WO2022108315A1 - 신장 오가노이드 배양 및 이식을 위한 탈세포 신장 조직 유래 지지체 및 이의 제조방법 - Google Patents
신장 오가노이드 배양 및 이식을 위한 탈세포 신장 조직 유래 지지체 및 이의 제조방법 Download PDFInfo
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- WO2022108315A1 WO2022108315A1 PCT/KR2021/016827 KR2021016827W WO2022108315A1 WO 2022108315 A1 WO2022108315 A1 WO 2022108315A1 KR 2021016827 W KR2021016827 W KR 2021016827W WO 2022108315 A1 WO2022108315 A1 WO 2022108315A1
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- kidney
- kem
- extracellular matrix
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- organoids
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Definitions
- the present invention relates to a scaffold derived from decellularized kidney tissue for culturing and transplanting kidney organoids and a method for preparing the same.
- Organoids are tissue analogs that can be used for various clinical applications such as drug screening, drug toxicity evaluation, disease modeling, cell therapy, and tissue engineering. It is a technology that is rapidly growing all over the world. Organoids are not only composed of various cells constituting specific organs and tissues of the human body within a three-dimensional structure, but also can implement complex interactions between them. Compared to that, it can be applied as a much more accurate in vitro model platform.
- Kidney organoids can be prepared from pluripotent stem cells, such as human induced pluripotent stem cells or embryonic stem cells, or by extracting and culturing adult stem cells from kidney tissue.
- pluripotent stem cells such as human induced pluripotent stem cells or embryonic stem cells
- Matrigel used for culturing kidney organoids does not implement a complex kidney tissue-specific microenvironment in vivo, there is a need to improve kidney organoid differentiation efficiency and function. Therefore, the development of a new culture system for producing more mature and functional kidney organoids is urgently required.
- kidney diseases such as end-stage renal disease and chronic nephritis
- the patient has to undergo hemodialysis for the rest of his life or undergo a kidney transplant. It is a serious disease that causes great suffering. Therefore, the development of a system that can efficiently cultivate kidney organoids as a precise in vitro model for the development of therapeutic agents for kidney disease is a very important issue from the point of view of health care.
- a hydrogel matrix composed of kidney tissue-specific extracellular matrix components was prepared through a decellularization process of kidney tissue, and this was applied to kidney organoid culture.
- the present invention not only simplifies the decellularization process, so that the matrix production process is very easy, but also the kidney tissue-specific extracellular matrix components and growth factors are well preserved, thereby enabling efficient growth and differentiation of kidney organoids.
- the potential as a culture matrix that can replace the existing Matrigel was verified.
- the present invention is to chemically process pig kidney tissue to obtain a large amount of decellularized tissue, and to prepare a hydrogel scaffold based on this to apply it to kidney organoid culture.
- One aspect of the present invention provides a scaffold for culturing and transplanting kidney organoids using a kidney tissue-derived extracellular matrix (KEM).
- KEM kidney tissue-derived extracellular matrix
- the kidney tissue-derived extracellular matrix may be prepared using a solution of Triton X-100 and ammonium hydroxide.
- the concentration of the kidney tissue-derived extracellular matrix in the scaffold may be 1 mg/ml to 10 mg/ml.
- Another aspect of the present invention comprises the steps of: 1) crushing the isolated kidney tissue; And 2) treating the shredded kidney tissue with Triton X-100 and ammonium hydroxide to decellularize to prepare a decellularized kidney tissue-derived extracellular matrix (KEM).
- KEM kidney tissue-derived extracellular matrix
- 3) lyophilizing the decellularized kidney tissue-derived extracellular matrix may further include the step of preparing a freeze-dried kidney tissue-derived extracellular matrix .
- the method may further include the step of 4) forming the lyophilized kidney tissue-derived extracellular matrix as a support for culturing and transplanting a kidney organoid in the form of a hydrogel.
- step 4) may be to dissolve the lyophilized kidney tissue-derived extracellular matrix in a pepsin solution to form a solution, and then to hydrogel by adjusting the pH.
