JP2003219873A - Immortalized cell derived from chinook salmon of salmonidae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell cultured in a serum-free medium in an ALPS medium system as a simply usable animal culture cell, and to provide an established animal cell culturable in the serum-free medium, a method for producing the cell, a method for culturing the cell, a method for preserving the cell, a kit for culturing cells including the cell, and the like. <P>SOLUTION: This cell is the established fish cell culturable in the serum-free medium, specifically, the established fish cell culturable in the serum-free medium containing a lactalbumin hydrolyzate, concretely, CHLS cell derived from a chinook salmon of Salmonidae. The method for producing the cell, the method for culturing the cell, the method for preserving the cell, the culture liquid for culturing the cell, the kit for culturing the cells including the cell, the method for proliferating a virus using the cell, a method for evaluating a specimen, a method for transforming the cell by a foreign gene, a transformant formed thereby, and a teaching aid comprising the kit for culturing the cell are also provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell line of fish that can be cultivated in a serum-free medium, more specifically, a cell line of fish that can be cultivated in a serum-free medium containing lactalbumin hydrolyzate, Production method, culture method thereof, storage method thereof, culture solution for culturing the same, a cell culture kit containing the same, a virus propagation method using the same, a test substance evaluation method, it The present invention relates to a method for transforming with a foreign gene, a transformant thereof, and an educational device comprising the culture kit.

[0002]

2. Description of the Related Art Fish cells have the same properties as warm-blooded animal cells and can be cultured in the same manner, but they are cultivated at a much lower temperature and in a wider temperature range than warm-blooded animal cells. It has the great feature that it is possible. Therefore, it can be said to be a valuable cell type as a cultured cell for experimental or industrial use. Although more than 100 kinds of cell lines of fish have been established, most of them are adhesion-dependent and were cultured by the static monolayer culture method. As described above, many cultured cells and excised cells of living tissues grow (scaffold-dependent) in a state of being adhered and extended to an object (culture surface, adhesive substrate).

For example, the isolated and dispersed floating cultured cells are spherical, but when they are transferred to a culture container as shown in FIG. 1 and left to stand, the spherical cells adhere to the substrate surface (see A in FIG. 1), It further extends (B) and divides and proliferates. Finally, the bottom surface of the incubator is flatly filled to form a monolayer cell sheet. This state is the state of C in FIG. 1, which is a proliferative state, that is, a confluent cell. The cells that have just become confluent are vigorous cells, but when continuously cultured, the cells increase to a fin-filled state (overgrowth: overgrowth state) and then stop growing (see D in FIG. 1). This is because "contact inhibition" due to contact with each other works. If this state is left as it is, cells gradually begin to die. If this state is continued, the cultured cells will die, so in the case of normal cell culture, it is necessary to carry out the passage operation of the seed cells in time. In other words, consider germ contamination in a clean bench (sterile operation)
However, a series of cell isolation / dispersion operations such as trypsin treatment must be applied to the cell sheet to carry out subculture.

On the other hand, in the suspension culture method, since cells can be suspended and cultured in a medium solution, the cells can be cultured in a large amount and with a relatively easy operation. To date, the following two types of fish cells that can be suspension-cultured have been reported. One is BCLidgerdin
g) reported CHSE-214 cells (King salmon embryo-derived cell line) in a suspended EMEM-S culture medium containing carboxymethyl cellulose (BCLidgerding, Develop. Biol. Standard, Four
9, 233-241 (1981)), the other one is CHSE-2.
14 is a method using the MB752 / 1 medium of CaCl ₂ -free Wei mouse called CHSE-sp developed by the present inventors (Hasobe M., et al., Bull. Eu.
r. Asso. Fish Pathol., 11, 142-144 (1991)). However, for the growth of this strain, about 160 μM Ca ²⁺ had to be continuously fed from the serum fraction in 5% FBS medium.

This cell line CHSE-sp became confluent when it was statically cultivated (state (C) in FIG. 1), and when the culture was further continued, the cells were fin-filled (overfilled). It is a cell prepared so that it will be in a state like E instead of the direction of D in FIG. 1 after increasing (growth: hyperproliferation state) (see D in FIG. 1). That is, CHSE-sp cells divide and proliferate even in a hyperproliferative state. The monolayer cell sheet attached to the bottom surface remains as it is, but the proliferated neoplastic cells continue to increase in the culture solution in a spherical suspension or light adhesion state. The floating cells taken out of the container were spherical on the underlying cell sheet, but when they were re-seeded in another culture container (cell-adhesive container), they immediately found the adhesive surface and quickly attached and proliferated. Start. That is, a general cell sheet is formed again.

[0006] One of the reasons why the CHSE-sp cells become as described above (E in Fig. 1) is calcium (Ca) in the medium.
The concentration. The medium of the supply cells is Waymout (Waymout
h's) MB752 / 1 (WM medium), but used with the formulation excluding CaCl ₂ . If the supply cell CHSE-s
When Ca is added to p at a normal concentration, that is, about 1 mM, meiotic floating spherical cells are not produced. As shown in Fig. 1-D, the culture system shows contact inhibition. Adhering cell layer produces suspension cells in which the above-mentioned underlying cells (adhesive sheet cells) produce floating cells.
The present inventors have designated s) as the ALPS culture system for convenience.

