WO2020005012A1

WO2020005012A1 - Method for producing human-derived three-dimensional organoid by non-surgical method

Info

Publication number: WO2020005012A1
Application number: PCT/KR2019/007894
Authority: WO
Inventors: 이현숙; 박지호; 이수현; 김성수
Original assignee: 서울대학교 산학협력단
Priority date: 2018-06-29
Filing date: 2019-06-28
Publication date: 2020-01-02

Abstract

Provided are a three-dimensional organoid obtained by in vitro culture of human organ tissue obtained by a non-surgical method, a producing method therefor, a culturing method thereof, and a use for genetic information analysis, karyotype analysis, drug effect validation, and/or drug screening of the organoid obtained as described above.

Description

Method for producing human-derived three-dimensional organoids by non-surgical method

Three-dimensional organoids obtained by culturing human organ tissue obtained by non-surgical method in vitro, preparation method thereof, culture method thereof, genetic information analysis, karyotype analysis, drug effect of the organoids obtained as described above Uses for validation, and / or drug screening are provided. In addition, the present invention allows for the early diagnosis of various diseases including cancer. That is, it is expected that the diagnosis of the disease can be made faster than the conventional method using drug screening.

As a conventional organ-derived organoid culture method, a technique of three-dimensional culture using cancer tissue extracted during surgery has been proposed. However, in some cancers, such as pancreatic cancer, more than half of the patients are inoperable, and it is very unlikely that a disease model using cancer-simulated organoids can be created using tissues removed during surgery as in the conventional method. .

On the other hand, the use of non-surgical methods such as endoscopic ultrasound guided acupuncture can be applied regardless of disease progression, and has much wider scalability than conventional surgical methods. .

However, there are some problems in making three-dimensional organoids from tissue samples obtained by non-surgical methods. First, since tissue samples obtained by non-surgical methods are much smaller in quantity than those obtained during surgery, the probability of success of three-dimensional organoid culture is lowered. This is believed to be due to the efficient dissociation of microstructure, but not the establishment of appropriate dissociation conditions that do not inhibit the growth of adult stem cell populations. In addition, there is a problem that long-term culture may not be made if the optimal culture conditions are not provided.

In order to overcome the above problems, there is a need for the development of a technique capable of efficiently producing organoids from non-surgically obtained tissues and enabling long-term culture. In addition, in order to be a technical platform for providing personalized medical care to patients and initial diagnosis of diseases including cancer, development of technology for more efficient immunostaining and karyotyping of long-term cultured three-dimensional organoids is necessary. Required.

The present invention has been made to solve the above conventional technical problems, a method for producing a human-derived three-dimensional organoid from tissue obtained by a non-surgical method and the step of removing the matrigel from the prepared three-dimensional organoid It further relates to a method for immunostaining and karyotyping, including.

This experimental technique using an organoid enables the identification of early stages of cancer and diseases that could not be identified by conventional pathological readings or by DNA sequencing of lesions taken during histological examination. As an initial diagnostic method, it is significant.

Application research using these three-dimensional organoids will be a springboard for providing customized medical care to actual patients by reflecting the in vivo situation where cells actually exist three-dimensionally.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

One example provides a method of preparing a three-dimensional organoid from biological tissue obtained by a non-surgical method. The biological tissue may be organ tissue isolated from a mammal, such as a human. The non-surgical method may be fine needle aspiration or the like.

Another example provides a three-dimensional organoid prepared by the above method.

Another example provides a composition for verifying drug effect comprising the three-dimensional organoid. Another example provides a method for verifying drug effect or providing information for drug responsiveness verification, comprising treating a drug with the three-dimensional organoid.

Another example provides a composition for drug screening comprising the three-dimensional organoid. Another example provides a drug screening method comprising treating a candidate substance to the three-dimensional organoid.

Another example provides a composition for analyzing genetic information or karyotype comprising the three-dimensional organoid. Another example provides a genetic information analysis or karyotype analysis method comprising measuring the genetic information or karyotype of the three-dimensional organoid.

Another example provides a composition for tissue or organ regeneration comprising the three-dimensional organoid.

Another example provides a method for producing or regenerating tissue or organ in vitro, comprising culturing the three-dimensional organoid.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for purposes of complete understanding of the invention, various specific details are set forth, such as specific forms, compositions, processes and the like. 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 particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "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, the context of “in one embodiment” or “embodiment” expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. In addition, particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined, 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.

Organs such as pancreas, small intestine, and stomach have been found to contain adult stem cell groups expressing Lgr5 markers in epidermal cells. The culturing of the adult stem cell group uses a medium for activating specific signaling systems (Wnt, EGFR, and / or SMAD, etc.) in the cells. In the present specification, based on a medium capable of selectively culturing such adult stem cell populations, a three-dimensional organoid (organoid) by selectively cultivating a sample obtained by endoscopically guided fine needle aspiration in a three-dimensional environment in vitro It provides a technique for manufacturing. This three-dimensional environment can be provided by extracellular matrix mimetics that mimic extracellular matrix (ECM), such as Matrigel.

More specifically, the extracellular matrix mimetic may be a porous medium such as a hydrogel or a porous membrane. The porous medium is initially liquid, and may be solidified by various conditions. The conditions include temperature, light, pH, pressure, vibration, and the like. For example, porous media that are liquid at high temperatures may gel at low temperatures. In addition, the hydrogel may be a natural or synthetic polymer material, collagen, fibrin, agarose, agar, atrigel, alginate, alginate, gelatin It is preferable that it is etc., It is most preferable that it is a matrigel. The Matrigel is commercially available as an artificially synthesized extracellular matrix, and includes, but is not limited to, laminin, collagen type IV, heparin sulfate proteoglycan and entaxin / nidogen.

In the present invention, an organoid may be understood as an example of an "ex vivo tissue construct." The "in vitro tissue structure" is a culture made by culturing or recombining cells isolated from stem cells or organ cells, and refers to a structure that maintains the form of tissue in vitro, and artificial organs, bio organs, mini organs, organs Used in the sense encompassing nooids, spheroids, glomeruli, and embryoid bodies.

