Culture medium composition for companion animal cells, method for producing companion animal cells, and screening method.

Abstract

This invention provides a culture medium composition for companion animal cells, a method for producing companion animal cells, and a screening method. [Solution] The culture medium composition is a culture medium composition for culturing companion animal cells, comprising at least one serum factor selected from the group consisting of 17-35 v / v% serum and serum substitutes, a growth factor of 3 ng / mL or more, and an antibiotic. Companion animal cells can be produced using this culture medium composition.

Description

[Technical Field]

[0001] The present invention relates to a culture medium composition for companion animal cells, a method for producing companion animal cells, and a screening method. [Background technology]

[0002] For the development and safety testing of new drugs for companion animals such as dogs, it is desirable to use the cells of the target companion animal to confirm the actual effects on the animal itself. However, the types of cultured cells that are readily available are limited compared to those for mice or humans.

[0003] For example, Patent Document 1 (Japanese Patent Publication No. 2022-027928) describes a process comprising: (i) obtaining an adipose tissue sample from a subject; (ii) incubating the adipose tissue under conditions suitable for at least partial digestion of the adipose tissue, wherein the conditions include incubation in a buffer containing calcium at a concentration between 50 mg / L and 500 mg / L, and in the presence of collagenase at a concentration between 0.2% wt / vol and 0.02% wt / vol; and (iii) the incubated adipose tissue A method is disclosed that includes the steps of: (iv) centrifuging tissue to obtain stromal vascular cells (SVF); (v) seeding the SVF cells into a tissue culture at a desired seeding concentration; (v) incubating the culture in a serum-supplemented medium under appropriate conditions to at least 85% flask confluence; (vi) recovering mesocellular cells (MSCs) from the culture; and (vii) subculturing the recovered MSCs or their descendants for at least 10 population doubling cycles to obtain expanded MSCs.

[0004] Furthermore, Patent Document 1 describes transferring mesenchymal stem cells (10g) from sickle adipose tissue collected from five female dogs to a tissue culture flask containing DMEM and 10% fetal bovine serum, and incubating them in a CO2 incubator at 37°C until a monolayer of confluent cells was present (7 to 10 days).

[0005] Furthermore, Patent Document 2 (JP 2007-513625) discloses a method for producing cells for transplantation into mammalian myocardial tissue, comprising the steps of: (a) supplying non-immortalized bone marrow stem cells; (b) subculturing the bone marrow stem cells twice under conditions that maintain the characteristics of bone marrow stem cells; (c) culturing the bone marrow stem cells in a myocardial-specific medium to differentiate them into myocardial blasts; and (d) collecting the cells when approximately 80-90% of the cells from step (c) have differentiated into myocardial blasts.

[0006] This document describes the collection of 15-20 mL of bone marrow stem cells from the ilium of dogs, and the subsequent culture of these stem cells in a medium containing 2-20% fetal bovine serum, 1-1,000 μM L-ascorbic acid-2-PO4, 5-15 ng / mL LIF (leukemia suppressor factor), and 1-200 nM dexamethasone.

[0007] Furthermore, Non-Patent Literature 1 (Int. J. Mol. Sci. 2023, 24, 2250. https: / / doi.org / 10.3390 / ijms24032250) describes that, in order to create immortalized canine adipose-derived mesenchymal stem cells, after collecting adipose tissue, the cells were digested with collagenase type I, filtered through a 100 μm nylon mesh, and cultured at 37°C in a humidified incubator containing 5% CO2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal solution. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2022-027928 [Patent Document 2] Special Publication No. 2007-513625 [Non-patent literature]

[0009] [Non-Patent Document 1] Int. J. Mol. Sci. 2023, 24, 2250. https: / / doi.org / 10.3390 / ijms24032250 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] While Patent Documents 1 and 2 both describe the collection of large quantities of adipose tissue cells or bone marrow stem cells from dogs and subsequent cell culture, they do not describe any technical concept that enables cell culture even from small amounts of collected cells by using a specific culture medium composition. Similarly, while Non-Patent Document 1 describes the collection of adipose cells from dogs, it also does not describe any technical concept that enables cell culture from small amounts of collected tissue by using a specific culture medium composition.