Needless to say, cultured cells are not only important for cells on an industrial scale, but also important materials for a wide range of biological research and educational materials. It is a matter of course that handling cultured cells requires equipment such as a clean bench and specialized techniques such as aseptic operation. Even if you have no experience in cell culture, or if you do not have specialized equipment or facilities, if you can easily use cultured animal cells for the time being, it will be useful for researchers. In addition, the story of cells is overflowing in science books for the general public and mass media, but high school students not only have cells with knowledge and schematic diagrams, but also animal cells that actually expand and proliferate in the red culture solution in the flask. If you have the opportunity to easily observe cells and think about the conditions / reasons for their survival, it may be a great motivation for you to have an interest in cell biology and biotechnology.

For this purpose, the CHSE-sp cells developed by the present inventors were extremely effective, but they required serum in the medium and required some expertise in their handling. I was there.

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a cell that can be easily cultured in an ALPS culture system in a serum-free medium as an animal culture cell. The present invention also provides a cell line that can be cultured in a serum-free medium, a method for producing the same, a method for culturing the same, a method for storing the same, a culture solution for culturing the same, a kit for cell culture containing the same, , A method for multiplying a virus using the method, a method for evaluating a test substance, a method for transforming it with a foreign gene,
An educational device comprising the transformant and the culture kit is provided.

[0010]

Means for Solving the Problems The present inventors have proposed a culture system (A) in which an underlying cell (adhesive sheet cell) produces a floating cell.
As a cell line capable of proliferating in the LPS culture system, CHSE-sp cells, which are cell lines derived from Salmonaceae genus Algae, have been developed. However, since CHSE-sp cells could be grown only in serum medium, their application range was limited. Therefore, it was possible to culture CHSE-sp cells in serum-free medium, and to study easily usable animal culture cells. It has been developed and can be cultivated in serum-free medium, and C
We have found a new cell line, CHLS, which has similar properties to HSE-sp cells. These cells were deposited on January 18, 2002 as FERM P-18681 at the Patent Microorganism Depositary Center, National Institute of Advanced Industrial Science and Technology.

That is, the present invention relates to a cell line of fish that can be cultured in a serum-free medium, and more specifically, a cell line of fish that can be completely serum-free cultured using lactalbumin hydrolyzate as a serum substitute. Regarding Further, the present invention, a cell line that can be cultivated in a suspension stirring culture system using a serum medium, is cultured in a suspension stirring culture system in which serum is gradually decreased, and a prototype that can be cultured in a culture system in which the amount of serum is reduced. The present invention relates to a method for producing or establishing a cell line of fish which can be cultured in the serum-free medium and which can be cultured in the serum-free medium by selecting the cell line and repeating the growth / selection of the cell line. Further, the present invention is
The present invention relates to a method of culturing a cell line of a fish that can be cultured in a serum-free medium in a serum-free medium, that is, the present invention is a serum-free medium containing lactalbumin hydrolyzate, wherein the calcium (Ca) concentration is It relates to a method of culturing the cell line of fish according to any one of claims 1 to 13, using a medium of 1 mM or less.

[0012] The present invention also provides a method for maintaining the temperature of a culture system containing a cell line of a fish that can be cultured in the serum-free medium of the present invention at a low temperature, specifically about 4 degrees or less. To a method for storing activated cells. Further, the present invention is
The present invention relates to a culture medium for culturing a cell line of fish that can be cultured in the serum-free medium of the present invention, and more particularly to a serum-free culture medium containing lactalbumin hydrolyzate. Further, the present invention is
A kit for culturing a cell line of a fish that can be cultivated in the serum-free medium of the present invention placed in a culture vessel and a cell line containing the culture solution, and for cell culture observation using the kit Of educational equipment. The present invention also relates to a method for evaluating a living cell of a test substance, which comprises introducing the test substance into a culture system of a cell line of fish that can be cultured in the serum-free medium of the present invention. The present invention also provides a method for transforming a cell line of a fish that can be cultured in the serum-free medium of the present invention, which comprises introducing a foreign gene into the cell line, and a transformant transformed by the method. Regarding the body Furthermore, the present invention is a method of inoculating a culture system of a cell line of a fish that can be cultured in the serum-free medium of the present invention with a virus, and growing the virus in a nutrient medium suitable for the growth of the virus, and The present invention relates to a method of introducing a test substance into a growing medium to evaluate the action of the test substance against the virus.

The present inventors have developed a cultured cell that can be easily used by anyone. The key points as a cultured cell that can be easily used by anyone are the unexpected properties of a cultured fish cell ((1) to (4) below) and the characteristics of the suspension-cultured fish cell CHLS developed by the present inventors (5). ) And (6). (1) Fish cells derived from a psychrothermic animal do not proliferate when cultured at a low temperature, and are in a hibernation state. (2) Fish cells that grow slowly at room temperature are
_{2 No} incubator required. (3) Detection of contaminants in fish cells is extremely low even without using a clean bench for cell manipulation. (4) Fish cells are highly unlikely to become a source of zoonotic pathogen contamination and are highly safe. (5) Fish cells in the floating agitation culture system show strong adhesion and spreading activity on a stationary culture substrate. (6) It can be cultured in a serum-free medium.

