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

Abstract

The purpose of the present invention is to provide a novel animal cell proliferation promoter. Specifically, a culture supernatant of an embryonic membrane of an egg of a bird or reptile is used as an animal cell proliferation promoter. For example, the proliferation of animal cells can be promoted by adding a cell proliferation agent, which contains a culture supernatant of an embryonic membrane of an egg of a bird or reptile as an active ingredient, to a culture medium for culturing animal cells.

Description

[0001] This case is a divisional application of a patent application with an application date of November 7, 2019 and an application number of 201980074375.3 (PCT / JP2019 / 043713) and an invention title of "Animal Cell Proliferation Promoter, Culture Medium for Animal Cell Culture, and Animal Cell Culture Apparatus". Technical Field

[0002] The present invention relates to an animal cell proliferation promoter, a culture medium for animal cell culture, and an animal cell culture apparatus. Background Art

[0003] In the culture of animal cells, generally, as a factor having the function of maintaining cell proliferation, a serum medium such as fetal bovine serum is added to the culture medium, or a serum-free medium to which hormones or proliferation factors are added instead of serum is used.

[0004] In addition to these factors, Japanese Patent Application Laid-Open No. 2013-247927 discloses that rice bran extract has a proliferation promoting effect on cultured animal cells. Further, Japanese Patent Application Laid-Open No. 2011-182736 discloses that when animal cells are cultured in a culture medium supplemented with a hydrolyzate of β-conglycinin concentrate, the cell proliferation rate can be increased. In addition, Japanese Patent Application Laid-Open No. 7-188292 discloses that a glycoprotein contained in the culture supernatant derived from human fibroblasts promotes the proliferation of human vascular endothelial cells and hepatocytes. In addition, Japanese Patent Application Laid-Open No. 5-301893 discloses that a protein contained in the cell culture supernatant obtained by culturing a human glioma cell line has a proliferation promoting effect on glial cells or fibroblasts. Summary of the Invention

[0005] Technical Problem to be Solved by the Invention

[0006] An object of the present invention is to provide a new animal cell proliferation promoter, a culture medium for animal cell culture, and an animal cell culture apparatus.

[0007] Technical Solution for Solving the Technical Problem

[0008] One embodiment of the present invention relates to an animal cell proliferation promoter containing a culture supernatant of the embryonic membrane of an egg of a bird or a reptile as an active ingredient. The above egg may be a chicken egg. The above animal cell proliferation promoter may be a cell proliferator for muscle cells, visceral cells, or nervous system cells, or may be a cell proliferator for liver cells, pancreatic cells, or fallopian tube cells.

[0009] Another embodiment of the present invention relates to a culture medium for cell culture containing the cell proliferation promoter described in any one of the above.

[0010] Another embodiment of the present invention relates to an animal cell culture device, which includes: a first culture tank for culturing cells derived from the embryonic membranes of eggs of birds or reptiles; a second culture tank for culturing animal cells for the purpose of proliferation; a first flow path for allowing a culture medium to flow from the first culture tank to the second culture tank; a second flow path for allowing the culture medium to flow from the second culture tank to the first culture tank; a culture medium flow rate control unit for causing the cell culture medium to flow back in the order of the first culture tank, the first flow path, the second culture tank, and the second flow path, 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 above-mentioned animal cells and / or the above-mentioned cell culture medium. In addition, the animal cell culture device further has: a culture medium introduction passage for introducing fresh cell culture medium into the above-mentioned cell culture medium flowing back; and a culture medium removal passage for removing the cell culture medium after culturing from the above-mentioned cell culture medium flowing back, and the above-mentioned culture medium flow rate control unit can also control the introduction of the above-mentioned fresh cell culture medium from the above-mentioned culture medium introduction passage and the removal of the above-mentioned cell culture medium after culturing from the above-mentioned culture medium removal passage according to the state of the above-mentioned animal cells and / or the above-mentioned cell culture medium.

[0011] ==Cross-reference to related literature==

[0012] This application is based on Japanese Patent Application No. 2018-210910 filed on November 8, 2018, claims priority, and incorporates it into this specification by reference. Description of the drawings

[0013] Figure 1 It is a schematic diagram of a cell proliferation device in one embodiment of the present invention.