[0011] Therefore, the first object of the present invention is to provide a culture medium composition that can efficiently culture cells even if they are taken from a small amount of tissue of a companion animal. A second object of the present invention is to provide a method for producing cells of various companion animals. A third object of the present invention is to provide a screening method using companion animal cells. [Means for solving the problem]

[0012] The inventors of this invention, after diligently studying to achieve the above objective, surprisingly discovered that companion animal cells can be successfully cultured by using a culture medium composition containing a specific high concentration of serum, a specific concentration of growth factors, and a specific concentration of antibiotics, thus completing the present invention.

[0013] In other words, the present invention may be configured in the following ways. [Aspect 1] A medium composition for culturing companion animal cells, comprising at least one serum factor selected from the group consisting of 17 to 35 v / v% (preferably 18 to 33 v / v%, more preferably 19 to 30 v / v%) of serum and serum substitutes, a growth factor of 3 ng / mL or more (preferably 3 to 50 ng / mL, more preferably 5 to 35 ng / mL, particularly preferably 5 to 10 ng / mL), and an antibiotic. [Aspect 2] The medium composition according to Aspect 1, wherein the ratio of the proportion X (ng / mL) of the growth factor to the proportion Y (v / v%) of the serum factor in the medium is X / Y = 5 / 95 to 65 / 35 (preferably 15 / 85 to 55 / 45, more preferably 20 / 80 to 45 / 55). [Aspect 3] The medium composition according to Aspect 1 or 2, wherein the cells are at least one adherent cell selected from the group consisting of fibroblasts, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, pericytes, keratinocytes, smooth muscle cells, and muscle cells. [Aspect 4] The medium composition according to Aspect 3, wherein the growth factor is a cell proliferation factor for adherent cells. [Aspect 5] The medium composition according to any one of Aspects 1 to 4, wherein the cells are non-stem cells. [Aspect 6] The medium composition according to any one of Aspects 1 to 5, wherein the serum factor is serum. [Aspect 7] The medium composition according to any one of Aspects 1 to 6, wherein the antibiotic contains at least a nucleic acid synthesis inhibitor. [Aspect 8] The medium composition according to any one of Aspects 1 to 6, wherein the antibiotic does not contain a nucleic acid synthesis inhibitor. [Aspect 9] The medium composition according to any one of Aspects 1 to 8, wherein the companion animal is a dog or a cat. [Aspect 10] A combined medium composition composed of the medium composition according to Aspect 7 as the first medium composition and the medium composition according to Aspect 8 as the second medium composition. 〔Aspect 11〕 A method for producing cells of a companion animal, comprising: As a first culture medium composition, in the culture medium composition described in Aspect 7, culturing cells collected from the tissue of a companion animal; After a predetermined period, changing the culture medium composition for culturing the cells to the culture medium composition described in Aspect 8 as a second culture medium composition and culturing the cells. 〔Aspect 12〕 The volume of the collected tissue is 50 mm 3 or less (preferably 30 mm 3 or less, more preferably 10 mm 3 or less), the production method according to Aspect 11. 〔Aspect 13〕 When the step of changing to the second culture medium composition is carried out on the 4th to 8th day (preferably the 5th to 7th day) with the day when culturing of the cells is started in the first culture medium composition as the first day, the production method according to Aspect 11 or 12. 〔Aspect 14〕 Furthermore, the production method according to any one of Aspects 11 to 13, including a step of subculturing. 〔Aspect 15〕 A method for screening an agent in vitro, comprising: applying an agent to a cell culture composed of cells obtained by the method according to any one of Aspects 11 to 14 or cells derived therefrom; and detecting the effect of the agent on the cells in the applied cell culture. The screening method. In this specification, the degree may include a range of ±10% of a numerical value, preferably a range of ±5%.

