JP2004350615A - Cryopreservation method of monocyte cell line and culture medium used in the method - Google Patents

Abstract

An object of the present invention is to provide a cryopreservation method suitable for a monocyte cell line and a medium used in the method.
A method for cryopreserving a monocyte cell line, comprising: (1) dispersing the monocyte cell line in a medium containing a monosaccharide and serum to prepare a dispersion liquid; The above-mentioned object is achieved by a method comprising the steps of :) freezing the dispersion and storing it under liquid nitrogen. Preferably, the monosaccharide is glucose, and the serum is fetal bovine serum, and the concentration of glucose is at least 5% by weight based on the total weight of the medium.
[Selection diagram] Fig. 1

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cryopreservation method of a cell line and a medium used in the method, and more particularly, to a cryopreservation method of an animal monocyte cell line and a medium used in the method.
[0002]
[Prior art]
Cryopreservation of cells is performed to prevent cell deterioration due to passage, to prevent contamination by bacteria, and to eliminate the complexity of maintaining passage.
[0003]
The culture media for cryopreservation of cells that have been used so far are generally referred to as fetal bovine serum (hereinafter abbreviated as “FBS”) and 5 to 15% dimethyl sorboxide (hereinafter abbreviated as “DMSO”). ) And glycerin are used (for example, see Non-Patent Document 1). It is known that many cells are sufficiently preserved in such a freezing medium. Then, the cells are suspended in the medium, packed in cryotubes or ampoules, cooled at about -1 ° C / min using a program freezer, and finally stored in liquid nitrogen (-196 ° C). Is common.
[0004]
In particular, the advantage that DMSO has been used for cryopreservation of cells for a long time as the medium is that when the crystals of ice present in the cells at the time of cryopreservation are increased and the cells are thawed from the preservation state, It is said that it is not to die.
[0005]
However, when the above-described freezing medium containing FBS and DMSO is applied for cryopreservation of monocyte cell lines such as dogs, pigs and cats, unlike other cells, a sufficient preservation effect is obtained. The inventor has found that it does not play. In addition, the monocyte cell line after cryopreservation grew in a growth medium containing 10% of heat-inactivated FBS, but this was complicated with the necessity of frequently refreshing the growth medium. there were.
[0006]
The establishment of the monocyte cell line of dog or pig was established by the present inventors (see Non-Patent Documents 2 and 3). In particular, since monocyte cell lines are extremely useful cells for studies in immunology and infectious diseases, if the cells can be stored for a long period of time, further progress in this research field is expected.
[0007]
Therefore, there is a strong demand for the development of a cryopreservation method for monocyte cell lines of animals including humans and a medium used in the method.
[0008]
[Non-patent document 1]
Journal of Immunological Methods 272 (2003) 35-48
[Non-patent document 2]
New Microbiological, 23: 441-444
[Non-Patent Document 3]
New Microbiological, 24: 243-247
[Problems to be solved by the invention]
In view of the above circumstances, an object of the present invention is to provide a cryopreservation method suitable for animal monocyte cell lines and a medium used in the method.
[0009]
[Means for Solving the Problems]
The above object is a method for cryopreservation of a monocyte cell line, comprising: (1) dispersing the monocyte cell line in a medium containing a monosaccharide and serum to prepare a dispersion liquid; C) freezing the dispersion and storing it under liquid nitrogen.
[0010]
According to a preferred aspect of the present invention, in the above method, the monosaccharide is glucose.
[0011]
According to a preferred aspect of the present invention, in the above method, the glucose concentration is at least 5% by weight based on the total weight of the medium.
[0012]
According to a preferred aspect of the present invention, in the above method, the serum is fetal bovine serum.
[0013]
Further, the above object is achieved by a medium for cryopreservation of a monocyte cell line, comprising a monosaccharide and serum.
[0014]
According to a preferred aspect of the present invention, in the medium, the monosaccharide is glucose.
[0015]
According to a preferred aspect of the present invention, in the medium, the concentration of the glucose is at least 5% by weight based on the total weight of the medium.
[0016]
According to a preferred aspect of the present invention, in the medium, the serum is fetal bovine serum.
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
Next, an embodiment of the present invention will be described. The following embodiments are exemplifications for describing the present invention, and are not intended to limit the present invention only to the embodiments.
