WO2024075941A1 - Technologie de fixation de surface à base de polymère superabsorbant pour vésicules ou cellules extracellulaires - Google Patents

Technologie de fixation de surface à base de polymère superabsorbant pour vésicules ou cellules extracellulaires Download PDF

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WO2024075941A1
WO2024075941A1 PCT/KR2023/009675 KR2023009675W WO2024075941A1 WO 2024075941 A1 WO2024075941 A1 WO 2024075941A1 KR 2023009675 W KR2023009675 W KR 2023009675W WO 2024075941 A1 WO2024075941 A1 WO 2024075941A1
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cancer
cells
extracellular vesicles
superabsorbent
superabsorbent resin
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English (en)
Korean (ko)
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이원종
함유민
이지윤
강유빈
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인천대학교 산학협력단
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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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/096—Polyesters; Polyamides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention relates to a surface immobilization technology based on a superabsorbent resin of extracellular vesicles or cells. More specifically, the present invention relates to a surface fixation technology based on a superabsorbent resin of extracellular vesicles or cells, and more specifically, by adding a solution containing extracellular vesicles or cells to the superabsorbent resin and incubating it. It relates to the technology of fixing to the surface of a superabsorbent polymer and its application.
  • Extracellular vesicles are nano-sized particles that contain biomolecules such as proteins, lipids, and nucleic acids from parent cells. Therefore, not only are various disease diagnosis methods using extracellular vesicles being developed, but extracellular vesicles are also being developed into pharmaceuticals, cosmetics, and food materials.
  • Size exclusion chromatography is widely used as a method to purify extracellular vesicles, but additional separation technology is still required because the purified extracellular vesicles are rather diluted in the eluate during the elution process.
  • the precipitation method using polyethylene glycol (PEG) has limitations because an impurity called PEG is added and artificial changes may occur in the properties of extracellular vesicles.
  • a method of separating extracellular vesicles from a solution using an ultra-high-speed centrifuge is sometimes used, but the yield of extracellular vesicles is very low with this method, and the strong centrifugal force may cause extracellular vesicles to be destroyed. It has the disadvantage of requiring expensive equipment and taking a lot of time to separate.
  • the present inventor has developed a method for concentrating extracellular vesicles using super absorbent polymer beads (SAP beads), which can easily concentrate extracellular vesicles from cell culture media or clinical samples in a short period of time and in a single process.
  • SAP beads super absorbent polymer beads
  • the purpose of the present invention is to provide a method of fixing extracellular vesicles or cells to the surface of a superabsorbent polymer and a method of utilizing the same based on the superabsorbent polymer.
  • the present invention provides a method for fixing extracellular vesicles or cells on the surface of a superabsorbent polymer comprising the following steps:
  • the superabsorbent polymer is a starch-based polymer obtained by copolymerizing starch with monomers of acrylonitrile, acrylic acid, acrylamide, or methacrylate; Cellulose-based polymer obtained by copolymerizing the above monomer with carboxymethyl cellulose (CMC); and polyvinyl alcohol-methacrylic acid block copolymer, styrene-maleic anhydride copolymer, polyacryl amide or polyoxyethylene. It may be characterized as being manufactured from one or more superabsorbent polymers (SAPs) selected from the group consisting of synthetic resin-based polymers including:
  • the superabsorbent resin may be a poly(acrylamide-co-acrylic acid)) superabsorbent resin.
  • the solution may include extracellular vesicles in the number of 1 ⁇ 10 6 to 1 ⁇ 10 13 particles or 1 ⁇ 10 4 to ⁇ 10 8 cells.
  • the superabsorbent polymer may be a superabsorbent resin bead having a diameter of 1 to 10 mm.
  • the present invention also provides a superabsorbent resin on which extracellular vesicles or cells prepared according to the above method are immobilized on the surface.
  • the present invention also provides a composition for diagnosing diseases comprising a superabsorbent resin on which extracellular vesicles or cells are fixed to the surface.
  • the present invention also provides a kit for diagnosing diseases comprising a superabsorbent resin on which extracellular vesicles or cells are immobilized on the surface.
  • the disease may be cancer.
