Animal cell proliferation promoter, animal cell culture medium and animal cell culture device

Abstract

To provide novel animal cell growth promoters, culture media for animal cell culture, and animal cell culture apparatuses.SOLUTION: A culture supernatant of an embryonic membrane from an avian or reptilian egg is used as an animal cell growth promoter. For example, adding, to an animal cell culture medium, a cell growth promoter comprising a culture supernatant of an embryonic membrane from an avian or reptilian egg as an active ingredient, can promote the animal cell growth.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an animal cell proliferation promoter, an animal cell culture medium, and an animal cell culture apparatus. [Background technology]

[0002] In the culture of animal cells, serum media containing fetal bovine serum or the like as a factor that functions to maintain cell growth, or serum-free media containing hormones or growth factors instead of serum, are generally used.

[0003] In addition to these factors, Japanese Patent Publication No. 2013-247927 discloses that rice bran extracts have a proliferation-promoting effect on cultured animal cells. Japanese Patent Publication No. 2011-182736 discloses that the cell proliferation rate can be increased by culturing animal cells in a medium to which a hydrolysate of β-conglycinin concentrate has been added. Japanese Patent Publication No. 7-188292 discloses that glycoproteins contained in the culture supernatant of human-derived fibroblasts promote the proliferation of human vascular endothelial cells and hepatic parenchymal cells. Japanese Patent Publication No. 5-301893 discloses that proteins contained in the cell culture supernatant of a human-derived glioma cell line have a proliferation-promoting effect on glial cells and fibroblasts. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a novel animal cell proliferation promoter, a medium for animal cell culture, and an animal cell culture apparatus. [Means for solving the problem]

[0005] One embodiment of the present invention is an animal cell proliferation promoter containing as an active ingredient a culture supernatant of embryonic membrane of an egg of a bird or reptile. The egg may be a chicken egg. The animal cell proliferation promoter may be a cell proliferation agent for muscle cells, visceral cells, or nervous system cells, or may be a cell proliferation agent for liver cells, pancreatic cells, or oviduct cells.

[0006] Another embodiment of the present invention is a cell culture medium containing any one of the cell growth promoters described above.

[0007] Another embodiment of the present invention is an animal cell culture device comprising: a first culture tank for culturing cells derived from the embryonic membrane of bird or reptile eggs; a second culture tank for culturing animal cells to be propagated; a first flow path for flowing a culture medium from the first culture tank to the second culture tank; a second flow path for flowing a culture medium from the second culture tank to the first culture tank; and a medium flow rate control unit for circulating the cell culture medium through the first culture tank, the first flow path, the second culture tank, and the second flow path in this order, and controlling the flow of the cell culture medium in the first flow path and the second flow path according to the state of the animal cells and / or the cell culture medium. The cell culture system may further include a medium inlet path for introducing fresh cell culture medium into the returning cell culture medium, and a medium removal path for removing the cultured cell culture medium from the returning cell culture medium, and the medium flow rate control unit may control the introduction of the fresh cell culture medium from the medium inlet path and the removal of the cultured cell culture medium from the medium removal path according to a state of the animal cells and / or the cell culture medium.

[0008] ==Cross references to related literature== This application claims priority based on Japanese Patent Application No. 2018-210910, filed on November 8, 2018, and the basic application is incorporated herein by reference. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1 is a schematic diagram of a cell proliferation device in one embodiment of the present invention. [Diagram 2] FIG. 1 shows photographs of the observation results indicating that when a culture supernatant of chicken egg chorioallantoic membrane was added to a medium in one embodiment of the present invention, cell proliferation was promoted in cells derived from the tissues shown in the figure (stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius, and bone). [Diagram 3] FIG. 2 is a graph quantitatively showing the results of FIG. 1 for cells derived from the liver, pancreas, and oviduct in one example of the present invention. [Figure 4] FIG. 1 is a graph showing that, in one embodiment of the present invention, when chicken egg yolk sac culture supernatant was added to a medium, cell proliferation was promoted in cells derived from the tissues shown in the figure (stomach, muscle, heart, liver, intestine, brain, bursa of Fabricius, and bone). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The objectives, features, advantages, and ideas of the present invention will be clear to those skilled in the art from the description of this specification, and those skilled in the art can easily reproduce the present invention from the description of this specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It is clear to those skilled in the art that various changes and modifications can be made based on the description of this specification within the intent and scope of the present invention disclosed in this specification.

[0011] ==Animal cell proliferation promoter== The animal cell proliferation promoter of the present disclosure contains a culture supernatant of embryonic membranes of bird or reptile eggs as an active ingredient.

