Immortalized corneal cell line derived from companion animals and its use

Abstract

To provide a companion animal-derived corneal cell line with well-preserved cell characteristics in vivo.SOLUTION: Provided are an immortalized companion animal-derived corneal cell line; a screening method comprising the steps of culturing a companion animal-derived corneal cell line and measuring the growth of the cell line, in the presence of a test substance, wherein the promoted growth of the cell line compared to the case without the test substance shows the test substance being a growth promoter for the companion animal-derived corneal cell; and a method for evaluating the toxicity of a test substance comprising the steps of culturing a companion animal-derived corneal cell line in the presence of the test substance and assessing the growth, viability, or function of the cell line, wherein the reduced growth, viability, or function of the cell line shows the test substance having toxicity to the companion animal-derived corneal cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an immortalized corneal cell line derived from a companion animal and uses thereof. More specifically, the present invention relates to an immortalized corneal cell line derived from a companion animal, a method for screening agents for promoting the proliferation of corneal cells derived from a companion animal, and a method for evaluating the toxicity of a test substance to a corneal cell line derived from a companion animal. [Background technology]

[0002] In recent years, companion animals have come to require the same advanced medical care as humans. However, medical care for companion animals has lagged behind. In recent years, due to ethical issues, it has become difficult to use companion animals such as dogs as experimental animals, which has also led to delays in veterinary research.

[0003] Because conducting experiments on humans is ethically unacceptable, human research often involves experiments at the cellular level. By isolating and growing cells from human tissue, it is possible to observe drug responses and cellular behavior. However, cells extracted from living organisms do not grow indefinitely; they usually reach the end of their lifespan after a certain number of divisions, resulting in a decline in the cells' proliferative capacity and a loss of their original cellular properties.

[0004] To solve this problem, a technique is used to immortalize cells by incorporating an oncogene into the cells (see, for example, Non-Patent Document 1). Many immortalized cells are actually commercially available. The use of immortalized cells makes it possible to analyze the properties of cells and their responses to drugs.

[0005] In contrast, immortalization technology for cells from companion animals such as dogs is still in its infancy, posing a barrier to conducting cell experiments similar to those in humans. Corneal endothelial cells, in particular, have poor proliferation ability when extracted from a living organism, so a large amount of fresh tissue is required to obtain the necessary number of cells for experiments. For example, in the case of dogs, multiple dog eyeballs are required, which means that eyeballs must be obtained from the dog. However, from an ethical standpoint, experiments using cells cannot be conducted.

[0006] In humans, there are organizations that preserve eye tissue (corneas) obtained from donated bodies, such as eye banks, and corneas can be purchased from these organizations. However, there are no eye banks for companion animals such as dogs, making it difficult to obtain corneas. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Alwin Prem Anand A., et al., Immortalization of neuronal progenitors using SV40 large T antigen and differentiation towards dopaminergic neurons, J Cell Mol Med., 16 (11), 2592-2610, 2012. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a companion animal-derived corneal cell line that well maintains the characteristics of cells in vivo. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] Immortalized corneal cell line derived from companion animals. [2] The corneal cell line derived from a companion animal described in [1], which is a corneal endothelial cell line. [3] The companion animal-derived corneal cell line described in [2], which exhibits a cobblestone-like shape when confluent. [4] Cultured to 1.0 × 10 5 ~5.0×10 5 pieces / cm 2 When the cell density reaches 100,000 μm, the area occupied by each cell is 200 to 1000 μm. 2The corneal cell line derived from a companion animal according to [2] or [3], [5] A companion animal-derived corneal cell line according to any one of [2] to [4], which expresses ZO-1 protein and Na / K-ATPase protein. [6] The companion animal-derived corneal cell line according to any one of [2] to [5], which is a canine corneal endothelial cell line. [7] The companion animal-derived corneal cell line described in [6], which is a cell line deposited under accession number NITE AP-03534. [8] A method for screening for a promoter of proliferation of companion animal-derived corneal cells, comprising the steps of culturing a companion animal-derived corneal cell line described in any one of [1] to [7] in the presence of a test substance, and measuring the proliferation of the companion animal-derived corneal cell line, wherein the promotion of proliferation of the companion animal-derived corneal cell line compared to that in the absence of the test substance indicates that the test substance is a promoter of proliferation of companion animal-derived corneal cells. [9] A method for evaluating the toxicity of a test substance to a companion animal-derived corneal cell line, comprising the steps of culturing a companion animal-derived corneal cell line described in any of [1] to [7] in the presence of a test substance, and measuring the proliferation, viability or function of the companion animal-derived corneal cell line, wherein a decrease in proliferation of the companion animal-derived corneal cell line compared to in the absence of the test substance, a decrease in viability of the companion animal-derived corneal cell line compared to in the absence of the test substance, or a decrease in function of the companion animal-derived corneal cell line compared to in the absence of the test substance indicates that the test substance is toxic to companion animal-derived corneal cells. [Effects of the Invention]