The present invention can be implemented in various forms without departing from the gist thereof.
[0018]
Animals used in the present invention are not particularly limited, and include dogs, pigs, cats, humans, and the like. Then, the cryopreservation of the monocyte cell line of such an animal is targeted.
[0019]
As the monocyte cell line used in the present invention, a cell obtained by passage of monocyte cells collected from peripheral blood of various healthy animals is used. For example, in the case of dogs, heparin was added to freshly collected blood from healthy young female beagle dogs, diluted with cold phosphate-buffered saline, and obtained according to a predetermined protocol. Monocyte cell lines obtained by passage of monocyte cells can be used in the present invention.
[0020]
The cryopreservation method according to the present invention will be specifically described. The monocyte cell line obtained as described above is dispersed in a medium containing a monosaccharide and serum, and then frozen. Then, it is frozen and stored under liquid nitrogen for a desired period.
[0021]
As serum used for the cryopreservation method according to the present invention, fetal bovine serum is preferably used because of its availability.
[0022]
In addition, glucose, fructose, galactose and the like can be mentioned as the monosaccharide used in the cryopreservation method according to the present invention. In particular, glucose is an example of a monosaccharide suitable for the culture medium of a monocyte cell line. The amount of the monosaccharide added to the medium used in the cryopreservation method according to the present invention needs to be at least 5% by weight or more based on the total weight of the medium. If the added amount of the saccharide is less than 5% by weight, the survival rate upon thawing after cryopreservation becomes low, which is not preferable.
[0023]
The medium used in the cryopreservation method according to the present invention may contain an inorganic component, vitamins, and amino acids as necessary. Examples of the inorganic component include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine, and cobalt. These components include, for example, potassium nitrate, sodium nitrate, calcium nitrate, potassium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, and sulfuric acid. It can be added as a compound such as manganese, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, and cobalt chloride.
[0024]
As the vitamins, for example, biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like can be added.
[0025]
As amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added.
[0026]
Generally, each of the above-mentioned components can be used at a concentration of about 0.1 μM to 100 μM of an inorganic component, and about 0.1 to about 100 mg / l of vitamins and amino acids, respectively.
[0027]
The medium used in the cryopreservation method according to the present invention is added with a cryoprotectant in advance, and after adding cells to be cryopreserved, the culture is frozen. Examples of the antifreeze include dimethyl sulfoxide, ethylene glycol, glycerin, glucose and the like.
[0028]
The freezing method used in the present invention includes: (1) a method in which monocyte cells are treated with a relatively high concentration of an antifreezing agent and then rapidly cooled with liquid nitrogen; (2) a method in which the cells are treated with an antifreezing agent. After that, a method of freezing to −40 ° C. to −80 ° C. using a program freezer or the like, if necessary, followed by rapid cooling with liquid nitrogen (preliminary freezing method) may be mentioned. The freezing method may be appropriately selected depending on the type of the cell.
[0029]
The cells cryopreserved according to the present invention can be used for reculturing after thawing and water supply. Monocyte cells stored in liquid nitrogen are rapidly thawed, for example, in warm water at 35 to 37 ° C., and then, after removing the cryoprotectant, a medium containing preferably 1.0 to 1.2 M sugar. To add water. Thereafter, the cells are re-cultured using a conventionally known medium for cell culture.
[0030]
The survival rate of the thawed monocyte cells after cryopreservation can be confirmed by a staining method.
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples.
[0031]
First, an example of an animal monocyte cell line will be described. In the present invention, a monocyte cell line collected from dog, pig, cat and human blood was used for the cryopreservation method according to the present invention.