In the organoid manufacturing technology provided herein, adult stem cells are cultured embedded in a three-dimensional environmental composition material, such as an extracellular matrix, and this culture method, unlike conventional two-dimensional cell culture, karyotype It is characterized by very little change and no spontaneous transformation. The three-dimensional organoid culture or manufacturing techniques provided herein provide for organs in living organisms (e.g., human body) compared to conventional cell culture methods (e.g., two-dimensional cell culture methods) from trace tissue samples obtained by non-surgical methods. It is characterized by the ability to obtain a three-dimensional organoid in vitro that has high mimicability and extremely low karyotype and does not have a spontaneous cancerous process. Due to these characteristics, the three-dimensional organoid can represent the genetic information of the donor of the tissue sample relatively accurately, and use it to analyze the cellular characteristics of individual tissue sample providers and to provide customized treatment by verifying drug reactivity. It can provide a cell platform that is the basis of, and can be usefully applied to various fields such as regenerative medicine such as tissue regeneration and long-term simulation in the future. In addition, the genetic information of the organoids representing the lesions more closely than the tissue specimens and the cytological characteristics of individual tissue sample providers through experimental methods applied to the organoids may be used for rapid initial diagnosis.

In addition, a tissue sample derived from a living body (human body) used in the three-dimensional organoid manufacturing technique provided herein may be a tissue obtained by a non-surgical method, such as an endoscopic derived tissue, and such endoscopic derived tissue may be surgically Unlike the case obtained by the method, the sampling can be performed at the same time as the patient's clinicopathological examination and / or diagnosis, providing the advantage of rapidly building an organoid platform that reflects the patient's genetic and histological information. do. By using such endoscopy-derived organoids, we can establish genome analysis, karyotyping, and drug screening techniques for the patients, which can be used as a platform for personalized medicine that can provide efficient diagnosis and treatment for patients in a relatively short time. Do.

In addition, the organoid culture technology used in the three-dimensional organoid production provided herein has the advantage of allowing long-term culture.

As used herein, the organoids are capable of self-renewal from adult stem cells (ASC), embryonic stem cells (ESC), induced pluripotent stem cells (iPSCs), and the like. It means a three-dimensional cell aggregate formed through self-organization. By re-aggregating and recombining cells in three-dimensional culture to resemble a living environment, it is possible to overcome the limitations of 2D cell lines cultured by 2D culture and to reproduce biophysiological functions of living organisms similarly, which is applied to disease modeling and drug screening. It is possible.

One embodiment of the present invention provides a method for producing a three-dimensional organoid from biological tissue obtained by a non-surgical method.

Specifically, the method

(1) dissociating the tissue sample isolated from the patient in a non-surgical manner;

(2) separating the cell pellet containing adult stem cells from the dissociated tissue sample; And

(3) culturing the cell pellet with the biological matrix material

It may include.

Such non-surgical methods are all non-invasive methods (eg, 0.1 mm to 2 mm in diameter of syringes or needles, etc.) to obtain small amounts of tissue samples in a method that does not involve relatively large area incisions and / or all or part of the organs. Needle aspiration (FNA); FNB (Fine Needle Biopsy), FNAB (Fine Needle Aspiration Biopsy), etc. May be used). The fine needle aspiration may be Endoscopic Ultrasound-Guided Fine Needle Aspiration (EUS-FNA) involving guiding with an endoscope. The fine needle used for fine needle aspiration has an outer diameter (OD) of 0.2 mm to 2 mm, 0.2 mm to 1.75 mm, 0.2 mm to 1.5 mm, 0.2 mm to 1.25 mm, 0.2 mm to 1 mm, and 0.2 mm to 0.75 mm 0.2 mm to 0.5 mm, 0.2 mm to 0.4 mm, 0.2 mm to 0.3 mm, 0.3 mm to 2 mm, 0.3 mm to 1.75 mm, 0.3 mm to 1.5 mm, 0.3 mm to 1.25 mm, 0.3 mm to 1 mm, 0.3 mm to 0.75mm, 0.3mm to 0.5mm, 0.3mm to 0.4mm, 0.4mm to 2mm, 0.4mm to 1.75mm, 0.4mm to 1.5mm, 0.4mm to 1.25mm, 0.4mm to 1mm, 0.4mm to 0.75mm, 0.4 mm to 0.5mm, 0.5mm to 2mm, 0.5mm to 1.75mm, 0.5mm to 1.5mm, 0.5mm to 1.25mm, 0.5mm to 1mm, or 0.5mm to 0.75mm, inner diameter (ID) of 0.1 to 1.75 mm, 0.1 to 1.5 mm, 0.1 to 1.25 mm, 0.1 to 1 mm, 0.1 to 0.75 mm, 0.1 to 0.5 mm, 0.1 to 0.4 mm, 0.1 to 0.3 mm, 0.1 to 0.2 mm, 0.15 to 1.75 mm, 0.15 to 1.5 in mm, 0.15 to 1.25 mm, 0.15 to 1 mm, 0.15 to 0.75 mm, 0.15 to 0.5 mm, 0.15 0.4 mm, 0.15 to 0.3 mm, 0.15 to 0.2 mm, 0.2 to 1.75 mm, 0.2 to 1.5 mm, 0.2 to 1.25 mm, 0.2 to 1 mm, 0.2 to 0.75 mm, 0.2 to 0.5 mm, 0.2 to 0.4 mm, or 0.2 to 0.3mm (external diameter> inner diameter). In one embodiment the microneedle may be of 19G (OD: 1.07mm, ID: 0.69mm), 22G (OD: 0.72mm, ID: 0.41mm), or 25G (OD: 0.51mm, ID: 0.26mm) However, it is not limited thereto.

The patient may be a mammal, such as a human, and may need to diagnose a disease, predict and / or verify drug efficacy, analyze karyotype or genetic information, or have a disease, such as a mammal, such as It can be human.