- Another aspect of the present invention provides a method for culturing kidney organoids on the support or the support prepared by the production method.
- the decellular support developed in the present invention enables efficient culturing of renal organoids, it is widely used in the medical industry such as new drug development, drug toxicity and efficacy evaluation, and patient-specific drug selection by replacing the existing Matrigel, which has various problems. It is expected to be useful. Through this, it is expected that it will improve the quality of life of the people in terms of health and society and create great added value in terms of economy and industry.
- Decellularized kidney tissue-derived artificial matrix scaffolds are expected to be widely used in various fields, such as disease modeling research that implements various intractable kidney diseases (acute/chronic nephritis, renal failure, etc.) in vitro and reveals the mechanism, and establishment of a transplant treatment platform. . Since the prevalence of such intractable kidney disease has recently increased significantly and a lot of research is needed, it is possible to generate profits as a research material for related basic research.
- intractable kidney diseases acute/chronic nephritis, renal failure, etc.
- Renal organoids have endless potential as a disease model as well as a cell therapy for regenerative medicine and tissue regeneration therapy.
- daily life is impossible due to hemodialysis, but the quality of life of the patient will be greatly improved if the fundamental kidney disease treatment is possible through renal organoid transplantation treatment using the decellularized kidney-derived scaffold developed in the present invention. and can significantly reduce related costs.
- the artificial scaffold developed in the present invention can be applied not only to stem cell-derived kidney organoids but also to kidney cancer organoid culture. Considering the size of the precision medicine market, it is expected to create enormous added value.
- the artificial scaffold developed in the present invention shows more functionality than Matrigel as a culture system, and is safer and very advantageous in terms of cost. It has advantages. Therefore, it is predicted that huge economic profits will be created only by the replacement effect of Matrigel.
- kidney tissue-derived extracellular matrix (KEM) scaffold for culturing kidney organoids.
- kidney tissue-derived extracellular matrix KEM
- Figure 5 shows the results of comparative analysis of the proteomic matrix of the decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM) for culturing kidney organoids.
- FIG. 6 shows the analysis results for selecting the optimal concentration of the decellularized kidney tissue-derived KEM hydrogel scaffold for culturing kidney organoids.
- FIG. 7 shows the results of expression analysis (cell immunostaining analysis) of the proliferation and differentiation markers of kidney organoids cultured on a KEM hydrogel support derived from decellularized kidney tissue.
- kidney organoids formed on a KEM hydrogel scaffold derived from decellularized kidney tissue.
- kidney Extracellular Matrix KEM
- kidney Extracellular Matrix 10 and 11 show the results of verifying the long-term storage possibility of a decellularized kidney tissue-derived extracellular matrix scaffold (Kidney Extracellular Matrix, KEM).
- kidney Extracellular Matrix KEM
- Kidney Extracellular Matrix, KEM shows the results of analysis of the functionality of kidney organoids cultured on a decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM).
- kidney fibrosis model is a result of constructing a kidney fibrosis model using kidney organoids cultured on a decellularized kidney tissue-derived extracellular matrix (KEM).
- KEM kidney tissue-derived extracellular matrix
- kidney Extracellular Matrix, KEM Kidney Extracellular Matrix
- molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing may be performed by conventional techniques commonly used in the art of recombinant DNA and within the abilities of those skilled in the art. Such techniques are known to those skilled in the art and are described in many standardized textbooks and reference books.
- One aspect of the present invention provides a scaffold for culturing and transplanting kidney organoids using a kidney tissue-derived extracellular matrix (KEM).
- KEM kidney tissue-derived extracellular matrix
- extracellular matrix refers to a natural scaffold for cell growth prepared through decellularization of tissues found in mammals and multicellular organisms.
- the extracellular matrix can be further processed through dialysis or crosslinking.
- the extracellular matrix is collagen, elastin, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines , and a mixture of structural and non-structural biomolecules, but not limited to growth factors.
- the extracellular matrix may contain about 90% collagen in various forms in mammals.
