
• 손영주
• 유혜린
• 이정훈
• 장유정
• 최지숙

Assignees

• 서울대학교산학협력단

Dates

Publication Date

20260507

Application Date

20250716

Priority Date

20241029

Claims (14)

1. A cutting step for cutting a biologically derived tissue into predetermined sizes; A mixing step of mixing the chopped biological tissue with a solid-state crystalline granular body at a predetermined content ratio; A freeze-grinding step of freeze-grinding a composition prepared by mixing; A washing step for washing the freeze-ground composition; and A method for preparing a tissue freeze-grinding composition, comprising a drying step of dissolving the washed composition in distilled water and freeze-drying it.
2. In Article 1, The above washing step is, A stirring step of stirring the freeze-ground composition with distilled water; and A method for preparing a tissue freeze-grinding composition comprising a separation step of centrifuging the stirred composition to remove the supernatant.
3. In Article 1, The above washing step is, A method for preparing a tissue freeze-grinding composition, which involves washing repeatedly a predetermined number of times.
4. In Article 1, The above organization is, A method for preparing a tissue freeze-grinding composition comprising at least one of the groups consisting of bone, cartilage, mucous membrane, blood vessel, liver, lung, stomach, heart, small intestine, large intestine, duodenum, skin, salivary gland, tongue, esophagus, muscle lymph node, fat, and cornea.
5. In Article 1, A method for preparing a tissue freeze-grinding composition having a predetermined size of 1 cm x 0.5 cm.
6. In Article 1, The above-mentioned biological tissue is, A method for preparing a tissue freeze-grinding composition, provided with at least one of hard tissue and soft tissue.
7. In Article 6, The above-mentioned biological tissue is, A method for preparing a tissue freeze-grinding composition, which is prepared as a hard tissue and mixed with the solid-state crystalline granular body in a predetermined ratio.
8. In Article 7, A method for preparing a tissue freeze-grinding composition, wherein the predetermined ratio of the above-mentioned biological tissue and the above-mentioned solid-state crystalline granular body is included in a weight ratio of 1:2.
9. In Article 6, The above-mentioned biological tissue is, A method for preparing a tissue freeze-grinding composition, which is prepared as a soft tissue and mixed with the solid-state crystalline granular body in a predetermined ratio.
10. In Article 9, A method for preparing a tissue freeze-grinding composition, wherein the predetermined ratio of the above-mentioned biological tissue and the above-mentioned solid-state crystalline granular body is included in a weight ratio of 1:2.
11. In Article 1 The above-mentioned solid-state crystalline granular body is, A method for preparing a tissue freeze-grinding composition capable of decellularizing the above-mentioned biological tissue.
12. In Article 11, The above-mentioned solid-state crystalline granular body is, A method for preparing a tissue freeze-grinding composition comprising: an ionic solid that is mixed with the tissue and used during freeze-grinding of the tissue, and capable of decellularizing the tissue.
13. In Article 11, The above-mentioned solid-state crystalline granular body is, A method for preparing a tissue freeze-grinding composition comprising: an ionic lattice structure material that is mixed with the tissue and used during freeze-grinding of the tissue, and capable of decellularizing the tissue.
14. In Paragraph 11, The above-mentioned solid-state crystalline granular body is, A method for preparing a tissue freeze-grinding composition comprising: a solid alkali metal salt or alkaline earth metal salt that is mixed with the tissue and used during freeze-grinding of the tissue, and capable of decellularizing the tissue.

Description

Method for producing a tissue freeze-grinding composition The present invention relates to a method for preparing a tissue freeze-grinding composition. Regenerative medicine refers to cell therapy, gene therapy, tissue engineering therapy, etc., performed using human cells, etc., to regenerate, restore, or form human body structures or functions, or to treat or prevent diseases. Regenerative medicine encompasses not only the structural replacement or restoration of human cells, tissues, and organs but also their functional replacement or restoration. It includes both technologies for producing these components in vitro to replace damaged body parts and technologies that promote the body's self-regeneration. Among these regenerative medicines, 3D bioprinting is a type of 3D printing technology that fabricates tissues or organs by layering living cells or biomaterials into desired shapes or patterns. The bioink utilized in this process refers to an ink-type material composed of living cells, substances (extracellular matrix), and growth factors, which can be used to fabricate structures such as artificial organs and tissues through bioprinting. To maintain the physiological characteristics of the extracellular matrix (ECM) to be used as a biomaterial, manufacturing methods must be applied to maximize the three-dimensional structure of the ECM and the bioactive substances. Key processes in the manufacturing process are decellularization, a technique for removing cells from tissues to develop biomaterials free from antigenicity that can cause immune rejection, and sterilization. However, in the production of extracellular matrix used as a biomaterial, a multi-step decellularization process is essential. Since the process involves multiple steps, it is time-consuming, and there are problems such as protein denaturation and protein loss caused by the long production time. FIG. 1 is a diagram showing the manufacturing process of a tissue freeze-grinding composition according to one embodiment of the present invention, and Figure 2 is a diagram showing the residual amount of DNA in the pulverized product according to the freeze-pulverization time, and Figure 3 is a graph showing DNA content according to tissue size, and Figure 4 is a graph showing the DNA content according to the type of crystal, and Figures 5 and 6 are graphs showing the DNA content according to the weight mixing ratio of tissue and crystals by tissue type, and FIGS. 7 and 8 are graphs comparing the DNA content, collagen, and elastin content of the final product after a decellularization process optimized by freeze-grinding according to the type of tissue, and Figure 9 is a graph showing the residual DNA content measured according to the type of tissue that underwent decellularization, and FIG. 10 is a scanning electron microscope image of the product immediately after the freeze-grinding step depending on the presence or absence of crystalline granules, and FIG. 11 is a scanning electron microscope image of the extracellular matrix obtained by freeze-drying the product obtained by freeze-grinding mucosal tissue through decellularization by osmotic action, and the cellular matrix obtained by freeze-grinding the product obtained by freeze-grinding mucosal tissue together with crystalline granules through a washing step. Figure 12 is a figure evaluating the ability to regulate chondrocyte differentiation by the final product after the decellularization process of stem cells, and FIGS. 13 and 14 are drawings evaluating the ability to regulate chondrocyte differentiation by the final product of bone tissue formation (in vivo), and Figure 15 shows the results of histoimmunochemical staining and protein quantification analysis to evaluate the ability of the final product after the decellularization process of stem cells to regulate chondrocyte differentiation, and FIG. 16 is a histochemical analysis and image quantification analysis performed to evaluate (in vivo) the expression patterns of inflammatory response-related factors after transplantation of stem cells and the final product after decellularization process, and FIG. 17 is a flowchart of a method for preparing a tissue freeze-grinding composition according to one embodiment of the present invention. Hereinafter, an embodiment of the method for preparing a tissue freeze-grinding composition according to the present invention will be described in detail with reference to the attached drawings. It should be noted that when assigning reference numerals to the components of each drawing, the same components are assigned the same reference numeral whenever possible, even if they are shown in different drawings. Furthermore, in describing the embodiments of the present invention, if it is determined that a detailed description of related known components or functions would hinder understanding of the embodiments of the present invention, such detailed description is omitted. In describing the componen