Medium for culturing and / or preserving cancer cells, method for primary culturing cancer cells, method for subculturing cancer cells, and method for preserving cancer cells

Abstract

The objective of the present invention is to provide a medium suitable for the primary culture of cancer cells. A medium for culturing and / or preserving cancer cells, the medium comprising: Contains at least a ROCK inhibitor and monothioglycerol The problem can be solved by using culture media.

Description

[Technical Field]

[0001] The disclosure in this application relates to a medium for culturing and / or preserving cancer cells, a method for primary culturing cancer cells, a method for subculturing cancer cells, and a method for preserving cancer cells. [Background technology]

[0002] Established cancer cell lines are commonly used in basic research into chemotherapy, including anticancer drugs. However, cancer cell lines that have been maintained and cultured in vitro for many years may have changed in properties from the original patient tumor tissue and may not adequately reflect their behavior in vivo. Therefore, primary culture of cancer cells is considered promising for more precise anticancer drug development and selection of optimal treatment for each patient.

[0003] Several primary culture methods are already known. For example, Patent Document 1 describes (1) a method for primary culturing cells in tissue (biological tissue) collected from a living body using a culture medium commonly used for cell culture without adding any special growth factors or inhibitors, and (2) a method for seeding and culturing cells in tissue collected from a living body on the top surface of a cell structure that contains cells constituting the interstitium and has a single layer or two or more cell layers stacked in the thickness direction.

[0004] Patent Document 2 describes a primary culture method that enables patient-derived cancer cells to be mass-cultured in vitro simply, quickly, and stably by (a) shredding cancer tissue derived from a living body, removing impurities from the shredded cancer tissue, (b) subjecting the tissue mass obtained in step (a) to suspension culture, and then (c) subjecting the culture obtained in step (b) to adhesion culture. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 039457 [Patent Document 2] Japanese Patent Application Publication No. 2018-011576 Summary of the Invention [Problem to be solved by the invention]

[0006] The success rate of primary culture of cancer cells from patient cancer tissue varies depending on the organ. While the success rate of primary culture of cancer cells from solid organs such as colon, stomach, and liver cancer is generally high, primary culture is not always successful. Lung cancer, on the other hand, is considered one of the most difficult cancer types to culture, with the success rate of primary culture of cancer cells from lung cancer tissue generally estimated to be around 10% (Giard DJ, et al., "In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors." J Natl Cancer Inst., 1973 Nov;51(5):1417-23. doi:10.1093 / jnci / 51.5.1417. PMID: 4357758.).

[0007] However, Patent Documents 1 and 2 do not mention that the composition of the medium is examined with a focus on the success rate of primary culture. In the field of primary culture of cancer cells, it is desired to provide a medium suitable for primary culture.

[0008] The present invention has been disclosed to solve the above-mentioned problems. As a result of extensive research, the present inventors have newly discovered that primary culture of cancer cells can be preferably carried out by using a medium containing at least a ROCK inhibitor and monothioglycerol.

[0009] That is, an object of the disclosure of the present application is to provide a medium suitable for the primary culture of cancer cells. [Means for solving the problem]

[0010] The disclosure of the present application relates to a medium for culturing and / or preserving cancer cells, a method for primary culturing cancer cells, a method for subculturing cancer cells, and a method for preserving cancer cells, which are shown below.

[0011] (1) A medium for culturing and / or preserving cancer cells, the medium comprising: Contains at least a ROCK inhibitor and monothioglycerol Culture medium. (2) The ROCK inhibitor is At least one selected from the group consisting of Y-27632 2HCl, Y-27632, H-1152 dihydrochloride, Hydroxyfasudil (HA-1100) HCl, GSK269962A HCl, Thiazovivin, Fasudil (HA-1077) HCl, GSK429286A, RKI-1447, Azaindole 1 (TC-S 7001), Y-39983 HCl, Netarsudil (AR-13324) 2HCl, Belumosudil (KD025), and AT13148. The medium described in (1) above. (3) The lock inhibitor is at least one selected from the group consisting of Y-27632 2HCl, Y-27632, H-1152 dihydrochloride, hydroxyfasudil (HA-1100) HCl, and GSK269962A HCl. The medium described in (2) above. (4) The final concentration of the ROCK inhibitor in the medium is 0.05 μmol / L or more and 100 μmol / L or less; The final concentration of monothioglycerol in the medium is 10 μmol / L or more and 5000 μmol / L or less. The medium described in (1) above. (5) The final concentration of the ROCK inhibitor in the medium is 0.05 μmol / L or more and 100 μmol / L or less; The final concentration of monothioglycerol in the medium is 10 μmol / L or more and 5000 μmol / L or less. The medium described in (3) above. (6) Further containing an antibiotic and / or antifungal agent The medium described in (1) above. (7) The cancer is lung cancer. The medium described in (1) above. (8) The medium is a medium for preserving cancer cells, Cancer cells are stored at room temperature The medium according to any one of (1) to (7) above. (9) A method for primary culture of cancer cells, the method comprising: A culture step of culturing cancer cells collected from a living body using the medium according to any one of (1) to (7) above. Primary culture method. (10) A method for subculturing cancer cells, the method comprising: The method includes a passaging step in which, during passaging, the cancer cells cultured by the primary culture method described in (9) above are removed from the culture vessel in a state in which adhesion of the cancer cells to the wall of the culture vessel is reduced by using a chelating agent that binds to divalent metal ions, and the cancer cells are transferred to a culture vessel containing a new medium. Subculture method. (11) A method for preserving cancer cells, the method comprising: A preservation step of immersing cancer cells collected from a living body in the medium according to any one of (1) to (7) above and preserving the cells at room temperature. How to save. (12) The medium contains fetal bovine serum The storage method described in (11) above. [Effects of the Invention]