- the extracellular matrix derived from various living tissues may have a different overall structure and composition due to the unique role required for each tissue.
- derived or “derived” means an ingredient obtained from the source mentioned by any useful method.
- the kidney tissue-derived extracellular matrix may be prepared by using a mixed solution of Triton X-100 and ammonium hydroxide.
- the concentration of the kidney tissue-derived extracellular matrix in the scaffold may be 1 mg/ml to 10 mg/ml, specifically 1 mg/ml to 7 mg/ml.
- the concentration of the renal extracellular matrix 1 mg/ml to 7 mg/ml, 1 mg/ml to 5 mg/ml, 1 mg/ml to 3 mg/ml, 3 mg/ml to 7 mg/ml, It may be 3 mg/ml to 5 mg/ml or 5 mg/ml to 7 mg/ml, and in one embodiment may be 1 mg/ml, 3 mg/ml, 5 mg/ml or 7 mg/ml.
- the support includes a three-dimensional hydrogel prepared based on the extracellular matrix derived from kidney tissue obtained by decellularization, and can be effectively used for culturing kidney organoids.
- kidney tissue contains an actual tissue-specific extracellular matrix component, it is possible to provide a physical, mechanical, and biochemical environment of the corresponding tissue, and is very effective in promoting differentiation into kidney tissue cells and tissue-specific functionality. to be.
- the “organoid” refers to a micro-organism manufactured in the form of an artificial organ by culturing cells derived from tissues or pluripotent stem cells in 3D form.
- the organoids are three-dimensional tissue analogs including organ-specific cells that arise from stem cells and self-organize (or self-pattern) in a manner similar to the in vivo state, and are limited to specific tissues by patterning of factors (Ex. growth factor). can develop into
- the organoid has the intrinsic physiological properties of a cell, and may have an anatomical structure that mimics the original state of a cell mixture (including not only defined cell types, but also residual stem cells, proximal physiological niche). .
- the organoid may have a shape and tissue-specific function, such as an organ, in which cells and cell functions are better arranged through a three-dimensional culture method, and have functionality.
- Another aspect of the present invention comprises the steps of: 1) crushing the isolated kidney tissue; And 2) treating the shredded kidney tissue with Triton X-100 and ammonium hydroxide to decellularize to prepare a decellularized kidney tissue-derived extracellular matrix (KEM).
- KEM kidney tissue-derived extracellular matrix
- Step 1) is a step of crushing the separated kidney tissue
- the kidney tissue may be isolated from a known animal, and specific examples of the animal may be cattle, pigs, monkeys, humans, and the like.
- the decellularization efficiency is high because the decellularization treatment is performed after crushing the isolated kidney tissue.
- the method of crushing the isolated kidney tissue may be made by a known method. In the present invention, more efficient and high-level cell removal is possible because the kidney tissue is subjected to a decellularization process by crushing the kidney tissue.
- Step 2) is a step of preparing the decellularized kidney tissue-derived extracellular matrix (KEM) by treating the crushed kidney tissue with Triton X-100 and ammonium hydroxide to decellularize it.
- the present invention treats only Triton X-100 and ammonium hydroxide, thereby minimizing tissue damage, so that more various proteins in kidney tissue can be preserved.
- the decellularization process can be accomplished while stirring 100 and ammonium hydroxide together.
- the method may further include the step of 3) lyophilizing the decellularized kidney tissue-derived extracellular matrix (KEM) to prepare a freeze-dried kidney tissue-derived extracellular matrix.
- KEM decellularized kidney tissue-derived extracellular matrix
- Step 3) is a step of lyophilizing the decellularized kidney tissue-derived extracellular matrix (KEM) to prepare a freeze-dried kidney tissue-derived extracellular matrix.
- KEM decellularized kidney tissue-derived extracellular matrix
- the lyophilized kidney tissue-derived extracellular matrix may be exposed to electron beam, gamma radiation, ethylene oxide gas or supercritical carbon dioxide after drying for sterilization.