The tissue sample may be any tissue sample derived from and isolated from tissue in vivo of the patient. The tissue may be tissue of an organ selected from the patient's organs, such as the pancreas, small intestine, stomach, esophagus, and the like, and if the patient has a disease, the tissue may be a tissue or tissue including the affected area or the affected area. It may be an organization. In another example, the tissue may be normal tissue, such as normal tissue from a patient or normal tissue outside the affected area in case of diseased organs.

The dissociating step may include treating the dissociation solution with a tissue sample. The dissociation solution is a solution such as HBSS (Hank's Balanced Salt Solution) or DMEM medium (eg, Advanced DMEM / F-12 media (Dulbecco's Modified Eagle Medium / Ham's F-12), etc.) (eg, 1 ml to 10 ml, 1 ml to 5 ml, 3 ml to 10 ml, or 3 to 5 ml), ROCK (rho-associated protein kinase) inhibitor (e.g., Y-27632 (UPAC name: (1R, 4r) -4-((R) -1-) aminoethyl) -N- (pyridin-4-yl) cyclohexanecarboxamide; Cas number: 146986-50-7) (e.g. 1-50 uM, 1-40 uM, 1-30 uM, 1-20 uM, 1-15 uM) , 1 to 12.5 uM, 2.5 to 50 uM, 2.5 to 40 uM, 2.5 to 30 uM, 2.5 to 20 uM, 2.5 to 15 uM, 2.5 to 12.5 uM, 5 to 50 uM, 5 to 40 uM, 5 to 30 uM , 5-20 uM, 5-15 uM, 5-12.5 uM, 7.5-50 uM, 7.5-40 uM, 7.5-30 uM, 7.5-20 uM, 7.5-15 uM, 7.5-12.5 uM, or 8-12 uM), 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), DNase ( DNase 1) (e.g. 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, 0.05 mg / ml to 0.2 mg / ml, or 0.08 mg / ml to 0.15 mg / ml), dispase (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 the like. It may be one containing one or more.

In one embodiment, the dissociation solution is 0.5-5 ml, 0.5-4 ml, 0.5-3 ml, 1-5 ml, 1-4 ml, or 1-3 ml with respect to tissue (Vpan = about 1.08 mg / mm ³ ). It may be used in an amount of, but is not limited thereto, and may be appropriately adjusted according to the amount of tissue. At this time, in order to more easily dissociate the tissue, physical dissociation (for example, rotating, slicing finely with scissors or a knife, etc.) may be performed simultaneously with the dissociation solution treatment or sequentially in any order. . When performing the step of rotating, the rotation speed is approximately 100 to 300 rpm, 100 to 250 rpm, 100 to 225 rpm, 100 to 200 rpm, 125 to 300 rpm, 125 to 250 rpm, 125 to 225 rpm, 125 to 200 rpm, 150 to 300 rpm, 150 to 250 rpm, 150 to 225 rpm, or 150 to 200 rpm.

The dissociation step is about 1 to 60 minutes, 1 to 50 minutes, 1 to 40 minutes, 1 to 30 minutes, 10 to 60 minutes, 10 to 50 minutes, 10 to 40 minutes, 10 to 30 minutes, 15 to 60 minutes. , 15 to 50 minutes, 15 to 40 minutes, or 15 to 30 minutes, the temperature conditions are not particularly limited, but in a range similar to the temperature in vivo, such as 30 to 40 ℃, 30 to 38 ℃, 32 to It may be carried out at 40 ℃, 32 to 38 ℃, 34 to 40 ℃, 34 to 38 ℃, 36 to 40 ℃, or 36 to 38 ℃ degree.

(2) separating the cell pellet including stem cells (eg, adult stem cells such as epithelial stem cells) from the dissociated tissue sample may be used to centrifuge the dissociated tissue to remove supernatant and / or pellets. It may include taking a step. The centrifugation may be performed at 2 to 6 ° C. (eg 4 ° C.) for 15 minutes to 1 hour at a speed of 800 to 1500 rpm, 800 to 1200 rpm, 1000 to 1500 rpm, or 1000 to 1200 rpm, but is not limited thereto. It can be carried out by appropriately adjusting according to the amount, property, etc. of the sample.

In the step (3) culturing the cell pellet with the biological matrix material, the biological matrix material is Matrigel, Extracellular matrix (ECM), Basement Membrane Extract (BME), Hyaluronic acid It may include one or more selected from the group consisting of lonic acid (hyaluronic acid). The Matrigel refers to a gelatin-like protein mixture secreted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. The extracellular matrix is a collection of biopolymers including molecules that are synthesized and secreted and accumulated in the cell, and are composed of fibrous proteins such as collagen and elastin, complex proteins such as glycosaminoglycan, fibronectin, and laminin. It may include one or more selected from the group consisting of adhesive proteins.

The culture can be carried out under conventional cell culture conditions, such as 35 to 38 ° C. and 3 to 12% CO ₂ conditions. In addition, the culture period may be carried out for 1 to 14 days, 1 to 10 days, 1 to 7 days, 3 to 14 days, 3 to 10 days or 3 to 7 days before passage culture, after that, 1 passage Up to 12 months, 9 months, or 6 with one or two repeated passages of 1 to 14 days, 1 to 10 days, 1 to 7 days, 3 to 14 days, 3 to 10 days, or 3 to 7 days It is also possible to continue culture for up to months. The medium used for the culture is DMEM / F-12 (Dulbecco's Modified Eagle Medium / Ham's F-12), penicillin / streptomycin, HEPES (Hank's Balanced Salt Solution), GlutaMAX, N-acetylcysteine (N-acetylcysteine), conditions At least one selected from the group consisting of media (eg, Rspo1-conditioned medium), nicotinamide, gastrin (eg, gastrin I), growth factors (eg, EGF, FGF, etc.), nogin, nogin-conditioning medium, and the like. It may be to include, but is not limited thereto.