[0012] The medium disclosed in the present application can be used to suitably carry out primary culture of cancer cells. The medium disclosed in the present application can also be used as a medium for subculture and preservation of cancer cells. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a photograph used as a drawing, showing lung cancer cells obtained by primary culture in Example 2. [Figure 2] FIG. 2 is a photograph used as a drawing, showing the third generation of lung cancer cells obtained by subculture in Example 4. [Figure 3] FIG. 3 is a photograph used as a drawing, showing the fourth generation of lung cancer cells obtained by subculture in Example 4. [Figure 4] FIG. 4 is a photograph used as a drawing, which shows lung cancer cells obtained by primary culture after storage at room temperature for 3 days in Example 6. [Figure 5] FIG. 5 is a photograph used as a drawing, showing lung cancer cells obtained by primary culture after storage in a refrigerator for three days in Comparative Example 4. [Figure 6] FIG. 6 is an electrophoresis photograph showing the results of examining the quality of DNA extracted from formalin-fixed, paraffin-embedded (FFPE), DNA extracted from Rapid-FFPE, and DNA extracted from Culture Cell in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0014] The following provides a detailed description of the culture medium for culturing and / or preserving cancer cells (hereinafter sometimes simply referred to as "culture medium"), the method for primary culturing cancer cells (hereinafter sometimes simply referred to as "primary culture method"), the method for subculturing cancer cells (hereinafter sometimes simply referred to as "subculturing method"), and the method for preserving cancer cells (hereinafter sometimes simply referred to as "preservation method") disclosed in the present application.

[0015] In addition, in this specification, (1) A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits. (2) Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field. (3) When describing something as "approximately ____ shape," it includes not only the exact ____ shape, but also a shape that can be understood as roughly ____ shape. This is interpreted as:

[0016] (Embodiments of culture medium) A medium according to an embodiment contains at least a ROCK inhibitor and monothioglycerol.

[0017] ROCK (Rho kinase; Rho-associated protein kinase) is a serine-threonine protein kinase identified as a target protein of the small GTP-binding protein Rho, and is involved in various physiological functions such as smooth muscle contraction and cell morphological changes. ROCK inhibitors are compounds that inhibit the activity of ROCK. There are no particular limitations on the ROCK inhibitors as long as they can inhibit ROCK activity. Commercially available ROCK inhibitors include, but are not limited to, Y-27632 2HCl, Y-27632, H-1152 dihydrochloride, Hydroxyfasudil (HA-1100) HCl, GSK269962A HCl, Thiazovivin, Fasudil (HA-1077) HCl, GSK429286A, RKI-1447, Azaindole 1 (TC-S 7001), Y-39983 HCl, Netarsudil (AR-13324) 2HCl, Belumosudil (KD025), AT13148, etc. The medium may contain one type of ROCK inhibitor, or two or more types may be combined.

[0018] The ROCK inhibitors exemplified above are the compounds shown below. JPEG0007735022000001.jpg214111JPEG0007735022000002.jpg215111

[0019] Among the ROCK inhibitors exemplified above, Y-27632 2HCl, Y-27632, H-1152 dihydrochloride, Hydroxyfasudil (HA-1100) HCl, and GSK269962A HCl are more preferred.

[0020] Monothioglycerol is a compound represented by the following formula (15), and is used as a stabilizer and antioxidant for antibiotics, as well as a curling agent (perm solution) and an antibacterial preservative due to its excellent reducing ability to cleave the SS bonds in hair. JPEG0007735022000003.jpg41108

[0021] As for the medium, a known cell culture medium can be used as the basal medium, except that it contains a ROCK inhibitor and monothioglycerol as essential ingredients. Examples include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM:F-12), Eagle's minimal essential medium (EMEM), Minimum Essential Medium Alpha (MEMα), Basal Medium Eagle (BME), and RPMI-1640. Various components, such as cell growth factors, can be added to these media as needed, and the composition can be adjusted to suit the cell type.

[0022] Examples of various components commonly added to culture media include cell growth factors such as FGF-2 (Fibroblast Growth Factor-2), TGF-β (Transforming Growth Factor-β), EGF (Epidermal Growth Factor), and VEGF (Vascular Endothelial Growth Factor); vitamins or vitamin derivatives such as ascorbic acid and retinoic acid; sugar sources such as glucose; amino acids; inorganic salts such as sodium selenite and sodium chloride; proteins such as transferrin; hormones such as insulin; differentiation inhibitors; differentiation inducers such as dexamethasone and oncostatin M; and antioxidants such as 2-mercaptoethanol and dithiothreitol.

[0023] Furthermore, depending on the type of cancer (particularly stomach cancer and colon cancer), if cells collected from a patient are cultured as is, bacteria or fungi attached to the collected cancer cells may proliferate. Therefore, antibiotics and / or antifungal agents may be added to the culture medium as needed. Antibiotics include, but are not limited to, penicillin, streptomycin, gentamicin, etc., and antifungal agents include amphotericin B, fluconazole, etc.