[0012] Examples of birds or reptiles from which eggs are obtained include quails, chickens, lizards, snakes, crocodiles, and turtles, but are not limited to these, and any bird or reptile that has an embryonic membrane of an egg may be used.

[0013] Here, the term "embryonic membrane" refers to membrane tissue that is formed outside the embryo during the development of birds and reptiles, performs functions such as protection, nutrition, and respiration for the embryo, and does not participate in the construction of the body after hatching or birth. Examples of such membranes include the chorion located at the outermost part of the embryo, the amnion that surrounds the embryo, the allantois that forms the allantois, the chorioallantoic membrane (CAM) that is formed by partial adhesion between the chorion and allantois, and the yolk sac that surrounds the yolk.

[0014] The culture supernatant of embryonic membrane refers to a medium in which embryonic membranes are isolated and cultured. The embryonic membranes to be cultured are the above-mentioned choriosperm, amniotic membrane, allantois, choriouria Among the membranes such as the oocyte membrane and the yolk sac, one type may be cultured, or a mixture of two or more types may be cultured.

[0015] Embryonic membranes can be collected from eggs by known methods (see, for example, Ichijima Eiji: Chemistry and Biology, Vol. 13, No. 8, pp. 489-497 (1975)). For example, specifically, embryonic membranes can be isolated by the following method. First, fertilized eggs are incubated for a predetermined period of time under conditions suitable for artificial incubation, and then the eggshell is cracked. (1) The allantois can be obtained by removing the swollen membrane arising from the ventral side of the posterior digestive tract of the embryo, (2) the amnion can be obtained by removing the transparent membrane tissue that does not have blood vessels running through it that is present around the fetus, (3) the yolk sac can be obtained by removing the membrane tissue that surrounds the yolk, and (4) the chorion can be obtained by removing the outermost membrane tissue that surrounds all the elements in the egg. If the incubation time is further extended, the chorion and allantois will partially fuse to form the chorioallantoic membrane. At this stage, the chorioallantoic membrane can be obtained by removing the membrane tissue that is in contact with the eggshell and has blood vessels running through it. The optimal incubation period may be appropriately determined depending on each animal and the target membrane tissue, and for example, in the case of chicken eggs, 4 to 21 days is preferable, 10 to 18 days is more preferable, and 13 to 15 days is even more preferable. When culturing these embryonic membranes, they may be cultured in a membranous state, but they may also be physically disrupted or subjected to an enzymatic treatment with a protease (e.g., collagenase, elastase, dispase, papain, etc.) to isolate embryonic membrane-derived cells from the embryonic membrane and culture them.

[0016] For culturing the embryonic membrane, a medium used in general animal cell culture can be used, for example, DMEM, F12, etc., but is not limited thereto. A supplement that is usually blended as a medium additive may be added to this medium. The culture conditions can also be those used in general animal cell culture, typically using a carbonate buffer-based medium, culturing at 5% CO2 and 37°C, but the culture conditions are not limited thereto and can be appropriately selected by those skilled in the art. The culture time is not particularly limited, but is preferably 1 to 7 days, more preferably 2 to 6 days. After culturing, the supernatant may be collected and added directly to the cell culture medium, or the medium obtained by removing solids such as cell debris by filtration or centrifugation may be added. The amount of culture supernatant added to the cell culture medium is not particularly limited as long as it is an effective amount that can show the proliferation effect of the cultured cells, but the final concentration in the medium is preferably 20% or more, more preferably 35% or more, and even more preferably 50% or more.

[0017] The type of animal cells to be cultured in the cell culture medium containing the culture supernatant is not particularly limited, and examples thereof include muscle cells such as smooth muscle cells, cardiac muscle cells, and skeletal muscle cells, visceral cells such as cardiac cells, hepatic cells, stomach-derived cells, and intestinal cells, and nervous system cells such as nerve cells and glial cells. The animal species from which the cells are derived is also not particularly limited, and examples thereof include humans, mice, rats, monkeys, and pigs.

[0018] In this way, the culture supernatant obtained by culturing cells derived from embryonic membranes can be added to a medium for culturing animal cells to promote the growth of the cultured animal cells, and therefore the culture supernatant can be used as an animal cell growth promoter.

[0019] When the culture supernatant of cells derived from embryonic membrane is used as an animal cell growth promoter, other cell growth factors may be used at the same time.