  • the cancer includes breast cancer, ovarian cancer, colon cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, Endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. It may be characterized
  • the present invention also provides a method for solvent replacement of a solution containing extracellular vesicles or cells comprising the following steps:
  • the replacement solvent may be PBS, distilled water, or culture medium.
  • step (b) may be characterized by resuspension by pipetting or vortexing.
  • the present invention also provides a method for preparing a solution containing extracellular vesicles or cells comprising the following steps:
  • the present invention by fixing extracellular vesicles or cells to the surface of a superabsorbent resin, it is possible to easily and quickly detect various biomarkers present in extracellular vesicles or cells while fixing the extracellular vesicles or cells to the surface of the superabsorbent resin.
  • the solvent that dissolves extracellular vesicles or cells can be easily replaced without contamination of the solvent before replacement, allowing for various research using extracellular vesicles or cells, medicines, cosmetics, etc. There are advantages that can be used in the development of food materials.
  • Figure 1 shows the results confirming that extracellular vesicles or cells can be immobilized on the surface of the superabsorbent resin after adding and incubating the PBS solution, free dye solution, extracellular vesicle solution, and cell solution to the superabsorbent polymer beads.
  • Figure 2 shows the results of comparing temperatures for recovering extracellular vesicles immobilized on the bead surface. Before fixation (Input) and after recovery (Output).
  • Figure 3 shows the expression of the fluorescent marker observed with (A) fixed on the bead surface or (B) without fixed on the bead surface after rolling circle amplification of the signal of the extracellular endoplasmic reticulum surface biomarker, and the bio-signal in the state fixed on the bead surface. This result confirmed that the effect of marker diagnosis was significantly superior.
  • concentration range described in this specification “to” is used to mean including (above and below) both critical ranges, and when both critical ranges are not included, the concentration range is described as “above” and “less than.” .
  • “about” used in numerical values is used to include a range expected to produce an effect substantially equivalent to the numerical value described by a person skilled in the art, for example, ⁇ 20% of the numerical value described. , ⁇ 10%, ⁇ 5%, etc., but is not limited thereto.
  • Extracellular vesicles are small endoplasmic reticulum (membrane vesicles) with a size of 30 to 1000 nm that exist in most cells. It exists in body fluids such as blood and urine, and in particular, exosomes contain various types of proteins, genetic materials (DNA, mRNA, miRNA), and lipids derived from the cell. Extracellular vesicles have a weakly negative surface charge. Extracellular vesicles are becoming increasingly important in this field as their potential for use in the diagnosis and treatment of various diseases has been revealed through various studies.
  • extracellular vesicles can be fixed to the surface of the superabsorbent polymer, extracellular vesicles present in biological clinical samples such as urine, sputum, blood, saliva, tears, and sweat can be easily diluted without separate separation and purification processes.
  • extracellular vesicles are fixed on the surface of the superabsorbent resin, and in that fixed state, various biomarkers that are known or may be discovered in the extracellular vesicles can be easily detected. The convenience and speed of molecular diagnosis can be improved.
  • the solution containing the extracellular vesicles can be easily replaced and recovered by resuspending in a desired solution depending on the purpose.
  • cells can also be immobilized on the surface of a superabsorbent polymer, and the solution containing the cells can be easily replaced and recovered by resuspending them in a desired solution depending on the purpose.
  • the present invention relates to a method for immobilizing extracellular vesicles and/or cells on a superabsorbent polymer surface comprising the following steps:
  • the incubation step may be incubation for about 1 hour to about 10 hours, for example, for about 1 hour to about 5 hours, for example, for about 1 hour to about 3 hours.
  • the extracellular vesicles and/or cells when the solution containing the superabsorbent polymer and extracellular vesicles and/or cells is incubated for less than 1 hour, the extracellular vesicles and/or cells may be concentrated, but are not fixed to the surface of the superabsorbent polymer. characteristics are not exhibited (see Republic of Korea Patent Publication No. 10-2022-0074738, Republic of Korea Patent No. 10-2329816, and Republic of Korea Patent No. 10-2118509), and when incubating for more than 1 hour as in the present invention, extracellular deposits appear on the surface of the superabsorbent resin. The characteristic of allowing endoplasmic reticulum and/or cells to be effectively fixed and maintained is exhibited.