In one embodiment, the step of culturing further comprises the step of culturing by adding a culture of fibroblasts or fibroblasts to the medium, or by replacing the medium with a culture medium containing a culture of fibroblasts or fibroblasts. It may include. The fibroblasts may be rodent-derived fibroblasts such as primates or mice, such as humans, for example, at least one selected from the group consisting of lung tissue-derived fibroblasts (eg, MRC5, MRC5-htert cell lines, etc.), The culture may be a cell-containing or cell-free culture in which the fibroblasts are cultured in a conventional medium. By culturing together with the addition of fibroblasts or fibroblast cultures in the culture step, the culture period of the organoids is at least 2 weeks, at least 3 weeks, at least 1 month, at least 1.5 months, at least 2 months, at least 3 months, 4 months It can increase to more than 5 months, or more than 6 months, and long-term culture is attained .

After the step of culturing, (4) may further comprise the step of separating the resulting organoids. The separating step is a step of separating a three-dimensional organoid having an average diameter of 0.1mm or more, 0.25mm or more, 0.5mm or more, 0.75mm or more, 1mm or more, or 1.25mm or more, 1.5mm or more, 1.75mm or more or 2mm or more. (The upper limit of the average diameter of the organoid may vary depending on the culture period, and may be about 15 mm, 12.5 mm, 10 mm, 7.5 mm, 5 mm, or 2.5 mm, but is not limited thereto).

Another example provides a three-dimensional organoid prepared by the above method. The three-dimensional organoids are derived from organ tissue obtained non-surgically from a patient (eg, a human) and have an average diameter of at least 0.1 mm, at least 0.25 mm, at least 0.5 mm, at least 0.75 mm, at least 1 mm, or at least 1.25 mm, 1.5 mm or more, 1.75 mm or more or 2 mm or more (the upper limit of the average diameter may be about 15 mm, 12.5 mm, 10 mm, 7.5 mm, 5 mm, or 2.5 mm, but is not limited thereto). The three-dimensional organoids may be characterized as being similar to the organs in vivo of the patient providing the organ tissues, and / or having very few karyotype changes, and / or having no spontaneous cancer. Further, in one embodiment, the three-dimensional organoid is at least two weeks, at least three weeks, at least one month, at least 1.5 months, at least two months, at least three months, at least four months, at least five months, or at least six months. It can be characterized by long-term culture.

Another example provides a composition for predicting or verifying a drug effect or a kit for predicting or verifying a drug effect including the three-dimensional organoid. Another example provides a method of predicting or verifying a drug effect (or a method of providing information to predicting or verifying drug reactivity) comprising treating a drug with the three-dimensional organoid.

The drug effect means a biological action to be achieved by the drug, and may mean an effect of alleviating, alleviating, or treating the desired disease (eg, cancer), and when the disease is cancer, the drug effect May mean reducing, eliminating, inhibiting the progression or metastasis of the cancer.

In the method for predicting or verifying the drug effect, the drug may be at least one selected from all drugs (for example, the drug is an anticancer agent when the desired disease is cancer) to predict or verify the effect on the desired disease. The organoid may be one derived from the affected (disease site) tissue of the disease of interest (eg, an organoid derived from cancer tissue if the disease of interest is cancer).

In the drug effect prediction or verification method, the step of treating the drug to the three-dimensional organoids, physical dissociation (eg 1000um, 750um or less) in order to facilitate the organoids to react with the drug , 500um or less, 100um or less, 75um or less, 50um or less, or 25um or less).

When the size (mass) and / or number of the organoids are reduced by drug treatment, it may be determined (or determined) that the drug exerts an effect on the desired disease in the patient from which the organoids are derived. Thus, the method of predicting or verifying the drug effect may include, after the step of treating the drug, measuring the size and / or number of drug-treated organoids and comparing the drug with the untreated organoids. Can be. To this end, the comparing may further comprise the step of measuring the size and / or number of organoids in which the drug has not been treated, the step of measuring the size and / or number of the drug-treated organoids. It may be performed simultaneously with the measuring step or sequentially. The organoids not treated with the drug may be a part of the organoids remaining after treating the drug to some of the organoids or organoids before the drug treatment. Further, in the drug effect prediction or verification method, after the comparing step, if the size and / or number of the organoids to which the drug is treated decreases in comparison with the organoids to which the drug is not treated, the drug may be And determining (or determining) that is effective in the treatment of the desired disease in the patient.

Another example provides a composition for drug screening comprising the three-dimensional organoid. Another example provides a method for drug screening comprising treating a candidate substance to the three-dimensional organoid.

In the drug screening method, treating the candidate material with the three-dimensional organoid may include physically dissociating the organoid (eg, 1000 μm or less, 750 μm or less), so that the organoid may be easily reacted with the candidate material. 500 um or less, 100 um or less, 75 um or less, 50 um or less, or 25 um or less).

In the drug screening method, the drug to be screened may be a drug having a therapeutic effect on a desired disease (eg, the drug is an anticancer agent when the desired disease is cancer), and the organoid may be a drug to be screened. It may be derived from the affected (disease site) tissue of the disease of interest (eg, an organoid derived from cancer tissue if the disease of interest is cancer).

The candidate may be selected from all bioactive materials, for example small biochemical small molecule chemicals, peptides, proteins (eg, antibodies, other protein drugs, etc.), nucleic acid molecules, extracts (eg, animals or Plant extracts) and the like. The drug screening method may be for screening drugs having a therapeutic effect on a desired disease.

In the screening method, when the candidate substance is reduced in its size (or volume, hereinafter 'size' is used to include volume) and / or the number of candidate substances, the candidate substance may be a target disease (eg, Can be determined (or judged) as a drug that is effective in cancer). Accordingly, the screening method may include measuring the size and / or number of candidate substance-treated organoids after comparing the candidate substance, and comparing the candidate substance with an untreated organoid. have. To this end, the comparing may further comprise measuring the size and / or number of the organoids to which the candidate material has not been treated, which step may include the size and / or size of the candidate material treated organoids. The number may be measured simultaneously with the measuring step or sequentially. The organoid untreated with the candidate substance may be a part of the organoid remaining after treating the candidate substance with some of the organoid or the organoid before the candidate substance treatment. In addition, the screening method, after the comparing step, if the size and / or number of the organoids treated with the candidate material is reduced compared to the organoids untreated with the candidate material, the disease for the drug Determining (or determining) a candidate drug that is effective (eg, cancer).