Advantages of the Invention

[0014] According to the culture medium composition for cells according to the present invention, even from a slightly collected tissue, cells of a companion animal can be cultured well.

Brief Description of the Drawings

[0015] This invention will be more clearly understood from the following description of preferred embodiments with reference to the accompanying drawings. However, the embodiments and drawings are for illustrative and explanatory purposes only and should not be used to define the scope of this invention.

[0016] [Figure 1] This is a photograph showing fibroblasts, which are a culture of primary cells obtained in Example 1, in a confluent state. [Modes for carrying out the invention]

[0017] [Culture medium composition] The culture medium composition is a culture medium composition for culturing companion animal cells, and comprises at least one serum factor selected from the group consisting of 17-35 v / v% serum and serum substitutes, a growth factor of 3 ng / mL or more, and an antibiotic.

[0018] Here, a companion animal is an animal kept by humans as a companion, and can also be called a pet animal. Examples include dogs, cats, rabbits, horses, and pigs. Of these, dogs and cats are preferred due to their high demand as companion animals.

[0019] Dog breeds include, for example, wolf-like breeds such as Siberian Huskies, Akita Inus, Shiba Inus, and Chow Chows; herding breeds such as Shetland Sheepdogs, German Shepherds, Collies, Border Collies, and Corgis; hound breeds such as Beagles, Miniature Dachshunds, Dachshunds, Italian Greyhounds, and Afghan Hounds; retriever breeds such as Labrador Retrievers and Golden Retrievers; spaniel breeds such as English Springer Spaniels, Brittany Spaniels, English Cocker Spaniels, American Cocker Spaniels, and Cavalier King Charles Spaniels; and Jack Russell Terriers. Examples include terrier breeds such as terriers, border terriers, West Highland White terriers, schnauzers, Cairn terriers, Scottish terriers, Norfolk terriers, Norwich terriers, Silky terriers, American Staffordshire terriers, and Staffordshire bull terriers; spitz breeds such as Samoyeds, spitzes, American Eskimo dogs, and pomeranians; poodle breeds such as standard poodles and toy poodles; mastiff-like breeds such as French bulldogs, bulldogs, boxers, great danes, and dobermans; and breeds such as Shih Tzus, Maltese, Havanese, Chihuahuas, Papillons, Pekingese, chins, and pugs. These can be purebred or mixed breeds. A purebred is a dog born from parents of the same breed, while a mixed breed is a dog born from parents of different breeds. In the case of mixed breeds, the breed of one of the parents may be used to refer to them. The definitions of purebred and mixed breeds are similar for other animals as well.

[0020] Examples of cat breeds include short-haired breeds such as the Japanese domestic cat mix, American Shorthair, British Shorthair, Scottish Fold, Munchkin, Exotic Shorthair, Burmese, Tonkinese, Chartreux, Korat, Oriental Shorthair, Abyssinian, Singapura, Japanese Bobtail, Ocicat, and Bengal; long-haired breeds such as the Ragdoll, Maine Coon, Norwegian Forest Cat, Siberian, Turkish Angora, Persian, Chinchilla mix, Himalayan, Minuret, Birman, Balinese, Somali, and LaPerm; Rex breeds such as the Devon Rex and Cornish Rex; and breeds such as the Egyptian Mau and Siberian.

[0021] Furthermore, the cells used can be cultured according to the type of animal, and may be adherent cells or suspension cells such as lymphocytes, leukocytes, and erythrocytes. However, adherent cells are preferred because they are cultured using a culture medium as a support. The adherent cells are not particularly limited as long as they are cells that proliferate while adhering to the culture vessel. Typical adherent cells include, for example, cells involved in the skin such as fibroblasts, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, pericytes, and keratinocytes; cells involved in muscles such as smooth muscle cells and muscle cells; cells involved in fat such as adipocytes; cells involved in the eye such as photoreceptor cells, corneal epithelial cells, and corneal endothelial cells; cells involved in bone such as osteoblasts, osteocytes, osteoclasts, chondrocytes, and chondrocytes; and cells involved in teeth such as periodontal ligament cells and osteoblasts. Furthermore, the culture medium composition of the present invention is preferable because it can be used to culture functional cells (i.e., non-stem cells).