Regarding (1), a method often used by researchers using cultured fish cells, for example, if cells of salmonid fish are stored at 4 ° C., they can be preserved without trouble for several months or more. (To be exact, they are dividing and multiplying at an extremely slow rate). If you return to the culture temperature when you want to use,
It also starts to grow. The story of the contamination in (3) is true, and even if contaminants are mixed in, the culture temperature is much lower than 37 ° C of warm-blooded animals, and because of the antibiotics in the medium, the proliferation of contaminants should be detected under a microscope. Is very few. This is very useful in student experiments for a large number of people. The story of biohazard in (4) is important. When students or non-professionals use animal cells, safety is paramount. (5) The present inventors have created CHSE-sp cells derived from King Salmon and FHM-sp cells of Cyprinidae as cell lines in a suspension culture system. Then, the present inventors cultivated cell lines that can be cultured in a suspension stirring culture system using a serum medium, for example, CHSE-sp cells, in a suspension stirring culture system in which serum was gradually decreased, and the amount of serum was reduced. A prototype cell line that can be cultivated in a culture system is selected, and the proliferation / selection of the cell line is repeated, and it can be cultivated in a serum-free medium, and has the same action and properties as those of CHSE-sp cells. The established cell line was established. Specifically, a cell line derived from Salmonaceae genus Algae (hereinafter referred to as CHLS cell) was found, and FERM P was established on January 18, 2002.
-18681 was deposited at the Patent Microorganism Depositary Center, National Institute of Advanced Industrial Science and Technology.

The characteristics of the deposited CHLS cells of the present invention are the cell line CHLS derived from Salmonaceae genus Astragalus,
Its scientific properties are: Suspension culture is possible. 2. Complete serum-free culture using lactalbumin hydrolyzate sterilized under high pressure as a serum substitute is possible. 3. It exhibits strong adhesion extension to static adhesive substrates. The taxonomic position is animal cells, and the medium is Weymouse medium MB752 / 1 (Ca-free), or in the short term, S-MEM, Gibco, BRL. 2180
It is also possible to substitute 0-081. The composition of the medium is C
aCl ₂ without added, Weymouth medium MB752 / 1
, 14 mM Hepes (pH adjusted with NaOH),
10% lactalbumin hydrolyzate (Lactoalbumin hydrolysa
te) aqueous solution 5% and kanamycin 0.06 g / L
The pH of the medium was 7.4, the sterilization condition of the medium was filter sterilization with a 0.2 μm filter, the culture temperature was 15-25 ° C., the culture period was 10 days, and the oxygen demand was It is aerobic and can be either suspension culture (spinner flask) or adherent culture.

The CHLS cells of the present invention are characterized by having the following various properties. Cell properties (culture method and cell properties enabled by production) 1. Serum-free culture is possible by adding lactalbumin hydrolyzate (LAH). In addition, conventional fish cells have 10% F
It was cultivated in the addition system of BS. 2. Floating agitation culture is possible. The conventional fish cells were an adhesion-dependent monolayer static culture system. 3. Roller bottle culture is possible. This culture method has been difficult with conventional fish cells. ) 4. Although it is in an isolated and dispersed state in the floating system, it retains a strong adhesive activity to the substrate. 5. In static culture, it rapidly spreads adhesion (weak adhesion activity in prototype). 6. Cultivation by the ALPS culture system is possible. In addition, A
The LPS culture system is a culture system that can be used by anyone, and is a novel culture method developed by the present inventors that is not found in conventional cultured cells. 7. A persistent cell adhesive sheet is formed by treatment with Ca or gelatin. 8. Regulation of Ca concentration can regulate the production of new cells in ALPS culture.

The CHLS cells of the present invention have the following characteristics. 1. It can be put to practical use at room temperature level. 2. Low temperature culture (preservation) is possible. 3. Various culture methods can be used. 4. It can be used without general cell culture technology and equipment. A
Anyone can use the LPS culture system. 5. There is no biorisk due to lactoalbumin hydrolyzate (LAH) that has been autoclaved. 6. Since it is an inexpensive LAH, the cost is about 1/10 of the conventional cost. 7. It has adhesive extensibility. It adheres and spreads rapidly on a stationary culture substrate. 8. Susceptible to fish disease virus. 9. High density mass culture is possible. 10. Sensing control is not required for high density mass culture. 11. It is possible to control growth using temperature. 12. The detection rate of contamination is extremely low due to the low temperature.

The CHLS cells of the present invention are conventional CHSE cells.
Like the -sp cell, it can be applied to the following fields. 1. As a teaching material for biology education. 2. As a substance evaluation system (production of bioactive substances and raw materials for viral vaccines) 3. As a test system for fish disease virus. 4. 4. As a useful substance production system (production of physiologically active substances and raw materials for viral vaccines) As an evaluation system for antiviral agents. 6. As an environmental pollutant evaluation system

The utility of the conventional CHSE-sp cells by the prototype cells has been already demonstrated in the following points. For example, 1. Use as a biological teaching material by ALPS culture system. 2. Toxic substance evaluation. 3. Evaluation of antiviral agents. 4. Virus production. 5. High density and large volume culture. 6. Gene transfer.

