KR20220021759A - Method for preparing organoids using microfluidic chip - Google Patents

Abstract

The present invention relates to a method for manufacturing an organoid, comprising a step of culturing a pre-organoid in a microfluidic chip. When using the method for manufacturing the organoid using the microfluidic chip provided in the present invention, it provides an environment close to the actual in vivo cell microenvironment by enabling culturing without a biological matrix material, thereby being able to be usefully applied to studies such as more accurate prediction of drug efficacy before drug treatment, validation of drug efficacy after drug treatment, karyotype analysis according to genotype, and observation of division rate and pattern.

Description

Method for preparing organoids using microfluidic chip {Method for preparing organoids using microfluidic chip}

The present invention relates to a method of manufacturing an organoid using a microfluidic chip, and can be used for research utilizing various organoids, such as drug testing, identification of mechanisms of cancer process, and tissue transplantation research.

The existing organoid culture method requires a support that can replace the extracellular matrix (ECM) such as Corning's Matrigel. However, in the case of Matrigel, it is derived from mouse sarcoma, and Corning did not disclose all the components of Matrigel, so the experimenter could not accurately determine which component of Matrigel affects organoid culture. In addition, since it cannot be said that organoids embedded and cultured in Matrigel mimic the original state of the existence of real human or mouse organs, culturing organoids without an ECM-simulating support such as Matrigel is conventional. It would be possible to dramatically improve the culture method. In order to check the reactivity of the actual drug to be tested, when the drug is treated on the organoid that grows on Matrigel, it is difficult to penetrate the Matrigel, so the drug concentration is 5 to 10 times higher than the concentration used in two-dimensional cell culture. There is a problem in using . This serves as a limit to the actual clinical application of drug reactivity results in organoids. In addition, when an experimenter uses genetic manipulation techniques such as small interfering RNA (siRNA) or CRISPR/Cas9 for genetic modification, it is difficult to penetrate Matrigel, so it is inconvenient to perform an experiment after removing it. In addition, there are many limitations in the presence of Matrigel in performing experiments such as immunofluorescence assay for molecular and cell biological analysis. In order to more closely mimic the organs of animal models such as humans or mice, a new culture method that can better reproduce the characteristics of organs derived from organoids with stem cell ability through self-structuring without Matrigel is essential. .

The present invention contains the contents of remarkably improving the disadvantages of organoid culture based on an ECM mimicking support such as Matrigel. The microfluidic chip-based matrigel-free organoid culture method developed in the present invention is a culture method capable of culturing organoids having stem cell ability of the derived organ without a separate ECM support, a biological matrix material. Therefore, applied research using these three-dimensional organoids is expected to become a stepping stone for providing customized medical care to actual patients.

One object of the present invention is to provide a method for manufacturing a three-dimensional organoid using a microfluidic chip that can be cultured without a biological matrix material.

Another object of the present invention is to provide an organoid manufactured using the microfluidic chip.

Another object of the present invention is to provide a method for verifying the effect of a candidate drug using the organoid prepared by the above method.

Another object of the present invention is to provide a method for preparing a genetically engineered organoid using the organoid prepared by the above method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable way in one or more embodiments.

Unless specifically defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

According to one embodiment of the present invention, it relates to a method for manufacturing an organoid using a microfluidic chip.

In the present invention, the "organoid" is self-renewal from adult stem cells (ASC), embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), etc. And it refers to a three-dimensional cell aggregate formed through self-organization, and may include an organoid or cell cluster formed from a suspension cell culture. The organoid may also be referred to as a small-like organ, an organ-like organ, or an organ-like organ. The organoid specifically includes one or more cell types among various types of cells constituting an organ or tissue, and should be able to reproduce the shape and function of the tissue or organ. In addition, by making the cells similar to the living environment by aggregating and recombination again using the three-dimensional culture method, it is possible to similarly reproduce the physiologically active function of the living body by overcoming the limitations of the 2D cell line cultured by the 2D culture method, so disease modeling and drug screening etc. can be applied.

Organoid as used herein may refer to a three-dimensional cell aggregate obtained by culturing the pancreas of a transgenic mouse and patient-derived pancreatic cells (eg, pancreatic adult stem cells) by a three-dimensional culture method. In addition, the organoid may be a cell aggregate cultured using a sample derived from a single adult stem cell or embryonic stem cell, and a spheroid as a cell aggregate simply aggregated from a single cell type or a multicellular mixture (eg, derived from a common tissue). It will be said that there is a difference between

The manufacturing method of the present invention may include first mixing a biological sample isolated from a target individual with a biological matrix material and culturing it as a pre-organoid.

In the present invention, the "individual" refers to mammals including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep and goats. It may be selected from, and preferably may be a human, but is not limited thereto.

In addition, in the present invention, the subject may have or may have a target disease, preferably cancer.

In the present invention, the biological sample refers to any material, biological fluid, tissue or cell obtained from or derived from an individual, and includes whole blood, leukocytes, and peripheral blood mononuclear cells. ), buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, respiration (breath), urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid , glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate (joint aspirate), organ secretions (organ secretions), tissue (tissue), cells (cell), cell extract (cell extract) and may be at least one selected from the group consisting of cerebrospinal fluid (cerebrospinal fluid), etc., preferably tissue may be, and more preferably may be a lesion tissue or a normal tissue, but is not limited thereto.

In the present invention, the step of culturing with the pre-organoid comprises the steps of: (a) dissociating a biological sample isolated from a target individual; (b) collecting a cell pellet from the dissociated sample; and (c) incubating the collected cell pellet with a biological matrix material.