  • the efficiency of fixing extracellular vesicles and/or cells is not significantly improved, so an incubation time of more than 10 hours is not desirable in terms of efficiency.
  • the superabsorbent polymer is a starch-based polymer obtained by copolymerizing starch with monomers of acrylonitrile, acrylic acid, acrylamide, or methacrylate; Cellulose-based polymer obtained by copolymerizing the above monomer with carboxymethyl cellulose (CMC); and polyvinyl alcohol-methacrylic acid block copolymer, styrene-maleic anhydride copolymer, polyacryl amide or polyoxyethylene.
  • CMC carboxymethyl cellulose
  • SAP superabsorbent polymer
  • synthetic resin-based polymers preferably poly(acrylamide-co-acrylic acid) ((Poly (acrylamide-co-acrylic acid))
  • It can be characterized as a highly absorbent resin.
  • the solution containing the extracellular vesicles or cells may include extracellular vesicles in the number of 1 ⁇ 10 6 to 1 ⁇ 10 13 particles or 1 ⁇ 10 4 to ⁇ 10 8 cells.
  • the extracellular vesicles or the cells may be included in a solution of 100 ⁇ L to 10 mL. In one embodiment, the extracellular vesicles or the cells may be contained in a solution of 100 ⁇ L to 5 mL, and in another embodiment, the extracellular vesicles or the cells may be contained in a solution of 100 ⁇ L to 3 mL, and in another embodiment, the extracellular vesicles or the cells may be contained in a solution of 100 ⁇ L to 3 mL. In one embodiment, the extracellular vesicles may be contained in a 100 ⁇ L to 2 mL solution.
  • a volume of 100 to 2000 ⁇ L containing the number of particles of 1 ⁇ 10 6 to 1 ⁇ 10 13 or the number of cells of 1 ⁇ 10 4 to ⁇ 10 8 It may be characterized as being manufactured from a solution of.
  • the solution containing the extracellular vesicles is a solution containing about 1 ⁇ 10 6 to about 1 ⁇ 10 13 extracellular vesicles in a volume of 100 to 2000 ⁇ L, for example, a volume of about 100 to about 500 ⁇ L. It may be characterized in that it is prepared as a solution, preferably in a volume of about 100 to about 500 ⁇ L containing about 5 ⁇ 10 9 to about 5 ⁇ 10 11 particles, for example, It may be characterized as being prepared as a solution with a volume of about 200 ⁇ L containing about 1 ⁇ 10 10 particles.
  • the solution containing the extracellular vesicles may be prepared as a solution containing 1 ⁇ 10 6 to 1 ⁇ 10 13 extracellular vesicles with a volume of 100 to 2000 ⁇ L.
  • the solution containing the cells may be prepared as a solution containing about 1 ⁇ 10 4 to 1 ⁇ 10 8 cells in a volume of 100 to 2000 ⁇ L, for example, a volume of about 100 to about 500 ⁇ L. , Preferably, it may be prepared as a solution with a volume of about 100 to about 500 ⁇ L containing about 5 ⁇ 10 4 to about 5 ⁇ 10 7 cells, for example, about 6 ⁇ 10 5 cells. It may be characterized as being prepared as a solution with a volume of about 200 ⁇ L.
  • the solution containing the cells may be prepared as a solution containing 1 ⁇ 10 4 to ⁇ 10 8 cells with a volume of 100 to 2000 ⁇ L.
  • the solution containing extracellular vesicles may be a solution containing low concentration of extracellular vesicles obtained using a kit for isolating extracellular vesicles from biological samples.
  • the kit for isolating extracellular vesicles is not particularly limited as long as it is a product focused on isolating extracellular vesicles from cell culture medium, and examples may include kits for isolating extracellular vesicles such as Exoquick and Exo-spin.