Furthermore, the CHLS cells of the present invention can be cultured in a serum-free medium, and the following applications are also considered. 1. Application to edible animal cells. 2. Application to edible drug production system. 3. Application to the production of fish-specific substances. 4. Application to gene expression system. 5. Application to test and research materials in areas without dedicated equipment. 6. Application of production of useful cells by cell fusion to counterpart cells. 7. Application of fish disease virus to vaccine production. 8. Application as a fine feed (cell feed by enhancing the function of useful organisms). 9. Application of space station as research material.

The CHLS cells of the present invention can be grown and grown under a simple culture condition which has not been possible with conventional vertebrate cells by repeating selection / proliferation on already known salmonid king salmon-derived cells (CHSE-214). It was established as a cell. The origin of the CHLS cells of the present invention is CHSE-214, which was established and established in the United States in the 1950s, but the CHSE-214 cells are cells that have many academically unclear parts. The CHLS cells of the present invention are conventional CHSE-21 cells that have been maintained and maintained by the present inventors for over 20 years.
Since 1989, the present inventors independently conducted a study and selection / amplification of various culturing methods, and after establishing various lines (prototypes), the establishment (establishment of culturing method) It is a fish cell. By the way, in order to maintain and establish stable properties, it is necessary to culture for long-term property maintenance. In general, animal cell culture has been carried out by adding fetal bovine serum or a special cell growth factor to a tissue culture solution, aeration with CO ₂ -air, static culture at 37 ° C. or roller bottle culture. Therefore, various substances produced by cell culture have a drawback of high cost. In addition, the culture operation required special equipment and skill.
Cultured cells of fish have strong FBS requirement and are dependent on external temperature, but are cells of vertebrate origin and have useful traits similar to those of cultured cells of homeothermic animals (compared to prokaryotic cells and unicellular organisms). ). However, the conventional cultured fish cells have not been within the scope of being able to list the basic properties and the characteristics that surpass the homeothermic animal cells in the culture method as practical materials. The CHLS cells of the present invention can be cultured in a tissue culture medium containing lactalbumin hydrolyzate at room temperature at agitation, and can be handled without a special culture device or specialized knowledge / technique. The CHLS cell of the present invention is a cell produced so that it can be cultured by various culturing methods, which are not possible with conventional fish cells. The useful characteristics of the CHLS cell are superior to those of cultured cells of warm-blooded animals. What can be done is as described above.

Comparing animal cell culture and microbial culture in terms of cost, operation, yield, etc., the latter is definitely advantageous.
However, since the CHLS cell of the present invention grows by a culture method similar to that of microbial culture, it is a cell line that is highly advantageous and highly practical for the production of substances that require animal cells, and is a biological teaching material, It can be used as a useful industrial material, research material, and educational material for evaluation of toxic substances and host cells for gene transfer and expression. In addition, a prototype of the present cell created during the development of the present cell, for example, CHSE-sp
As described above, its usefulness has already been proved in various practical tests. In consideration of the characteristics of fish cells not found in warm-blooded animal cells, for example, they can be cultured at room temperature, can be stored at low temperature, and have no biorisk. Practicality is expected to increase further.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The CHLS cells of the present invention can be used in the same manner as conventional CHSE-sp cells and can be more simply and practically used because serum is not required as a medium. Has become. CHL of the present invention
A case where S cells are used as a "cultured cell kit as a teaching material for biology education / environmental education" will be described. This kit is designed to allow suspension culture using a spinner flask. The extra cell suspension, which appears during subculture of suspension cells in a spinner flask, is stored in an ordinary container. This is so-called static suspension culture, but if it is cultured at low temperature, it can be stored for a long period of time in a cell-dispersed state, and it can be used for experiments when necessary. If the lid is opened and transferred to a culture vessel, it will adhere quickly and can be used the same as normal monolayer cells on the next day (see Fig. 1). Anyone can use the cultured cells of fish regardless of the specialization and preparation of the cell culture technique, that is, regardless of "time, person, place".

Next, a culture system (adhering cell layer produces suspension c) in which an underlaying cell (adhesive sheet cell) produces a floating cell using such a cell culture kit
The outline and usage of the ALPS culture system based on the ALPS culture system (ells) will be explained based on FIG. Step I (Handling of Cell Flask) The CHLS cells are in the ALPS state (FIG. 1-E) because they are shipped by cold courier (4 ° C. refrigerated storage). Take it out and tap the container lightly, the cells will float in the culture solution, so check for turbidity and cell grains. Since the cells are prepared by aseptic technique, it is necessary to be careful not to open the cap easily to avoid contamination by contaminants.
The container is a culture flask for adherent cells. Since cells are alive, they change every day. It is important to check the cell condition in advance when using it for experiments. If you are accustomed to it, you can judge it from the face of the cell under an inverted microscope,
In the case of beginners, a part of the cells is actually collected to check the cell state and cell concentration (see supplement below).