[0032]
[Example 1]
It was collected from a healthy young female beagle dog and diluted with about three times the volume of cold Dulbecco's phosphate-buffered saline (hereinafter referred to as "PBS") to about 30 ml of heparinized blood. The blood thus obtained was kept at 10 ° C. for 3 hours, and the supernatant was gently layered on an equal volume of specific gravity solution (ρ = 1.0077), followed by centrifugation (800 × g for 15 minutes). As a result, a band consisting of lymphocytes and monocytes was formed almost at the center of the centrifuge tube, and the leukocyte fraction was separated. The leukocyte fraction thus obtained is suspended in a basic cell culture medium (hereinafter referred to as “CGM”) described below so that the number of cells per ml becomes about one million, and 10 ml is added to a glass plate. The mixture was distributed into flasks (culture area: 4 cm x 8 cm), and statically cultured at 36.5 ° C. The basic cell culture medium used was a slightly modified Dulbecco's modified Eagle medium. In addition, the basic cell culture medium contains 1 g glucose, 2 g galactose, 1 g fructose, 0.3 g N-acetyl-D (+)-glucosamine, 0.2 g HEPES, and 0.22 g per 1000 ml. Of sodium pyruvate was further added. CGM contained 10-20% of heat-inactivated fetal bovine serum (hereinafter referred to as "FBS"). Antibiotics were added according to a conventional method. After one day of culture, the primary culture was rinsed with warm CGM to remove non-adherent cells, and then one third of the CGM was replaced during culture at intervals of three to four days. In this way, the cells were cultured for 4 weeks. Then, a part of the cells cultured in this manner was removed with a pipette and moved to a petri dish to secure in vitro growth. One of them was designated Cn / K99 and was subcultured. In the present invention, cells cultured for the 12th and 15th passages were used in the following experiments.
[0033]
[Experimental example 2]
About 50 ml of blood collected from a healthy adult female pig immediately after addition of heparin was diluted with cold PBS not containing calcium and magnesium salts. The leukocyte fraction mainly containing lymphocytes and monocytes was obtained by centrifugation (800 × g for 15 minutes). The leukocyte fraction thus obtained is suspended in the above-mentioned CGM so that the number of cells per ml becomes about one million, and the cell culture glass flask is used for static culture at 36.5 ° C. And dispersed. To maintain only adherent cells, the primary culture was rinsed with warm CGM during the 20 hour culture. After three weeks of culture, a number of adherent cells were identified. Indeed, after the addition of conditioned medium removed from porcine monocyte cell culture, cell growth was significantly improved. One of them, designated as SW / K99, was subcultured in glass flasks and plastic flasks. In the present invention, cells subcultured in the 48th and 49th passages were used in experiments described below.
[0034]
[Example 3]
Approximately 10 ml of blood from a healthy female cat (2 years old), immediately supplemented with heparin and free of retrovirus and coronavirus, was diluted with cold PBS without calcium and magnesium salts. A leukocyte fraction mainly containing lymphocytes and monocytes was obtained by concentration gradient centrifugation under the same conditions as in Example 1. The leukocyte fraction thus obtained was suspended in the above-mentioned CGM so that the number of cells per 1 ml became about 1 million, and dispersed in a glass flask for cell culture. Static culture was performed. The primary culture was rinsed with warm CGM in the culture for 20 hours. In this way, non-adherent cells were removed. During the two-week culture, the CGM was replaced twice a day to initiate the growth of a number of epithelial cells, one of which was designated FL / K02 and was subcultured in a glass flask to form a thirteenth generation. The 14th and 14 th subcultured cells were subjected to the experiments described below.
[0035]
[Example 4]
Heparin was added to about 30 ml of blood immediately after collection of a human, and diluted with cold bivalent cation-free cold dabelco phosphate buffered saline (hereinafter, referred to as “PBS”). The blood thus obtained was gently layered with the supernatant on an equal volume of a specific gravity solution (ρ = 1.077 g / ml) and centrifuged (800 xg for 15 minutes). A band consisting of lymphocytes and monocytes was formed almost at the center of the tube. This band was suspended in CGM, and the centrifugation was repeated. Thereafter, the suspension was again suspended in GM in 10 ml portions so that the number of cells per 1 ml was about 2 million, and distributed in a glass cell culture flask (culture area: 32 cm ² ), and 36.5 ° C. Was statically cultured. To obtain only adherent cells, half of the GM was refreshed at least twice a day after 20 hours of culture. Two weeks later, the growth of several adherent clonal cells was confirmed. One of the lines having particularly excellent proliferative properties was named "K63" and was subcultured up to the 26th generation. In the cryopreservation experiment described later, cells that were subcultured with the 12th and 24th generations were used.