[0020] ==Cell proliferation device== The cell proliferation device of the present invention comprises a first culture tank for culturing cells derived from the embryonic membrane of bird or reptile eggs, a second culture tank for culturing animal cells to be propagated, a first flow path for flowing a culture medium from the first culture tank to the second culture tank, a second flow path for flowing a culture medium from the second culture tank to the first culture tank, and a medium flow rate control unit that circulates the cell culture medium through the first culture tank, the first flow path, the second culture tank, and the second flow path in that order, and controls the flow of the cell culture medium in the first flow path and the second flow path according to the state of the animal cells and / or the cell culture medium.

[0021] In the first culture tank, the cells are cultured according to the method for culturing cells derived from embryonic membranes carried out when preparing the animal cell proliferation promoter as described above. In the second culture tank, the cells are cultured according to the method for culturing animal cells in a medium containing the animal cell proliferation promoter as described above.

[0022] The first culture tank and the second culture tank are fluidically connected by the first flow path and the second flow path. For example, as shown in FIG. 1(A), the first culture tank 11 and the second culture tank 12 may be directly connected by the first flow path 21 and the second flow path 22. As long as the first culture tank 11 and the second culture tank 12 are fluidically connected, one or more other culture tanks may be provided, and various configurations are possible for connecting the other culture tanks to the first culture tank 11 or the second culture tank 12. For example, as shown in FIG. 1(B), a third culture tank 13 may be provided in the middle of the first flow path 21, and the flow path may be connected from the first culture tank 11 to the second culture tank 12 through the third culture tank 13. 1(C), a third culture tank 13 may be provided independently of the second culture tank 12, and a third flow path 31 and a fourth flow path 32 may be provided to fluidly connect the first culture tank 11 and the third culture tank 13, separate from the first flow path 21 and the second flow path 22. In the cases of (B) and (C), by appropriately selecting cells to be cultured in the third culture tank 13, the cells can be influenced by the culture in the first culture tank 11 or the second culture tank 12, or can influence those cultures.

[0023] Here, there are various possible influences that the culture in the first culture tank 11 or the culture in a culture tank other than the second culture tank 12 may have from the culture in the first culture tank or the second culture tank, and various influences that the culture in the first culture tank or the second culture tank may have. Examples of such influences include cell proliferation, cell differentiation, tissue morphogenesis, adjustment of the pH of the culture medium, and prevention of bacterial contamination of the culture medium.

[0024] Specifically, for example, by placing cells in the third culture tank 13 that are different from the cells in the second culture tank 12 and can grow in a medium in which cells derived from an embryonic membrane have been cultured, cells can be grown simultaneously in the second culture tank 12 and the third culture tank 13. Alternatively, by culturing cells derived from an embryonic membrane that are different from the cells derived from the first culture tank 11 in the third culture tank 13, the growth of the cells cultured in the second culture tank 12 can be further promoted. Alternatively, the cells cultured in the third culture tank 13 may be cells that can grow in a medium in which cells derived from an embryonic membrane have been cultured and that secrete a factor that causes the cells cultured in the second culture tank 12 to grow.

[0025] The tanks may be connected by tubes that form flow paths. Valves may be installed in these tubes. The medium flow rate control unit may monitor the state of the cultured cells and / or cell culture medium, and control the flow (e.g., flow rate, flow rate, etc.) of the cell culture medium in each flow path via the valves according to the state.

[0026] The structure of the valve is not particularly limited, and may be a known structure such as a flange type or a screw type, and the method of adjusting the valve is also not particularly limited, and may be a known adjustment method such as an air-driven type, an electromagnetic type, an electric type, a hydraulic type, etc. In addition, the tube may be provided with a check valve to prevent backflow of the cell culture medium in the tube.

[0027] Examples of the state of the cells and / or cell culture medium monitored by the medium flow rate control unit include, but are not limited to, the state of cell proliferation, the state of cell differentiation, and the pH of the medium. The state of cell proliferation can be determined, for example, by counting the number of cells per unit area, or by measuring the ratio of the area occupied by the cells to the bottom area of ​​the plate. The state of cell differentiation can be determined, for example, by measuring fluorescence after introducing a marker gene into the cells that is expressed and fluoresces upon differentiation. The pH of the medium may be determined, for example, by directly measuring the pH, or by adding a dye that changes depending on the pH to the medium and determining the color of the dye. EXAMPLES

[0028] (1) Method for isolating chorioallantoic membrane or yolk sac The chorioallantoic membrane or yolk sac used in the test was isolated by the following method.

