Medium for culturing and / or storing cancer cells, method for primary culture of cancer cells, method for subculture of cancer cells, and method for storing cancer cells

Abstract

The present invention addresses the problem of providing a medium appropriate for primary culture of cancer cells.　This problem can be solved by a medium for culturing and / or storing cancer cells, the medium comprising at least ROCK inhibitor and monothioglycerol.

Description

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

[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.

[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 culture of cells in tissue (biological tissue) collected from a living body using a culture medium generally 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 containing cells constituting the interstitium and having 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.

[0005] International Publication No. 2019 / 039457 Japanese Patent Application Laid-Open No. 2018-011576

[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.

[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 culture medium for culturing and / or preserving cancer cells, the culture medium comprising at least a ROCK inhibitor and monothioglycerol. (2) The medium according to (1), wherein 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. (3) The medium according to (2), wherein 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. (4) The medium according to (1), wherein the final concentration of the ROCK inhibitor in the medium is 0.05 μmol / L or more and 100 μmol / L or less, and the final concentration of monothioglycerol in the medium is 10 μmol / L or more and 5000 μmol / L or less. (5) The medium according to (3) above, wherein the final concentration of the ROCK inhibitor in the medium is 0.05 μmol / L or more and 100 μmol / L or less, and the final concentration of monothioglycerol in the medium is 10 μmol / L or more and 5000 μmol / L or less. (6) The medium according to (1) above, further comprising an antibiotic and / or an antifungal agent. (7) The medium according to (1) above, wherein the cancer is lung cancer. (8) The medium according to any one of (1) to (7) above, wherein the medium is a medium for preserving cancer cells, and the cancer cells are preserved at room temperature. (9) A method for primary culture of cancer cells, the primary culture 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.(10) A method for subculturing cancer cells, the subculturing method comprising a passaging step of removing cancer cells cultured by the primary culture method described in (9) above 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 transferring the cancer cells to a culture vessel containing a new culture medium. (11) 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 described in any one of (1) to (7) above and preserving them at room temperature. (12) The preservation method described in (11) above, wherein the culture medium contains fetal bovine serum.

[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.

[0013] Figure 1 is a photograph, substituted for a drawing, of lung cancer cells obtained by primary culture in Example 2. Figure 2 is a photograph, substituted for a drawing, of third-generation lung cancer cells obtained by subculture in Example 4. Figure 3 is a photograph, substituted for a drawing, of fourth-generation lung cancer cells obtained by subculture in Example 4. Figure 4 is a photograph, substituted for a drawing, of lung cancer cells obtained by primary culture after storage at room temperature for three days in Example 6. Figure 5 is a photograph, substituted for a drawing, of lung cancer cells obtained by primary culture after storage in a refrigerator for three days in Comparative Example 4. Figure 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.

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

[0015] Furthermore, in this specification, (1) a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" 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 that are generally acceptable in the technical field, and (3) when it is written "approximately ____-shaped," it is interpreted as including not only the exact ____-shaped, but also a shape that is understood to be roughly ____-shaped.

[0016] (Embodiment of 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. A ROCK inhibitor is a compound that inhibits the activity of the above-mentioned ROCK. There are no particular limitations on the ROCK inhibitor, as long as it can inhibit the activity of ROCK. 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 ROCK inhibitor contained in the medium may be one type, or two or more types may be combined.

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

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

[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 S—S bonds in hair.

[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, including, but not limited to, DMEM (Dulbecco's Modified Eagle's Medium), DMEM:F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), EMEM (Eagle's minimal essential medium), MEMα (Minimum Essential Medium Alpha), BME (Basal Medium Eagle), and RPMI-1640. Various components such as cell growth factors may be added to these media as needed, and the media may be used with their composition adjusted to suit the cell type.