[0024] The cancer may be of any type, including, but not limited to, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, anal cancer, pancreatic cancer, liver cancer, bile duct cancer, gastrointestinal endocrine tumor, gastrointestinal stromal tumor (GIST), breast cancer, lung cancer, mesothelioma, thymic cancer, kidney cancer, urothelial cancer, testicular tumor, prostate cancer, endometrial cancer, cervical cancer, uterine sarcoma, ovarian malignant tumor, tongue cancer, gum cancer, floor of mouth cancer, pharyngeal cancer, laryngeal cancer, salivary gland cancer, thyroid cancer, osteosarcoma, Ewing's sarcoma, soft tissue sarcoma, myelodysplastic syndrome, skin cancer, neuroblastoma, malignant glioma (glioblastoma), malignant lymphoma, and multiple myeloma.

[0025] The culture medium according to the embodiment has been able to achieve a high success rate of primary culture of not only stomach cancer, liver cancer, and pancreatic cancer, but also lung cancer, which has traditionally been considered difficult to culture, as will be shown in the examples described below.

[0026] The concentrations of the ROCK inhibitor and monothioglycerol added to the medium are not particularly limited as long as they are within a range that allows suitable primary culture of cancer cells. The final concentration of the ROCK inhibitor in the medium per 1 L of medium can be, but is not limited to, a lower limit of 0.05 μmol or more, 0.06 μmol or more, 0.07 μmol or more, 0.08 μmol or more, 0.1 μmol or more, 0.2 μmol or more, 0.4 μmol or more, 0.6 μmol or more, 0.8 μmol or more, 1 μmol or more, 2 μmol or more, 3 μmol or more, 4 μmol or more, 5 μmol or more, 6 μmol or more, 7 μmol or more, 8 μmol or more, 9 μmol or more, 10 μmol or more, and an upper limit of 100 μmol or less, 90 μmol or less, 80 μmol or less, 70 μmol or less, 60 μmol or less, 50 μmol or less, and the like.

[0027] The final concentration of monothioglycerol in the medium is not limited, and examples of the lower limit per 1 L of medium include 10 μmol or more, 20 μmol or more, 30 μmol or more, 40 μmol or more, 50 μmol or more, 60 μmol or more, 70 μmol or more, 80 μmol or more, 90 μmol or more, and 100 μmol or more, and examples of the upper limit include 5000 μmol or less, 4000 μmol or less, 3000 μmol or less, 2000 μmol or less, 1000 μmol or less, 750 μmol or less, and 500 μmol or less.

[0028] Furthermore, as shown in the examples described below, the culture medium according to the embodiment can be suitably used not only for primary culture but also for subculture and preservation of cancer cells collected from patients. In particular, cancer cells do not need to be refrigerated when being stored; they can be stored at room temperature. This improves the convenience of handling collected cancer cells. As used herein, "room temperature" refers to the temperature at which cancer cells are stored without using a cooling or heating method, and generally refers to a temperature between approximately 15°C and 30°C, although this varies depending on the season. As used herein, "preservation" refers to maintaining the viability of collected cancer cells while minimizing their death during the period from when the cancer cells are collected from a living body until the primary culture method is performed (the preservation period). During the preservation period, the cancer cells may be left in place or transported (for example, from the hospital where the cancer cells were collected to a facility where the primary culture is performed).

[0029] The medium disclosed in the present application has the following effects. (1) This method not only increases the success rate of primary culture of lung cancer, which has traditionally been difficult to achieve, but can also be used for primary culture of cancer cells other than lung cancer. Therefore, because it can be used for primary culture of cancer cells of any type, it is useful for clinical applications such as genetic analysis for diagnosis and treatment and selection of anticancer drugs, as well as basic research into the molecular mechanisms of cancer, gene mutation analysis, and drug discovery research. (2) In addition to primary culture and subculture, it can also be used as a medium for preserving cancer cells. Therefore, a single medium can be used for multiple purposes, making it useful in clinical settings.

[0030] (Embodiment of Primary Culture Method) Next, a primary culture method according to an embodiment will be described. The primary culture method includes a culture step of culturing collected cancer cells using any of the media described in the above medium embodiments.

[0031] The subject from which cancer cells are to be collected may be any living organism (living animal), preferably a human with cancer. Cancer tissue from a living organism may be collected by a known method, including, but not limited to, collection or excision during surgery, biopsy, etc.

[0032] The culture method for the culture step is not particularly limited as long as it can culture cancer cells collected from a living body. Typically, tissue containing cancer cells collected from a living body is shredded before culture. The shredding can be performed using a knife, scissors, or other tools until the tissue mass is no longer visible to the naked eye (approximately 1 mm square or less). The culture method can be explant culture, in which the shredded tissue containing cancer cells is cultured as is, or enzyme-treated dispersed culture, in which the shredded tissue containing cancer cells is separated into cells using an enzyme and then cultured. Note that the term "culturing cancer cells" used herein encompasses both the culturing of tissue containing shredded cancer cells and the culturing of cancer cells separated into cells. Furthermore, the term "cancer cells" used herein encompasses both "isolated cancer cells" and "tissue containing cancer cells." Enzymes used in enzyme-treated dispersed culture are not particularly limited as long as they are commonly used in the field of cell culture, and examples include collagenase, trypsin, papain, dispase, etc.