  • the biological sample is not particularly limited as long as it contains or is likely to contain extracellular vesicles in the liquid, but is preferably composed of prokaryotic cells, eukaryotic cells, bacteria, fungi, yeast, stem cells, and cells isolated from plants and animals. It may be a culture medium obtained by culturing one or more selected from the group, or it may be a body fluid such as blood, saliva, urine, milk, amniotic fluid, or ascites, or a culture medium obtained by culturing the same.
  • the extracellular vesicles collectively refer to biological nanoparticles derived from cells of Archaea, Prokarya, or Eukarya, and include exosomes, argosomes, and dexosomes. , ectosomes, exovesicle, oncosome, prominosome, prostasome, tolerosome, microparticle, microvesicle ( microvesicle, nanovesicle, blebbing vesicle, budding vesicle, exosome-like vesicle, matrix vesicle, membrane vesicle ), shedding vesicle, membrane particle, shedding microvesicle, membrane bleb, epididimosome, promininosome, texosome ( It may include texosome) or archeosome, preferably exosome, microvesicle, and microparticle, and most preferably exosome. .
  • the cells may be Archaea, Prokarya, or Eukarya cells.
  • Resins made from the above-mentioned high-absorbency polymer can absorb solvents tens to hundreds of times their weight (up to 300 times), and since the material itself absorbs solvents, the absorption amount is greater than that of cotton wool or cheesecloth, and it can absorb solvents at a level of up to 300 times its own weight. Since it does not emit solvents, it is most often used in diapers for infants, adults, and animals.
  • the superabsorbent polymer is usually a white powder, and when manufactured to have a certain volume, for example, in the form of a spherical bead, it has the property of absorbing a solvent, swelling, and gelling. In the present invention, it is added to the surface of the swollen bead.
  • a white powder when manufactured to have a certain volume, for example, in the form of a spherical bead, it has the property of absorbing a solvent, swelling, and gelling. In the present invention, it is added to the surface of the swollen bead.
  • One of its main characteristics is that it was the first to discover that extracellular vesicles or cells can exist in a fixed state.
  • the superabsorbent polymer may be characterized as a superabsorbent polymer bead having a diameter of 0.5 to 10 mm, preferably about 1 mm to about 3 mm.
  • the present invention relates to a superabsorbent resin on which extracellular vesicles and/or cells prepared according to the above method are immobilized on the surface.
  • the superabsorbent resin on which extracellular vesicles and/or cells are immobilized on the surface can be used for disease diagnosis.
  • the present invention can provide a superabsorbent resin for diagnosing diseases in which extracellular vesicles and/or cells are immobilized on the surface.
  • the present invention can provide a composition for diagnosing diseases comprising a superabsorbent resin on which extracellular vesicles and/or cells are fixed to the surface.
  • the present invention can provide a use for diagnosing diseases of a superabsorbent resin on which extracellular vesicles and/or cells are immobilized on the surface.
  • the present invention can provide a method for diagnosing a disease comprising measuring the amount of a biomarker in extracellular vesicles and/or a superabsorbent polymer on which cells are immobilized on the surface.
  • the amount of the biomarker may be significantly up- or down-regulated compared to the control group.
  • the present invention can provide the use of a superabsorbent resin on which extracellular vesicles and/or cells are immobilized on the surface for manufacturing a kit for diagnosing diseases.
  • the present invention can provide a kit for diagnosing diseases including a superabsorbent resin on which extracellular vesicles and/or cells are immobilized on the surface.
  • the kit includes a superabsorbent resin together with a reagent for separating extracellular vesicles and/or cells from a sample, and additionally includes a method for isolating extracellular vesicles and/or cells from a sample and/or isolated extracellular vesicles and/or Alternatively, a manual instructing how to fix cells to superabsorbent resin may be additionally included.
  • the disease may be cancer.
  • cancer refers to or refers to a physiological condition typically characterized by uncontrolled cell growth in mammals.
  • Cancers subject to diagnosis in the present invention include breast cancer, ovarian cancer, colon cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, and non-Hodgkin lymphoma.
  • Hodgkin's lymphoma Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine.
  • Cancer endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and It may be selected from the group consisting of pituitary adenoma, but is not limited thereto, and cancers that can be diagnosed using extracellular vesicles or biomarkers contained in cells can be diagnosed using the composition or kit according to the present invention without limitation.