[0022] Of these, cells involved in skin, such as fibroblasts, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, pericytes, and keratinocytes, and cells involved in muscle, such as smooth muscle cells and muscle cells, are preferred due to the high demand for use in animal studies.

[0023] (serum factor) The culture medium composition contains a relatively large amount of serum factors. The serum factors may be at least one selected from the group consisting of serum and serum substitutes, and may be used alone or in combination of two or more. Examples of serum include serums that can be collected from various animals, such as fetal bovine serum (FBS), horse serum, horse serum, pig serum, and dog serum. Examples of serum substitutes include StemSure (Fujifilm Wako Pure Chemical Industries, Ltd.), Artificial Serum (Thermo Fisher Scientific, Inc.), KnockOut (trademark), Serum Replacement (Thermo Fisher Scientific), Chemically Defined Lipid Concentrate (Thermo Fisher Scientific), and XerumFree (trademark) (TNCBio). Of these, serum is preferred from the viewpoint of ease of availability, and fetal bovine serum is particularly preferred.

[0024] The concentration of serum factors is 17-35 v / v%, preferably 18-33 v / v%, and more preferably 19-30 v / v%, based on the volume of the culture medium composition. Including serum factors at a higher concentration than usual in the culture medium can promote the proliferation of cells collected from tissue. On the other hand, if the amount of serum factors is too high, the viscosity may increase, which may impair cell mobility during proliferation and adversely affect cell morphology and function.

[0025] (growth factor) Examples of growth factors include fibroblast growth factor, keratinocyte growth factor, epidermal growth factor, transforming growth factor, heparin-binding epidermal growth factor, connective tissue growth factor, nerve growth factor, neurotrophic factor, bone morphogenetic factor, insulin-like growth factor, hepatocyte growth factor, angiopoietin, adrenamodulin, platelet-derived growth factor, vascular endothelial growth factor, stem cell factors, and leukemia inhibitors. These growth factors may be used individually or in combination of two or more. Preferably, the growth factors may be selected according to the type of cells being targeted.

[0026] Among these growth factors, cell growth factors for adherent cells are preferred, and in particular, fibroblast growth factor, keratinocyte growth factor, and epidermal growth factor are preferred.

[0027] The growth factor content is 3 ng / mL or more, based on the volume of the culture medium composition, preferably 3 to 50 ng / mL, more preferably 5 to 35 ng / mL, and particularly preferably 5 to 10 ng / mL. By combining high concentrations of serum factors with growth factors, it is possible to obtain cell cultures from animal cells that previously did not proliferate.

[0028] The ratio of the growth factor X (ng / mL) to the serum factor Y (v / v%) in the culture medium composition may be, for example, X / Y = 5 / 95 to 65 / 35, preferably 15 / 85 to 55 / 45, and more preferably 20 / 80 to 45 / 55.

[0029] (antibiotics) Antibiotics are active substances produced by microorganisms that inhibit the growth of other microorganisms such as bacteria and viruses, and / or have bactericidal effects. Examples of antibiotics include cell wall synthesis inhibitors (e.g., penicillins such as penicillin, ampicillin, and amoxicillin; β-lactams such as cefazolin, ceftriaxone, and cefpodoxime; glycopeptides such as vancomycin; fosfomycins such as fosfomycin), cell membrane dysfunction inhibitors (azoles such as itraconazole, voriconazole, and posaconazole; polyenes such as amphotericin, natamycin, hamycin, perimycin, rimocidine, and candiosin; fluoropyrimidines such as flucytosine), and protein synthesis inhibitors. Examples of harmful drugs include macrolides such as erythromycin, clarithromycin, and azithromycin; diterpenes such as thiamrin fumarate; lincomycins such as clindamycin; oxazolidinones such as linezolid; tetracyclines such as tetracycline and doxycycline; and aminoglycosides such as gentamicin, streptomycin, and amikacin, as well as small subunit inhibitors. Nucleic acid synthesis inhibitors include old quinolones; fluoroquinolones, ciprofloxacin, and levofloxacin. These antibiotics may be used alone or in combination of two or more.