[0033] Furthermore, if necessary, the cancer cells may be sieved using a washed and sterilized filter to remove contaminants.

[0034] Known cancer cell culture methods include adherent culture, in which cancer cells are grown in a monolayer on an artificial substrate, and suspension culture, in which cancer cells are grown in a free-floating state in a medium. Adherent culture is suitable for most cell types, including primary culture, while suspension culture is primarily suitable for non-adherent cells. Therefore, while adherent culture is a preferred example of the primary culture method according to the present embodiment, cancer cells may be initially cultured in suspension culture to form cell aggregates, followed by adherent culture.

[0035] Culture vessels used in the culture process may be well-known vessels commonly used in the field of cell culture. For adherent culture, commercially available vessels with surfaces treated to facilitate cell or cell aggregate adhesion may be used, although this is not limited thereto. Depending on the type and tissue type of cancer, cells may be seeded on plates immobilized with laminin 511, laminin 411, soluble E-cadherin, or the like. The shape and size of the culture vessel can also be selected depending on the type of cancer cells, the required amount, and other conditions. Flasks, bottles, dishes, tubes, plates, and other vessels of various sizes are commercially available, and these can be used.

[0036] For suspension culture, commercially available culture vessels for suspension culture have surfaces treated to prevent cells or cell aggregates from adhering, and these may be used. The shape and size of the culture vessel can also be selected depending on conditions such as the type of cancer cells and the required amount. Flasks, bottles, dishes, tubes, plates, and other vessels of various sizes are commercially available, and these can be used.

[0037] The culture conditions, such as culture time and temperature, may be appropriately determined by the person carrying out the culture depending on the type of cancer.

[0038] The primary culture method according to the embodiment has the following effects. (1) The primary culture of cancer cells grown in the culture process resembles the clinical tissue of the living body from which it was collected. In other words, the state of cancer tissue in a living body can be reproduced in vitro. Therefore, the primary culture obtained by the primary culture method according to the embodiment can be used to elucidate the molecular mechanisms of cancer, drug sensitivity tests, screening of pharmaceuticals suitable for the living body (patient) from which it was collected, genetic analysis of cancer cells, etc. It can also be used in research and development, such as drug discovery. (2) By using the medium disclosed in this application, the success rate of lung cancer cells, which are considered particularly difficult to culture as primary cultures, is improved to approximately 70%. In this specification, "success rate" means that the number of cancer cells increases by approximately 100 times through primary culture. By improving the success rate, the probability of performing the elucidation of the molecular mechanisms of cancer, drug sensitivity testing, screening of appropriate pharmaceuticals for the collected living body (patient), genetic analysis of cancer cells, etc., as described in (1) above, increases. Therefore, personalized medical care specific to each patient can be realized. (3) Cancer tissues collected during surgery or other procedures are generally fixed in formalin, and paraffin blocks are prepared for use in pathological observations. However, the formalin used for tissue fixation causes cross-linking between nucleic acids (e.g., DNA) and proteins, making DNA susceptible to physical stress. Furthermore, formalin can be oxidized to form formic acid, which depurinates DNA and causes DNA strand breaks. Therefore, even if formalin-fixed cancer tissues are used for DNA analysis, the quality of the DNA is very poor. On the other hand, as shown in the examples below, cancer cells obtained by the primary culture method can yield high-quality gDNA, improving the accuracy of genetic analysis.

[0039] (Embodiment of Subculture Method) Next, a subculture method according to an embodiment will be described. The subculture method includes a subculture step in which, during subculture, cancer cells cultured by the primary culture method described above are removed from the culture vessel in a state in which adhesion of the cancer cells to the wall of the culture vessel is reduced by using a chelating agent that binds to divalent metal ions, and the cancer cells are then transferred to a culture vessel containing a new medium.

[0040] The medium used in the subculture method may be the same as the medium used in the primary culture method. Alternatively, the medium components used in the subculture method may be different from those used in the primary culture method, as long as they contain a ROCK inhibitor and monothioglycerol. Furthermore, the subculture performed after the subculture step is preferably an adherent culture, but suspension culture is also acceptable. The subculture step and subculture may be repeated multiple times.

[0041] There are no particular limitations on the chelating agent, as long as it binds to divalent metal ions (calcium ions, zinc ions, copper ions, magnesium ions, iron ions, etc.) contained in the medium. Examples include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), and 1,2-bis(o-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid (BAPTA).

[0042] The concentration of the chelating agent added during subculture is not particularly limited as long as it allows the cultured cells to be removed from the culture vessel and does not cause significant damage to the cells. Although not limited, approximately 0.5 to 1 mL of a 1 mM chelating agent solution may be added.

[0043] In conventional cell culture methods, enzymes are used to detach cells from the culture vessel during passaging. However, the use of enzymes can weaken the cell membrane and receptors on the cell membrane surface, weakening the cells and reducing proliferation. Furthermore, weakly expressed receptors on the cell membrane surface can disappear, making them undetectable during cell membrane surface analysis. In contrast, the passaging method according to the embodiment uses a chelating agent that binds to divalent metal ions, allowing cancer cells to be easily removed from the culture vessel. In other words, the passaging process can be considered a process of removing cancer cells from the culture vessel without using enzymes and transferring them to a culture vessel containing new medium.