[0022] Examples of various components that are generally 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 gastric 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, and examples thereof include, but are 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, uterine 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. Examples of the final concentration of the ROCK inhibitor in the medium per 1 L of medium include, but are not limited to, lower limits 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 upper limits 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 can be stored at room temperature without the need for refrigeration. 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 the use of cooling or heating means, and generally refers to approximately 15°C to 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 (e.g., 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 advantages. (1) It not only increases the success rate of primary culture of lung cancer, which has traditionally been considered difficult to achieve, but can also be used for primary culture of cancer cells other than lung cancer. Therefore, since it can be used for primary culture of cancer cells regardless of the type of cancer, it is useful for clinical applications such as genetic analysis for diagnosis and treatment and anticancer drug selection, 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, since a single type of medium can be used for different purposes, it is useful in clinical settings, etc.

[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 the 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] The culture vessel used in the culture step may be a known vessel commonly used in the field of cell culture. For adhesion culture, commercially available vessels with surfaces treated to facilitate cell or cell aggregate adhesion may be used, although 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 may also be selected depending on conditions such as the type and required amount of cancer cells. Flasks, bottles, dishes, tubes, plates, and the like are commercially available in various sizes, and these may 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 achieves the following advantages: (1) The primary culture of cancer cells grown in the culture step resembles the clinical tissue of the collected living body. 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 for elucidating the molecular mechanisms of cancer, drug sensitivity testing, screening for drugs suitable for the collected living body (patient), genetic analysis of cancer cells, and the like. It can also be used in research and development, such as drug discovery. (2) By using the medium disclosed in the present application, the success rate of primary culture of lung cancer cells, which is considered particularly difficult, is improved to approximately 70%. Note that, as used herein, "success rate" means that the number of cancer cells increases by approximately 100-fold through primary culture. An improved success rate increases the probability of performing the elucidation of the molecular mechanisms of cancer, drug sensitivity testing, screening for drugs suitable for the collected living body (patient), genetic analysis of cancer cells, and the like, as described in (1) above. Therefore, personalized medical care tailored to each patient can be realized. (3) Cancer tissue collected during surgery or other procedures is generally fixed in formalin, and paraffin blocks are prepared and used for pathological observation. However, formalin, which is 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 tissue is 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 Passage Culture Method) Next, a passage culture method according to an embodiment will be described. The passage culture method includes a passaging step in which, during passaging, cancer cells cultured by the above-described primary culture method 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 significantly damage the cells. Although not limited, it is sufficient to add about 0.5 to 1 mL of a 1 mM chelating agent solution.

[0043] In conventional cell culture methods, enzymes are used to detach cells from culture vessels during passaging. However, using enzymes can weaken cell membranes 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 Preservation Method) Next, a preservation method according to an embodiment will be described. The preservation method according to the 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 a storage period of 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 preferable 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, and 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 contained in the storage medium, there is a risk of the cultured cancer cells being contaminated with viruses, etc. Even if the cultured cancer cells are contaminated with viruses, there is no particular problem if the cultured cancer cells are used for research purposes or screening for drug discovery, etc. However, from the perspective of improving safety, if the storage period of the collected cancer cells is short, it is desirable 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.