In the present invention, the step of dissociating the biological sample may be performed by treating the biological sample with a dissociation solution, wherein the dissociation solution is HBSS (Hank's Balanced Salt Solution) (eg, 1 ml to 10 ml, 1 ml to 5 ml, 3 ml to 10 ml, or 3 ml to 5 ml), collagenase (eg, collagenase P) (eg, 0.1 mg/ml to 5 mg/ml, 0.1 mg/ml to 3 mg/ml , 0.5 mg/ml to 5 mg/ml, 0.5 mg/ml to 5 mg/ml, 0.5 mg/ml to 2 mg/ml, or 0.8 mg/ml to 1.5 mg/ml) and DNase (eg, DNase 1) ( For example, 0.01 mg/ml to 1 mg/ml, 0.01 mg/ml to 0.5 mg/ml, 0.01 mg/ml to 0.2 mg/ml, 0.05 mg/ml to 1 mg/ml, 0.05 mg/ml to 0.5 mg/ml ml, 0.05 mg/ml to 0.2 mg/ml, or 0.08 mg/ml to 0.15 mg/ml).

In addition, in the present invention, the dissociation solution is 0.5 ml to 5 ml, 0.5 ml to 4 ml, 0.5 ml to 3 ml, 1 ml to the obtained sample, preferably tissue (Vpan = about 1.08 mg/mm ³ ) It may be used in an amount of 5 ml, 1 ml to 4 ml, or 1 ml to 3 ml, but is not limited thereto, and may be appropriately adjusted according to the amount of the tissue.

In the present invention, in order to facilitate the dissociation of the sample, physical dissociation (eg, cutting finely with scissors, a knife, etc.) may be additionally performed, and the physical dissociation is performed simultaneously with the treatment of the dissociation solution, Alternatively, it can be performed at this time regardless of order.

In the present invention, the step of collecting a cell pellet from the dissociated sample as described above may be performed. Here, the collecting of the cell pellet may include separating the cells from the dissociated sample and collecting the cell pellet by centrifuging the separated cells.

In the present invention, the step of separating the cells from the dissociated sample may be performed by filtering the dissociated sample through a filter and separating and taking the cells that did not pass, or by conventional cell identification means such as microscopic observation, which is limited thereto. it is not

In the present invention, the isolated cell may be selected from all stem cells capable of forming a three-dimensional organoid through self-renewal and self-organization, for example, adult stem cells (ASC), embryonic stems, etc. It may be at least one selected from the group consisting of embryonic stem cells (ESC), induced pluripotent stem cells (iPSCs), and progenitor cells.

In the present invention, the adult stem cells are stem cells extracted from umbilical cord, umbilical cord blood (umbilical cord blood), or adult bone marrow, blood, nerves, organs, epithelium, muscle, etc., immediately before differentiation into cells of specific organs. primordial cells of The adult stem cells may be one or more selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and the like. The mesenchymal stem cells (MSC) are also called mesenchymal stromal cells (MSC), osteoblasts, chondrocytes, muscle cells (myocytes), adipocytes (adipocytes) , refers to a multipotent stromal cell that can differentiate into various types of cells such as epithelial cells and nerve cells. Mesenchymal stem cells are epithelial tissue, placenta, umbilical cord, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, and milk teeth. It may be selected from among pluripotent cells derived from non-marrow tissues, such as dental pulp.

In the present invention, the embryonic stem cells (embryonic stem cells) are stem cells derived from a fertilized egg, and are stem cells having the property of being able to differentiate into cells of all tissues.

In the present invention, the induced pluripotent stem cells (iPS cells) are also called dedifferentiated stem cells, and by injecting cell differentiation-related genes into somatic cells that have been differentiated and returning them to the cell stage prior to differentiation, like embryonic stem cells Cells that induce pluripotency.

In the present invention, the progenitor cells have the ability to differentiate into a specific type of cell similar to stem cells, but are more specific and targeted than stem cells, and unlike stem cells, the number of divisions is limited. The progenitor cells may be mesenchymal-derived progenitor cells, but are not limited thereto. In the present specification, progenitor cells are included in the category of stem cells, and unless otherwise specified, 'stem cells' is interpreted as a concept that also includes progenitor cells.

In the present invention, the filter has a size that does not pass cells, for example, 50 μm to 200 μm, 50 μm to 170 μm, 50 μm to 150 μm, 50 μm to 120 μm, 80 μm to 200 μm, 80 μm to 170 μm, 80 μm to 150 μm, 80 μm to 120 μm, or 90 μm to 110 μm) may be a conventional cell filter having pores, but is not limited thereto.

In the present invention, the step of collecting the cell pellet by centrifuging the separated cells as described above may be performed. Here, the conditions of the centrifugation are not particularly limited, but, for example, 1000 rpm to 2000 rpm, 1000 rpm to 1800 rpm, 1000 rpm to 1600 rpm, 1200 rpm to 2000 rpm, 1200 rpm to 1800 rpm, 1200 rpm to 1600 rpm, 1400 rpm to 2000 rpm, 1400 rpm to 1800 rpm, or 1400 rpm to 1600 rpm 1 to 20 minutes, 1 to 15 minutes, 1 to 12 minutes, 5 to 20 minutes, 5 to 15 minutes, 5 to 12 minutes, 8 to 20 minutes, 8 to 15 minutes, or 8 to 12 minutes.

In the present invention, the step of culturing the cell pellet collected as described above with a biological matrix material may be performed.

In the present invention, the biological matrix material may include one or more selected from the group consisting of Matrigel, Basement Membrane Extract (BME), hyaluronic acid, and the like. The Matrigel refers to a gelatin-like protein mixture secreted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. The extracellular matrix is an aggregate of biopolymers including molecules synthesized in cells and secreted and accumulated outside the cells, fibrous proteins such as collagen and elastin, complex proteins such as glycosaminoglycans, and cells such as fibronectin and laminin It may include one or more selected from the group consisting of adhesive proteins and the like, but is not limited thereto.

In the present invention, the culture conditions of the cell pellet are not particularly limited, and may be performed under conventional cell culture conditions, for example, at a temperature of 35 to 37 ℃ and 8 to 12% by volume CO ₂ under conditions of 1 to 10 days. , 1 to 7 days, 3 to 10 days, or 3 to 7 days.