[0014] Figure 2 It is a photograph showing the observation results that the cell proliferation of cells derived from the tissues (stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius, and bone) shown in the figure is promoted in the case of adding the culture supernatant of the chicken embryo chorioallantoic membrane to the culture medium in one example of the present invention.

[0015] Figure 3 In one example of the present invention, for cells derived from the liver, pancreas, and oviduct, it quantitatively represents Figure 1 The results of the

[0016] Figure 4 It is a graph showing that the cell proliferation of cells derived from the tissues (stomach, muscle, heart, liver, intestine, brain, bursa of Fabricius, and bone) shown in the figure is promoted in the case of adding the culture supernatant of the egg yolk sac of an egg to the culture medium in one example of the present invention. Detailed implementation mode

[0017] The purpose, features, advantages and ideas of the present invention are obvious to those skilled in the art from the description in this specification. If one is skilled in the art, the present invention can be easily reproduced from the description in this specification. The following-described embodiments and specific examples of the invention represent preferred embodiments of the present invention and are shown for illustration or explanation purposes. The present invention is not limited to these. Within the scope and intention of the present invention disclosed in this specification, various changes and modifications can be made based on the description in this specification, which are obvious to those skilled in the art.

[0018] ==Animal cell proliferation promoter==

[0019] The animal cell proliferation promoter of the present invention contains the culture supernatant of the embryonic membrane of the eggs of birds or reptiles as an active ingredient.

[0020] Examples of the birds or reptiles that are the sources of the eggs from which the embryonic membranes are collected include quails, chickens, lizards, snakes, crocodiles, turtles, etc., but are not limited to these. Any bird or reptile animal having an embryonic membrane of an egg is acceptable.

[0021] Here, the embryonic membrane refers to: during the development of birds and reptiles, it is formed outside the embryo and plays functions such as protecting, nourishing, and breathing for the embryo, and does not participate in the construction of the body after hatching or birth. Examples include: the serosa (chorion) located on the outermost side of the embryo, the amnion that wraps the embryo body, the allantoic membrane that forms the allantois, the chorioallantoic membrane (chorioallantoic membrane: CAM) formed by partial healing of the serosa and the allantoic membrane, the yolk sac that wraps the yolk, etc.

[0022] The culture supernatant of the embryonic membrane refers to the culture medium obtained by separating and culturing the embryonic membrane. The cultured embryonic membrane can be one of the above-mentioned membranes such as the serosa, amnion, allantoic membrane, chorioallantoic membrane, and yolk sac, or a mixture of multiple types can be cultured.

[0023] The embryonic membranes can be collected from eggs by known methods (for example, refer to Eiji Ichijima: Chemistry and Biology, Vol. 13, No. 8, p489 - 497 (1975), etc.). For example, specifically, the embryonic membranes can be separated by the following method. First, after incubating the fertilized eggs under conditions suitable for artificial hatching for a specified time, the eggshell is broken. (1) The allantoic membrane can be obtained by taking out the swollen membrane generated from the ventral side of the posterior part of the digestive tract of the embryo; (2) The amnion can be obtained by taking out the avascular transparent membrane tissue present around the fetus; (3) The yolk sac can be obtained by taking out the membrane tissue that wraps the yolk; (4) The serosa can be obtained by taking out the outermost membrane tissue that surrounds all the elements inside the egg. In addition, when the incubation time is extended, the serosa and the allantoic membrane partially fuse to form the chorioallantoic membrane. At this stage, the chorioallantoic membrane can be obtained by taking out the membrane tissue that contacts the eggshell and has blood vessels. The incubation time can be appropriately determined according to each animal and also according to the target membrane tissue. For example, in the case of chicken eggs, it is preferably 4 to 21 days, more preferably 10 to 18 days, and further preferably 13 to 15 days. When culturing these embryonic membranes, they can be directly cultured in a membranous state, or the cells derived from the embryonic membranes can be separated from the embryonic membranes by physically breaking the membrane or by enzymatic treatment with proteases (such as collagenase, elastase, dispase (neutral protease), papain, etc.) and then cultured.