Another example provides a composition or kit for genetic information analysis or karyotype analysis comprising the three-dimensional organoid. Another example provides a genetic information analysis or karyotype analysis method comprising measuring the genetic information or karyotype of the three-dimensional organoid. Determining the genetic information or karyotype of the three-dimensional organoids is extracted nucleic acid (DNA and / or RNA) from the organoids to obtain the genetic information (gene or genomic sequence, etc.) by conventional methods (e.g. WGS, etc.) Conventional genetic information analysis or karyotype analysis, such as analyzing, and / or visualizing the organoid by immunofluorescence staining or the like.

Through such genetic information analysis and / or karyotype analysis, it is possible to diagnose the pathological state of the patient or organ from which the organoid is derived. Accordingly, another example of the present invention provides a composition or kit for diagnosing a disease of a patient derived from the organoid, the other example comprises the three-dimensional organoid. Another example includes a method of diagnosing a disease or disease, comprising performing morphological analysis, histological analysis, and / or genetic analysis (such as genetic information or karyotyping in a conventional manner) on the three-dimensional organoid. Provides information on the diagnosis of the. The disease may be a disease (eg cancer) associated with morphological abnormalities, histological abnormalities, and / or genetic abnormalities of the organ from which the organoids are derived.

Another example provides a composition for organ or organ production or regeneration comprising the three-dimensional organoid. Another example provides a method for producing or regenerating tissue or organ in vitro, comprising culturing the three-dimensional organoid.

If the three-dimensional organoid is derived from normal tissue, such as normal tissue of a patient or normal tissue other than the affected part of the diseased organ, the tissue or organ produced or regenerated therefrom may cause disease or pathological symptoms. It may be a normal tissue or organ that does not have. Such tissues or organs produced or regenerated from three-dimensional organoids derived from normal tissues can be used for living grafts.

If the three-dimensional organoid is a diseased tissue, such as a diseased tissue of an organ of a patient, the tissue or organ produced or regenerated therefrom may be a tissue or organ capable of modeling the disease. Such tissues or organs produced or regenerated from three-dimensional organoids derived from diseased tissue may be used for disease modeling.

In one embodiment of the invention, (a) mixing the cells isolated from the individual with a hydrogel and cultured in vitro tissue constructs; And, (b) removing the hydrogel from the ex vivo tissue structure; provides a method for confirming the expression of the biomarker in the ex vivo tissue structure, the hydrogel is collagen, fibrin, agarose, agar It provides a method for confirming the expression of a biomarker in an in vitro tissue construct comprising at least one selected from the group consisting of, matrigel, alginate, gelatin, extracellular matrix, or hyaluronic acid, The hydrogel provides a method for confirming the expression of the biomarker in the ex vivo tissue structure, characterized in that the matrigel, the ex vivo tissue structure is artificial organs, bio organs, mini organs, organoids, spheroids, spheres In vitro tissues characterized in that at least one selected from the group consisting of upper body, and embryonic body Provided are methods for identifying the expression of a biomarker in a construct, wherein the biomarker is an antibody, antigen, aptamer, receptor, ligand, enzyme substrate that specifically binds to a nucleic acid, protein, sugar, or lipid expressed in a cell. It provides a method for confirming the expression of the biomarker in an in vitro tissue construct, characterized in that at least one selected from the group consisting of enzyme inhibitors, enzyme cofactors, and enzymes, the biomarker is a protein expressed in cells It provides a method for confirming the expression of the biomarker in an in vitro tissue construct, characterized in that the antibody specifically binding, wherein the antibody is treated in 2 to 8 days of the biomarker in the ex vivo tissue construct Provided are methods for identifying expression.

In another embodiment of the present invention, the method comprising the steps of: (a) mixing the cells isolated from the individual with a hydrogel to culture the ex vivo tissue construct; And, (b) removing the hydrogel from the ex vivo tissue structure; provides a method for determining the karyotype in the ex vivo tissue structure comprising, and after the step (a) colcemide 200 in the ex vivo tissue structure It provides a method for identifying karyotype in the in vitro tissue structure further comprising the step of treating to 400ng / mL, the hydrogel is collagen, fibrin, agarose, agar, matrigel, alginate, gelatin, extracellular matrix (extracellular matrix), or in vitro tissue structure characterized in that it comprises one or more selected from the group consisting of hyaluronic acid, the method for identifying karyotype, wherein the hydrogel is an ex vivo tissue characterized in that the matrigel Provided is a method for confirming expression of a biomarker in a construct, wherein the ex vivo tissue construct comprises an artificial organ, Bio-organs, mini-organs, organoids, spheroids, globulars, and embryonic body provides a method for identifying karyotypes in an in vitro tissue construct, characterized in that the karyotype, the karyotype qualitative Provided is a method for identifying karyotypes in an ex vivo tissue construct characterized by identifying abnormalities, or numerical abnormalities.

In another embodiment of the invention, the method comprises the steps of: (a) obtaining a tissue isolated from the subject by a non-surgical method; (b) mixing the cells isolated from the tissue with a hydrogel and culturing the in vitro tissue construct; (c) removing the hydrogel from the ex vivo tissue construct; And, (d) confirming the expression of the biomarker in the hydrogel removed ex vivo tissue structure; provides a method for providing information for diagnosing the disease of the tissue comprising a rule.

In another embodiment of the invention, the method comprises the steps of: (a) obtaining a tissue isolated from the subject by a non-surgical method; (b) mixing the cells isolated from the tissue with a hydrogel and culturing the in vitro tissue construct; (c) removing the hydrogel from the ex vivo tissue construct; And, (d) identifying the karyotype in the hydrogel removed ex vivo tissue structure; provides a method for providing information for diagnosing the disease of the tissue comprising a rule.

Hereinafter, the present invention will be described in detail step by step.