[0044] The subculture method according to the embodiment provides the effect of improving the efficiency of subculture.

[0045] (Embodiment of storage method) Next, a preservation method according to an embodiment will be described. The preservation method according to an embodiment includes a preservation step of immersing cancer cells collected from a living body in the medium described in the embodiment of the medium and preserving the cells at room temperature.

[0046] The culture medium has already been described in the above-mentioned embodiment of the culture medium, and the cancer cells collected from a living body have already been described in the above-mentioned embodiment of the primary culture method. Therefore, detailed description will be omitted to avoid redundancy.

[0047] Storing cancer cells in a refrigerator damages the cells. Therefore, even if cancer cells stored in a refrigerator are subjected to primary culture, they may not grow. On the other hand, the storage method according to the embodiment allows storage at room temperature by using the culture medium disclosed in the present application. Therefore, the effect of improving the convenience of handling cells when primary culturing cancer cells collected by surgery is obtained. Furthermore, since the cells can be stored at room temperature, for example, cancer cells collected at a clinic that does not have a cell culture facility can be transported at room temperature to a facility capable of primary culture, thereby improving the convenience of transportation.

[0048] The storage period is not particularly limited as long as the cancer cells are not killed or their proliferation ability is not significantly reduced during storage, but approximately 1 to 4 days is preferable. Optionally, antibiotics such as penicillin, streptomycin, and gentamicin may be added to the medium.

[0049] When cancer cells collected using a storage medium are stored for a long period of time, the storage medium preferably contains FBS (fetal bovine serum). In addition to FBS, it is also desirable to contain a serum substitute (serum-free supplement) such as KSR. The inclusion of FBS (and serum-free supplements) ensures stable storage of cancer cells. The concentration of FBS contained in the storage medium is not limited, but examples of lower limits include 0.5% by volume or more, 0.75% by volume or more, 1.0% by volume or more, 1.5% by volume or more, 2.0% by volume or more, 2.5% by volume or more, and 3.0% by volume or more. Examples of upper limits include 20% by volume or less, 18% by volume or less, 16% by volume or less, 14% by volume or less, 12% by volume or less, and 10% by volume or less.

[0050] The concentration of the serum-free supplement is not limited, but examples of lower limits include 0.5 vol% or more, 0.75 vol% or more, 1.0 vol% or more, 1.5 vol% or more, 2.0 vol% or more, 2.5 vol% or more, and 3.0 vol% or more, and examples of upper limits include 20 vol% or less, 18 vol% or less, 16 vol% or less, 14 vol% or less, 12 vol% or less, and 10 vol% or less.

[0051] If animal-derived components are included in the storage medium, there is a risk of the cultured cancer cells becoming contaminated with viruses, etc. Even if the cultured cancer cells are contaminated with viruses, this does not pose a particular problem if they are used for research purposes or screening for drug discovery, etc. However, from the perspective of increasing safety, if the storage period of the collected cancer cells is short, it is preferable that the storage medium does not contain FBS (and serum-free supplements). In this specification, "long-term storage" means storage for 3 days or more, and "short-term storage" means storage for less than 3 days.

[0052] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the technical scope of the disclosure in the present application. [Example]

[0053] [Preparation of culture medium] Example 1 A medium was prepared with the following composition. 1. Media for primary culture and subculture (common to both adherent and suspension culture), storage media (for short-term storage) 1 μM A83-01 (TGF-β inhibitor, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) ·50ng / mL EGF (epidermal growth factor. Manufactured by Sigma-Aldrich) 2mM CHIR99021 (GSK-3 inhibitor, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 1 μmol / L Y-27632 (ROCK inhibitor, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) ·×100 N2 Supplement (Thermo Fisher) ·×50 B27 Supplement (Thermo Fisher) 20ng / mL bFGF (basic fibroblast growth factor, Sigma-Aldrich) 2mM Glutamax TM Supplement (glutamic acid, manufactured by Thermo Fisher Scientific) 50 μmol / L monothioglycerol (Fujifilm Wako Pure Chemical Industries, Ltd.) ·×100 MEM Non-Essential Amino Acids Solution (manufactured by Thermo Fisher) 0.5mg / mL BSA (bovine serum albumin, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 100 units / mL Penicillin G (Fujifilm Wako Pure Chemical Industries, Ltd.) 100 μg / mL streptomycin sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0054] 2.Long-term storage medium 5% by volume FBS (fetal bovine serum, Thermo Fisher Scientific) 2.5% by volume KSR (serum-free supplement, manufactured by Thermo Fisher Scientific) 1 mmol / L sodium pyruvate (Thermo Fisher) 2.5% by volume Human Serum (Sigma-Aldrich) 2mM Glutamax TM Supplement (glutamic acid, manufactured by Thermo Fisher Scientific) 1 μmol / L Y-27632 (ROCK inhibitor, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 50ng / mL SAG (Smoothened agonist, manufactured by Abcam) 50 μmol / L monothioglycerol (Fujifilm Wako Pure Chemical Industries, Ltd.) RPMI1640 medium (Thermo Fisher) 100 units / mL Penicillin G (Fujifilm Wako Pure Chemical Industries, Ltd.) 100 μg / mL streptomycin sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0055] [Primary culture] <Example 2> 1.Culture container In Example 2, the following culture vessels were used. Multi-well plate for adherent culture (1 / 12 well). The multi-well plate for adherent culture was coated with iMatrix-511 Coat (Takara Bio) overnight in a refrigerator. Culture medium 1mL / well. HydroCell plates (24 wells) for suspension culture. Note that plates for suspension culture are not coated with iMatrix-511 coat (manufactured by Takara Bio Inc.). Culture medium 1mL / well.