[0029] Fertilized chicken eggs were incubated at 37°C, and after 14 days, the eggshell was cracked and the membrane tissue that was in contact with the eggshell and through which blood vessels ran was taken out as the chorioallantoic membrane, while the membrane tissue that surrounded the egg yolk was taken out as the yolk sac, and these membrane tissues were immersed in HBSS (Hank's Balanced Salt Solution).These membrane tissues were physically cut into small pieces using tweezers, centrifuged, and the supernatant was removed.

[0030] Collagenase solution (200 IU / mL) was added to each isolated membrane tissue, and the tissue was kept at 37°C for 1 hour, then centrifuged again and the supernatant was removed. HBSS was added to each collagenase-treated membrane tissue, and the tissue was filtered through a 40 μm filter. The filtrate was centrifuged and the supernatant was removed to recover chorioallantoic membrane-derived cells or yolk sac-derived cells. When chorioallantoic membrane-derived cells or yolk sac-derived cells were to be preserved, they were suspended in a cell freezing solution (10% DMSO solution), frozen by gradually cooling to -80°C, and preserved at -80°C.

[0031] (2) Method for culturing chorioallantoic membrane or yolk sac The chorioallantoic membrane-derived cells or yolk sac-derived cells obtained in (1) were added to DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution (hereinafter referred to as FBS-PS-containing DMEM) at a concentration of 4 × 10 6 The cells were seeded onto a cell culture dish (10 cm in diameter) at a concentration of 10 cells / dish and incubated at 37°C, 5% CO 2 The cells were incubated under ambient conditions for 2 to 4 days.

[0032] (3) Method for collecting culture supernatant The chorioallantoic membrane culture medium or yolk sac culture medium prepared by the culture method shown in (2) was filtered through a 0.22 μm filter, and the obtained filtrate was used as an animal cell growth promoter for animal cell culture. When the prepared culture supernatant was to be stored, it was frozen at -20°C.

[0033] (4) Animal cell culture (4-1) Method for isolating cells from each tissue Chicken cells used for culture were isolated by the following method.

[0034] First, chicken fertilized eggs were incubated at 37°C, and after 14 days, the eggshell was broken to remove the chicken embryo. The stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius, and bone were isolated from the 14-day chicken embryo and immersed in HBSS solution. Then, cells derived from each tissue were isolated in the same manner as in (1).

[0035] (4-2) Cultivation methods for cells from various tissues Cells derived from each tissue were cultured in the same manner as in (2) using a culture medium prepared by adding an equal volume of chorioallantoic membrane culture supernatant or yolk sac culture supernatant prepared in (3) to FBS / PS-containing DMEM. As a control, cells were cultured under the same conditions except that the medium was FBS / PS-containing DMEM alone without the addition of chorioallantoic membrane culture supernatant or yolk sac culture supernatant.

[0036] (4-3) Method for counting cell numbers in each culture The results of (4-2) were quantitatively evaluated for cells derived from the liver, pancreas, oviduct, stomach, muscle, heart, intestine, brain, bursa of Fabricius, and bone. The number of cells derived from each tissue was counted by the following method.

[0037] First, the culture medium of the cells derived from each tissue was removed from the dish, and PBS was added to wash the cells. After removing the PBS, a trypsin-EDTA solution was added and incubated at 37°C for 1-2 minutes to detach the cells, and then FBS-PS-containing DMEM was added to stop the reaction. All the solution containing the detached cells was collected and centrifuged to obtain cells. Furthermore, the cells were suspended in FBS-PS, and 20 μL of this cell suspension was mixed with 20 μL of trypan blue solution. The number of live cells not stained with trypan blue in this cell suspension was counted under a microscope using a hemocytometer.

[0038] (4-4) Results First, cells were observed under a phase-contrast microscope after being cultured for about 3 to 5 days in medium supplemented with chorioallantoic membrane culture supernatant. Photographs of the cells are shown in Figure 2. No matter which cells were cultured, the number of cells was greater when the chorioallantoic membrane culture supernatant prepared in (3) was added than when it was not added. This indicates that the addition of the chorioallantoic membrane culture supernatant promoted cell proliferation.

[0039] To quantitatively demonstrate these results, chorioallantoic membrane culture supernatant was used for cells derived from the liver, pancreas, and oviduct, and the average cell counts and standard errors for experiments performed in three petri dishes are shown in Figure 3. In the cases of cells derived from the liver, pancreas, and oviduct, the addition of chorioallantoic membrane culture supernatant resulted in approximately 2.5- to 4-fold cell proliferation compared to the absence of addition of chorioallantoic membrane culture supernatant.