Patient-derived three-dimensional organoids obtained by non-surgical methods in accordance with the manufacturing methods provided herein can represent genetic information of the donor of a tissue sample relatively accurately, and can be used to analyze the cellular characteristics of individual tissue sample providers. It can be used for initial diagnosis, and can provide a cell platform that is the basis of customized treatment through verifying drug responsiveness, and can be used in various fields such as tissue regeneration using stem cells and regenerative medicine fields such as long-term simulation in the future. Can be applied.

1A and 1B illustrate various techniques ranging from cryopreservation and tissue bank construction, genome analysis, karyotyping, three-dimensional imaging, drug reactivity verification, and tissue regeneration through three-dimensional organoid cultures from human organ samples according to one embodiment. It is a system diagram that illustrates derivation.

Figure 2 is a schematic diagram showing the culturing method of cells obtained from human organ samples by the fine needle aspiration method according to an embodiment and the change over time of the cells accordingly.

FIG. 3 is a 3D fluorescence immunoassay image of a 3D organoid labeled with DAPI, tubulin, and phalloidin.

4 is a fluorescence in situ using a label of DAPI, TTAGGG, CCCTAA to label telomeres of three-dimensional organoids having normal cells or abnormal telomeres (Telomere-Sister Chromatid Exchange) according to an embodiment. Hybridization (FISH) is a fluorescence image and graph showing the results of the experiment.

5A and 5B show the results of genomic analysis by extracting DNA and RNA from an organoid derived from a human organ sample according to one embodiment.

Figure 6a shows the result of immunostaining after 3 days of primary antibody, 3 days of secondary antibody after the step of removing the matrigel from the human organ sample-derived organoids according to an embodiment.

Figure 6b shows the result of immunostaining after 3 days of primary antibody, 3 days of secondary antibody without removing the matrigel from human organ sample-derived organoids according to one embodiment.

Figure 6c shows the results of immunostaining the antibody treatment time after 1 day, the second day 3 hours antibody treatment without removing the matrigel from the human organ sample-derived organoid according to an embodiment.

Figure 7 shows the results of karyotyping after the step of removing the matrigel from the human organ sample-derived organoid according to an embodiment and the result of karyotype without removing the matrigel.

The present invention has been made to solve the limitations of the prior art, and is for the early diagnosis of more efficient cancer and disease. The present invention further provides a step of removing Matrigel from three-dimensional organoids prepared from tissues obtained by non-surgical methods to provide optimized results in immunostaining and karyotype analysis. In addition, this experimental technique applying the organoids can identify early stages of cancer and disease that could not be identified by conventional pathological readings or by DNA sequencing of lesions taken during histological examination.

Hereinafter, the present invention will be described in more detail with reference to examples, which are merely illustrative and are not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below may be modified without departing from the essential gist of the invention.

실시예 1. 인체 장기로부터 수득한 세포의 3차원 오가노이드의 배양 Example 1 Culture of Three-Dimensional Organoids of Cells Obtained from Human Organs

Organ tissue samples (Vpan = 1.08mg / mm ³ ) obtained from the pancreas of pancreatic cancer patients by fine needle aspiration method (19G, 22G, or 25G needles) were obtained. HBSS with 10 μM of Y-27632 and 1 mg / ml of collagenase (Roche), 1 mg / ml of dispase (Sigma Aldrich), and 0.1 ml / ml of DNAase (Sigma Aldrich) (Hank's Buffered Saline Solution) Cells obtained through the cell lysis step after lysis at a rate of 180 rpm on a rotator at a temperature of 37 ° C. for 15 to 30 minutes using an amount as a dissociation solution in an amount Centrifuged and washed once with PBS (Phosphate Buffer Saline). The washed cells obtained above were seeded on Matrigel (Corning), the amount of which was adjusted according to the number of cells, and cultured with three-dimensional organoids (see FIGS. 1 and 2).

실시예 2. 3차원 오가노이드의 핵형분석Example 2. Karyotyping of Three-Dimensional Organoids

In order to prevent mitosis of the cells constituting the three-dimensional organoid obtained in Example 1, the cell cycle was detained by treating colcemide (colcemide, 100 ng / mL) for 2 to 12 hours. Discard the liquid culture solution to dissolve the matrigel in which the cell cycles are detained, and add a solution containing Dispase and Trypsin to the cells on the matrigel, and pipetting 20 Once matrigel was debrided and washed with PBS. The washed cell pellets are preferably suspended in a solution in a ratio of trypsin: PBS = 1: 1 (w / w) and pipetted at intervals of 5 minutes until all the cells are separated by the disappearance of visible pellets. Single cell suspensions were obtained. The obtained single cells were resuspended in 0.56% (w / v) KCl stock solution and allowed to stand for 20 minutes in water at 37 ° C., followed by mixing in a methanol / acetic acid = 3: 1 ratio (v / v). 1 mL of solution was added to fix. The fixed cells were removed by KCl solution after centrifugation using a 15 mL conical tube, 5-6 mL of the fixed solution was added, and stored at -20 ° C. After the fixed cells were popped on the slides, the telomeres of the unfolded chromosomes were labeled with fluorescence, followed by karyotyping. The results are shown in FIG. 4.

실시예 3: 인체 장기로부터 수득한 세포의 3차원 오가노이드의 배양 Example 3: Cultivation of three-dimensional organoids of cells obtained from human organs

1 mg / ml of collagenase (Roche) and DNase (Sigma Aldrich) 0.1ml / ml to tissue samples collected from the pancreas of pancreatic cancer patients by non-surgical fine needle aspiration HBSS formulated with 3 ml of dissociation solution was used as a dissociation solution, and dissociated at 37 ° C. for 30 minutes at a rotator 180 rpm.

The tissue that has undergone the dissociation step is washed once using PBS (Phosphate-Buffered Saline), and then planted in a matrix gel, a substrate mimetic whose amount is controlled according to the number of cells washed, and stored together in a chemically defined liquid medium. Was incubated. The medium composition used at this time is as follows:

1% (vol / vol) penicillin / streptomycin, 10 mM HEPES, 1% GlutaMAX, 1:50 B27 Additive (Gibco), 1 in DMEM / F-12 (Dulbecco's Modified Eagle Medium / Ham's F-12; Thermo Fisher Scientific) mM N-acetylcysteine, 5% (vol / vol) Rspo1 containing 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 recombinant human FGF10 (Peptron), 25 ng / ml recombinant human nogin (Peptron) or 5% (vol / vol) nogin-containing culture (Hans Clevers lab), and 10 uM Y -27632.