Step II (Dispersion Treatment of Suspended Cells) For use in experiments and confirmation of the cell state, suspended cells are homogenized. That is, as shown in FIG. 2, the liquid is sprayed onto the cell adhesion surface (bottom surface of the container) to suspend the spherical cells that are lightly adhered, and to disperse the cell aggregates,
Pipette solution 5 or 6 times. The underlying cell layer will not be peeled off even if it is blown a little strongly, but caution should be exercised because excessive pipetting and use of a tip that is too thin damages floating cells.

Step III (Collection and Correction of Cell Suspension) The cell suspension uniformly dispersed in Step II is collected in a container. PH correction if necessary (p
Since H decreases and the color tone of the culture solution containing phenol red tends to be yellow, a few drops of 7.5% NaHCO ₃ are added dropwise to correct the color tone to a red light, that is, about pH 7.4. ), Cell concentration, and Ca concentration are corrected.
Alternatively, the medium is exchanged (the cells are allowed to settle and the supernatant culture solution is removed and replaced with the target medium).

Step IV (Use of Recovered Cells) The cell suspension of Step III is placed in a commercially available culture container (for example,
1. Culture flask, 2. Culture Petri dish, 3. Inoculate into a multi-well culture plate, etc. and use for the experiment (detailed description of inoculation volume, cell concentration, etc. is omitted). Step V (Replenishment of the medium to the underlying cells) A new medium is supplied to the flask from which the cell suspension has been removed. In the ALPS culture system of CHLS cells, the amount is larger (about 20 ml) than a general liquid volume (about 5 ml / 25 cm ² ).
ml / 25 cm ² flask). The medium is a commercially available medium such as MEM without CaCl ₂ (14 mM
Hepes buffer system, containing about 3% fetal calf serum).

Step VI (Floating growth of mitogenic neoplastic cells) When the cell container newly supplemented with the medium was cultured at about 20 ° C., it was left in a stationary floating state as 1 → 3 as shown in the right frame of FIG. Spherical cells are born. After a few days about 40-5
A cell suspension of 0,000 cells / ml is obtained. Since CHLS cells can be cultivated at low temperature, they can be cultivated leisurely according to the experiment. Step VII (ALP
After cryopreservation of S cells) After culturing at an appropriate temperature and confirming that the number of spherical floating cells has increased, store them in a refrigerator or the like until the next experiment. It can be stored for more than a few weeks and can be used again immediately after returning it to an appropriate temperature.

ALPS Cycle The ALPS cycle is the continuous reproduction of stationary floating cells by collecting and consuming only the spherical cells that have proliferated and regenerated and floated while keeping the adherent cell sheet on the bottom of the flask intact. It is a system that does. Management and maintenance can be easily performed by using low-temperature culture and preservation at appropriate times. Supplementary explanation Confirmation of cell state: Confirm the state and activity of spherical cells that have been increased in the ALPS culture system by inoculating a small amount on a petri dish after suspension dispersion. Cell adhesion and spreading activity: 1 for healthy CHLS cells
It adheres within a period of time (cells do not move even if the liquid moves), and spreads and adheres flatly within a few hours. If the cells are spherical even after overnight culture, they are weak cells and should be discarded. Estimation of cell concentration: For example, 0.5 in a 24-well culture plate
When the cell solution of ml / well is added and the spread cells are in a state where there is no gap, it is about 500,000 cells / ml. Cell aggregate / dispersion state: If there is an extremely large cell aggregate, it is inconvenient for some experiments, so pipette operation is performed. However, even if some of the cells adhere to the bottom surface of the static incubator, they will spread into a monolayer sheet the next day.

Suspension culture system The static suspension culture and cryopreservation of CHLS cells will be described with reference to FIG. (1) The CHLS cells of this kit, at the supplier of the kit, have been subjected to suspension stirring culture at a culture temperature of 20 ° C. continuous passage in a spinner flask as shown at the lower left of FIG. The suspended cells are refilled in a culture container (upper left end of FIG. 3) to start ALPS culture (or the second container (II) from the left side of FIG. 3 is packed and shipped). (2) When sending cells to the cultured cell kit user,
LPS type (above (II) in FIG. 3) or a spinner flask floating cells (under (II) in FIG. 3: Polycarbonate rectangular container, middle stage in (II) in FIG. 3: polymethylpentene) (Centrifugal tube). The cells are shipped by low-temperature courier in a stationary floating state (above and below (II) in FIG. 3) in which they cannot adhere to the bottom surface in the container or in the ALPS state.
After receiving the cells, cryopreserve the container until the day of use. (3) The cells are collected from the cell storage container, the cell state is confirmed (adhesion extension activity, estimation of cell concentration, etc.), and a cell plate for experiment is prepared. Available immediately. (4) If the prepared cell container / plate (cells in a state where adhesion and extension have been completed) is stored at low temperature, it can be stored for several weeks or longer.
Since it is in a "combat state", it can be used for various tests immediately after returning to the culture temperature.

The present inventors are investigating whether a cell line derived from a cyprinid fish (FHM-sp: capable of growing even at 30 ° C. or higher) can be used as a similar “cultured cell kit”. . In addition, fertilized fish eggs (such as medaka blastomere and rainbow trout eyed eggs) may become "cultured cells that anyone can handle." For example, the early blastomere cells (blastomere) that can be easily removed from medaka eggs that have been disinfected with isodine, etc., are sterile cultured cells that divide easily under a microscope, and their appearance is certainly interesting. It is also meaningful to think of its application while comparing it with the CHLS cells mentioned above. In addition, it is also meaningful to observe the whole body of small fish (larvae of rainbow trout and medaka fish) with whole tissue prepared as a preparation for staining tissue sections.