In the present invention, the post-treatment step of removing the biological matrix material from the pre-organoid cultured as described above may be further performed.

In the present invention, the post-treatment step of removing the biological matrix material from the pre-organoids includes: crushing the cultured pre-organoids by mechanically applying stress; culturing in the presence of a recovery solution (Corning product number 354253, USA); and centrifuging to obtain a pre-organoid pellet.

In the present invention, the culturing step may be performed under low temperature conditions, preferably at 3 to 7° C. for 30 minutes to 72 hours, 1 to 48 hours, 1 to 24 hours, or 1 to 12 hours.

Prior to the culturing step in the present invention, it may further include a washing step. Here, the washing may be performed using phosphate-buffered saline (PBS), and may be repeated at least once, preferably 1 to 10 times.

In the present invention, the performing conditions of the step of obtaining pre-organoid pellets by centrifugation are not particularly limited, but, for example, at 2 to 6 ° C., preferably at 3 to 5 ° C., at 1000 to 1500 rpm, preferably is performed at 1100 to 1300 rpm, 3 to 10 minutes, preferably 4 to 7 minutes.

In the present invention, the pre-organoid obtained as described above, preferably the pre-organoid pellet from which the biological matrix material is removed after culturing may include the step of culturing in a microfluidic chip.

In the present invention, the "microfluidic chip" is a type of 3D cell culture device and is used as a support plate for seeded cells, and ultimately enables three-dimensional culture of organoids through aggregation, proliferation and migration. corresponding to the culture plate

The microfluidic chip of the present invention may include a plate provided with a chamber, and the chamber may be formed in the shape of a square column.

In addition, in the present invention, the chamber may include at least one shell capable of accommodating cells, preferably pre-organoids, and culturing them as organoids in three dimensions.

In the present invention, the shell may include an inlet part open to accommodate the pre-organoids, and a closed accommodation part in which the pre-organoids are supported and cultured.

In the present invention, the inlet may have a hexagonal structure in cross section.

In the present invention, the accommodating part has a space in which pre-organoids or cultured organoids are accommodated. there is.

In the present invention, 20 to 1000 shells may be included in the chamber, but the present invention is not limited thereto.

In the present invention, a plurality of the shells are included in the chamber, and the interval between any one corner of the inlet part of the shell and any one corner of the neighboring shell may be 0.01 mm to 1.0 mm, but is not limited thereto.

In the present invention, the material of the microfluidic chip is PDMS (Polydimethylsiloxane), Polycarbonate, PTMSP (poly(1-trimethylsilyl-1-propyne)), PTFE (Polytetrafluoroethylene), urethane, PET (Polyethylene terephthalate) and agarose. It may correspond to any one or more materials selected from the group consisting of, and is not limited thereto if it corresponds to a conventional material capable of maintaining a barrier to the outside.

In the present invention, by using the microfluidic chip described above, there is an advantage in that it is possible to specifically 3D culture pre-organoids into organoids without a biological matrix material.

In the present invention, prior to the step of culturing the pre-organoids from which the biological matrix material has been removed in the microfluidic chip, the method may further include pre-treating the microfluidic chip.

In the present invention, the pre-treating of the microfluidic chip may include: culturing by adding a medium to the microfluidic chip; and removing the medium.

In the present invention, before performing the step of culturing by adding a medium to the microfluidic chip, the method may further include washing the microfluidic chip with alcohol. Here, the alcohol may be ethanol, preferably ethanol diluted to 60 to 80% by weight in a solvent, more preferably ethanol diluted to 65 to 75% by weight, but is not limited thereto.

In the present invention, the medium added to the microfluidic chip is Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium, Advanced DMEM, advanced DMEM), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI-1640 and GMEM ( It may be one or more selected from the group consisting of Glasgow's Minimal essential Medium), but is not limited thereto.

In the present invention, the culture performed after adding the medium to the microfluidic chip may be performed at 35 to 40° C., but is not limited thereto.

In the present invention, it may include the step of removing the medium after culturing by adding a medium to the microfluidic chip.

In the present invention, the pre-treated microfluidic chip may be inoculated with the pre-organoid pellet and cultured.

In the present invention, the step of inoculating and culturing the pre-organoid pellet may include the step of culturing the organoids by primary culture, and then re-inoculating the obtained organoids in a microfluidic chip to secondary culture.

In the present invention, the second culturing step may be performed by subculture by repeating one or more times, preferably two or more times, more preferably two to ten times.

In the present invention, the primary or secondary culture conditions are not particularly limited, and may be performed under conventional cell culture conditions, for example, 1 to 10 days at 35 to 37° C. and 8 to 12% by volume CO ₂ conditions. to 7 days, 3 to 10 days or 3 to 7 days.

Prior to the secondary culturing in the present invention, it may further include washing the organoids obtained by the primary culturing.

Prior to the secondary culturing in the present invention, the method may further include centrifuging the washed organoids as described above. Here, the conditions for performing the centrifugation are not particularly limited, but, for example, may be performed at 1000 to 1500 rpm, preferably 1150 to 1300 rpm, for 3 to 10 minutes, preferably for 4 to 7 minutes.

Prior to the secondary culturing in the present invention, the method may further include resuspending the centrifuged organoids in a new culture medium as described above.

In the present invention, the new culture medium may be a medium containing a biological matrix material, and the biological matrix material is a group consisting of Matrigel, Basement Membrane Extract (BME), hyaluronic acid, etc. It may be to include one or more selected from.

In the present invention, the Matrigel may include one or more selected from the group consisting of fibrous proteins such as collagen and elastin, complex proteins such as glycosaminoglycan, and cell adhesion proteins such as fibronectin and laminin. However, the present invention is not limited thereto.

In the present invention, it will be said that the subculture is characterized in that it is performed on a microfluidic chip.

In the present invention, the organoids manufactured using the microfluidic chip as described above have excellent ability to simulate the living environment compared to the existing organoids, and by optimizing the culture growth conditions to ensure long-term viability, various candidate drug reactivity tests such as anticancer drugs and/or more advantageously applied to drug screening.