Continuous division by introducing hTERT gene into MRC5 (CCL-171) (https://www.atcc.org/products/all/CCL-171.aspx) for long-term culture to express activated telomerase An immortalized MRC5-htert cell line containing 1% (vol / vol) penicillin / streptomycin, 10% (vol / vol) Fetal Bovine Serum (Gibco) in DMEM (Dulbecco Modified Eale Medium, Thermo fisher scidentific) After culturing for 2 days in the culture, and then subcultured with human pancreatic organoid culture while further passage for 2 days, the culture was collected and used as a culture medium for organoid organ culture. The composition of the human pancreatic organoid culture is as follows.

1% (vol / vol) penicillin / streptomycin, 10 mM HEPES, 1% GlutaMAX, 1:50 B27 Additive (Gibco), 1 mM in DMEM / F-12 (Dulbecco's Modified Eagle Medium / Ham's F-12; Thermo Fisher Scientific) N-acetylcysteine, 5% (vol / vol) Rspo1-containing 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 recombinant human FGF10 (Peptron), 25 ng / ml recombinant human nogin (Peptron) or 5% (vol / vol) nogin-containing medium (Hans Clevers lab), and 10 uM Y -27632.

After digesting a portion of the cultured organoids to the single cell level, knockout serum (Thermo scientific) 80% (v / v), DMEM / F12 10% (v / v), and DMSO 10% (w / v) It was put in a solution containing and stored frozen, thawed and cultured if necessary.

The obtained organoids can be subjected to the following assays:

Without degrading the form of the organoids to be cultured, after removing the matrigel, fixation of 4% paraformaldehyde, staining the nuclei with DAPI, and tubulin and phalloidin by immunofluorescence staining After staining, confocal microscopy was used to implement three-dimensional organoid imaging. (See Figure 3)

Matrigel was removed from human organoids grown in Matrigel, and genomic DNA and RNA were extracted from the conventional method and subjected to genome analysis (see FIGS. 5A and 5B).

The pancreatic cancer-derived three-dimensional organoids were divided into small units (less than 22 μm) and then divided into small wells capable of cell culture to select drugs responsive to pancreatic cancer patients.

Organoids derived from normal pancreas tissues were selected and normal pancreas tissues were used.

실시예 4: 3차원 오거노이드의 면역염색Example 4 Immunostaining of Three-Dimensional Organoids

After removing the media from the organoids held in 12 well plates, the Matrigel was removed for 20 minutes to 1 hour using a recovery solution (354253, Corning, 1 ml / well). I went through the steps. The matrigel-free organoids were transferred to a 15 ml conical tube and washed three times with PBS (Phosphate Buffer Saline) on ice (4 ° C). The organoids retaining the washed spherical form were transferred to 2 ml vials using a pipette and filled with 4% PFA (paraformaldehyde) and fixed on ice for 1 hour. At this time, 4% PFA was changed three times in 10 minute intervals for the first 30 minutes so that the final concentration was 4%. After washing three times at room temperature, 1% Triton X-100 was dissolved in PBS (1% PBS-T) and infiltrated at room temperature for 1 hour. At this time, 1% PBS-T was changed three times at 10 minute intervals for the first 30 minutes so that the final concentration was 1%.

The cells were washed twice with 0.2% PBS-T, then treated with blocking buffer (3% BSA in 0.2.% PBS-T) and left at room temperature for 30 minutes. After the blocking buffer to which the primary antibody was added 400ul into a 2ml vial containing cells, and placed at room temperature for 3 days. After washing the cells three times with 0.2% PBS-T, the blocking buffer to which the secondary antibody was added was added to 400 ul as in the primary antibody and left at room temperature for 3 days. After washing three times with 0.2% PBS-T, 5 mg / ml DAPI was diluted in PBS 1: 1: 5000 to 1: 10000 and placed at room temperature for 3 to 12 hours. After washing three times with 0.2% PBS-T again, and transferred to eight well glass bottom chambers, a 3D organoid imaging was implemented using a confocal microscopy with a vectashield (FIG. 6A). Reference).

Further experiments were performed with organoids that did not remove Matrigel to compare the significance of the immunostaining effect due to Matrigel removal.

	제조예 1Preparation Example 1	제조예 2Preparation Example 2	제조예 3Preparation Example 3
1차 항체 처리시간Primary antibody treatment time	3일3 days	3일3 days	1일1 day
2차 항체 처리시간Secondary Antibody Treatment Time	3일3 days	3일3 days	3시간3 hours
매트리젤 Matrigel	제거remove	존속Survival	존속Survival

In the case of Preparation Examples 2 and 3 of Table 1, only the antibody treatment time was changed in the presence of Matrigel by the conventional immunostaining method. The result of the said manufacture example 1 was shown to FIG. 6A, the result of the manufacture example 2 to FIG. 6B, and the result of the manufacture example 3 is shown to FIG. 6C. As a result of further experiments on the organoid without preparation of Matrigel (Preparation Example 2) under the same antibody treatment conditions as those of Preparation Example 1 in Table 1 (primary antibody 3 days / secondary antibody 3 days), In contrast, it was clearly confirmed that there was no immunostaining (see FIG. 6B). In order to confirm whether the antibody time factor affects immunostaining, even when the antibody treatment time was relatively short with the first antibody one day and the secondary antibody 3 hours (Preparation Example 3) without removing the materigel, the preparation example As in the result of 2, the protein in the nucleus was not stained at all, and only non-specifically stained foci outside the organoid could be identified (see FIG. 6C). Referring to Figure 6 it can be seen that the result of staining remarkably effectively when removing the Matrigel and at the same time prolong the antibody treatment time.