- the method may further include 4) forming the lyophilized kidney tissue-derived extracellular matrix as a support for culturing and transplanting a kidney organoid in the form of a hydrogel.
- Step 4) is a step of forming the lyophilized kidney tissue-derived extracellular matrix into a hydrogel-type kidney organoid culture and support for transplantation.
- the step may be made through gelation, specifically, the lyophilized kidney tissue-derived extracellular matrix may be dissolved in a pepsin solution to form a solution, and then hydrogelled by adjusting the pH.
- a three-dimensional hydrogel-type scaffold can be prepared by crosslinking the extracellular matrix derived from the decellularized kidney tissue, and the gelled scaffold can be used in various fields related to experiments and screening as well as organoid culture.
- the “hydrogel” is a material that loses fluidity and forms a porous structure by solidifying a liquid using water as a dispersion medium through a sol-gel phase change. can be formed.
- the gelation is performed by dissolving the lyophilized kidney tissue-derived extracellular matrix in an acidic solution with a proteolytic enzyme such as pepsin or trypsin, and adjusting the pH, specifically, using 10X PBS and 1 M NaOH to neutral pH and 1X PBS buffer. It may be set to an electrolyte state and made for 30 minutes at a temperature of 37°C.
- a proteolytic enzyme such as pepsin or trypsin
- Another aspect of the present invention provides a method for culturing kidney organoids on the support or the support prepared by the production method.
- the existing Matrigel-based culture system is an extract derived from animal cancer tissue, and the difference between batches is large and does not mimic the actual liver environment, and the efficiency of differentiation and development into kidney organoids is insufficient, whereas the scaffold creates a kidney tissue-like environment. Therefore, it is suitable for culturing kidney organoids.
- the culture refers to a process of maintaining and growing cells under suitable conditions, and suitable conditions include, for example, the temperature at which the cells are maintained, nutrient availability, atmospheric CO 2 content, and cell density.
- Conditions suitable for the formation of the organoid may be conditions that facilitate or allow cell differentiation and formation of multicellular structures.
- the decellularized kidney tissue-derived extracellular matrix scaffold developed in the present invention can be manufactured through a minimized chemical treatment method, there is less damage to tissue-specific components compared to the scaffold prepared by the existing decellularization method, and the production time and It is more efficient in terms of cost reduction and has the advantage of easy mass production. Therefore, it is expected to be more advantageous in terms of commercialization compared to the existing decellular matrix.
- kidney tissue-derived scaffold developed in the present invention, it was confirmed that, while all immunogenic cells were removed, various extracellular matrix components and growth factors contained in the actual kidney tissue were well preserved. Through proteomic analysis, important extracellular matrix components and related proteins in kidney tissue were identified. Therefore, the decellularized kidney tissue-derived matrix provided a kidney tissue-specific microenvironment, enabling efficient culturing of kidney organoids.
- kidney organoids was successfully induced in the developed decellularized kidney tissue-derived hydrogel.
- Decellular scaffolds of various extracellular matrix concentrations were prepared and applied to kidney organoid culture to select the optimal concentration conditions for the most efficient kidney organoid culture.
- kidney organoids cultured on the developed decellular support and the kidney organoids cultured on the Matrigel support used as a control were compared and analyzed, it was confirmed that the differentiation of kidney organoids cultured on the decellularized support was further enhanced. Based on these results, it was confirmed that kidney organoids cultured on decellularized scaffolds can reproduce the actual kidney tissue more accurately and precisely than kidney organoids cultured using the conventional method. As a result, it was confirmed that the decellularized kidney tissue-derived scaffold actually contributes to the efficient differentiation and development of kidney organoids, demonstrating its potential as a substitute for Matrigel.
- pig kidney tissue was subjected to a decellularization process to prepare an extracellular matrix-based scaffold (Kidney Extracellular Matrix; KEM).