[0010] According to the present invention, a companion animal-derived corneal cell line can be provided that well maintains the characteristics of cells in vivo. [Brief explanation of the drawings]

[0011] [Figure 1] 1(a) and (b) show micrographs of corneal endothelial cells in Experimental Example 1. [Figure 2]2(a) to (c) are representative micrographs of canine corneal endothelial cells in Experimental Example 2. FIG. [Figure 3] 3(a) and (b) are representative micrographs of immortalized canine corneal endothelial cells at passage number 20 (P20) taken in Experimental Example 4. FIG. [Figure 4] 4(a) to 4(c) are fluorescence micrographs taken in Experimental Example 5 showing the results of immunostaining of immortalized canine corneal endothelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Companion animal-derived corneal cell line] In one embodiment, the present invention provides an immortalized companion animal-derived corneal cell line. The companion animal-derived corneal cell line of this embodiment maintains the characteristics of cells in vivo and can be used as a model of in vivo corneal cells for research, drug screening, toxicity evaluation, etc. Furthermore, because the cell line can be cultured and expanded, there are few ethical issues with its use.

[0013] Examples of companion animals include dogs, cats, rabbits, etc. Examples of corneal cell lines include corneal epithelial cells, corneal endothelial cells, etc.

[0014] The method for immortalizing companion animal-derived corneal cells is not particularly limited, but immortalization is preferably achieved by introducing an immortalizing gene, such as the SV40 Large T antigen gene or the human telomere reverse transcriptase (hTERT) gene.

[0015] The NCBI accession number of the SV40 Large T antigen gene is M99347.1, etc. As the SV40 Large T antigen gene, for example, a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 can be used. The NCBI accession numbers of the cDNA of the hTERT gene are NM_001193376, NM_198253, NM_198254, NM_198255, etc.

[0016] The companion animal-derived corneal cell line of this embodiment closely reflects the characteristics of cells in a living organism. For example, when the cell line is a corneal endothelial cell line, the cell line closely reflects the characteristics of cells in a living organism, such that the cell line exhibits a cobblestone-like shape when confluent.

[0017] Here, the term "a cobblestone-like shape at confluence" refers to a culture of 1.0 × 10 5 ~5.0×10 5 pieces / cm 2 When the cell density reaches 100,000 μm, the area occupied by each cell is 200 to 1000 μm. 2 It can also be said that...

[0018] Alternatively, "well reflecting the characteristics of cells in a living organism" may mean, for example, when the cell line is a corneal endothelial cell line, that the cell line expresses ZO-1 protein and Na / K-ATPase protein.

[0019] ZO-1 and Na / K-ATPase are known to be corneal endothelial cell markers. ZO-1 (also known as TJP1) is a tight junction-associated protein. Tight junctions are a type of intercellular junction that connects adjacent cells and prevents various molecules from passing between them. NCBI accession numbers for the canine ZO-1 gene cDNA include NM_001003140.1.

[0020] Na / K-ATPase is a membrane transport enzyme and transmembrane protein in cells. This enzyme is also called the sodium-potassium pump or simply the sodium pump because it pumps sodium ions out of the cell and takes in potassium ions in conjunction with intracellular ATP hydrolysis. The NCBI accession numbers for the canine Na / K-ATPase gene cDNA are L42173.1, etc.