According to another embodiment of the present invention, it relates to an organoid prepared by the method of the present invention.

In the present invention, the organoid may be an organoid cultured in a state in which the biological matrix material is removed or a biological matrix material-free organoid, more preferably a Matrigel free organoid.

According to another embodiment of the present invention, it relates to a method for screening a candidate drug using the organoid of the present invention.

In the present invention, the "screening" refers to selecting a substance having a specific target property from a candidate group consisting of several substances by a specific manipulation or evaluation method.

The screening method provided by the present invention may include, first, treating a candidate drug to the organoid provided by the present invention.

In the present invention, the method may further include the steps of the method for preparing an organoid described above before treating the drug.

Here, the candidate material is preferably any one selected from the group consisting of natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, bacterial or fungal metabolites and bioactive molecules , but not limited thereto.

According to another embodiment of the present invention, it relates to a method for producing a genetically engineered organoid comprising the step of manipulating a gene encoding a target protein or a target gene in the organoid prepared by the method of the present invention.

In the present invention, the genetic manipulation may be performed on a microfluidic chip. It will be said that the present invention is characterized in that it is possible to genetically manipulate organoids on a microfluidic chip, unlike the conventional case in which genetic manipulation is performed in a normal medium.

In the present invention, the term "genetic engineering" refers to a process of introducing foreign genetic material into a host cell, and examples of the genetic material may be DNA or RNA. In order to induce gene expression, the process of transferring the genetic material to the nucleus of a host cell is essential, and for successful genetic manipulation, it is required that the foreign genetic material be stably maintained in the host cell.

In the present invention, the genetic manipulation may be performed as a series of processes in which a gene encoding a desired protein or a gene encoding a desired gene is specifically targeted and cut, and the edited gene is transferred. . The gene editing refers to a type of genetic engineering in which DNA is inserted, replaced, or deleted from a genome using artificially engineered nucleases or gene scissors.

In the present invention, during the genetic manipulation, transfection in which an external genetic material is injected into eukaryotic cells according to the type of host cell, transformation in which nucleic acids are injected into prokaryotic cells with a plasmid, etc., and a virus are used. Transduction exists to inject nucleic acids into prokaryotic cells. The present invention can be carried out by transfecting eukaryotic cells.

In the present invention, the genetic manipulation may be performed by transfecting a gene encoding a target protein or a target gene into the organoid.

In the present invention, the transfection method may be performed using a viral vector or a non-viral vector. Here, the viral vector has the advantage of high delivery efficiency and long duration, retroviral vector, adenoviral vector, vaccinia virus vector, adeno-associated viral vector (adeno-associated viral vector) , cancer cell lytic viral vectors, and the like, but are not limited thereto. In addition, the nonviral vector may include a plasmid, and in addition, various formulations such as liposomes, cationic polymers, micelles, emulsions, solid lipid nanoparticles, etc. may be used, Cationic polymers for nucleic acid delivery include natural polymers such as chitosan, atelocollagen, and cationic polypeptide, poly (L-lysin), linear or branched PEI (polyethylene imine), and cyclodextrin series. Synthetic polymers such as polycations (cyclodextrin-based polycation) and dendrimers may also be included, but are not limited thereto.

In the present invention, upon transfection of the vector, microinjection, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene -Mediated transfection (polybrene-mediated transfection), electroporation (electroporation), gene gun (gene gun) or other known methods for introducing nucleic acid into the cell can be introduced into the cell, but is limited thereto No (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).

In addition, in the present invention, the genetic manipulation may use a gene encoding a target protein or a gene editing technique that allows editing of a target gene. At this time, depending on whether the substance recognizing the target sequence is a protein or a guide RNA, a zinc finger nuclease (ZFN), a transcription activator-like effector that uses a gene scissors that binds to a target gene sequence in the form of a protein The nuclease; TALEN) method and the CRISPR gene scissors protein can be performed by the CRISPR (clustered regularly interspaced short palindromic repeat) method, which uses only one type of protein and uses only a guide RNA according to the sequence of the target gene, but is limited thereto. However, it may be included without limitation as long as it is a gene editing method generally performed in the art.

As an example of the present invention, among the gene editing technologies, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) method, Cas9 or Cpf protein as a gene scissors protein of the CRISPR class may be used, preferably CRISPR/Cas9 method using Cas9 protein In this case, when introducing the nuclease and gRNA gene into the organoid cell, various introduction methods and forms can be used. For example, a vector including the nuclease and the gRNA gene is applied to the organoid. Transfection can be performed.

In the present invention, the gRNA (guide RNA) is also called sgRNA (single-guide RNA), and refers to an RNA that is complementary to a DNA of a site to be edited. It can be used for transfection by designing an sgRNA that complementarily binds to a target gene sequence, for example, an sgRNA having a sequence complementary to a gene such as PLK1 and p53 as a target gene, preferably PLK1 It may be the sgRNA represented by SEQ ID NO: 1 that complementarily binds to a gene, but is not limited thereto, and any sgRNA sequence that is complementary to a target gene that requires editing is selected and may be included without limitation.

In the present invention, the nuclease includes Cas protein, DNA sequence specific endonuclease, RNA guided DNA endonuclease (eg, Cpf1), restriction endonuclease, meganuclease, homing endonuclease , TAL effector nucleases, and zinc finger nucleases. Specific examples include, but are not limited to, type I, type II, type III, type IV, and type V endonucleases as nucleases. Preferably, the CRISPR nuclease may be a Cas nuclease or a Cpf1 nuclease. More preferably, Cas9, e.g., bacteria (such as S. pyogenes, S. pneumoniae, S. aureus, or Cas9 cloned or derived from S. thermophilus).

In the present invention, the method may further include the step of confirming whether or not the organoid is genetically manipulated after the genetic manipulation.