실시예 5: 3차원 오가노이드의 핵형분석Example 5: karyotyping of three-dimensional organoids

Three-dimensional organoids were treated with colcemide (200 ng / ml-400 ng / mL) for 12-16 hours to stop the cell cycle. After removal of the culture from the organoids maintained in 12 well plates, the Matrigel was removed for 30 minutes using a recovery solution (354253, Corning, 1 ml / well). The cells were washed twice with 4 ° C. PBS. The pellets were collected using a centrifuge and then resuspended in 1 ml of TrypLE (manufactured by Thermomofisher). The released cells were transferred to 12 well plates and pipetting for 1 to 2 minutes at 37 ° C. After confirming with a microscope, the cells were confirmed to be single cells and transferred to a 15 ml conical tube and washed twice with PBS. The obtained single cells were resuspended in 0.56% (w / v) potassium chloride (KCl) stock solution, and after 20 minutes in 37 ° C water, 1 mL of fixed solution (methanol: acetic acid = 3: 1) was added. Mix slowly. After centrifugation, the supernatant was discarded, and the process of adding 10 ml of the fixed solution was repeated three times. The fixed cells were centrifuged using a 15 mL conical tube to remove KCl solution, and then 5-6 mL of the fixed solution was added and stored at -20 ° C. After the immobilized cells were popped on the slide, the chromosomes unfolded and the telomeres were labeled with fluorescence, followed by karyotyping. The karyotyping results at this time can be confirmed through the upper photo of FIG. 7. By removing the Matrigel, the karyotype was clearly visible.

In order to compare the significance of karyotyping effects due to Matrigel removal, additional experiments were conducted with organoids without Matrigel removal. The results are shown in the bottom photo of FIG. Referring to Figure 7, it can be confirmed that the karyotype does not come out. Through this experiment it can be seen that there is a significant difference in the presence or absence of Matrigel as in Example 4 when karyotype analysis. The present invention is distinguished from the method used in the conventional organoid karyotype analysis.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

The present invention overcomes the limitations of the prior art and is for early diagnosis of more effective cancers and diseases. In order to provide a personalized medical service to patients and to be a technological platform for early diagnosis of diseases including cancer, it is necessary to develop a technology for more efficient immunostaining and karyotype analysis of long-term cultured three-dimensional organoids. . This experimental technique applying the organoids of the present invention enables the identification of early stages of cancer and disease that could not be identified by conventional pathological readings or by DNA sequencing of lesions taken during histological examination. Application research using these three-dimensional organoids will be a springboard for providing customized medical care to actual patients by reflecting the in vivo situation where cells actually exist three-dimensionally.

Claims

(a) mixing the cells isolated from the individual with a hydrogel and culturing the in vitro tissue construct; And,

(b) removing the hydrogel from the ex vivo tissue construct; a method of confirming expression of a biomarker in an ex vivo tissue construct.
The method of claim 1,

The hydrogel is characterized in that it comprises one or more selected from the group consisting of collagen, fibrin, agarose, agar, matrigel, alginate, gelatin, extracellular matrix, or hyaluronic acid, in vitro tissue structure To confirm the expression of a biomarker in the.
The method of claim 2,

The hydrogel is characterized in that the matrigel, the method for confirming the expression of the biomarker in the tissue structure ex vivo.
The method of claim 1,

The ex vivo tissue construct is characterized in that at least one selected from the group consisting of artificial organs, bio organs, mini organs, organoids, spheroids, globulars, and embryos, the expression of the biomarker in the ex vivo tissue constructs How to.
The method of claim 1,

The biomarker is selected from the group consisting of antibodies, antigens, aptamers, receptors, ligands, enzyme substrates, inhibitors, enzyme cofactors, and enzymes that specifically bind to nucleic acids, proteins, sugars, or lipids expressed in cells. At least one selected, characterized in that the expression of the biomarker in an in vitro tissue construct.
The method of claim 5,

The biomarker is an antibody that specifically binds to a protein expressed in the cell, characterized in that the method for confirming the expression of the biomarker in an in vitro tissue construct.
The method of claim 6,

The antibody is treated for 2 to 8 days, the method of confirming the expression of the biomarker in the tissue structure ex vivo.
(a) mixing the cells isolated from the individual with a hydrogel and culturing the in vitro tissue construct; And,

(b) removing the hydrogel from the ex vivo tissue construct.
The method of claim 8,

After the step (a) further comprising the step of treating the ex vivo tissue structure to the colseamide 200 to 400ng / mL; further comprising, the method for determining karyotype in the in vitro tissue structure.
The method of claim 8,

The hydrogel is characterized in that it comprises one or more selected from the group consisting of collagen, fibrin, agarose, agar, matrigel, alginate, gelatin, extracellular matrix, or hyaluronic acid, ex vivo tissue structure To check for karyotypes.
The method of claim 10,

The hydrogel is characterized in that the matrigel, the method for confirming the expression of the biomarker in the tissue structure ex vivo.
The method of claim 8,

The ex vivo tissue structure is characterized in that at least one selected from the group consisting of artificial organs, bio organs, mini organs, organoids, spheroids, globulars, and embryos, karyotype in the ex vivo tissue structure.
The method of claim 8,

The karyotype identification is characterized in that for identifying the qualitative abnormality, or numerical abnormality of the chromosome, the method for identifying karyotype in the ex vivo tissue structure.
(a) obtaining tissue isolated from the subject in a non-surgical manner;

(b) mixing the cells isolated from the tissue with a hydrogel and culturing the in vitro tissue construct;

(c) removing the hydrogel from the ex vivo tissue construct; And,

(d) confirming the expression of the biomarker in the hydrogel removed ex vivo tissue construct; a method comprising providing a rule for diagnosing a disease of a tissue.
(a) obtaining tissue isolated from the subject in a non-surgical manner;

(b) mixing the cells isolated from the tissue with a hydrogel and culturing the in vitro tissue construct;

(c) removing the hydrogel from the ex vivo tissue construct; And,

(d) identifying karyotypes in the hydrogel-depleted ex vivo tissue construct; a method comprising providing a rule for diagnosing a disease of a tissue.