[0056] 2. Primary Culture Procedure (1) Lung cancer tissue collected during surgery was placed in a 15 mL tube containing 2 mL of short-term storage medium and stored for 2 hours. (2) The tissue was transferred to a 10 cm dish using a wide-tipped tip, and a small amount of short-term storage medium was added. The tissue was then cut into small pieces (approximately 0.5-1 mm square) using a scalpel blade. The cut pieces were then placed back into the short-term storage medium and incubated at 37°C for 30 minutes. (3) The mixture was diluted to 10 mL with PBS and washed by centrifugation (1,000 rpm, 5 min). (4) The supernatant was removed by suction and subjected to enzyme treatment. Specifically, 2 mL of 0.1% Collagenase I was added, vortexed, and then subjected to enzyme reaction (37°C, 20-30 min, shaking). Collagenase I (SIGMA) was diluted 5-fold from 0.5% with HBSS (containing Mg and Ca). (5) The mixture was diluted to 10 mL with 9 mL of PBS and 1 mL of CELLOTION (manufactured by Takara Bio Inc.) and centrifuged for washing (1,000 rpm, 5 minutes). (6) 2 mL of PBS was added, and the tissue was dissociated by pipetting. The resulting 2 mL solution was filtered into a new 15 mL tube, along with the tissue debris. The tissue debris remaining on the filter was gently crushed with a pipette, and the tube was washed with approximately 7 mL of PBS and filtered. 1 mL of CELLOTION was added, and the mixture was centrifuged (1,000 rpm, 5 min). (7) The supernatant was removed by suction to the last drop, and 3 mL of hemolytic agent was added. The mixture was vortexed to perform hemolysis (4°C, 15 minutes). (8) The mixture was diluted to 10 mL with 1 mL of PBS and CELLOTION, and then centrifuged (1,000 rpm, 5 min) for washing. (9) The supernatant was removed by aspiration, and the volume was again increased to 10 mL with PBS and 1 mL of CELLOTION, followed by centrifugation (1,000 rpm, 5 min). Before centrifugation, 12 μL of the cell suspension was added to a counting chamber and the number of cells was counted. (10) The supernatant was removed by suction to 100 μL, and the cancer cells were seeded (number of cells to be seeded was determined appropriately) onto a medium for primary culture (a well plate for adhesion culture or a plate for suspension culture described in "1. Culture vessel" above). (11) The cells were cultured for approximately 7 days in a cell culture incubator at 37°C with 5% carbon dioxide. The medium was changed approximately once every 2 to 3 days.

[0057] In Example 2, lung cancer tissues collected from 62 patients (N = 62) were divided into two halves, and experiments were performed on each half using adherent culture and suspension culture. In other words, samples from the same patient were examined using two culture methods: adherent culture and suspension culture. The results showed that primary culture was successful in 30 cases using both adherent and suspension culture, 4 cases using adherent culture alone, and 12 cases using suspension culture alone. In other words, of the 62 cases, primary culture was successful (a 100-fold or greater increase in lung cancer cell numbers) in both adherent and suspension culture or either one of them in 46 cases. Approximately 74% of lung cancer tissues collected from living subjects were successfully cultured. Figure 1 shows a photograph of lung cancer cells obtained by primary culture (adherent culture).

[0058] <Comparative Example 1> An experiment was conducted using the same procedure as in Example 2 (N=11), except that the ROCK inhibitor was not added to the medium composition of Example 1. As a result, cancer cells hardly proliferated in either adherent culture or suspension culture.

[0059] <Comparative Example 2> An experiment was conducted in the same manner as in Example 2 (N=8), except that monothioglycerol was not added to the medium composition of Example 1. The cancer cells were cultured in both adherent and suspension culture media, and their numbers decreased by 20% within one week.

[0060] The results of Example 2 and Comparative Examples 1 and 2 confirmed that the use of a medium supplemented with a combination of a ROCK inhibitor and monothioglycerol enables primary culture with a high success rate, even for lung cancer cells, which are known to have a very low success rate for primary culture. Furthermore, it was confirmed that the medium disclosed in the present application is useful for both adherent culture and suspension culture.