In the present invention, a marker protein may be used to determine whether the gene is manipulated, preferably transfection, and the marker protein can be visualized by specifically targeting a nucleic acid sequence and generating an optical signal. Examples of the marker protein in the present invention include a fluorescent reporter protein, more specifically, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) or mCherry. can, but is not limited thereto.

In the present invention, the determination of whether the genetic manipulation is performed may be performed by a method of confirming by western blot or indirect intracellular staining flow cytometry, and may be performed by various alternative methods known in the art. .

As described above in the present invention, sgRNA, Cas9 protein or homology target repair (HDR) template can be used in various forms (DNA, RNA, Protein) and in various methods (transfection, electroporation, viral gene delivery, Gesicle, etc.) to target cells or tissues. It can be introduced into the NHEJ pathway, and it can be confirmed whether the gene is manipulated through electrophoresis confirmation or sequence analysis of indel mutations or HDR-inserted genes or SNPs.

When using the method for manufacturing organoids using the microfluidic chip provided in the present invention, it provides an environment close to the actual in vivo cellular microenvironment by enabling culture without a biological matrix material to more accurately predict drug efficacy before drug treatment; It can be usefully applied to studies such as verification of drug efficacy after drug treatment, karyotype analysis according to genotype, and observation of cleavage rates and patterns.

1A to 1C are diagrams showing the results of primary culture or subculture of mouse or patient-derived pancreatic organoids using a microfluidic chip according to an embodiment of the present invention.
2 is a diagram showing the results of culturing mouse-derived pancreatic organoids using an A1 chip according to an embodiment of the present invention.
3 is a diagram showing the results of drug screening in 2D breast cancer cell lines (MCF10A and MCF7) according to an embodiment of the present invention.
4A and 4B are diagrams showing the results of drug screening on pancreatic organoids derived from mice or patients containing Matrigel according to an embodiment of the present invention.
5 is a diagram showing the results of drug screening in mouse or patient-derived pancreatic organoids using the microfluidic chip according to an embodiment of the present invention.
6A and 6B are diagrams showing the results of measuring cell viability by performing an MTT assay before and after drug treatment using a microfluidic chip according to an embodiment of the present invention.
7 is a diagram illustrating a fluorescence image showing the results of immunofluorescence staining analysis of patient-derived organoids using a microfluidic chip according to an embodiment of the present invention.
8 is a diagram showing the results of immunofluorescence staining to confirm stem cell ability (stemness) of patient-derived organoids according to an embodiment of the present invention.
9 is a diagram showing the results of genome analysis using patient-derived organoids according to an embodiment of the present invention.
10 is a diagram showing the results confirmed through fluorescence analysis and Western blot after transfection using patient-derived organoids on a microfluidic chip according to an embodiment of the present invention.

<Description of the project supporting the present invention>

The present invention is a private support project of a private institution (task number: 0413-20190020, research project name: development of a three-dimensional pancreatic organoid culture protocol on standardized plates, host institution: Next&Bio Co., Ltd., research period: 2019-07-01~ 2020-06-30) was developed with the support of

<Specific contents of the present invention>

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Subculture of mouse and patient-derived pancreatic organoids using a microfluidic chip

1.1 Preparation of biological samples

The present inventors obtained biological samples from mice and patients, which are target subjects, for culturing organoids using a microfluidic chip.

First, in order to culture an organoid derived from a KRAS ^G12D mouse with a point mutation in the oncogene KRAS gene, the mouse was euthanized and then dissected to obtain a pancreatic tissue. As a dissociation solution, HBSS (Hank's Balanced Salt Solution; GIBCO ^® ) 3 to 5 ml, collagenase P (Roche) 1 mg / ml, and DNase 1 (Sigma Aldrich) 0.1 mg / ml of a mixture of 0.1 mg / ml was prepared by warming in a 37 ℃ water bath. The whole mouse pancreas tissue obtained above (Vpan = 1.08 mg/mm ³ ) was transferred to a 100 mm Petri dish, 100 ul of the prepared dissociation solution was added, and finely cut 5 to 10 times using scissors or a knife. The chopped pancreatic tissue (Vpan = 1.08 mg/mm ³ ) is transferred to a 50 ml conical tube, and 3 to 5 ml of the dissociation solution is added thereto, and the tube is placed in a shaking incubator at 230 rpm and 37 ° C. Incubated and taken out after 10 to 15 minutes, 10 to 15 ml of cold FBS was put. The obtained dissociated pancreatic tissue was passed through a 100 um pore cell strainer, and washed 2-3 times using HBSS. Pancreatic duct cells were selected and selected by observing the pancreatic tissue that did not pass through the cell filter under a microscope. The selected pancreatic duct cells were centrifuged at 1500 rpm for 10 minutes to collect a pellet, and then washing with HBSS was repeated twice.

The step for primary culture was performed by repeating the same process with the sample obtained from the patient-derived pancreatic tissue in the same way as above.

1.2 Primary culture of organoids from each sample (pre-organoid culture)

After mixing the obtained cell pellet and 200 ul of Matrigel (Matrigel, Corning) so that the volume ratio of the pellet and Matrigel is 1: 5, it is placed in a 12-well or 24-well plate, respectively, in an amount of 100 to 150 ul based on a 12-well plate per well. cells were inoculated with About an hour after cell inoculation, when Matrigel hardens, the medium was put in an amount of 1 ml based on a 12-well plate and 500 ul based on a 24-well plate, and incubated at 37° C. and 10% CO ₂ in an incubator for 48 to 72 hours or more. . At this time, DMEM/F-12 (Dulbecco's Modified Eagle Medium/Ham's F-12; Thermo Fisher Scientific) was supplemented with 1% (vol/vol) penicillin/streptomycin, 10 mM HEPES, 1% GlutaMAX, 1:50 B27 supplement (Gibco). ), 1 mM N-acetylcysteine, 5% (vol/vol) Rspo1-culture (Hans Clevers lab), 10 mM nicotinamide, 10 nM recombinant human [Leu15]-gastrin I (Sigma Aldrich), 50 ng/ml Recombinant mouse EGF (Peptron), 100 ng/ml human recombinant FGF10 (Peptron), and 25 ng/ml recombinant human Noggin (Peptron) or 5% (vol/vol) Noggin (Hans Clevers lab) This mixed culture medium composition was used. The results of primary culture from KRAS ^G12D mouse tissue were observed with an inverted microscope (Zeiss) and are shown in FIG. 1A.