[0024] In the culture of embryonic membranes, the media used in ordinary animal cell culture can be used. Examples include DMEM, F12, etc., but are not limited to these. Supplements usually formulated as media additives can be added to this medium. The culture conditions can also be those used for ordinary animal cell culture. Typically, a carbonate buffer-based medium is used, and the culture is carried out at 5% CO2 and 37 °C, but the culture conditions are not limited to this, and those skilled in the art can appropriately select. The culture time is not particularly limited, preferably 1 to 7 days, more preferably 2 to 6 days. After culturing, the supernatant is recovered and can be directly added to the medium for cell culture, or the medium obtained by removing solid substances such as cell residues by filtration or centrifugation can be added. The addition amount of the culture supernatant to the medium for cell culture is not particularly limited as long as it is an effective amount that can confirm the proliferation effect of the cultured cells. The final concentration in the medium is preferably 20% or more, more preferably 35% or more, and further preferably 50% or more.

[0025] The type of animal cells cultured in the cell culture medium supplemented with the culture supernatant is not particularly limited, and examples include: muscle cells such as smooth muscle cells, cardiomyocytes, and skeletal muscle cells; cardiac cells; hepatocytes; visceral cells such as cells derived from the stomach and cells derived from the intestine; or nervous system cells such as nerve cells or glial cells. The animal species from which the cells are derived is also not particularly limited, and examples include humans, mice, rats, monkeys, pigs, etc.

[0026] Thus, by adding the culture supernatant obtained by culturing cells derived from the embryonic membrane to the animal cell culture medium, the proliferation of the cultured animal cells can be promoted. Therefore, the culture supernatant can be used as an animal cell proliferation promoter.

[0027] In addition, when the culture supernatant of cells derived from the embryonic membrane is used as an animal cell proliferation promoter, other cell proliferation factors can also be used simultaneously.

[0028] ==Cell Proliferation Device==

[0029] The cell proliferation device of the present invention includes: a first culture tank for culturing cells derived from the embryonic membrane of an egg of a bird or reptile; a second culture tank for culturing animal cells for the purpose of proliferation; a first flow path for allowing the culture medium to flow from the first culture tank to the second culture tank; a second flow path for allowing the culture medium to flow from the second culture tank to the first culture tank; and a culture medium flow rate control unit for circulating the cell culture medium in the order of the first culture tank, the first flow path, the second culture tank, and the second flow path, 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 above-mentioned animal cells and / or the above-mentioned cell culture medium.

[0030] In the first culture tank, the cells derived from the embryonic membrane are cultured according to the culture method of the cells derived from the embryonic membrane as described above when preparing the animal cell proliferation promoter. In the second culture tank, the animal cells are cultured according to the culture method of culturing animal cells in the medium supplemented with the animal cell proliferation promoter as described above.

[0031] The first culture tank and the second culture tank are fluidly connected by the first flow path and the second flow path. For example, as shown in (A) of Figure 1 , the first culture tank 11 and the second culture tank 12 can 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 fluidly connected, one or more other culture tanks can also be provided, and various configurations can also be considered for the connection between the other culture tanks and the first culture tank 11 or the second culture tank 12. For example, as shown in (B) of Figure 1 , a third culture tank 13 can be provided in the middle of the first flow path 21, and the flow path is connected from the first culture tank 11 to the second culture tank 12 through the third culture tank 13. Or, as shown in Figure 1As shown in (C), a third culture tank 13 is provided independently of the second culture tank 12, and flow paths are also provided for fluidly connecting the first culture tank 11 and the third culture tank 13, namely, a third flow path 31 and a fourth flow path 32, which are different from the first flow path 21 and the second flow path 22. In the case of (B) or (C), by appropriately selecting the cells cultured in the third culture tank 13, these cells can be affected by the culture in the first culture tank 11 or the second culture tank 12, or can affect these cultures.

[0032] Here, the culture in the first culture tank 11 or the culture in a culture tank other than the second culture tank 12 being affected by the culture in the first culture tank or the second culture tank, or having an impact on the culture in the first culture tank or the second culture tank, various impacts can be considered. For example, they can include: cell proliferation, cell differentiation, tissue morphogenesis, pH regulation of the culture medium, prevention of bacterial contamination in the culture medium, etc.