[0061] Example 3 Primary culture was performed using the same procedures as in Example 2, except that cancer tissue collected during surgery for osteosarcoma, gastric cancer, ovarian cancer, colon cancer, pancreatic cancer, and esophageal cancer was used instead of lung cancer tissue (except that 2.5 μg / mL amphotericin B (Fujifilm Wako Pure Chemical Industries, Ltd.) and 5 μg / mL fluconazole (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to gastric cancer and colon cancer). The success rates of primary culture (cell numbers proliferating 100-fold or more) were as follows. Primary culture was successful in both adherent culture and suspension culture, but unlike in Example 2, only the results of the culture method with the highest success rate are shown. Osteosarcoma: Successful in 3 out of 4 cases. Success rate 75%. Gastric cancer: Successful in 7 out of 10 cases. Success rate: 70%. The 3 failed cases were discontinued due to fungal contamination, but the proliferation of cancer cells was confirmed. Ovarian cancer: Successful in 2 out of 3 cases. Success rate 67%. Colon cancer: Successful in 4 out of 5 cases, a success rate of 80%. Pancreatic cancer: Successful in 6 out of 10 cases, a success rate of 60%. Of the 4 unsuccessful cases, it was separately confirmed that the resected tissue in 3 cases was free of cancer cells. Esophageal cancer: Successful in 2 out of 2 cases. Success rate 100%.

[0062] From the above results, it was confirmed that the medium disclosed in the present application is useful for the primary culture of not only lung cancer but also many other types of cancer. In particular, the success rate of primary culture of lung cancer cells was reported to be about 10% using conventional methods, but by using the medium disclosed in the present application, the success rate was significantly improved to about 74%.

[0063] [Subculture] Example 4 Following the primary culture in Example 2 above, subculture was carried out according to the following procedure. For the subculture, lung cancer cells that had been primarily cultured by adhesion culture were used. The subculture process is described below. (1) The medium was aspirated and washed with PBS. (2) 1 mL of 1 mM EDTA solution was added as a chelating agent, and the cells were allowed to react for 5 to 15 minutes in a 37°C carbon dioxide incubator. The cells were observed under a microscope every 5 minutes, and when the cells began to detach from the dish, PBS was added and the cells were recovered by pipetting. (3) The mixture was transferred to a 15 mL tube, and the contents were diluted to 10 mL with PBS and 1 mL of CELLOTION, followed by centrifugation washing (1,000 rpm, 5 min).

[0064] Some of the second-generation lung cancer cells were further subcultured. Figure 2 shows a photograph of the third generation, and Figure 3 shows a photograph of the fourth generation. The success rate of subculture from the second to fourth generations was approximately 90%.

[0065] <Comparative Example 3> Subculture was carried out in the same manner as in Example 4, except that an enzyme (0.05% Trypsin, 1 mL) was added instead of the chelating agent used in Example 4. As a result, many cancer cells died, and 50% to 70% of the cells could not be subcultured.

[0066] [Primary culture and subculture using other ROCK inhibitors] <Example 5> Primary culture was performed in the same manner as in Example 2, except that four ROCK inhibitors were used instead of Y-27632 in Example 2: Y-27632 2HCl (MedChemExpress), H-1152 dihydrochloride (MedChemExpress), Hydroxyfasudil (HA-1100) HCl (MedChemExpress), and GSK269962A HCl (MedChemExpress). The success rates of primary culture were as follows. Note that primary culture was successful in both adherent and suspension culture, but unlike in Example 2, only the results of the culture method with the highest success rate are shown. Y-27632 2HCL: Successful in 2 out of 3 cases. Success rate: 67%. H-1152 dihydrochloride: Successful in 3 out of 4 cases. Success rate 75%. ·Hydroxyfasudil (HA-1100) HCL: Successful in 3 out of 4 cases. 75% success rate. GSK269962A HCL: Successful in 2 of 3 cases. Success rate: 67%.

[0067] Furthermore, lung cancer (3 cases), pancreatic cancer (2 cases), and gastric cancer (3 cases) were selected from the lung cancer, pancreatic cancer, and gastric cancer cells cultured to the second generation using the four types of ROCK inhibitors in Example 5, and further subcultured. All of these were successfully subcultured to the fourth generation. The cell proliferation rate after subculture was 0.99 for H-1152 dihydrochloride, 1.13 for Hydroxyfasudil (HA-1100) HCl, and 1.03 for GSK269962A HCl, with Y-27632 2HCl as the standard (1), indicating similar proliferation rates.

[0068] From the above results, it was confirmed that the use of the medium disclosed in the present application enables primary culture and subculture with a high success rate regardless of the type of cancer cells.

[0069] [Preservation medium] <Example 6, Comparative Example 4> Lung cancer cells collected from a living body were immersed in a long-term storage medium and stored at room temperature (Example 6) or in a refrigerator (Comparative Example 4) for three days, after which primary culture (adherent culture only) was performed using the same procedure as in Example 2. Figure 4 shows a photograph of lung cancer cells stored at room temperature on day 7 of primary culture. Figure 5 shows a photograph of lung cancer cells stored in a refrigerator on day 7 of primary culture. As is clear from the photograph in Figure 4, cancer cells collected from a living body proliferated even when stored at room temperature in a long-term storage medium. On the other hand, as is clear from the photograph in Figure 5, when cancer cells collected from a living body were stored in a refrigerator, the proliferation of the cancer cells was inferior to that when stored at room temperature. Generally, cancer cells collected from a living body are stored in a refrigerator if they are not immediately cultured. By using the medium disclosed in the present application, collected cancer cells can be stored at room temperature, thereby improving the convenience of handling the collected cells.