1.3 Secondary Culture of Organoids on Microfluidic Chips

The present inventors tried to confirm the effect of culturing organoids during secondary culture in the present invention, a microfluidic chip (MicroFIT, StemFIT 3D ^® ). First, to prepare the chip, wash the wells with 70% ethanol so that no bubbles are generated, remove the ethanol, replace the media with Advanced DMEM (basic media for organoid culture medium), and then place the organoids in an incubator at 37 ° C. was incubated while it was being prepared. The organoids to be inoculated are mechanically dissociated, washed with cold 1x PBS on ice, and a recovery solution of about 500 ul based on the organoid cultured in 12 wells is added, and then at 4°C for 1 hour. The incubation was performed to remove Matrigel, and after removal, the cells were pelleted by centrifugation at 1200 rpm at 4°C for 5 minutes. The existing Advanced DMEM was removed, and the pelleted organoids were resuspended in organoid media at 1 ml per chip, and inoculated to evenly enter the wells.

After resuspension of the organoids collected in the above method in the organoid culture medium, seeding on the prepared microfluidic chip, overnight overnight, the cultured organoids were examined under an inverted microscope; Zeiss) was observed and shown in Figure 1b. In the order shown in FIG. 1b, the results of comparing the growth degree by passage after transferring from the mouse and patient-derived pancreatic organoids to the microfluid chip are shown.

It can be seen that not only genetically modified mouse pancreatic organoids but also patient-derived organoids are grown in a uniform size with well-formed spherical morphology in the microfluidic chip.

1.4 Organoid subculture on microfluidic chips

Patient-derived pancreatic organoid cultured in Matrigel is mechanically dissociated, washed with cold 1x PBS or recovery solution at 4°C, and centrifuged at 1200 rpm for 5 minutes. was removed. The collected organoids were resuspended in the organoid culture medium and then inoculated into the prepared microfluidic chip, and the state after one day corresponds to the passage 1 - 1 day (day 1) of FIG. 1c. In addition, the organoids in the microfluidic chip of Passage 1 = 2 days (day 2) were collected by pipetting, washed with 1x PBS, centrifuged at 1200 rpm for 5 minutes, and the pelleted organoids were collected After resuspending in an organoid culture medium containing 2% Matrigel, it was again inoculated into a microfluidic chip. The state after one day after inoculation corresponds to the passage 2 = 1 day (day 1) of Figure 1c, and the state after one more passage culture in the same way as above is passage 3 in Figure 1c (passage 3) = 1 corresponds to day 1 As such, it can be confirmed that organoids are well formed even when subculture on the microfluidic chip is repeated several times (see FIG. 1c ).

Comparative Example 1: Subculture of mouse pancreatic organoids using A1 chip

1.1 Organoid secondary culture

In order to comparatively verify the excellent organoid culture effect during secondary culture of the microfluidic chip of Example 1.3, as another chip, an A1 microfluidic chip used for conventional cell aggregate culture was used. to conduct the experiment. The A1 fluid chip used in this experiment is composed of 12 wells, each of which has a shape of a straight structure with a flat lower surface in a well made of a 2 mm circumference of an inlet circle. There is a difference from the shape of the inlet hexagonal perimeter 2.1 mm structure of the present invention, the 0.5 mm width well, and the hemispherical structure with the lower surface. Mouse-derived pancreatic organoids were secondary cultured on the A1 chip by the method of Example 1.3 (see FIG. 2).

As a result, unlike the case of culturing in the microfluidic chip according to an embodiment of the present invention, the size of the organoids did not grow evenly in the A1 chip, and poor results in both morphology and viability It can be seen that appears (see FIGS. 1b and 2). It was confirmed that sustainable culture was no longer possible due to a significant decrease in growth in the A1 chip. Compared with the result of culturing in a microfluidic chip, the efficacy was significantly higher despite the fact that it had to be prepared in a uniform size to be useful for drug screening. was confirmed to be lowered.

Example 2. Drug reactivity test of mouse or patient-derived pancreatic organoids

Referring to Example 1, after culturing mouse and patient-derived pancreatic organoids using a microfluidic chip, the following steps were performed to test drug reactivity. Specifically, after dissociating the pancreatic tissue isolated from the mouse or patient, and collecting the cell pellet containing the pancreatic duct cells from the dissociated pancreatic tissue, the collected cell pellet together with the biomaterial in a 37°C, 5% carbon dioxide gas cell incubator was brought up in From the time point 24 to 48 hours after the establishment of culture with the organoid until just before the time point of drug treatment for the following drug screening, the organoids were further cultured. Thereafter, drugs from mouse or patient-derived organoids were diluted in the organoid culture medium and then treated with the organoids.

2D breast cancer cell lines MCF10A, MCF7, Kras ^G12D mutant mice and wild type mice or pancreatic organoids prepared from patient-derived tissues were treated with 0 uM, 100 uM, 200 uM, 500 uM of anticancer drugs and then 3 to Photos observed with an inverted microscope (Zeiss) on the fifth day are shown in FIGS. 3 to 5 .

As a 2D cell line, the results of comparing the groups in which the cancer cell lines MCF10A and MCF7 were not treated with or treated with anticancer drugs are shown in FIG. 3 . In addition, the drug screening results of organoids derived from Kras ^G12D mutant mice and wild type mice in the presence of Matrigel are shown in FIGS. 4A and 4B . Figure 5 shows the results of treatment with the drug in Kras ^G12D mutant mice, wild type mice, and patient-derived organoids that were secondary-cultured using a microfluidic chip without Matrigel.