In addition to the educational equipment described above, such a culture kit is used in addition to the purposeful researches such as the cell physiology of fish, the fish gene and its expression system, the fish disease virus, and the experimental system for evaluating water environment pollution. It can also be used for evaluation of physiologically active substances, screening of antiviral agents in combination with fish viruses, and the like.

The present invention provides a cell line of fish that can be cultivated in a serum-free medium, for example, a cell line of CHLS cells (accession number FERM;
P-18681). The cell line of fish that can be cultured in the serum-free medium of the present invention is preferably a cell line of fish that is capable of complete serum-free culture using lactalbumin hydrolyzate as a serum substitute, and is cultured in suspension stirring culture. Is possible and has a property of rapidly adhering and extending in static culture. Further, it is also possible to culture in a culture system (ALPS culture system) in which the adhesive sheet cells produce floating cells, and it is also possible to culture by roller bottle culture. The fish cell line of the present invention is in an isolated and dispersed state in a suspension system, but retains a strong adhesive activity to the substrate. Furthermore, it has the property of forming a persistent cell adhesive sheet by treatment with calcium (Ca) or gelatin.

The fish cell line that can be cultured in the serum-free medium of the present invention can be cultured in a medium having a calcium (Ca) concentration of 1 mM or less, preferably 500 μM or less,
It is also possible to culture in a Ca-free medium from 100 μM or less. In addition, the production of neoplastic cells in ALPS culture can be regulated by regulating the calcium (Ca) concentration in the medium. For example, at around 1 mM, floating spherical neoplastic cells are hardly generated, and the number of floating spherical neoplastic cells can be increased by lowering the Ca concentration. The fish cell line that can be cultured in the serum-free medium of the present invention is preferably one that is sensitive to fish disease virus. For example, CHLS cells are conventional CHS
It has the same susceptibility to fish disease virus as E-sp cells.

Utilizing the susceptibility of the fish cell line culturable in the serum-free medium of the present invention to the fish disease virus, the virus is cultivated in the culture system of the fish cell line culturable in the serum-free medium of the present invention. Can be propagated. The virus can be inoculated into a culture system of a cell line of fish that can be cultured in the serum-free medium of the present invention, and the virus can be propagated in a nutrient medium suitable for the growth of the virus. If necessary, the propagated virus can be purified to remove the virus. The virus is preferably fish disease virus, but is not limited thereto. It is also possible to introduce a test substance into the above-mentioned medium in which the virus is growing to evaluate / screen the action of the test substance on the virus. Therefore, the present invention provides a method for inoculating a culture system of a cell line of a fish that can be cultured in the serum-free medium of the present invention with a virus, and growing the virus in a nutrient medium suitable for growing the virus, and It is intended to provide a method for evaluating or screening the action of a test substance on the virus by introducing the test substance into a growing medium.

The fish cell line that can be cultivated in the serum-free medium of the present invention is the cell line that can be cultivated in the conventional suspension stirring culture system using a serum medium in the suspension stirring culture system in which serum is gradually decreased. After culturing, a prototype cell line that can be cultivated in a culture system with a reduced amount of serum is selected, and growth / selection of the cell line is repeated to establish a cell line that can be cultured in the serum-free medium of the present invention. be able to. Selection of prototypes,
Careful attention must be paid to the culture and proliferation of the established cell line, and long-term culture is necessary to obtain a stable established cell line.

The present invention also provides a method for culturing a cell line of fish which can be cultured in the serum-free medium of the present invention. In this culture method of the present invention, lactalbumin hydrolyzate is preferably 1 to 20%, more preferably 3 to
A method for culturing a cell line of the fish of the present invention using a serum-free medium containing about 10% and having a calcium (Ca) concentration of 1 mM or less, preferably 500 μM or less, more preferably 100 μM or less is Can be mentioned.
As the culture system, suspension stirring culture system, static culture system, ALP
Various culture systems such as S culture system can be selected.
In addition, these culture systems can be combined and cultured. Further, as described above, it is also possible to regulate the calcium (Ca) concentration in the medium to regulate the production of neoplastic cells in ALPS culture. The culture temperature may be an appropriate temperature of room temperature or higher, around room temperature or lower, but is usually 0 ° C. or higher and 30 ° C. or lower. Moreover, the growth rate of the established cell line can be adjusted by adjusting the culture temperature. Almost hibernation can be achieved by setting the culture temperature to 10 ° C or lower, preferably 5 ° C or lower, and usually around 4 ° C.

The present invention also provides a method for preserving fish cell lines that can be cultured in the serum-free medium of the present invention. As the preservation method of the present invention, the cell line can be preserved by keeping the culture system containing the cell line of the fish of the present invention at a low temperature as it is. The temperature for storage varies depending on the type of fish, but is preferably a temperature at which the fish can hibernate. The storage temperature is, for example, 10 ° C. or lower, preferably about 4 ° C.
Temperatures near or below are included.

