Animal cell growth stimulants, cell culture media containing these animal cell growth stimulants, and animal cell culture equipment.

Abstract

The invention relates to an animal cell growth stimulant. Specifically, a supernatant of embryonic membrane culture derived from fertilized eggs of poultry or reptiles is used as an animal cell growth stimulant. For example, by adding, to an animal cell culture medium, an animal cell growth stimulant comprising a supernatant of embryonic membrane culture derived from fertilized eggs of poultry or reptiles as the active substance, the growth of animal cells can be stimulated.

Description

Animal cell growth promoter, medium for animal cell culture, and animal cell culture apparatus

[0001] The present invention relates to an animal cell growth promoter, a medium for animal cell culture, and an animal cell culture apparatus.

[0002] In the culture of animal cells, generally, a serum medium supplemented with fetal bovine serum or the like in the medium or a serum-free medium supplemented with hormones, growth factors, etc. instead of serum is used as a factor having a function of maintaining cell growth.

[0003] In addition to those factors, Japanese Patent Application Laid-Open No. 2013-247927 discloses that rice bran extract has a growth promoting effect on cultured animal cells. Japanese Patent Application Laid-Open No. 2011-182736 discloses that culturing animal cells in a medium supplemented with a hydrolyzate of β-conglycinin concentrate can increase the cell growth rate. Further, Japanese Patent Application Laid-Open No. 7-188292 discloses that a glycoprotein contained in the culture supernatant of human-derived fibroblasts promotes the growth of human vascular endothelial cells and hepatocytes. Further, Japanese Patent Application Laid-Open No. 5-301893 discloses that a protein contained in the cell culture supernatant obtained by culturing a human-derived glioma cell line has a growth promoting effect on glial cells and fibroblasts.

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

[0005] One embodiment of the present invention is an animal cell growth promoter containing the culture supernatant of the embryonic membrane of the eggs of birds or reptiles as an active ingredient. The egg may be a chicken egg. The animal cell growth promoter may be a cell growth agent for muscle cells, visceral cells, or nerve cells, or may be a cell growth agent for liver cells, pancreatic cells, or fallopian tube cells.

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

[0007] Another embodiment of the present invention is an animal cell culture apparatus comprising: 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 intended for proliferation; a first channel for flowing culture medium from the first culture tank to the second culture tank; a second channel for flowing culture medium from the second culture tank to the first culture tank; and a culture medium flow rate control unit that perfuses the cell culture medium in the order of the first culture tank, the first channel, the second culture tank, and the second channel, and controls the flow of the cell culture medium in the first channel and the second channel according to the state of the animal cells and / or the cell culture medium. Furthermore, the system may further include a medium introduction channel for introducing fresh cell culture medium into the perfusion cell culture medium, and a medium removal channel for removing the cultured cell culture medium from the perfusion cell culture medium, wherein the medium flow rate control unit controls the introduction of the fresh cell culture medium from the medium introduction channel and the removal of the cultured cell culture medium from the medium removal channel according to the state of the animal cells and / or the cell culture medium.

[0008] ==Cross-reference to related literature== This application claims priority based on Japanese Patent Application No. 2018-210910, filed on 8 November 2018, which is incorporated herein by reference.

[0009] This is a schematic diagram of a cell proliferation apparatus in one embodiment of the present invention. This is a photograph showing the results of observations in one embodiment of the present invention, where cell proliferation is promoted in cells derived from the tissues shown in the figure (stomach, skeletal muscle, heart, gallbladder, intestine, brain, bursa of Fabricius, and bone) when the culture supernatant of chicken egg yolk sac is added to the culture medium. This is a graph quantitatively showing the results of Figure 1 for cells derived from the liver, pancreas, and oviduct in one embodiment of the present invention. This is a graph showing that cell proliferation is promoted in cells derived from the tissues shown in the figure (stomach, muscle, heart, liver, intestine, brain, bursa of Fabricius, and bone) when the culture supernatant of chicken egg yolk sac is added to the culture medium in one embodiment of the present invention.

[0010] The object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and will be easily reproducible to those skilled in the art from the description herein. The embodiments and specific examples of the invention described below are examples of preferred embodiments of the present invention and are provided for illustrative or explanatory purposes only, and do not limit the present invention thereto. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein, within the intent and scope of the present invention as disclosed herein.