Summarizing the obtained results, as shown in FIG. 5 , it was observed that the reactivity to the tested drug was different depending on whether the microfluidic chip of the present invention was used. In particular, it was confirmed that the drug reactivity of the 3D organoid was better compared to the 2D cell line, and furthermore, it was confirmed that the same drug screening effect could be obtained at a relatively low concentration in the state without Matrigel compared to the state with Matrigel. In other words, when a microfluidic chip without Matrigel was used in pancreatic organoids derived from mice and patients, the same effect was achieved even at a lower concentration of 50 uM compared to 100 uM treatment of the drug in the state with Matrigel. It can be seen that . Through this, the microfluidic chip of the present invention can be usefully used in confirming the effect of a candidate drug and/or in drug screening.

Example 3. Various uses of organoids manufactured using microfluidic chips

3.1 Cell viability assay (MTT assay) in organoids manufactured using microfluidic chips

MTT assay is a method of measuring the metabolic activity of cells, and shows purple color in living cells. In the same manner as in Example 1, a patient-derived organoid was prepared in an untreated state, and then viability was measured with MTT reagent, and is shown in FIG. 6a. In addition, the results of measuring viability with MTT reagent on the 4th day after each yap inhibitor (0 uM, 5 uM) and AP-1 inhibitor (0 uM, 200 uM) drugs were treated in patient-derived organoids are shown in FIG. 6b shown in The figure is a photograph taken after applying the MTT assay technique after inoculating the microfluidic chip with organoids.

As a result, it was confirmed that it was possible to culture organoids with viability in the microfluidic chip of the present invention (see Fig. 6a), and in the case of organoids treated with drugs, cells died and the shape of the organoids could not be maintained, It can be confirmed that the dye is not purple (see FIG. 6b). As such, there was a problem in that the reagent did not penetrate well into the organoid, so that the dye did not work well in the normal general organoid culture method. This suggests that the penetration of the drug as well as the reagent is easy. Using this technique, the microfluidic chip is applied to the drug test process to enable tracking of drug treatment response, drug treatment in an even size state, and further quantification after drug treatment.

3.2 Confirmation of stemness through immunofluorescence assay (IFA)

As in 3.1 above, the microfluidic chip was inoculated with organoids the next day, and the cells were fixed by adding 4% paraformaldehyde (PFA), and after removing the PFA, they were washed 3 times with 1x PBS. After permeabilizing for 1 hour at RT with 1% PBS-T, it was further washed 3 times with 0.2% PBS-T. After blocking with 3% BSA under RT conditions for 1 hour, primary antibodies, alpha-tubulin, CD44, and CD24, were attached at RT conditions for one day. After washing 3 times with 0.2% PBS-T, a fluorescence secondary antibody was attached at RT for one day. After washing 3 times with 0.2% PBS-T and DAPI staining, the organoids were transferred from the microfluidic chip to the 8-well chamber, and then PBS-T was removed. After mounting with Vectashield, fluorescence images were observed by photographing with a confocal microscope (see FIGS. 7 and 8). In the case of imaging under a microscope after immunofluorescent staining of existing organoids, Matrigel had to be removed as a separate procedure, the treatment period of primary and/or secondary antibodies was long, and the washing process had to be submerged by gravity. The disadvantage of taking a long time and the disadvantage of having to process a large amount of antibody existed. When this inconvenience is dyed by using the microfluidic chip of the present invention, time and economic utility are improved, and there is an advantage of more convenience.

3.3 Genome analysis of organoids

In addition, genomic analysis was performed using the patient-derived organoids prepared above. Referring to FIG. 9 , it is a result of genomic analysis using organoids cultured without a biological matrix material using the microfluidic chip of the present invention (see FIG. 9 ).

Referring to FIG. 9 , it can be seen that when genome analysis is performed using organoids, cellularity is remarkably superior when compared to cases using tissue (fine-needle biopsy; FNB).

3.4 Organoid Transfection Using Microfluidic Chips

As in Example 1, the microfluidic chip was inoculated with organoids and transfected by transfection on the organoids after one day. First, in order to select a sgRNA that can be recognized by the CRISPR/Cas9 enzyme of the human PLK1 gene region, each exon region was inserted into the CRISPR RGEN tools to serve as a guide RNA hPLK1 exon2 gRNA (5'-CGTAGGTAGTATCGGGCCTC-3' sequence (SEQ ID NO: 1) )) was designed. The gRNA was cloned into pCMV, a vector containing a Cas9 enzyme, by site-directed mutagenesis after synthesizing the gene to prepare a pCMV-hPLK1-Cas9-RFP vector. According to a protocol published in the art, the prepared CRISPR/Cas9 recombinant vector was introduced into the organoid tissue in the microfluidic chip and transfected. At this time, Etube A contained 200 ul of OptiMEM and 5 ug of DNA, and Etube B was prepared to contain 200 ul of OptiMEM and 20 ul of PEI, and then, Etube A and Etube B were mixed and incubated for 5 to 10 minutes. After that, CRISPR/Cas9 was transfected by removing the existing media, mixing 800 to 1000 ul of new organoid medium and the incubated mixture, and pouring it onto the microfluidic chip. On the second day after transfection with CRISPR/Cas9, red fluorescent protein (RFP) detection was performed on DNA to confirm transfection.

As a result, it was confirmed that the transfection was well performed as the red fluorescent protein caused by RFP was detected in the patient-derived pancreatic organoid (see FIG. 10). In addition, the corresponding organoids were collected and loaded on a 10% SDS-PAGE gel to separate proteins, and as a result of Western blot analysis, it was confirmed that PLK1 was reduced by CRISPR/Cas9 in the PDO_SgPLK1 group (see FIG. 10 ).