[0021] In recent years, the level of animal medical care has improved, and some medical departments offer treatments similar to those for humans. In the field of veterinary ophthalmology, treatments were previously performed using eye drops for humans, but in recent years, many eye drops specifically for animals approved by the Ministry of Agriculture, Forestry and Fisheries have become commercially available. Furthermore, many intraocular surgeries, such as cataract surgery and vitreoretinal surgery, are now performed in Japan, and intraocular lenses specifically for animals are also commercially available. In the future, newly developed eye drops and intraocular drugs and products will require safety testing before being marketed. However, there is a shortage of cells that can be used to perform these tests in vitro. In response to this, these tests can be performed in vitro using the cell line of the present embodiment.

[0022] Furthermore, it is known that corneal endothelial cells have low proliferation ability in vivo and cannot regenerate once damaged. There are many diseases in which corneal endothelial cells are damaged, and in many cases this leads to blindness.

[0023] For example, canine corneal endothelial cells have low proliferation properties, and conventionally, it has been almost impossible to conduct experiments on them. In contrast, by using the cell line of this embodiment, it becomes possible to elucidate, at the cellular level, the mechanism by which corneal endothelial cells do not proliferate. Furthermore, because corneal endothelial cells are also cells that are easily damaged during intraocular surgery, it becomes possible to screen drugs and the like that can reduce damage.

[0024] The companion animal-derived corneal cell line of this embodiment is preferably a canine corneal endothelial cell line. Furthermore, the canine corneal endothelial cell line is preferably the cell line deposited under accession number NITE AP-03534.

[0025] As described below in the Examples, it has been confirmed that the cell line deposited under Accession Number NITE AP-03534 has high proliferation potential and maintains this proliferation potential even after more than 20 passages. Furthermore, this cell line maintains the characteristic of exhibiting a cobblestone-like morphology when confluent. Furthermore, this cell line maintains the expression of ZO-1 protein and Na / K-ATPase protein, which are corneal endothelial cell markers. Thus, this cell line well maintains the characteristics of corneal endothelial cells in vivo. Furthermore, many of this cell line with a low passage number have been cryopreserved, and it has been confirmed that they can be recultured from the cryopreserved state. Therefore, this cell line is highly promising as a research material for companion animal-derived corneal cells, and in the future, these cells can be used for pharmaceutical development, etc.

[0026] [Method for screening agents that promote proliferation of corneal cells derived from companion animals] In one embodiment, the present invention provides a screening method for a proliferation promoter of companion animal-derived corneal cells, comprising the steps of culturing the above-mentioned companion animal-derived corneal cell line in the presence of a test substance and measuring the proliferation of the companion animal-derived corneal cell line, wherein promotion of proliferation of the companion animal-derived corneal cell line compared to that in the absence of the test substance indicates that the test substance is a proliferation promoter of companion animal-derived corneal cells.

[0027] As described above, for example, canine corneal endothelial cells have low proliferation ability even in vivo and are known to be unable to regenerate once damaged. In contrast, the screening method of the present embodiment makes it possible to screen for agents that promote the proliferation of companion animal-derived corneal cells.

[0028] The test substance is not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, and the like.

[0029] In the screening method of this embodiment, various commonly used measurement methods can be used to measure cell proliferation. Specific examples include direct cell counting; cell viability assays using tetrazolium compounds such as MTT, MTS, and WST-1; mitochondrial membrane potential-dependent dye assays that measure damage to mitochondrial membranes using dyes that accumulate in the mitochondrial membrane in a mitochondrial membrane potential-dependent manner, such as TMRE, TMRM, JC-1, JC-10, and Mitotracker Red; esterase-cleavage dye assays using dyes such as calcein AM and calcein violet AM; assays based on the measurement of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels; glycolytic activity assays; oxygen consumption assays; and methods for detecting cells in the cell proliferation phase by immunostaining with cell proliferation markers such as ki67.

[0030] Measurement of cell proliferation may be carried out after culturing the companion animal-derived corneal cell line in the presence of the test substance for a predetermined period of time, or may be carried out over time while culturing the above-mentioned companion animal-derived corneal cell line in the presence of the test substance.

[0031] When cell proliferation is promoted in the presence of the test substance compared to the absence of the test substance, the test substance can be said to be an agent for promoting the proliferation of companion animal-derived corneal cells.