[0033] For example, specifically, by placing in the third culture tank 13 cells that are different from the cells in the second culture tank 12 and can proliferate in a culture medium for culturing cells derived from the embryonic membrane, cells can be made to proliferate simultaneously in the second culture tank 12 and the third culture tank 13. Or, by culturing in the third culture tank 13 cells derived from the embryonic membrane that are different from those in the first culture tank 11, the proliferation of the cells cultured in the second culture tank 12 can be further promoted. Or, the cells cultured in the third culture tank 13 can also be cells that can proliferate in a culture medium for culturing cells derived from the embryonic membrane and secrete factors that cause the cells cultured in the second culture tank 12 to proliferate.

[0034] The tanks are connected by tubes, and these tubes can form flow paths. Additionally, valves can be provided on these tubes. The culture medium flow control unit can monitor the state of the cultured cells and / or the cell culture medium, and based on this state, control the flow of the cell culture medium in each flow path (such as flow rate, flow volume, etc.) via the valve.

[0035] The structure of the valve is not particularly limited, as long as it is a known structure such as a flange type or a threaded type. The adjustment method of the valve is also not particularly limited, as long as it is a known adjustment method such as air-driven, electromagnetic, electric, or hydraulic. Additionally, in order to prevent the backflow of the cell culture medium in the tube, a check valve can also be provided in the tube.

[0036] The states of cells and / or cell culture media monitored by the culture medium flow control unit can be exemplified by, for example, the situation of cell proliferation, or the situation of cell differentiation, the pH of the culture medium, etc., but are not limited to these. The situation of cell proliferation can be judged by, for example, counting the number of cells per unit area, or measuring the proportion of the area occupied by the cells relative to the bottom area of the culture dish, etc. The situation of cell differentiation can be judged, for example, by pre-introducing a fluorescently labeled gene expressed during differentiation into the cells and measuring the fluorescence. The pH of the culture medium can be directly measured, for example, or a pigment that changes due to pH can be added to the culture medium and judged according to its color.

[0037] Example

[0038] (1) Method for separating the chorioallantoic membrane or yolk sac

[0039] The chorioallantoic membrane or yolk sac for the test was separated by the following methods respectively.

[0040] Fertilized chicken eggs were incubated at 37 °C. After 14 days, the eggshells were broken, and the membrane tissue in contact with the eggshell and having blood vessels was taken out as the chorioallantoic membrane. At the same time, on the other hand, the membrane tissue wrapping the egg yolk was taken out as the yolk sac, and these membrane tissues were separately immersed in HBSS (Hank’s Balanced Salt Solution). After physically chopping these membrane tissues with forceps, etc., centrifugation was carried out to remove the supernatant.

[0041] Collagenase solution (200 IU / mL) was added to each of the separated membrane tissues, and after maintaining at 37 °C for 1 hour, centrifugation was carried out again to remove the supernatant. HBSS was added to each of the membrane tissues treated with collagenase, and filtered through a 40 μm filter. By centrifuging the obtained filtrate and removing the supernatant, cells derived from the chorioallantoic membrane or cells derived from the yolk sac were recovered. In addition, when storing cells derived from the chorioallantoic membrane or cells derived from the yolk sac, they were suspended in a cell freezing solution (10% DMSO solution), slowly cooled to -80 °C and frozen, and stored at -80 °C.

[0042] (2) Method for culturing the chorioallantoic membrane or yolk sac

[0043] Cells derived from the chorioallantoic membrane or cells derived from the yolk sac obtained in (1) were added to DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (PS) solution (hereinafter referred to as FBS·PS-containing DMEM) at 4×10 6Cells / dish were inoculated into a cell culture dish (10 cm in diameter) and cultured for 2 - 4 days at 37°C in a 5% CO₂ environment.

[0044] (3) Method for recovering culture supernatant

[0045] The chorioallantoic membrane culture fluid or yolk sac culture fluid prepared by the culture method shown in (2) was filtered through a 0.22 μm filter, and the resulting filtrate was used as an animal cell proliferation promoter for animal cell culture. In addition, when storing the prepared culture supernatant, it was stored frozen at -20°C.

[0046] (4) Animal cell culture

[0047] (4-1) Method for separating cells from each tissue

[0048] The chicken cells for culture were separated by the following method.