As such, it suggests that organoid transfection on the microfluidic chip from which Matrigel is removed as in the present invention is also easy. It is expected that this technique can be used for various purposes by applying the transfection technique using a microfluidic chip.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Seoul National University R&DB Foundation Next&Bio Inc. <120> Method for preparing organoids using microfluidic chip <130> PDPB201842 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> sgRNA <400> 1 cgtaggtagt atcgggcctc 20

Claims (32)

A method for producing an organoid, comprising the step of culturing a pre-organoid in a microfluidic chip. The method of claim 1,
The microfluidic chip includes a plate provided with a chamber,
The chamber is made in the shape of a square pillar,
The method of claim 1, wherein the chamber includes at least one shell capable of accommodating and culturing pre-organoids. 3. The method of claim 2,
The shell comprises an inlet that is opened to accommodate the pre-organoids, and a closed receiver in which the pre-organoids are supported and cultured. 4. The method of claim 3,
The inlet section has a hexagonal structure,
The accommodating part has a space in which the pre-organoids are accommodated, is configured in a columnar shape to have a predetermined depth from the inlet part, and the bottom surface is a hemispherical shape protruding downwards, the method of manufacturing an organoid. 5. The method of claim 4,
A plurality of the shells are included in the chamber, and the distance between any one edge of the neighboring shell from any one edge of the inlet part of the shell is 0.01 mm to 1.0 mm, the method of manufacturing an organoid. The method of claim 1,
The material of the microfluidic chip is from the group consisting of PDMS (Polydimethylsiloxane), Polycarbonate, PTMSP (poly(1-trimethylsilyl-1-propyne)), PTFE (Polytetrafluoroethylene), urethane, PET (Polyethylene terephthalate) and agarose. Any one or more selected, the method for producing an organoid. The method of claim 1,
The pre-organoid is a method for producing an organoid, obtained by mixing and culturing a biological sample isolated from a target individual with a biological matrix material. 8. The method of claim 7,
The pre-organoids are
(a) dissociating the biological sample isolated from the subject of interest;
(b) collecting a cell pellet from the dissociated sample; and
(c) a method for producing an organoid, which is obtained from the step of incubating the collected cell pellet with a biological matrix material. 8. The method of claim 7,
The biomaterial is collagen, fibrin, agarose, agar, matrigel, alginate, gelatin, or a method of producing an organoid comprising at least one selected from the group consisting of hyaluronic acid. 8. The method of claim 7,
The biological sample includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears ( tears), mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, Ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, tissue, cell, cell extract extract) and at least one selected from the group consisting of cerebrospinal fluid, a method for producing an organoid. 8. The method of claim 7,
The method for producing an organoid, further comprising a post-treatment step of removing the biomaterial from the pre-organoid, after the step of culturing by mixing with the biomaterial. 12. The method of claim 11,
The post-treatment step of removing the biomaterial from the pre-organoid is,
crushing the cultured pre-organoids by mechanically applying stress;
culturing in the presence of a recovery solution; and
A method for producing organoids, comprising the step of centrifuging to obtain a pre-organoid pellet. 13. The method of claim 12,
The step of culturing in the presence of the recovery solution (recovery solution) is performed at 3 to 7 ℃ for 30 minutes to 72 hours, the method for producing an organoid. 13. The method of claim 12,
The centrifugation is performed at 2 to 6 ℃ 1000 to 1500 rpm for 3 to 10 minutes, the method for producing an organoid. The method of claim 1,
The culturing in the step of culturing the pre-organoids in the microfluidic chip is 35 to 37° C. and 8 to 12 vol% CO ₂ The method for producing an organoid, which is performed for 1 to 10 days. The method of claim 1,
The method for producing an organoid, further comprising pre-treating the microfluidic chip prior to culturing the pre-organoid in the microfluidic chip. 17. The method of claim 16,
The step of pre-processing the microfluidic chip,
culturing by adding a medium to the microfluidic chip; and
A method for producing an organoid comprising removing the medium. 18. The method of claim 17,
The medium is at least one selected from the group consisting of DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI-1640 and GMEM (Glasgow's Minimal essential Medium), a method for producing an organoid . The method of claim 1,
The culturing of the pre-organoids may include: culturing the pre-organoids primarily; and
Method for producing an organoid comprising; reinoculating the organoid obtained by primary culturing in a microfluidic chip and culturing secondary. 20. The method of claim 19,
Prior to the second culturing step, the method for producing an organoid further comprising the step of centrifuging at 1000 to 1500 rpm for 3 to 10 minutes. 20. The method of claim 19,
Prior to the second culturing step, the method for producing an organoid, further comprising the step of resuspending the centrifuged organoid in a new culture medium. 20. The method of claim 19,
The second culturing step can be subcultured by repeating one or more times, a method for producing an organoid. 23. An organoid prepared by the method of any one of claims 1 to 22. (a) treating the organoid of claim 23 with a candidate drug; and,
(b) measuring the cell viability (viability) of the organoid; comprising, a method of screening a candidate drug. 24. A method for producing a genetically engineered organoid, comprising the step of engineering a gene encoding a protein of interest or a gene of interest in the organoid of claim 23. 26. The method of claim 25,
wherein the method is on a microfluidic chip. 26. The method of claim 25,
The method of manipulating the gene is performed in such a way that the organoid is transfected with a gene encoding a target protein or a gene of interest. 28. The method of claim 27,
The method of transfection will be performed using a viral vector or a non-viral vector. 29. The method of claim 28,
For transfection of the vector, microinjection, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection The method, which is performed by any one method selected from the group consisting of transfection (polybrene-mediated transfection), electroporation (electroporation) and gene gun (gene gun). 26. The method of claim 25,
The genetic manipulation is any one gene selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR (clustered regularly interspaced short palindromic repeat; CRISPR). The method of any one of the preceding claims. 31. The method of claim 30,
The method is to be carried out by the CRISPR (clustered regularly interspaced short palindromic repeat) method, the method. 26. The method of claim 25,
Confirmation of whether the genetic manipulation will be performed in a manner that is confirmed by western blot or indirect intracellular staining flow cytometry analysis.

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