[0011] ==Animal Cell Proliferation Promoter== The animal cell proliferation promoter of this disclosure contains the culture supernatant of the embryonic membrane of an egg of a bird or reptile as an active ingredient.

[0012] The birds or reptiles from which the eggs from which the embryonic membranes are collected may include, but are not limited to, quail, chickens, lizards, snakes, crocodiles, and turtles; any bird or reptile that has an embryonic membrane in its egg will suffice.

[0013] Here, embryonic membrane refers to membrane tissue that forms outside the embryo during the development of birds and reptiles, performing functions such as protecting, nourishing, and respiration of the embryo, and not participating in the construction of the body after hatching or birth. Examples include the serosa located on the outermost part of the embryo, the amnion surrounding the embryonic body, the allantoate forming the urinary sac, the chorioallantoic membrane (CAM) where the serosa and allantoate are partially fused, and the yolk sac surrounding the yolk.

[0014] The culture supernatant of embryonic membranes is the culture medium obtained by isolating and culturing embryonic membranes. The embryonic membranes to be cultured can be one type or a mixture of several types from the aforementioned membranes such as the serous membrane, amniotic membrane, allanine, serous membrane, and yolk sac.

[0015] The embryonic membrane can be collected from the egg by known methods (see, for example, Eiji Ichijima: Chemistry and Biology, Vol. 13, No. 8, pp. 489-497 (1975)). Specifically, the embryonic membrane can be isolated by the following method: First, after incubating a fertilized egg under conditions suitable for artificial incubation for a predetermined period, the eggshell is cracked open. (1) The allantoamnios can be obtained by removing the swollen membrane that arises from the ventral side of the posterior part of the digestive tract of the embryo. (2) The amnion can be obtained by removing the transparent membrane tissue without blood vessels that surrounds the fetus. (3) The yolk sac can be obtained by removing the membrane tissue that surrounds the yolk. (4) The serous membrane can be obtained by removing the outermost membrane tissue that surrounds all the elements of the egg. Furthermore, if the incubation time is extended, the serous membrane and allantoamnios will partially fuse to form the serosa. At this stage, the serosa can be obtained by removing the membrane tissue that is in contact with the eggshell and has blood vessels. The incubation period can be appropriately determined depending on the animal and the target membrane tissue. For example, in the case of chicken eggs, 4 to 21 days is preferred, 10 to 18 days is more preferred, and 13 to 15 days is even more preferred. When culturing these embryonic membranes, they may be cultured as membranes, but embryonic membrane-derived cells may also be isolated and cultured from the embryonic membrane by physically disrupting the membrane or by enzymatic treatment with proteases (e.g., collagenase, elastase, dispase, papain, etc.).

[0016] For culturing embryonic membranes, culture media commonly used for animal cell culture can be used, such as DMEM and F12, but are not limited to these. Supplements that are normally included as culture medium additives may be added to this medium. Culture conditions can also be those commonly used for animal cell culture, typically using a carbonate-buffered medium at 5% CO2 and 37°C, but the culture conditions are not limited to this and can be appropriately selected by those skilled in the art. The culture time is not particularly limited, but 1 to 7 days is preferred, and 2 to 6 days is more preferred. After culturing, the supernatant may be collected and added directly to the cell culture medium, or the medium obtained by removing solid matter such as cell residue 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 shows a proliferation effect on 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 types of animal cells cultured in the cell culture medium to which this culture supernatant is added are not particularly limited, but examples include muscular cells such as smooth muscle cells, cardiomyocytes, and skeletal muscle cells; visceral cells such as cardiac cells, hepatocytes, stomach-derived cells, and intestinal-derived cells; and nervous system cells such as nerve cells and glial cells. The animal species from which the cells originate are also not particularly limited, but examples include humans, mice, rats, monkeys, and pigs.

[0018] Thus, by adding the culture supernatant obtained from culturing germ membrane-derived cells to the animal cell culture medium, the proliferation of the cultured animal cells can be promoted. Therefore, this culture supernatant can be used as an animal cell proliferation promoter.

[0019] Furthermore, when using the culture supernatant of germ membrane-derived cells as an animal cell growth promoter, other cell growth factors may be used simultaneously.