[0030] The culture medium composition may be broadly classified into a first culture medium composition containing a nucleic acid synthesis inhibitor and a second culture medium composition not containing a nucleic acid synthesis inhibitor. Furthermore, the amount of antibiotic in the first culture medium composition may be relatively greater than the amount of antibiotic in the second culture medium composition.

[0031] For example, the first culture medium composition may contain, as an antibiotic, a nucleic acid synthesis inhibitor and at least one selected from the group consisting of cell wall synthesis inhibitors, cell membrane dysfunction agents, large protein subunit synthesis inhibitors, and small protein subunit synthesis inhibitors. Preferably, the first culture medium composition may contain, as an antibiotic, a fluoroquinolone nucleic acid synthesis inhibitor and at least one selected from the group consisting of penicillin-based cell wall synthesis inhibitors, polyene-based cell membrane dysfunction agents, lincomycin-based large protein subunit synthesis inhibitors, and aminoglycoside-based small protein subunit synthesis inhibitors.

[0032] For example, the second culture medium composition may contain at least one antibiotic selected from the group consisting of cell wall synthesis inhibitors, cell membrane dysfunction agents, and small protein subunit synthesis inhibitors. Preferably, the second culture medium composition may contain at least one antibiotic selected from the group consisting of penicillin-based cell wall synthesis inhibitors, polyene-based cell membrane dysfunction agents, lincomycin-based protein synthesis inhibitors, and aminoglycoside-based protein synthesis inhibitors.

[0033] In the second culture medium composition, the antibiotic content is, for example, 900 μg / mL or less, preferably 700 μg / mL or less, and more preferably 500 μg / mL or less, based on the volume of the culture medium composition. The lower limit is not particularly limited, but may be 100 μg / mL or more.

[0034] In the second culture medium composition, the ratio of the antibiotic Z (μg / mL) to the serum factor Y (v / v%) may be, for example, Z / Y = 99 / 2 to 70 / 30, preferably 98 / 2 to 80 / 20, and more preferably 97 / 3 to 90 / 10.

[0035] The culture medium composition may include commercially available culture media depending on the organism being cultured. Examples of commercially available culture media include Eagle MEM (Minimum Essential Medium), α-MEM, DMEM (Dulbecco's Modified Eagle Medium) and other Eagle MEM-based media; HAM-based media such as F10 HAM, F12 HAM, F12K HAM; Iscove's-based media such as IMDM (Iscove's Modified Dulbecco's Medium); RPMI-based media such as RPMI-1640; and BME (Basal Medium Eagle). These media may be used individually or in combination of two or more. Examples of mixed media include Eagle MEM-based / HAM-based media.

[0036] Furthermore, the culture medium composition may optionally contain water, phosphate-buffered saline (PBS), amino acids (e.g., essential amino acids, non-essential amino acids, etc.), vitamins (e.g., riboflavin, biotin, cyanocobalamin, ascorbic acid, etc.), trace elements (e.g., selenium, iron, zinc, copper, etc.), buffering agents (e.g., HEPES, etc.).

[0037] The culture medium composition of the present invention makes it possible to obtain cell cultures from even small amounts of tissue. In particular, it is possible to obtain cell cultures from various cells of companion animals (for example, non-stem cells such as fibroblasts), which were not possible with conventional methods.

[0038] The culture medium composition of the present invention may be a combined culture medium composition comprising the first culture medium composition and the second culture medium composition described above. Here, a combined culture medium composition means two culture medium compositions that are used at different times during cell culture, and preferably the first culture medium composition is used first and the second culture medium composition is used later.