  • the present invention may be applied to any disease or condition that can be diagnosed using biomarkers contained in extracellular vesicles and/or cells.
  • the "diagnosis” refers to determining the subject's susceptibility to a specific disease or condition, determining whether the subject currently has a specific disease or condition, and determining whether the subject currently has a specific disease or condition. Determining a subject's prognosis (e.g., identifying a pre-metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment), or therametrics (e.g., determining the efficacy of a treatment) includes monitoring the state of an object to provide information. For the purpose of the present invention, the diagnosis is to confirm whether or not the cancer has developed or the possibility (risk) of developing it.
  • a subject's prognosis e.g., identifying a pre-metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment
  • therametrics e.g., determining the efficacy of a treatment
  • extracellular vesicles and/or cells immobilized on the surface of a superabsorbent polymer can be used for biomarker diagnosis using a nucleic acid molecule amplification reaction.
  • the single nucleic acid molecule amplification reaction is an exponential amplification reaction.
  • EXPAR exponential amplification reaction.
  • RCA rolling circle amplification
  • IDE aptamer inhibitor-DNA-enzyme
  • aptamer-IDE system aptamer-IDE system.
  • nucleic acid targets present in extracellular vesicles or cells immobilized on the surface of a superabsorbent polymer can be amplified using rolling circle amplification (RCA) or EXPAR and used for diagnosis.
  • RCA rolling circle amplification
  • EXPAR electrospray amplification
  • the content of extracellular vesicles fixed to the surface of the superabsorbent polymer is preferably 1 ⁇ 10 3 to 1 ⁇ 10 13 particles, preferably 1 ⁇ 10 6 to 1 ⁇ 10 13 particles in terms of diagnostic sensitivity.
  • the present invention relates to a method for solvent replacement of a solution containing extracellular vesicles and/or cells comprising the following steps:
  • the replacement solvent may be a solvent suitable for dissolving the extracellular vesicles and/or cells or for use in subsequent experiments or diagnostic steps, such as PBS, distilled water, or culture medium. It is not limited to this.
  • the solvent may be a buffer solution or an experimental reagent.
  • step (b) may be characterized by resuspending by a physical method, such as pipetting or vortexing, but is not limited thereto.
  • the present invention also provides a method for preparing a solution containing extracellular vesicles and/or cells comprising the following steps:
  • Example 1 Isolation of extracellular vesicles derived from HEK293T cells
  • HEK293T cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% Exosome-Depleted Fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco) in a 100 mm cell culture dish (SPL Life Sciences, Korea). , Cellgro, USA) and cultured for 2 to 3 days, maintained at 37°C and 5% CO 2 conditions.
  • Cell culture medium was collected in a 50 mL conical tube (SPL Life Sciences) and centrifuged at 3,000 xg for 15 minutes to remove cell debris. Filtered with a 0.22- ⁇ m cellulose acetate syringe-filter (GVS, Italy).
  • ExoQuickTM Exosome Precipitation Solution (System Bioscience, USA) was mixed with the above cell culture medium in a 1:5 volume ratio in a 15 ml conical tube (SPL Life Sciences) and incubated at 4°C for 16 hours. The above solution was centrifuged at 1,500
  • a fluorescently stained extracellular vesicle solution with a concentration of 5 ⁇ 10 10 particles/200 ⁇ L was prepared using PBS.
  • PBS 198 ⁇ L of PBS mixed with 2 ⁇ L of BODIPYTMC5-Ceramide (free dye solution) and PBS were used.
  • the above superabsorbent polymer beads were cut into 2 mm thick pieces using a knife, placed on a 24 well plate (SPL Life Sciences), and analyzed under a fluorescence microscope.
  • a fluorescently stained cell solution was prepared using PBS to provide HEK293T cells at a concentration of 3 ⁇ 10 3 cells/ ⁇ L.
  • 16 mg (1 piece) of superabsorbent resin beads (Poly(acrylamide-co-acrylic acid) sap beads, CAS no. 9003-06-9) was added to 200 ⁇ L of the solution and incubated for 2 hours.