[0020] ==Cell Proliferation Apparatus== The cell proliferation apparatus of the present invention comprises: 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 intended for proliferation; a first channel for flowing culture medium from the first culture tank to the second culture tank; a second channel for flowing culture medium from the second culture tank to the first culture tank; and a culture medium flow rate control unit that perfuses the cell culture medium in the order of the first culture tank, the first channel, the second culture tank, and the second channel, and controls the flow of the cell culture medium in the first channel and the second channel according to the state of the animal cells and / or the cell culture medium.

[0021] In the first culture tank, culture is carried out in accordance with the method for culturing germ membrane-derived cells used when preparing animal cell proliferation promoters, as described above. In the second culture tank, culture is carried out in accordance with the method for culturing animal cells in a culture medium to which animal cell proliferation promoters have been added, as described above.

[0022] The first culture tank and the second culture tank are fluidly connected by the first channel and the second channel. For example, as shown in Figure 1(A), the first culture tank 11 and the second culture tank 12 may be directly connected by the first channel 21 and the second channel 22. As long as the first culture tank 11 and the second culture tank 12 are fluidly 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 Figure 1(B), a third culture tank 13 may be provided in the middle of the first channel 21, and the channel may be connected from the first culture tank 11 through the third culture tank 13 to the second culture tank 12. Alternatively, as shown in Figure 1(C), a third culture tank 13 may be provided independently of the second culture tank 12, and in addition to the first channel 21 and the second channel 22, a third channel 31 and a fourth channel 32 may be provided to fluidly connect the first culture tank 11 and the third culture tank 13. In cases (B) and (C), by appropriately selecting the cells to be cultured in the third culture tank 13, those cells can be affected by or influence the culture in the first culture tank 11 and the second culture tank 12.

[0023] Here, various influences can be considered on the culture in the first culture tank 11 and culture in culture tanks other than the second culture tank 12, as well as on the culture in the first culture tank and culture in the second culture tank. Examples include cell proliferation, cell differentiation, tissue morphogenesis, pH adjustment of the culture medium, and prevention of bacterial contamination of the culture medium.

[0024] For example, specifically, by placing cells in the third culture tank 13 that are different from those in the second culture tank 12 and can grow in the culture medium used to cultivate germ membrane-derived cells, cells can be grown simultaneously in both the second culture tank 12 and the third culture tank 13. Alternatively, by culturing germ membrane-derived cells different from those in the first culture tank 11 in the third culture tank 13, the growth of 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 the culture medium used to cultivate germ membrane-derived cells and that secrete factors that promote the growth of cells cultured in the second culture tank 12.

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

[0026] The valve structure is not particularly limited and may be any known structure such as flange type or threaded type. The valve adjustment method is also not particularly limited and may be any known adjustment method such as air-driven, electromagnetic, electric, or hydraulic. Furthermore, a check valve may be provided in the tube to prevent backflow of the cell culture medium within the tube.

[0027] The state of cells and / or cell culture medium monitored by the culture medium flow control unit may include, but is not limited to, the state of cell proliferation, the state of cell differentiation, and the pH of the culture 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 cells to the bottom area of ​​the plate. The state of cell differentiation can be determined, for example, by introducing a marker gene that is expressed and emits fluorescence upon differentiation into the cells and measuring the fluorescence. The pH of the culture medium may be determined, for example, by directly measuring the pH, or by adding a dye that changes with pH to the culture medium and determining the pH by its color.

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

[0029] Fertilized chicken eggs were incubated at 37°C. After 14 days, the eggshells were cracked open, and the membrane tissue in contact with the shell and containing blood vessels was removed as the serosa, while the membrane tissue surrounding the yolk was removed as the yolk sac. These membrane tissues were then immersed in HBSS (Hank's Balanced Salt Solution). After physically cutting these membrane tissues into small pieces with tweezers, they were centrifuged, and the supernatant was removed.

[0030] Each isolated membrane tissue was treated with collagenase solution (200 IU / mL) and held at 37°C for 1 hour. The tissue was then centrifuged again, and the supernatant was removed. HBSS was added to each collagenase-treated membrane tissue, filtered through a 40 μm filter, and the resulting filtrate was centrifuged to remove the supernatant, thereby recovering either chaloallantoic membrane-derived cells or yolk sac-derived cells. For preservation of chaloallantoic membrane-derived or yolk sac-derived cells, they were suspended in cell freezing solution (10% DMSO solution), gradually cooled to -80°C, and then stored at -80°C.