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

[0054] 2. Medium for long-term storage 5% by volume FBS (fetal bovine serum, manufactured by Thermo Fisher Scientific) 2.5% by volume KSR (serum-free supplement, manufactured by Thermo Fisher Scientific) 1 mmol / L sodium pyruvate (manufactured by Thermo Fisher Scientific) 2.5% by volume Human Serum (manufactured by Sigma-Aldrich) 2 mM 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.) 50 ng / mL SAG (smoothened agonist, manufactured by Abcam) 50 μmol / L monothioglycerol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) RPMI1640 medium (manufactured by Thermo Fisher Scientific) 100 units / mL penicillin G (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 100 μg / mL streptomycin sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0055] [Primary Culture] Example 2 1. Culture Vessels In Example 2, the following culture vessels were used. - Multiwell plate for adherent culture (1 / 12 well). The multiwell plate for adherent culture was coated overnight in a refrigerator with iMatrix-511 Coat (manufactured by Takara Bio Inc.). Culture medium: 1 mL / well. - HydroCell plate for suspension culture (24 well). The plate for suspension culture was not coated with iMatrix-511 Coat (manufactured by Takara Bio Inc.). Culture medium: 1 mL / 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 shredded with a scalpel blade (approximately 0.5-1 mm square). The shredded tissue pieces were then placed back into the short-term storage medium and incubated at 37°C for 30 minutes. (3) The volume was increased to 10 mL with a scalpel and centrifuged for 5 minutes at 1,000 rpm. (4) The supernatant was aspirated 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 minutes, shaking). Collagenase I (SIGMA) was diluted 5-fold from 0.5% with HBSS (containing Mg and Ca). (5) The solution was made up to 10 mL with 9 mL of PBS and 1 mL of CELLOTION (manufactured by Takara Bio Inc.) using a measuring tape, and then centrifuged for 5 minutes at 1,000 rpm. (6) 2 mL of PBS was added, and the tissue was lysed by pipetting. The lysed 2 mL solution was filtered into a new 15 mL tube, along with the tissue residue. The tissue residue 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 centrifugally washed for 5 minutes at 1,000 rpm. (7) The supernatant was aspirated to the last drop, 3 mL of hemolytic agent was added, and the mixture was vortexed to perform hemolysis (4°C, 15 min). (8) The mixture was made up to 10 mL with 1 mL of PBS and CELLOTION, and then centrifuged (1,000 rpm, 5 min). (9) The supernatant was aspirated, and the mixture was made up to 10 mL with 1 mL of PBS and CELLOTION, and then centrifuged (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 aspirated to 100 μL, and the cancer cells were seeded into primary culture medium (well plates for adherent culture or plates for suspension culture described in "1. Culture Vessel" above) (the number of cells to be seeded was determined appropriately). (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 case 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, 46 cases achieved successful primary culture (a 100-fold or greater increase in lung cancer cell numbers) using both adherent culture and suspension culture, or either one. Approximately 74% of lung cancer tissues collected from living subjects achieved successful primary culture. Figure 1 shows a photograph of lung cancer cells obtained by primary culture (adherent culture).

[0058] Comparative Example 1 An experiment was conducted in the same manner 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 the adherent culture or the suspension culture.

[0059] Comparative Example 2 An experiment was conducted in the same manner as in Example 2, except that monothioglycerol was not added to the medium composition of Example 1 (N=8). In both adherent culture and suspension culture, the cancer cells were reduced by 20% in 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 cultures were performed using the same procedures as in Example 2, except that cancer tissues collected during surgery for osteosarcoma, gastric cancer, ovarian cancer, colon cancer, pancreatic cancer, and esophageal cancer were used instead of lung cancer tissue (with the exception of adding 2.5 μg / mL amphotericin B (Fujifilm Wako Pure Chemical Industries, Ltd.) and 5 μg / mL fluconazole (Fujifilm Wako Pure Chemical Industries, Ltd.) to gastric cancer and colon cancer). The success rates of primary cultures (cell numbers growing 100-fold or more) were as follows. Primary cultures were successful in both adherent and suspension cultures, but unlike Example 2, only the results of the culture method with the highest success rate are shown. Osteosarcoma: Success in 3 out of 4 cases, success rate 75%. Gastric cancer: Success in 7 out of 10 cases, success rate 70%. The three failed cases were discontinued due to fungal contamination, but cancer cell proliferation was confirmed. Ovarian cancer: Success in 2 out of 3 cases, success rate 67%. Colon cancer: Successful in 4 out of 5 cases. Success rate: 80%. Pancreatic cancer: Successful in 6 out of 10 cases. Success rate: 60%. Of the 4 unsuccessful cases, in 3 cases it was separately confirmed that there were no cancer cells in the resected tissue. 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, subculture was performed according to the following procedure. For subculture, lung cancer cells that had been primarily cultured using adherent culture were used. The subculture process is as follows: (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 incubated in a 37°C carbon dioxide incubator for 5 to 15 minutes. The cells were observed under a microscope every 5 minutes. When the cells began to detach from the dish, PBS was added and the cells were recovered by pipetting. (3) The cells were transferred to a 15 mL tube, and the volume was increased to 10 mL with PBS and 1 mL of CELLOTION, followed by centrifugation (1,000 rpm, 5 minutes).