[Cryopreservation]
The cells obtained as described above were grown in a glass flask using a nutrient medium containing 10% FBS (hereinafter referred to as "GM"). The cells after culturing were dispersed even in an EDTA-tyrosine mixed solution, washed twice with cold GM, and then resuspended in a cryopreservation medium described later.
[0036]
The cryopreservation medium used for the experiment of the present invention is shown below.
(1) Medium 1: The medium according to the present invention, comprising 75 volumes of FBS + 25 volumes of 20% (w / v) glucose.
(2) Medium 2: As a comparative example, it is composed of 65 volumes of FBS + 25 volumes of 20% by weight glucose + 10 volumes of DMSO.
(3) Medium 3: As a comparative example, consisted of 65 volumes of FBS + 25 volumes of 20% by weight glucose + 10 volumes of glycerin.
(4) Medium 4: As a comparative example, the medium is composed of 90 volumes of a nutrient solution containing 10% FBS + 10 volumes of DMSO.
[0037]
Various monocyte cells were suspended in a cryotube (a serum tube manufactured by Sumitomo Bakelite Co., Ltd.) containing the above medium so that the number of cells per 1.5 ml became about 2 to 3.5 million cells. Immediately after, the tubes were maintained at 4 ° C. for 40 minutes, then at −80 ° C. for 40 minutes, and then stored in liquid nitrogen.
[0038]
After storage in liquid nitrogen for 30 days, the cells were thawed in a 38 ° C. water bath for 5 minutes, and then the cells were washed once with sufficient cold GM. To examine the cell viability by the trypan blue dye exclusion test, the washed cells were suspended and diluted again with the above GM.
[0039]
Further, this cell suspension was diluted with GM and seeded on a glass petri dish (6 cm in diameter) at 10 ml per dish or 1.5 ml per slip. These media were incubated for 3 days at a temperature of 36.5 ° C. and a humidity of 99% while refluxing 0.1% CO ₂ gas. After staining with 0.1% crystal violet solution containing 5% formalin for 2 minutes, the number of cell clones in the Petri dish was counted. The slip culture was fixed with methanol, and Giemsa staining was performed.
[0040]
FIG. 1 shows the results of the recovery rate after thawing of all monocyte cell lines after cryopreservation performed in the experiment of the present invention. As is clear from the results shown in FIG. 1, the medium 1 according to the present invention showed 97.5% and 95% of each of the monocyte cell lines SW / K99, Cn / K99, FL / K02 and K63, respectively. It was demonstrated that it was possible to recover with a high viability of 0.5%, 98.5% and 92.5%.
[0041]
On the other hand, as shown in the results of the culture media 2 to 4, the recovery ratio of each monocyte cell line by thawing after cryopreservation was considerably lower than that of the culture medium 1 as compared with the culture medium 1. Specifically, with respect to SW / K99, the recovery rates of 72.5%, 70.5% and 73.6% are respectively obtained in the medium 2, the medium 3 and the medium 4 which are the medium containing DMSO and glycerin. It proved to be impractical. In contrast, the culture medium 1 according to the present invention was found to be a practically useful cryopreservation medium for monocyte cell lines.
[0042]
【The invention's effect】
Use of the medium containing serum and glucose as a monosaccharide according to the present invention provides a cryopreservation method and a cryopreservation medium that are useful for monocyte cell lines.
[Brief description of the drawings]
FIG. 1 shows the results of the recovery rate after thawing of all monocyte cell lines after cryopreservation performed in the present invention.

Claims (8)

A cryopreservation method for a monocyte cell line,
(1) a step of preparing a dispersion by dispersing the monocyte cell line in a medium containing a monosaccharide and serum; and (2) a step of freezing and storing the dispersion under liquid nitrogen. A method comprising: The method of claim 1, wherein the monosaccharide is glucose. The method according to claim 1, wherein the concentration of the glucose is at least 5% by weight based on the total weight of the medium. 2. The method of claim 1, wherein said serum is fetal calf serum. A medium for cryopreservation of monocyte cell lines, comprising a monosaccharide and serum. The culture medium according to claim 5, wherein the monosaccharide is glucose. The medium according to claim 5 or 6, wherein the concentration of glucose is at least 5% by weight based on the total weight of the medium. The medium according to claim 5, wherein the serum is fetal bovine serum.

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