[0031] (2) Method for culturing the serosa or yolk sac The serosa-derived cells or yolk sac-derived cells obtained in (1) are added to DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution (hereinafter referred to as FBS-PS-containing DMEM), and 4 × 10 ６ Seeds were seeded in a cell culture dish (10 cm in diameter) at a concentration of cells / dish, and cultured at 37°C and 5% CO2. ２ The cells were cultured in an environmental environment for 2 to 4 days.

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

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

[0034] First, fertilized chicken eggs were incubated at 37°C, and after 14 days, the eggshells were cracked open to extract chicken embryos. From these 14-day-old chicken embryos, the stomach, skeletal muscle, heart, gallbladder, intestines, brain, bursa of Fabricius, and bones were isolated and each was immersed in HBSS solution. Subsequently, cells derived from each tissue were isolated using the same method as in (1).

[0035] (4-2) Method of culturing cells from each tissue 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 the euphoric membrane culture supernatant or yolk sac culture supernatant prepared in (3) to DMEM containing FBS and PS. As a control, cells cultured under the same conditions were used, except that a medium containing only FBS and PS without the addition of euphoric membrane culture supernatant or yolk sac culture supernatant was used.

[0036] (4-3) Method for measuring cell count in each culture The results from (4-2) were quantitatively evaluated for cells derived from the liver, pancreas, fallopian tube, 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.

[0037] First, the cell culture medium from each tissue was removed from the dish, and the cells were washed with PBS. After removing the PBS, trypsin-EDTA solution was added, and the cells were incubated at 37°C for 1-2 minutes to detach them. Then, DMEM containing FBS and PS was added to stop the reaction. The entire solution containing the detached cells was collected, and the cells were obtained by centrifugation. Furthermore, FBS and PS were added to these cells and they were suspended. 20 μL of this cell suspension was mixed with 20 μL of trypan blue solution. In this cell suspension, the number of living cells that were not stained with trypan blue was counted under a microscope using a hemocytometer.

[0038] (4-4) Results First, cells cultured for approximately 3 to 5 days in a medium supplemented with the serum allantoic membrane culture supernatant were observed using a phase-contrast microscope. Figure 2 shows photographs of these cells. In all cases of cell culture, the number of cells was higher when the serum allantoic membrane culture supernatant prepared in (3) was added compared to when it was not added. This indicates that cell proliferation was promoted by the added serum allantoic membrane culture supernatant.

[0039] To quantitatively demonstrate these results, Figure 3 shows the average cell count and standard error when experimenting with liver, pancreas, and fallopian tube-derived cells using chaloalunol culture supernatant in three petri dishes. In all cases of liver, pancreas, and fallopian tube-derived cells, adding chaloalunol culture supernatant resulted in approximately 2.5 to 4 times more cell proliferation compared to when chaloalunol culture supernatant was not added.

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

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

Claims

1. An animal cell culture proliferation promoter containing as an active ingredient the culture supernatant of embryonic membranes derived from fertilized eggs of birds or reptiles 2. The animal cell proliferation promoter according to claim 1, wherein the embryonic membrane is at least one selected from the group consisting of amnion, chorioallantoic membrane and yolk sac.

3. The animal cell proliferation promoter according to claim 1 or 2, wherein the eggs are chicken eggs.

4. The animal cell proliferation promoter according to any one of claims 1 to 3, which is a cell proliferation agent for muscle cells, visceral cells, or nervous system cells.

5. The animal cell proliferation promoter according to any one of claims 1 to 3, which is a cell proliferation agent for liver cells, pancreatic cells, and oviduct cells.

6. A cell culture medium containing the cell growth promoter according to any one of claims 1 to 5.

7. An animal cell culture device comprising: a first culture tank for culturing cells derived from the embryonic membrane of avian 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 culture 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.

8. An animal cell culture device according to claim 7, further comprising: a medium inlet path for introducing fresh cell culture medium into the circulating cell culture medium; and a medium removal path for removing post-culture cell culture medium from the circulating cell culture medium, wherein the medium flow rate control unit controls the introduction of the fresh cell culture medium from the medium inlet path and the removal of the post-culture cell culture medium from the medium removal path according to the state of the animal cells and / or the cell culture medium.

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