[0039] [Method for producing companion animal cells] A method for producing companion animal cells includes the steps of culturing cells from tissue collected from a companion animal in a first culture medium composition, The method includes the step of changing the culture medium composition for culturing cells to a second culture medium composition having a lower concentration of antibiotic than the first culture medium composition after a predetermined period, and culturing the cells.

[0040] The first and second culture medium compositions may be the same as the culture medium compositions described above, and the second culture medium composition may have the same or different composition as the first culture medium composition, except that it contains a lower concentration of antibiotic.

[0041] Tissue can be collected from companion animals by methods commonly used by those skilled in the art. For example, tissue collected from companion animals may be subdivided by cutting or other means.

[0042] Even with a small amount of tissue collected, cell culture can be successfully performed using the culture medium composition of the present invention. For example, if the volume of tissue collected is 50 mm³, 3 It may be less than 30 mm, preferably 30 mm 3 More preferably 10mm 3 The following may also be used. The lower limit of tissue volume is determined appropriately depending on the type of tissue, etc., but for example, 5 mm 3 That's fine too.

[0043] The cells of the tissue are cultured for a predetermined period of time in contact with the first culture medium composition. Examples of culture vessels used for culturing include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, etc.

[0044] The culture vessel may be pre-coated with a cell-supporting substrate, such as collagen.

[0045] The culture conditions are not particularly limited, as long as they allow the target cells to be cultured. The carbon dioxide (CO2) concentration may be, for example, about 5% by volume. The culture temperature may be, for example, 5 to 45°C, preferably 10 to 40°C, and more preferably 35 to 37°C. The culture medium may be changed, for example, every hour to 5 days, or every 1 to 3 days as needed. It is preferable to use the first culture medium composition as the replacement medium until the predetermined period has elapsed.

[0046] After a predetermined period, the culture medium composition for culturing cells is switched to a second culture medium composition having a lower concentration of antibiotic than the first culture medium composition, and the cells are cultured. The predetermined period can be determined according to the size of the culture vessel and the state of cell proliferation, but if the day on which cells are cultured in the first culture medium composition is considered day 1, the culture medium composition may be changed to the second culture medium composition on, for example, days 4 to 8, preferably days 5 to 7.

[0047] In the second culture medium composition, the primary cells may be in a subconfluent or confluent state on the surface of the culture vessel. Here, subconfluent means, for example, that the companion animal cells occupy 70-95% of the adhesion surface of the culture vessel in a plan view, and confluent means that the companion animal cell culture occupies 95% or more of the adhesion surface of the culture vessel in a plan view.

[0048] Furthermore, primary cells may be further subcultured. In this case, a portion of the cell culture containing primary cells from the companion animal that has reached confluence may be taken and subcultured, but it is preferable to take a portion of the cell culture that has reached subconfluence and subculture it.

[0049] In subculturing, the second culture medium composition may be used in the culture vessel. Furthermore, the carbon dioxide (CO2) concentration may be, for example, about 5% by volume. The temperature during cultivation may be, for example, 5 to 45°C, preferably 10 to 40°C, and more preferably 35 to 37°C. The culture medium can be changed, for example, every hour to 5 days, or every 1 to 3 days as needed.

[0050] [Screening Method] This invention also includes screening methods. The screening method is an in vitro screening method for an agonist, comprising the steps of: applying the agonist to a cell culture composed of cells produced by the method described above or cells derived from such cells (e.g., immortalized cells immortalized by a known method); and detecting the effect of the agonist on the cells in the cell culture that have been in contact with the agonist.