[0070] <Comparative Example 5> An experiment was carried out in the same manner as in Example 6, except that a medium was used that was the same as in Example 2 but without the ROCK inhibitor and monothioglycerol, i.e., a conventionally commonly used medium. In Comparative Example 5, an experiment was also carried out using ovarian cancer cells in addition to lung cancer cells. The results were as follows. Lung cancer: 0 out of 3 cases were successful. The success rate is 0%. Ovarian cancer: 0 out of 2 cases were successful. Success rate 0%.

[0071] From the above results, it was confirmed that the medium disclosed in the present application can be used for primary culture, subculture, and storage. Furthermore, it was confirmed that storage at room temperature is preferable for storing cancer cells.

[0072] [About DNA quality] Example 7 Next, we conducted an experiment to examine the DNA quality of cancer cells obtained from primary culture. The experimental procedure is as follows.

[0073] <Sample preparation> (1) Formalin-fixed, paraffin-embedded (FFPE) DNA Lung cancer tissue collected during surgery from four patients (referred to as Samples 1, 2, 3, and 4 in Figure 4) was fixed in formalin using standard procedures and for a standard processing time. The tissue was then sectioned and prepared into paraffin blocks for pathological diagnosis using standard processes (formalin-fixed paraffin-embedded: FFPE). Genomic DNA (gDNA) was extracted from these blocks using a commercially available DNA extraction kit from paraffin sections prepared using a microtome using standard techniques.

[0074] (2) Rapid-FFPE DNA Lung cancer tissues collected during surgery from the three patients (Sample 3 was not included) were immediately cut into pieces of approximately 5-10 mm in size and formalin-fixed paraffin blocks were prepared using standard methods. Paraffin sections were then prepared in the same manner as for FFPE, and gDNA was extracted.

[0075] (3) Culture Cell DNA Lung cancer tissues collected from the four patients during surgery were placed in short-term storage medium and transported to a cell culture room. Primary culture was then performed according to the procedure described in Example 2. gDNA was extracted from the cancer cells grown in the culture.

[0076] <Electrophoresis> The quality of the gDNA extracted in the sample preparation section above was confirmed using a TapeStation system (Agilent). Figure 6 shows the results. As shown in the gel electrophoresis results in Figure 6, almost no bands were visible in the FFPE samples. On the other hand, bands were visible in the Rapid-FFPE samples, but they were broad. This is likely due to the formalin used in tissue fixation causing cross-linking between proteins and DNA or DNA degradation. In contrast, the primary culture (Culture Cell) samples showed clean bands without DNA degradation. The DNA Integrity Number (DIN) value used to determine the relative quality of the genomic DNA (gDNA) in each sample was highest in Culture Cell samples, as can be seen in Figure 6. The DIN rating ranges from 1 to 10, with higher DIN values ​​indicating less gDNA degradation.

[0077] These results indicate that high-quality gDNA can be obtained from primary cultured cancer cells, and that this can be used for genetic analysis of individual patients. [Industrial Applicability]

[0078] The medium disclosed in the present application allows for the primary culture of cancer cells with a high success rate, and is therefore useful for the medical industry.

Claims

1. A medium for culturing and / or preserving cancer cells, the medium comprising: At least a ROCK inhibitor and monothioglycerol are included, the final concentration of the ROCK inhibitor in the medium is 0.05 μmol / L or more and 100 μmol / L or less; The final concentration of monothioglycerol in the medium is 10 μmol / L or more and 5000 μmol / L or less. Culture medium.

2. The ROCK inhibitor is At least one selected from the group consisting of Y-27632 2HCl, Y-27632, H-1152 dihydrochloride, Hydroxyfasudil (HA-1100) HCl, GSK269962A HCl, Thiazovivin, Fasudil (HA-1077) HCl, GSK429286A, RKI-1447, Azaindole 1 (TC-S 7001), Y-39983 HCl, Netarsudil (AR-13324) 2HCl, Belumosudil (KD025), and AT13148. The medium according to claim 1.

3. The ROCK inhibitor is at least one selected from the group consisting of Y-27632 2HCl, Y-27632, H-1152 dihydrochloride, hydroxyfasudil (HA-1100) HCl, and GSK269962A HCl. The culture medium according to claim 2.

4. The final concentration of monothioglycerol in the medium is 10 μmol / L or more (excluding 10 μmol / L) and 5000 μmol / L or less. The medium according to claim 1.

5. Further containing an antibiotic and / or antifungal agent The medium according to claim 1.

6. The cancer is lung cancer The medium according to claim 1.

7. the medium is a medium for preserving cancer cells, Cancer cells are stored at room temperature The culture medium according to any one of claims 1 to 6.

8. A method for primary culture of cancer cells, the method comprising: A culture step of culturing cancer cells collected from a living body using the medium according to any one of claims 1 to 6. Primary culture method.

9. A method for subculturing cancer cells, the method comprising: The method includes a passaging step in which, during passaging, the cancer cells cultured by the primary culture method according to claim 8 are removed from the culture vessel in a state in which adhesion of the cancer cells to the wall of the culture vessel is reduced by using a chelating agent that binds to divalent metal ions, and the cancer cells are then transferred to a culture vessel containing a new medium. Subculture method.

10. A method for preserving cancer cells, the method comprising: A preservation step of immersing cancer cells collected from a living body in the culture medium according to any one of claims 1 to 6 and preserving the cells at room temperature. How to save.

11. The medium contains fetal bovine serum The storage method according to claim 10.

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