- KEM Kid Extracellular Matrix
- the decellularization process used in the present invention is differentiated from the existing decellularization methods in that it uses only a solution mixed with 1% Triton X-100 and 0.1% ammonium hydroxide, which is a more relaxed condition than the conventional method, and thus tissue damage. It was possible to preserve more various extracellular matrix and growth factor proteins in kidney tissue by minimizing In addition, since the decellularization process was performed after the kidney tissue was chopped, it had the advantage of more effective and reliable removal of cellular components (DNA) from the tissue.
- DNA cellular components
- Kidney Extracellular Matrix (KEM) derived from decellularized kidney tissue was prepared from pig kidney tissue and the properties were analyzed.
- KEM hydrogel can provide a structural microenvironment suitable for culturing kidney organoids.
- Hydrogel scaffolds of 4 concentrations were prepared using extracellular matrix (KEM) derived from decellularized kidney tissue, and changes in physical properties according to concentrations were measured. Gels of all concentration conditions were prepared through crosslinking reaction for 30 minutes in an incubator at 37°C. It was confirmed that the storage modulus (G′) value was consistently higher than the loss modulus (G′′) value under all concentration conditions including the lowest concentration of 1 mg/ml. As a result, it was confirmed that a stable polymer network was formed through crosslinking in the hydrogel, and as the KEM concentration increased, the mechanical properties (modulus) also increased.
- KEM extracellular matrix
- Proteomics was performed to confirm the extracellular matrix component contained in the KEM scaffold derived from decellularized kidney tissue.
- KEM hydrogel a scaffold derived from decellularized kidney tissue produced through proteomic analysis, contains various types of extracellular matrix components such as collagens, glycoproteins, and proteoglycans. It was confirmed that various secreted factors, such as growth factors, coagulation factors, and cytokines, that exist in a pre-existing state are also contained.
- Collagen type VI (COL6), which occupies a large proportion among the 10 major ECMs constituting KEM hydrogel, is a major component of the renal glomerular extracellular matrix and plays an important role in the formation of kidney tissue, and laminin (LAM) glycoprotein of the basement membrane As a major component, it was involved in renal cell and tissue differentiation, homeostasis and survival.
- LAM laminin glycoprotein of the basement membrane
- BGN biglycan
- kidney organoid formation and development Therefore, it is expected that these various proteins present in the actual kidney tissue observed in the KEM hydrogel can induce kidney organoid formation and development.
- KEM derived from decellularized kidney tissue for culturing kidney organoids and the proteomic matrix of Matrigel support were comparatively analyzed. Specifically,
- Tubular fragments were extracted from mouse kidney tissue.
- KEM hydrogels of various concentration conditions were first prepared. The extracted tubular fragments were uniformly mixed with KEM hydrogel scaffolds derived from decellularized kidney tissue at each concentration, and then three-dimensional culture was performed to induce the formation of kidney organoids. Subculture was carried out on the 7th day of culture and further cultured for 5 days, and on the 12th day of total culture, the form, formation efficiency, and gene expression of kidney organoids formed under each KEM concentration condition were confirmed. Matrigel was used as a control.
- Kidney organoids were formed in KEM hydrogels under the concentration conditions other than 1 mg/ml, and it was confirmed that they were formed in a spherical shape similar to the organoids cultured in Matrigel used as a control. No organoids were formed in the hydrogel at a concentration of 1 mg/ml.
- organoids could be cultured at all concentrations except KEM hydrogel at a concentration of 1 mg/ml. .
- ZO-1 a marker involved in tight-junction between cells, was also well expressed in kidney organoids cultured in KEM hydrogels of all concentrations.
- KI67 a marker related to organoid proliferation, was also well expressed in kidney organoids cultured in KEM hydrogels of all concentrations.
- kidney organoids can be continuously cultured for more than 2 months in KEM hydrogel, and the various cell type markers of the kidney are similar or higher than Matrigel, a commercially available organoid culture matrix. was confirmed to be expressed.