[0032] [Method for assessing the toxicity of test substances to companion animal-derived corneal cell lines] In one embodiment, the present invention provides a method for evaluating the toxicity of a test substance to a companion animal-derived corneal cell line, comprising the steps of culturing the above-mentioned companion animal-derived corneal cell line in the presence of a test substance and measuring the proliferation, viability, or function of the companion animal-derived corneal cell line, wherein a decrease in proliferation of the companion animal-derived corneal cell line compared to in the absence of the test substance, a decrease in the viability of the companion animal-derived corneal cell line compared to in the absence of the test substance, or a decrease in function of the companion animal-derived corneal cell line compared to in the absence of the test substance indicates that the test substance is toxic to companion animal-derived corneal cells.

[0033] In the toxicity evaluation method of this embodiment, the test substance and the method for measuring cell proliferation are the same as those described above.

[0034] Measurement of cell viability (viability), which can also be considered as a measurement of cytotoxicity, can be performed using a variety of commonly used methods, including detection of leaked enzymes such as lactate dehydrogenase (LDH), staining with dyes such as trypan blue, apoptosis detection methods such as annexin V binding assays, detection of DNA aggregation and fragmentation, and detection of activated caspases, as well as assays related to oxidative stress, such as quantification of reactive oxygen species (ROS), detection of ROS-induced protein modifications, and measurement of antioxidant capacity.

[0035] The cell proliferation or viability may be measured after culturing the companion animal-derived corneal cell line in the presence of the test substance for a predetermined period of time, or may be measured over time while culturing the above-mentioned companion animal-derived corneal cell line in the presence of the test substance.

[0036] A test substance can be said to be toxic to companion animal-derived corneal cells if cell proliferation is reduced in the presence of the test substance compared to its absence, or if cell viability (survival) is reduced in the presence of the test substance compared to its absence.

[0037] Furthermore, the functions of companion animal-derived corneal cell lines include, for example, the maintenance of tight junctions and the maintenance of pump function. Whether or not tight junctions are maintained can be determined, for example, by measuring electrical resistance or the permeability of low-molecular-weight substances. Whether or not pump function is maintained can be determined by an assay using an Ussing chamber. An Ussing chamber is a device that electrophysiologically measures electrolyte transport in the epithelial membrane using the short-circuit current method.

[0038] The cell function may be measured after culturing the companion animal-derived corneal cell line in the presence of the test substance for a predetermined period of time, or may be measured over time while culturing the above-mentioned companion animal-derived corneal cell line in the presence of the test substance.

[0039] If cell function is reduced in the presence of the test substance compared to its absence, i.e., if tight junctions are disrupted, pump function is reduced, etc., the test substance can be said to be toxic to companion animal-derived corneal cells. [Example]

[0040] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0041] [Experimental Example 1] (In vivo observation of corneal endothelial cells) Corneal endothelial cells extracted from a dog's eyeball were fixed, stained with alizarin red, and observed under a microscope. Microscopic photographs are shown in Figures 1(a) and (b). Figure 1(a) is a photograph taken at a magnification of 20x, and Figure 1(b) is a photograph taken at a magnification of 40x. As a result, it was confirmed that corneal endothelial cells in vivo have a hexagonal shape.

[0042] [Experimental Example 2] (Primary culture of canine corneal endothelial cells) Canine corneal endothelial cells were primary cultured and the cell morphology was observed. Figures 2(a) to 2(c) are microscopic photographs of representative cells. Figure 2(a) is a photograph of the first passage (P1), Figure 2(b) is a photograph of the third passage (P3), and Figure 2(c) is a photograph of the fifth passage (P5).

[0043] As a result, it was revealed that canine corneal endothelial cells maintained a cobblestone-like shape at low passage numbers, but as the passage number increased, the cells became larger, lost their cobblestone-like shape, and their proliferation ability significantly decreased.

[0044] [Experimental Example 3] (Establishment of a canine corneal endothelial cell line) Canine corneal endothelial cells were immortalized by transfection with a retroviral vector encoding the SV40 Large T antigen. The retroviral vector was prepared by transfecting packaging cells with pBABE-puro SV40 LT (catalog number 13970, Addgene) and pCMV-VSV-G (catalog number 8454, Addgene), which encodes the pantropic envelope. The nucleotide sequence of the SV40 Large T antigen gene contained in pBABE-puro SV40 LT is shown in SEQ ID NO: 1.