[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 drugs, Y-27632 2HCl (manufactured by MedChemExpress), H-1152 dihydrochloride (manufactured by MedChemExpress), Hydroxyfasudil (HA-1100) HCl (manufactured by MedChemExpress), and GSK269962A HCl (manufactured by MedChemExpress), were used as ROCK inhibitors instead of Y-27632 in Example 2. The success rates of primary culture were as follows. Note that 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.・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. Success rate 75%. ・GSK269962A HCL: Successful in 2 out 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 up to the second generation using the four types of ROCK inhibitors in Example 5, and further subcultured, and all were able to be subcultured up to the fourth generation. The cell proliferation rate by 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.

[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] [Storage 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. Subsequently, 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, their proliferation was inferior to that of cells 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 performed in the same manner as in Example 6, except that a medium was used that was the same as that used in Example 2 but without the ROCK inhibitor and monothioglycerol, i.e., a medium that has been commonly used in the past. In Comparative Example 5, an experiment was also performed with ovarian cancer cells in addition to lung cancer cells. The results were as follows: Lung cancer: 0 out of 3 cases were successful. Success rate: 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] [DNA Quality] Example 7 Next, an experiment was carried out to examine the DNA quality of cancer cells obtained by 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, which shows the results) was fixed in formalin using standard procedures and for a standard processing time. The tissue was then excised and paraffin blocks for pathological diagnosis were prepared using a standard process (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 standard method using a microtome.

[0074] (2) Rapid-FFPE DNA Lung cancer tissues collected during surgery from the three patients (none in Sample 3) were immediately cut into pieces of approximately 5-10 mm, and formalin-fixed paraffin blocks were prepared using standard methods. Paraffin sections were then prepared in the same manner as in the FFPE method described above, 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 in Example 2. gDNA was extracted from the cancer cells grown in the culture.

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

[0077] From the above results, it can be seen that high-quality gDNA can be obtained from primary cultured cancer cells, and therefore it can be used for genetic analysis of individual patients.

[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.

2. The medium according to claim 1, wherein 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.

3. The medium according to claim 2, wherein 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.

4. The medium according to claim 1, wherein the final concentration of the ROCK inhibitor in the medium is 0.05 μmol / L or more and 100 μmol / L or less, and the final concentration of monothioglycerol in the medium is 10 μmol / L or more and 5000 μmol / L or less.

5. The medium according to claim 3, wherein the final concentration of the ROCK inhibitor in the medium is 0.05 μmol / L or more and 100 μmol / L or less, and the final concentration of monothioglycerol in the medium is 10 μmol / L or more and 5000 μmol / L or less.

6. The medium according to claim 1, further comprising an antibiotic and / or an antifungal agent.

7. The medium according to claim 1, wherein the cancer is lung cancer.

8. The medium according to any one of claims 1 to 7, wherein the medium is a medium for preserving cancer cells, and the cancer cells are preserved at room temperature.

9. A primary culture method for cancer cells, the primary culture 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 7.

10. A method for subculturing cancer cells, the subculturing method comprising: removing cancer cells cultured by the primary culture method according to claim 9 from a culture vessel in a state where adhesion of the cancer cells to the wall surface of the culture vessel is reduced by using a chelating agent that binds to a divalent metal ion during subculture, and transferring the cancer cells to a culture vessel containing a new medium, the subculturing method including a subculture step.

11. A method for preserving cancer cells, the preserving method comprising: a preserving step of immersing cancer cells collected from a living body in the medium according to any one of claims 1 to 7 and preserving the cells at room temperature.

12. The preserving method according to claim 11, wherein the medium contains fetal bovine serum.

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