[0040] Next, we similarly investigated the proliferation-promoting effect on cells derived from stomach, muscle, heart, liver, intestine, brain, bursa of Fabricius, and bone using a medium supplemented with yolk sac culture supernatant (n=2), and in all cases, the number of cells was greater when yolk sac culture supernatant was added than when chorioallantoic membrane culture supernatant was not added, as shown in Figure 4. This indicates that the proliferation of cells was promoted by the addition of yolk sac culture supernatant. [Industrial Applicability]

[0041] The present invention makes it possible to provide a novel animal cell proliferation promoter, a medium for animal cell culture, and an animal cell culture device.

Claims

1. A method for producing a culture supernatant of any one of the mixed cultures selected from the group consisting of: 1) amnion-derived cells, chorioallantoic membrane-derived cells, and yolk sac-derived cells, 2) amnion-derived cells and chorioallantoic membrane-derived cells, 3) amnion-derived cells and yolk sac-derived cells, or 4) chorioallantoic membrane-derived cells and yolk sac-derived cells, obtained from an embryonic membrane derived from a fertilized egg of a bird.

2. The method for producing a culture supernatant according to claim 1 , wherein the fertilized avian egg is a chicken egg.

3. A method for producing an animal cell proliferation promoter, comprising: Obtaining a culture supernatant by the method for producing a culture supernatant according to claim 1 or 2; A step of producing an animal cell proliferation promoter containing the culture supernatant; A manufacturing method comprising:

4. A method for producing an animal cell proliferation promoter, comprising: Obtaining a culture supernatant of amnion-derived cells obtained from an embryonic membrane derived from a fertilized egg of a bird; A step of producing an animal cell proliferation promoter containing the culture supernatant; A manufacturing method comprising:

5. A method for producing an animal cell proliferation promoter as described in claim 4, wherein the avian fertilized egg is a chicken egg.

6. The manufacturing method described in any one of claims 3 to 5, wherein the animal cell proliferation promoter is an animal cell proliferation promoter for one or more types of animal cells selected from the group consisting of liver, pancreas, oviduct, stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius and bone.

7. A method for producing a medium for animal cell culture, comprising the steps of: Obtaining an animal cell proliferation promoter by the production method according to any one of claims 3 to 6; A step of producing a medium for animal cell culture containing the animal cell proliferation promoter; A manufacturing method comprising:

8. a first culture vessel in which any one of 1) amnion-derived cells, chorioallantoic membrane-derived cells, and yolk sac-derived cells, 2) amnion-derived cells and chorioallantoic membrane-derived cells, 3) amnion-derived cells and yolk sac-derived cells, or 4) chorioallantoic membrane-derived cells and yolk sac-derived cells, which are obtained from avian fertilized eggs, is co-cultured; a second culture tank in which one or more animal cells to be grown are cultured, the animal cells being selected from the group consisting of liver, pancreas, oviduct, stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius, and bone; A first flow path for flowing a culture medium from the first culture tank to the second culture tank; A second flow path for flowing a culture medium from the second culture tank to the first culture tank; an animal cell culture device comprising: a medium flow rate control unit that circulates animal cell culture medium through a first culture tank, a first flow path, a second culture tank, and a second flow path in that order, and controls the flow of the animal cell culture medium in the first flow path and the second flow path according to a state of the animal cells and / or the animal cell culture medium.

9. A first culture tank in which amnion-derived cells obtained from avian fertilized eggs are cultured; a second culture tank in which one or more animal cells to be grown are cultured, the animal cells being selected from the group consisting of liver, pancreas, oviduct, stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius, and bone; A first flow path for flowing a culture medium from the first culture tank to the second culture tank; A second flow path for flowing a culture medium from the second culture tank to the first culture tank; an animal cell culture device comprising: a medium flow rate control unit that circulates animal cell culture medium through a first culture tank, a first flow path, a second culture tank, and a second flow path in that order, and controls the flow of the animal cell culture medium in the first flow path and the second flow path according to a state of the animal cells and / or the animal cell culture medium.

10. a medium introduction path for introducing fresh animal cell culture medium into the refluxing animal cell culture medium; and a medium removal path for removing the cultured animal cell culture medium from the circulating animal cell culture medium, 10. The animal cell culture device according to claim 8 or 9, wherein the culture medium flow rate control unit controls the introduction of the fresh animal cell culture medium from the culture medium inlet path and the removal of the cultured animal cell culture medium from the culture medium removal path according to a state of the animal cells and / or the animal cell culture medium.

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