[0045] As a result, 99 immortalized cell lines were obtained. These clones were selected based on cell morphology, resulting in the establishment of four canine corneal endothelial cell lines. Of these four clones, one clone was selected that best reflected the characteristics of corneal endothelial cells in vivo using immunostaining and mRNA expression levels for ZO-1 and Na / K-ATPase. The selected canine corneal endothelial cell line was deposited at the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (received on September 9, 2021, accession number NITE AP-03534, cell name "CCEC-M1").

[0046] [Experimental Example 4] (Study of canine corneal endothelial cell lines 1) The canine corneal endothelial cell line established in Experimental Example 3 (accession number NITE AP-03534, cell name "CCEC-M1") was cultured, and the cell morphology was observed. Figures 3(a) and (b) are micrographs of representative cells at passage 20 (P20). Figure 3(b) is an enlarged image of the boxed area in Figure 3(a).

[0047] As a result, while the primary culture of canine corneal endothelial cells in Experimental Example 2 had a limit of proliferation at around P5, CCEC-M1 cells maintained proliferation ability even at P20. Furthermore, even at P20, they maintained the same paving stone-like shape as in primary culture.

[0048] [Experimental Example 5] (Study of canine corneal endothelial cell lines 2) The CCEC-M1 cells established in Experimental Example 3 were fixed and immunostained for corneal endothelial cell markers ZO-1 protein and Na / K-ATPase protein. Figures 4(a) to 4(c) are fluorescence micrographs showing the results of immunostaining. Figure 4(a) shows the results of staining for ZO-1 protein. Figure 4(b) shows the results of staining for Na / K-ATPase protein. Figure 4(c) shows the results of staining the nucleus with 4',6-diamidino-2-phenylindole (DAPI).

[0049] As a result, it was confirmed that the obtained immortalized canine corneal endothelial cells expressed ZO-1 protein and Na / K-ATPase protein, which are corneal endothelial cell markers. [Industrial Applicability]

[0050] According to the present invention, a companion animal-derived corneal cell line can be provided that well maintains the characteristics of cells in vivo.

Claims

1. A canine or feline-derived corneal endothelial cell line immortalized by introduction of the SV40 Large T antigen gene.

2. The corneal endothelial cell line according to claim 1 , which exhibits a cobblestone-like shape when it reaches confluence.

3. Cultured to 1.0 × 10 ５ ~5.0 x 10 ５ pieces / cm ２ When the cell density reaches 100,000 μm, the area occupied by each cell is 200 to 1000 μm. ２ The corneal endothelial cell line according to claim 1 or 2,

4. The corneal endothelial cell line according to any one of claims 1 to 3, which expresses a ZO-1 protein and a Na / K-ATPase protein.

5. The corneal endothelial cell line according to any one of claims 1 to 4, which is a canine corneal endothelial cell line.

6. The corneal endothelial cell line according to claim 5, which is the cell line deposited under accession number NITEP-03534.

7. A step of culturing the corneal endothelial cell line according to any one of claims 1 to 6 in the presence of a test substance; measuring the proliferation of the corneal endothelial cell line; A method for screening for a proliferation promoter of canine- or feline-derived corneal endothelial cells, wherein the promotion of proliferation of the corneal endothelial cell line compared to that in the absence of the test substance indicates that the test substance is a proliferation promoter of canine- or feline-derived corneal endothelial cells.

8. A step of culturing the corneal endothelial cell line according to any one of claims 1 to 6 in the presence of a test substance; measuring the proliferation, viability or function of the corneal endothelial cell line; A method for evaluating the toxicity of a test substance to a canine or feline-derived corneal endothelial cell line, wherein a decrease in proliferation of the corneal endothelial cell line compared to the absence of the test substance, a decrease in the viability of the corneal endothelial cell line compared to the absence of the test substance, or a decrease in the function of the corneal endothelial cell line compared to the absence of the test substance indicates that the test substance is toxic to canine or feline-derived corneal endothelial cells.

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