  • the above superabsorbent polymer beads were cut into 2 mm thick pieces using a knife, placed on a 24 well plate (SPL Life Sciences), and analyzed under a fluorescence microscope.
  • Example 5 Comparison of recovery temperatures of extracellular vesicles immobilized on the bead surface
  • Extracellular vesicles were immobilized on the bead surface as described in Example 3.
  • the extracellular vesicle fixation temperatures were set to 4°C and 25°C, respectively.
  • 1 mL of PBS solution was added to the beads on which the extracellular vesicles were immobilized, and the immobilized extracellular vesicles were recovered through pipetting.
  • the number of extracellular vesicles in the recovered solution was analyzed using nanoparticle tracking analysis.
  • the number of extracellular vesicles (Input) used for fixation was set as 100%, and the number of recovered extracellular vesicles (Output) was converted to a percentage.
  • Example 6 Diagnosis through amplification of extracellular vesicle surface biomarker signal and immobilization on bead surface
  • the surface protein biomarker (CD63) of extracellular vesicles was fluorescently detected using rolling circle amplification (RCA).
  • DNA aptamers CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAT TTT TCG
  • TAT CTG TGC TCA GTA AGA, Bioneer was targeted at 100 nM.
  • the DNA aptamer is hybridized to the same concentration of complementary designed Padlock probe (Phosphate-CAC AGA TAC GTT ATA CCC GGT CGC TTT AGC CTT CTT ACT GAG, Bioneer), and the Padlock probe is lysed by T4 DNA ligase (Thermo Scientific). were linked to form single-stranded circular DNA that served as a reaction template.
  • Padlock probe Phosphate-CAC AGA TAC GTT ATA CCC GGT CGC TTT AGC CTT CTT ACT GAG, Bioneer
  • the experimental group was analyzed using a fluorescence microscope without reacting with superabsorbent resin beads.

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Abstract

La présente invention concerne une technologie de fixation de surface à base de polymère superabsorbant pour des vésicules ou des cellules extracellulaires et, plus spécifiquement, une technologie et des applications de celle-ci, la technologie fixant, à la surface d'un polymère superabsorbant, des vésicules extracellulaires ou des cellules par ajout d'une solution qui comprend des vésicules extracellulaires ou des cellules et l'incubation de celles-ci. La présente invention fixe des vésicules extracellulaires ou des cellules à la surface d'un polymère superabsorbant, permettant ainsi la détection simple et rapide de divers biomarqueurs présents dans les vésicules extracellulaires ou les cellules tandis que les vésicules ou cellules extracellulaires sont fixées à la surface du polymère superabsorbant, et permettant le remplacement simple d'un solvant, qui dissout des vésicules extracellulaires ou des cellules, selon l'objectif d'application des vésicules ou cellules extracellulaires, et peut ainsi être appliqué à diverses recherches et développement, dans lesquels des vésicules ou cellules extracellulaires sont utilisées, de produits pharmaceutiques, de produits cosmétiques et de matériaux alimentaires.
PCT/KR2023/009675 2022-10-04 2023-07-07 Technologie de fixation de surface à base de polymère superabsorbant pour vésicules ou cellules extracellulaires WO2024075941A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3361570B2 (ja) * 1993-06-24 2003-01-07 株式会社荏原総合研究所 高分子ヒドロゲル粒状物を利用する微生物固定化方法
JP2004035639A (ja) * 2002-07-01 2004-02-05 Nippon Kayaku Co Ltd 吸水性樹脂
JP4854669B2 (ja) * 2004-09-23 2012-01-18 トリパス イメージング， インコーポレイテッド 生物学的試料を固定化するためのポリカチオンポリマーコーティング
JP2016510976A (ja) * 2013-03-01 2016-04-14 サイモン ゴールズボロウ，アンドリュー 試料固定及び安定化
WO2022189645A1 (fr) * 2021-03-11 2022-09-15 Chemometec A/S Évaluation de particules biologiques immobilisées dans un hydrogel
CN114231517A (zh) * 2021-12-06 2022-03-25 上海纳米技术及应用国家工程研究中心有限公司 一种大规模生产外泌体的细胞固定化材料的制备方法及其产品和应用

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