[0051] For example, the activating agent may be applied to the cell culture by coating or spraying, or the cell culture may be immersed in the activating agent. Furthermore, the effect of the drug on cells in a cell culture may be detected by measuring cell viability or cell proliferation rate. These detections may involve counting the number of cells or measuring the amount of DNA synthesis. Furthermore, the effects may be detected by observing changes in the appearance of the cell culture. For example, the effects of the agonist may be detected by observing changes in the shape or structure of the cells. These detections may also be performed by evaluating the state of the cell culture when the agonist is applied compared to when the agonist is not applied. Alternatively, the agonist and the control drug may be applied separately to the cell culture, and the effects of each drug on the cell culture may be compared to detect the effect of the agonist. Screening agonists in vitro is preferable because it allows for the selection of agonists without imposing the burden of bioassays on animals. [Examples]

[0052] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited in any way by these examples and comparative examples. In the following examples and comparative examples, various physical properties were measured by the methods described below.

[0053] [Example 1] (Preparation of basic culture medium) A basic culture medium was prepared by mixing 500 mL of DMEM / F12 Ham medium, 5 mL of antibiotic solution (product name: Antibiotics Mix, manufactured by Gibco, containing 10,000 units / mL of penicillin, 10,000 μg / mL of streptomycin, and 25 μg / mL of amphotericin B), 500 μL of thiamrin fumarate (100 μg / mL), and 500 μL of ciprofloxacin (10 μg / mL) in a clean bench.

[0054] (Culture of primary cells) Epididymal tissue (approximately 5 mm) collected during neutering surgery from a male dog (Toy Poodle mix, 1 year old). 3 The tissue was washed with a basic culture medium, then shredded and made into a paste. Basic culture medium was then added, the whole mixture was centrifuged, and the supernatant was aspirated. Basic culture medium was added to the pellet remaining after aspiration, and the pellet was broken up to form a suspension.

[0055] On the other hand, a basic culture medium was placed in a collagen-coated dish, and then the suspension was added dropwise. After that, fetal bovine serum (FBS), bFGF, and an antibiotic mixture (trade name: Zell Shield, manufactured by Minerva Biolabs GmbH, containing ciprofloxacin, clindamycin, and natamycin) were mixed to prepare the first culture medium composition. In the first culture medium composition, the final concentration of fetal bovine serum was 20 v / v%, the final concentration of bFGF was 5 ng / mL, the final concentration of thiamrin fumarate was 100 ng / mL, the final concentration of ciprofloxacin was 10 ng / mL, the final concentration of penicillin was 100 μg / mL, the final concentration of streptomycin was 100 μg / mL, and the final concentration of amphotericin B was 25 ng / mL. The dish containing the cell suspension was cultured in a wet incubator (37°C, CO2 concentration: 5 vol%).

[0056] The culture medium was changed daily using the first culture medium composition, and on the seventh day, it was switched to the second culture medium composition. The second culture medium composition was obtained by mixing fetal bovine serum and bFGF with a basic medium. In the second culture medium composition, the final concentration of fetal bovine serum was 20 v / v%, the final concentration of bFGF was 5 ng / mL, the final concentration of penicillin was 100 μg / mL, the final concentration of streptomycin was 100 μg / mL, and the final concentration of amphotericin B was 25 ng / mL.

[0057] After 19 days, we confirmed that the epididymal tissue-derived fibroblasts had reached subconfluent to confluent (the adhesion surface of the culture vessel was 80-90% or more in a planar view).

[0058] (Culture of subcultured cells) The culture medium was removed, washed with PBS, and 5 mL of acutase (Innovative Cell Technologies, Inc.) was added. After standing at 37°C for 30 minutes, the detached cells were collected. The cells were divided into two 10 cm dishes and cultured using the second culture medium composition. Once confluent, the cells were detached again using acutase, cell preservation solution was added, and the cells were stored in liquid nitrogen until use.

[0059] [Comparative Example 1] In the first culture composition and the second culture composition, culture was performed in the same manner as in Example 1 except that fetal bovine serum was 10 v / v%, but primary cells could not be obtained from the tissue.

[0060] [Examples 2 to 4 and Comparative Example 2] Instead of the testicular tissue (about 5 mm 3 ) collected during castration from a dog (Toy Poodle Mix, 1 year old, male) cultured in Example 1, ovarian tissue (about 5 mm 3 ) collected during contraception from a cat (Japanese Cat Mix, 6 months old, female) was used, and primary cell culture was performed in the same manner as in Example 1 except for culturing as shown in Table 1. The number of fibroblasts derived from ovarian tissue 10 days after the culture is shown in Table 1.