[0049] First, fertilized chicken eggs were incubated at 37°C. After 14 days, the eggshells were broken and the chicken embryos were taken out. The stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius, and bone were separated from the 14-day-old chicken embryos and immersed in HBSS solution. Then, the cells derived from each tissue were separated by the same method as in (1).

[0050] (4-2) Method for culturing cells from each tissue

[0051] The cells derived from each tissue were cultured using a culture medium prepared by adding an equal amount of the chorioallantoic membrane culture supernatant or yolk sac culture supernatant prepared in (3) to DMEM containing FBS·PS, by the same method as in (2). In addition, as a control, a culture medium of DMEM containing only FBS·PS without adding the chorioallantoic membrane culture supernatant or yolk sac culture supernatant was used, and otherwise, cells cultured under the same conditions were used.

[0052] (4-3) Method for measuring the number of cells in each culture

[0053] The results of (4-2) were quantitatively evaluated for the cells derived from the liver, pancreas, oviduct, stomach, muscle, heart, intestine, brain, bursa of Fabricius, and bone. Here, the number of cells derived from each tissue was measured by the following method.

[0054] First, remove the culture medium of cells from each tissue from the culture dish, and add PBS to wash the cells separately. After removing the PBS, add trypsin-EDTA solution, incubate at 37 °C for 1 - 2 minutes to detach the cells, and then add DMEM containing FBS·PS to stop the reaction. Recover all the solution containing the detached cells, and obtain the cells by centrifuging it. In addition, add FBS·PS to the cells to suspend them, and mix 20 μL of the cell suspension and 20 μL of trypan blue solution. In this cell suspension, use a hemocytometer under a microscope to measure the number of viable cells not stained by trypan blue.

[0055] (4 - 4) Results

[0056] First, observe the cells cultured in the medium supplemented with the chorioallantoic membrane culture supernatant for about 3 - 5 days using a phase-contrast microscope. Figure 2 The photograph of the cells is shown. In any case of culturing cells, compared with the case without adding the chorioallantoic membrane culture supernatant, the number of cells is larger when the chorioallantoic membrane culture supernatant prepared in (3) is added. It shows that the addition of the chorioallantoic membrane culture supernatant promotes cell proliferation.

[0057] To quantitatively represent this result, the average and standard error of the cell numbers when experiments were carried out on cells derived from the liver, pancreas, and fallopian tube using the chorioallantoic membrane culture supernatant in 3 culture dishes are shown in Figure 3 In any case of cells derived from the liver, pancreas, and fallopian tube, compared with the case without adding the chorioallantoic membrane culture supernatant, the cells proliferated to about 2.5 - 4 times in the case where the chorioallantoic membrane culture supernatant was added.

[0058] Next, using the medium supplemented with the yolk sac culture supernatant, the proliferation-promoting effect on cells derived from the stomach, muscle, heart, liver, intestine, brain, bursa of Fabricius, and bone was also studied in the same way (n = 2). As a result, as shown in Figure 4 In any case, compared with the case without adding the chorioallantoic membrane culture supernatant, the number of cells is larger when the yolk sac culture supernatant is added. It shows that the addition of the yolk sac culture supernatant promotes cell proliferation.

[0059] Industrial applicability

[0060] According to the present invention, a new animal cell proliferation promoter, a culture medium for animal cell culture, and an animal cell culture device can be provided.

Claims

1. A culture supernatant obtained by mixed culturing of any one of the following cells (1) to (4) selected from cells derived from the embryonic membranes of fertilized eggs of birds, (1) cells derived from the amnion, cells derived from the chorioallantoic membrane, and cells derived from the yolk sac; (2) cells derived from the amnion and cells derived from the chorioallantoic membrane; (3) cells derived from the amnion and cells derived from the yolk sac; (4) cells derived from the chorioallantoic membrane and cells derived from the yolk sac.

2. The culture supernatant according to claim 1, wherein: the fertilized egg of the bird is a chicken egg.

3. An animal cell proliferation promoter, wherein: it contains the culture supernatant according to claim 1 or 2 as an active ingredient.

4. A culture medium for animal cell culture, wherein: it contains the animal cell proliferation promoter according to claim 3.

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