- Kidney Extracellular Matrix (KEM) derived from decellularized kidney tissue ( FIGS. 10 and 11 )
- kidney Extracellular Matrix KEM
- A Decellularized kidney tissue-derived KEM solution (concentration 7 mg/ml) is refrigerated at 4 °C for up to one month, and it is used for culturing kidney organoids to check whether the KEM hydrogel can be stored for a long time and whether it is stable or not did
- kidney-specific markers AQP3 collected duct cell
- CALB1 distal tubule cell
- PAX8 renal progenitor cell
- kidney Extracellular Matrix, KEM Kid Extracellular Matrix
- A Decellularized kidney tissue-derived KEM solution (concentration 7 mg/ml) is stored frozen at -80 °C for up to 3 months, and then re-thawed and used for kidney organoid culture to determine whether KEM can be stored for a long time and whether it is stable or not. Confirmed.
- the KEM hydrogel solution derived from decellularized kidney tissue can be stored stably for at least 3 months without affecting its activity and function even if it is stored frozen in a freezing condition (-80 °C).
- kidney organoids After culturing kidney organoids on decellularized scaffolds from other organs to demonstrate the tissue-specific effects of kidney-specific extracellular matrix components contained in decellularized kidney tissue-derived KEM scaffolds on the formation and development of kidney organoids Comparative analysis was performed.
- kidney organoids As a result of culturing kidney organoids on decellularized hydrogel scaffolds derived from kidney (KEM), intestine (IEM), muscle (MEM), skin (SkEM), and heart (HEM), skin and heart-derived dehydration on day 7 It was confirmed that kidney organoids were not properly formed in the cell scaffold. In addition, when subculture was performed once and additional culture was performed for 4 days (a total of 11 days of culture), it was observed that all of the decellularized tissue scaffolds except for the decellularized kidney tissue-derived scaffolds were formed with small organoid sizes, and cardiac-derived In the support, it was confirmed that organoids were not formed in the same way as on the 7th day.
- Kidney Extracellular Matrix (KEM) derived from decellularized kidney tissue (FIG. 13)
- kidney organoids cultured in KEM hydrogel derived from decellularized kidney tissue could well implement kidney function in vivo.
- P-gp P-glycoprotein
- kidney organoids cultured in KEM hydrogel have the function of efflux pump to discharge foreign substances.
- kidney fibrosis disease model was constructed using kidney organoids cultured in KEM hydrogel. To this end, renal fibrosis was induced by treatment with TGF-b drug in mouse kidney organoids. On the 9th day of renal organoid culture (passage 1 on the 7th day, additional culture for 2 days), each concentration was treated for 3 days, the analysis was performed on the 12th day, and kidney organoids not treated with TGF- ⁇ (NT, No treatment) was compared as a control.
- C Expression of renal fibrosis-related markers Vimentin, COL1 (collagen type 1), and ⁇ -SMA (alpha smooth muscle actin) was confirmed through immunostaining.
- TGF- ⁇ was treated with kidney organoids cultured in 7 mg/ml KEM, it was confirmed that the KEM hydrogel contracted and the fibrosis-induced organoids merged into one mass.
- the expression of all fibrosis markers was increased in kidney organoids treated with TGF- ⁇ , and it was confirmed that all of the kidney organoids in Matrigel and KEM support showed similar fibrosis marker expression patterns.
- KEM hydrogel scaffold is suitable as a material for transplantation of kidney organoids
- 5 mg/ml KEM scaffold derived from the decellularized kidney tissue was transplanted into the mouse subcutaneous tissue, followed by histological analysis.
- H&E staining was performed to check the degree of infiltration of inflammatory cells into the transplanted KEM hydrogel scaffold
- Toluidine blue staining was performed to check the presence or absence of mast cells caused by an immune response.
- kidney organoids were transplanted into the mouse kidney capsule using KEM hydrogel.
- KEM hydrogel was mixed 1:20 (v/v ) (KEM: culture medium composition) ratio was mixed and transplanted into the living body.
- kidney tissue was obtained on the 4th day after transplantation and histological analysis was performed. As a result, as shown in FIG. 16 , the presence of a fluorescence signal inside the renal capsule was confirmed, and thus, it was confirmed that the transplanted organoid was well engrafted in the tissue.