The present invention provides a culture medium for culturing a cell line of fish which can be cultured in the serum-free medium of the present invention.
The culture solution of the present invention, lactalbumin hydrolyzate,
A culture medium comprising a serum-free medium containing preferably 1 to 20%, more preferably 3 to 10%, and having a calcium (Ca) concentration of 1 mM or less, preferably 500 μM or less, more preferably 100 μM or less. It consists of If necessary, an antibiotic may be added to the culture medium of the present invention.

The present invention further provides a kit for culturing a cell line of a fish, which can be cultured in the serum-free medium of the present invention in a culture medium, in a culture vessel. The culture medium in the kit of the present invention is preferably a serum-free culture medium containing the above-mentioned lactalbumin hydrolyzate. The culture container of the kit of the present invention includes (I
Examples thereof include ALPS type as shown in I), a rectangular container made of polycarbonate, and a centrifuge tube made of polymethylpentene. Further, in the kit of the present invention, the cells of the present invention are cultured, various operations are performed, and supplemental culture solutions necessary for observing the cells, various dropper, slit culture cases, and It is also possible to attach a material that serves as a substrate for cell line culture. Examples of the material that serves as a substrate for culturing the cell line of the present invention include, but are not limited to, one or more materials selected from gelatin, albumin, collagen, and fibronectin.

The above-mentioned kit of the present invention is useful as an educational device, particularly an educational device for observing cell proliferation. The above-mentioned kit of the present invention is also useful as a culture kit for research and development. The present invention also provides a method for evaluating the biochemical properties of various chemical substances using the cell line of fish or the transformant thereof that can be cultured in the serum-free medium of the present invention. That is, the present invention provides a method for evaluating a living cell of a test substance, which comprises introducing the test substance into a culture system of a cell line of fish that can be cultured in the serum-free medium of the present invention. By introducing the test substance at various concentrations and observing the state of the cell line of the present invention in culture, the biochemical properties of the test substance with respect to living cells can be evaluated.

Furthermore, the cell line of fish which can be cultured in the serum-free medium of the present invention can also be used as a host cell in genetic engineering. Although the cells of the present invention are vertebrate cells, they can be used as vertebrate cells with improved productivity because they can be subjected to suspension stirring culture by a method substantially similar to that of microorganisms. That is, the present invention provides a method for transforming a cell line of the present invention, which comprises introducing a foreign gene into the cell line of the fish, and a transformant introduced with the foreign gene transformed by the method. I will provide a.

[0043]

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

Example 1 (Establishment of CHLS cells) King SEALON-derived CHSE-214 cells established and established in the United States in the 1950s were examined and tested for various culture methods and selected / amplified to obtain various strains (prototypes). ) Was established (establishment of culture method). In order to maintain and establish stable properties, culture for long-term property maintenance was performed. Established CHLS cells, 2002 1
It was deposited on the 18th of March as the deposit number FERM P-18681.

Example 2 (Culture of CHLS cells) Wey mouse medium MB752 / 1 (Ca-free) medium (medium composition is Wey mouse medium MB752 / 1 without addition of CaCl ₂ and 14 mM Hepes (NaO)
PH adjusted with H), 10% lactalbumin hydrolyzate (Lactoa
lbumin hydrolysate) 5% aqueous solution and kanamycin 0.06 g / L added)
Is 7.4, the culture temperature is about 20 ° C, and 1 under aerobic conditions.
The suspension culture was carried out for 0 days. After the completion of this culture, no change was observed in the cell trait.

Example 3 (Viral susceptibility test of CHLS cells) A pathogenic virus, an infectious hematopoietic necrosis virus (I), which still causes great damage to fish, especially salmon and trout, for fish farming / culture.
The sensitivity to nfectious hematopoietic encrosis virus (IHNV) (TCID50 method) was 10 ⁸ level, which was the highest level as a cultured cell.

Example 4 (CHLS cell virus production test) In order to develop the IHNV viral vaccine described in Example 3, the amount of virus production was tested in a suspension agitation culture system from the viewpoint of mass culture. 2x at cell concentration (1.7 million cells / ml)
It was 10 ⁸ / ml, which was the highest level of known cultured cells. By the way, in the suspension culture, since it is easy to increase the cell density (10 million cell level), it is easy to significantly increase the production amount.

[0048]

EFFECTS OF THE INVENTION The fish cell line of the present invention, which can be cultivated in the serum-free medium, is a vertebrate cell, but it can be cultivated by a method similar to that of a microorganism and can be easily cultivated and manipulated. It is useful as a vertebrate cell. Further, the cell line of the present invention is a cell that can be suspension-cultured in a serum-free medium, can be easily cultured by anyone without requiring a specialized device or technique, and is an educational material or a research material. Or, it is useful as a cultured cell for industrial use.

[Brief description of drawings]

FIG. 1 is a schematic view showing the proliferation of cells in a culture container.

FIG. 2 schematically shows a specific method of ALPS culture using the cell line of the present invention.