[0061] [Table 1]

[0062] When the following animals were cultured in the same manner as in Example 1, various primary cells could be obtained. (Dog) Toy Poodle, Jack Russell, Chihuahua, Italian Greyhound, Husky, Chihuahua, Italian Greyhound, Husky, Havanese, Labrador, Welsh Corgi, Shiba Inu, French Bulldog, Golden Retriever, Miniature Dachshund, Pomeranian, Shih Tzu, Dalmatian (Cat) Japanese Domestic Cat Mix, Minuet, Chinchilla Mix, Persian, Siberian, American Shorthair, Scottish Fold, Ragdoll, Munchkin

[0063] In addition, primary cell culture was performed in the same manner as in Example 2 except for the conditions described in the following table. In the table, when cell culture reaches confluence, it is indicated by 〇, and when it does not reach confluence, it is indicated by ×.

[0064] [Table 2] [Industrial applicability]

[0065] According to the cell culture medium composition of the present invention, companion animal cells can be successfully cultured from a small amount of tissue, and the resulting companion animal cells can be used, for example, for screening.

[0066] As described above, preferred embodiments of the present invention have been explained, but those skilled in the art will readily anticipate various changes and modifications within the obvious scope by reviewing this specification. Therefore, such changes and modifications shall be construed as falling within the scope of the invention as defined by the claims.

Claims

1. A culture medium composition for culturing companion animal cells, comprising at least one serum factor selected from the group consisting of 17-35 v / v% serum and serum substitutes, a growth factor of 3 ng / mL or more, and an antibiotic.

2. The culture medium composition according to claim 1, wherein the ratio of the proportion X (ng / mL) of the growth factor to the proportion Y (v / v%) of the serum factor in the culture medium is X / Y = 5 / 95 to 65 / 35.

3. The culture medium composition according to claim 1, wherein the cells are at least one adherent cell selected from the group consisting of fibroblasts, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, pericytes, keratinocytes, smooth muscle cells, and muscle cells.

4. The culture medium composition according to claim 1, wherein the growth factor is a cell proliferation factor for adherent cells.

5. The culture medium composition according to claim 1, wherein the cells are non-stem cells.

6. The culture medium composition according to claim 1, wherein the serum factor is serum.

7. The culture medium composition according to claim 1, comprising at least a nucleic acid synthesis inhibitor as an antibiotic.

8. The culture medium composition according to claim 1, wherein the antibiotic does not contain a nucleic acid synthesis inhibitor.

9. The culture medium composition according to any one of claims 1 to 8, wherein the companion animal is a dog or a cat.

10. A combined culture medium composition comprising the culture medium composition according to claim 7 as a first culture medium composition and the culture medium composition according to claim 8 as a second culture medium composition.

11. A method for producing companion animal cells, As the first culture medium composition, the culture medium composition according to claim 7 comprises the step of culturing cells taken from the tissue of a companion animal, A manufacturing method comprising the step of, after a predetermined period, changing the culture medium composition for culturing cells to the culture medium composition described in claim 8 as a second culture medium composition and culturing cells.

12. The volume of the collected tissue was 50 mm ３ The manufacturing method according to claim 11, which is as follows:

13. The manufacturing method according to claim 11, wherein the step of changing to a second culture medium composition is performed on the 4th to 8th day, with the day on which cells were started to be cultured in the first culture medium composition being considered as day 1.

14. Furthermore, the manufacturing method according to claim 11, further comprising the step of subculturing.

15. A method for screening agonists in vitro, The step of applying an agonist to a cell culture composed of cells obtained by the method described in any one of claims 11 to 14 or cells derived therefrom; and A step of detecting the effect of the agonist on cells in the cell culture to which the drug has been applied, A screening method that includes this.

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