- kidney organoids Animal experiments and histological analysis were performed to confirm that the KEM hydrogel scaffold derived from decellularized kidney tissue can be used as a material for transplantation of kidney organoids.
- AKI acute kidney injury
- 10 mg/kg cisplatin drug was injected intraperitoneally once into the mouse intraperitoneal cavity and 25 ⁇ l of 7 mg/ml KEM hydrogel to enhance transplantation efficiency and engraftment the next day 600-700 kidney organoids were transplanted into mouse kidney capsules using On the 14th day of transplantation, kidney tissues from each group of mice were obtained and subjected to H&E staining and immunostaining analysis.
- A As a result of H&E staining, areas of acute tubular necrosis (ATN) and hyperemic kidney, which are typical aspects of acute kidney injury, were extensively observed in damaged kidney tissue that was not transplanted with organoids. In contrast, when kidney organoids were transplanted using KEM scaffolds, it was confirmed that the kidney organoids were well engrafted in the cortical area of the damaged kidney tissue, and the damaged area was significantly reduced compared to the control kidney tissue that was not treated. In addition, as a result of quantitative analysis of the glomerular area to confirm the degree of contraction of the glomerular structure, which is one of the symptoms of acute kidney injury, the kidney tissue transplanted with kidney organoids maintains a less damaged glomerular structure than the non-transplanted tissue. was confirmed.
- the KEM hydrogel scaffold can be used as a material for organoid transplantation to treat a kidney disease model, and can improve the engraftment efficiency of organoids in damaged tissue and at the same time induce renal structure recovery. .
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Abstract
Description
Claims (8)
- 신장 조직 유래 세포외기질(Kidney Extracellular Matrix; KEM)을 이용한 신장 오가노이드 배양 및 이식용 지지체.
- 제1항에 있어서,상기 신장 조직 유래 세포외기질은 Triton X-100 및 수산화암모늄을 혼합한 용액을 이용하여 제조된 것인, 지지체.
- 제1항에 있어서,상기 지지체 내 상기 신장 조직 유래 세포외기질의 농도는 1 mg/ml 내지 10 mg/ml인, 지지체.
- 1) 분리된 신장 조직을 파쇄하는 단계; 및2) 상기 파쇄된 신장 조직에 Triton X-100 및 수산화 암모늄을 처리하여 탈세포하여 탈세포된 신장 조직 유래 세포외기질 (KEM)을 제조하는 단계를 포함하는 신장 오가노이드 배양 및 이식용 지지체 제조방법.
- 제4항에 있어서,상기 2) 단계 이후 3) 상기 탈세포 신장 조직 유래 세포외기질 (KEM)을 동결건조 하여 동결건조 신장 조직 유래 세포외기질을 제조하는 단계를 더 포함하는 신장 오가노이드 배양 및 이식용 지지체 제조방법.
- 제5항에 있어서,상기 3) 단계 이후 4) 상기 동결건조 신장 조직 유래 세포외기질을 하이드로젤 형태의 신장 오가노이드 배양 및 이식용 지지체로 형성하는 단계를 더 포함하는 신장 오가노이드 배양 및 이식용 지지체 제조방법.
- 제 6항에 있어서,상기 4) 단계는 상기 동결건조 신장 조직 유래 세포외기질을 펩신 용액에 용해시켜 용액화 한 뒤 pH를 조정하여 하이드로젤화 하는 것인 신장 오가노이드 배양 및 이식용 지지체 제조방법.
- 제1항의 지지체 또는 제4항의 제조방법에 의해 제조된 지지체에서 신장 오가노이드를 배양하는 방법.
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US18/252,895 US20240026300A1 (en) | 2020-11-17 | 2021-11-17 | Kidney extracellular matrix-derived scaffold for culture and transplantation of kidney organoid and method of preparing the same |
EP21895083.0A EP4230727A1 (en) | 2020-11-17 | 2021-11-17 | Decellularized kidney extracellular matrix-derived scaffold for culturing and transplanting kidney organoid, and preparation method therefor |
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