[Fig. 3] Fig. 3 schematically shows the culture, division, and transportation forms of the cell line of the present invention and the method of using the same.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/70 C12N 5/00 B G09B 23/36 E (72) Inventor Takaaki Shirato 5 Kobuchi, Sagamihara City, Kanagawa Prefecture -11-9 F term (reference) 2C032 CB01 4B024 AA20 CA01 DA02 GA18 4B063 QA05 QA18 QR77 QR79 4B065 AA90 AA95 AB04 AC20 BA01 BB02 BB19 BB32 BC08 CA46 CA60

Claims (41)

[Claims] 1. A cell line of fish that can be cultured in a serum-free medium. 2. The cell line according to claim 1, which is capable of complete serum-free culture using a lactalbumin hydrolyzate as a serum substitute. 3. The cell line according to claim 1 or 2, which is capable of suspension stirring culture. 4. The cell line according to any one of claims 1 to 3, which rapidly adheres and extends in stationary culture. 5. The adhesive sheet cells can be cultured in a culture system that produces floating cells (ALPS culture system).
The cell line according to any one of 1. 6. The cell line according to claim 1, which can be cultured in a roller bottle. 7. The cell line according to any one of claims 1 to 6, which is in an isolated and dispersed state in a suspension system, but retains strong adhesive activity to a substrate. 8. The cell line according to any one of claims 1 to 7, which forms a persistent cell adhesion sheet by treatment with calcium (Ca) or gelatin. 9. The cell line according to claim 1, which has sensitivity to a fish disease virus. 10. The cell line according to claim 1, which can be cultured in a medium having a calcium (Ca) concentration of 1 mM or less. 11. ALP by controlling calcium (Ca) concentration
The established cell line according to claim 10, which is capable of regulating the production of neoplastic cells in S culture. 12. The cell line according to any one of claims 1 to 11, wherein the cell line of fish is a cell line derived from Salmonaceae trout. 13. The cell line has an accession number FERM P-.
The cell line according to claim 12, which is 18681. 14. A prototype strain which can be cultured in a suspension stirring culture system using a serum medium, is cultured in a suspension stirring culture system in which serum is gradually reduced, and can be cultured in a culture system in which the amount of serum is reduced. 2. A cell line capable of culturing in a serum-free medium by selecting activated cells, repeating growth / selection of cell lines, and culturing.
A method for producing the cell line described in any one of 3 above. 15. The method according to claim 14, wherein the cell line that can be cultured in a suspension stirring culture system using a serum medium is CHSE-214 cells. 16. The cell line that can be cultured in a suspension stirring culture system using a serum medium is CHSE-sp cells.
The method according to 4. 17. The fish cell line according to claim 1, which is a serum-free medium containing a lactalbumin hydrolyzate and has a calcium (Ca) concentration of 1 mM or less. Method of culturing. 18. The method according to claim 17, wherein the culture system is a suspension stirring culture system. 19. The method according to claim 17, wherein the culture system is an ALPS culture system. 20. The calcium (Ca) concentration is adjusted to A
20. The method of claim 19, wherein the production of neoplastic cells in LPS culture is regulated. 21. The method according to claim 17, wherein the culture temperature is a temperature around room temperature or lower. 22. The method according to claim 21, wherein the culture temperature is controlled to control the growth rate of the cell line. 23. A method for preserving the cell line of a fish by maintaining the culture system containing the cell line of the fish according to any one of claims 1 to 13 at a low temperature. 24. The method of claim 23, wherein the temperature is about 4 ° C. or lower. 25. A culture medium for culturing the cell line of the fish according to any one of claims 1 to 13. 26. The culture medium according to claim 25, which is a serum-free culture medium containing a lactalbumin hydrolyzate. 27. The culture medium according to claim 25 or 26, which has a calcium (Ca) concentration of 1 mM or less. 28. The method according to any one of claims 1 to 13, which is placed in a culture container.
A kit for culturing the established cell line of the fish according to any one of 1 to 3 and a culture solution. 29. The kit according to claim 28, wherein the culture medium is a serum-free culture medium containing lactalbumin hydrolyzate. 30. The calcium (Ca) concentration of the culture solution is 1
The kit according to claim 28 or 30, which is at most mM. 31. The kit according to claim 28, wherein the culture medium further contains an antibiotic. 32. The kit according to any one of claims 28 to 31, further comprising a supplemental culture medium and a dropper. 33. The kit according to claim 28, further comprising a slit culture case for observing cell growth. 34. The kit according to any one of claims 28 to 32, further comprising a material serving as a substrate for cell line culture. 35. The kit according to claim 34, wherein the substrate is one or more of gelatin, albumin, collagen, and fibronectin. 36. An educational device for observing cell proliferation, which comprises the kit according to any one of claims 28 to 35. 37. A method for evaluating a living cell of a test substance, which comprises introducing the test substance into the culture system for the cell line of fish according to any one of claims 1 to 13. 38. A method for transforming a cell line of a fish according to any one of claims 1 to 13, which comprises introducing a foreign gene into the cell line. 39. A transformant in which a foreign gene is introduced into the fish cell line according to any one of claims 1 to 13. 40. A method of inoculating the culture system of the cell line of fish according to any one of claims 1 to 13 with a virus and allowing the virus to grow in a nutrient medium suitable for growing the virus. 41. A method for evaluating the effect of a test substance on the virus by introducing the test substance into the medium in which the virus according to claim 40 is growing.