KR20150144057A

KR20150144057A - Method For Warming Cell Culture Media And Cell Culturing Method Using The Same

Info

Publication number: KR20150144057A
Application number: KR1020140072688A
Authority: KR
Inventors: 이원구; 박용; 윤종규
Original assignee: 경희대학교 산학협력단
Priority date: 2014-06-16
Filing date: 2014-06-16
Publication date: 2015-12-24

Abstract

The present invention relates to a cell culture medium heating method capable of shortening the time required for warming a cell culture medium to a target temperature and further preventing pollution of the medium during an exhaust heating process, A cell culture method using the same is disclosed. The method for heating a medium for cell culture according to the present invention is characterized by heating the cell culture medium to a target temperature by irradiating the cell culture medium with microwave. According to the present invention, since the medium for cell culture can be warmed up to the target temperature in a very short time as compared with the medium heating method using the constant temperature water tank, the time required for cell culture preparation can be shortened considerably, Contamination of the medium due to fungi that may occur in the constant temperature water tank can be prevented and a clean culture environment can be provided through sterilization of the medium. In addition, the cell culture is carried out with the medium heated by microwave, and the result can indirectly confirm the influence of the food heated by the microwave on the human body, especially the cells.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for heating a culture medium for cell culture,

The present invention relates to a cell culture medium heating method for warming a cell culture medium to a target temperature and a cell culture method using the same, and more particularly, to a cell culture medium heating method for heating a culture medium using a microwave, And a cell culture method using the same.

Cell culture refers to the process of culturing cells. Cell culture is a process in which single cells are dispersed and cultured by treatment with a protease or the like. More specifically, a living tissue is sterilely selected and treated with a digestive enzyme such as trypsin or proanase to separate into single cells and to perform primary culture, and a single cell obtained by dispersing a subculturing cell line or cell line by the same enzyme treatment Transplantation and inoculation are carried out in the growth medium, and the following subculture is performed.

The development of the cell culture method has made it possible to treat the cells constituting the living body as a single cell organism and the flow of analyzing the animal cells based on the knowledge and the technology obtained from E. coli etc. through the cell culture has been established, And can be quantitatively treated at the cellular level, such as basal metabolism, proliferation, differentiation, aging, and cancer virus infection.

Cell culture includes monolayer culture in which the cells adhere to the culture vessel and proliferate, and suspension culture in which the cells proliferate in a floating state. There is also a single cell culture in which a single cell is cultured to form a colony, or a mass culture in which a large number of cells are cultured. Cultured cells have epithelial, fibroblastic, and lymphoid morphology. Primary cultured cells, which have been separated and cultured in vivo, constitute the chromosomes of normal diploid chromosomes.

In addition, primary cultured cells have normal characteristics, but they have a positive phenotype with transformation (transformation) of their replication ability, chromosome, and morphology according to the passage, and sometimes the differentiation function in the derived tissue of the cell sometimes disappears.

Almost all types of cells are treated with trypsin or EDTA solution, floated in a suitable culture medium, and then placed in a glass or plastic incubator to maintain the temperature around 37 ° C. . Non-tumor cells form a cell layer of a monolayer.

On the other hand, when cells are cultured in a culture container, for example, a flask, the number of cells (cell number) therein increases over time. Therefore, the cells should be placed in a new flask so that they can grow in a new environment, and a new culture medium (culture medium) should be added thereto.

The medium is also referred to as a culture medium as described above. In order to cultivate a microorganism or a tissue of animals or plants, the culture medium, that is, a nutrient substance required for the cell is used as a main component and the material used for cell proliferation, storage, do.

In the conventional medium heating method, the medium is heated to a suitable temperature (about 37 캜) by heating for about 60 minutes using a constant temperature water bath, and the medium is heated in a heated medium The culture was continued.

1 and 2, the medium heating method using the constant temperature water tank 10 is a technique for increasing distilled water W into the water tank and using the thermal conductivity of the distilled water to raise the temperature of the medium. A tube holder 11 is disposed and the tube holder 11 supports the tube 1 containing the medium to prevent conduction of the tube 1.

However, in the medium heating method using the constant temperature water tank 10 described above, heating of the medium (50 mL) takes a long time of about 60 minutes (3,600 seconds). During the cell culture process or during the experiment, Water fungus or the like generated in the constant temperature water tank 10 may be generated on the surface of the tube 1, and there is a high risk of contamination of the medium due to mold or the like. Further, The tube 1 may be contaminated when the tube 1 is conveyed and immersed in the distilled water W when the tube 1 is handled. Therefore, when the heated medium is transferred to a cell culture container (for example, a flask) .

Korean Patent Publication No. 10-2011-0074564, June 30, 2011 Korean Patent Publication No. 10-2009-0086414, August 12, 2009

SUMMARY OF THE INVENTION The present invention has been proposed in order to solve the above problems. It is an object of the present invention to shorten the time required for warming the cell culture medium to the target temperature, Which is capable of preventing the contamination of the medium in the middle, and a cell culture method using the same.

The present invention relates to a culture medium heating method for culturing a cell culture medium, wherein the culture medium is heated by irradiating a microwave to the culture medium for cell culture and warming the culture medium to a target temperature, &Lt; / RTI >

In another aspect, the present invention provides a method for culturing a cell culture comprising the steps of: (a) irradiating a cell culture medium with a microwave to warm the culture medium to a target temperature; And (b) introducing the culture medium for cell culture into a culture container containing the cells.

The frequency of the microwave is 2.4 GHz to 2.5 GHz and the wavelength is 0.11 m to 0.12 m, but is not limited thereto. The target temperature is not limited to 37 ° C to 38 ° C.

According to the present invention, since the medium for cell culture can be warmed up to the target temperature in a very short time as compared with the medium heating method using the constant temperature water tank, the time required for cell culture preparation can be shortened considerably, Contamination of the medium due to fungi that may occur in the constant temperature water tank can be prevented and a clean culture environment can be provided through sterilization of the medium. In addition, the cell culture is carried out with the medium heated by microwave, and the result can indirectly confirm the influence of the food heated by the microwave on the human body, especially the cells.

BRIEF DESCRIPTION OF THE DRAWINGS The features and advantages of the present invention will become better understood with reference to the following description taken in conjunction with the following detailed description of embodiments of the invention,
FIG. 1 and FIG. 2 are a perspective view and a cross-sectional view schematically showing a conventional hot water bath for a medium heating method for cell culture;
3 is a perspective view showing an embodiment of a medium heating apparatus for a medium heating method for cell culture according to the present invention;
Fig. 4 is a configuration diagram of the medium heating apparatus shown in Fig. 3; Fig.
5 is a view showing a time required for heating the medium;
FIG. 6 is a flow chart showing one embodiment of a cell culture method using a medium heating method for cell culture according to the present invention;
7 is a graph showing a change in temperature of water heated by a microwave;
8 is a graph showing the time required for heating the water (a) and the cell culture medium (b) to 37 占 폚 using a microwave for volume change;
FIG. 9 is a photograph of Hela cells (HeLa Cell) after cell culture by an experimental group and a control group;
10 is a photograph showing the viability of the cells by the experimental group and the control group; And
FIG. 11 is a graph showing changes in cell density by the experimental group and the control group.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In describing embodiments of the present invention, the same reference numerals are used for the same configurations.

The present invention relates to a cell culture medium heating method for culturing a cell culture medium and a cell culture method using the same, wherein the cell culture medium is irradiated with a microwave to heat the cell culture medium to a target temperature There are technical features to warm up and can give improved results in biological cells or tissue culture.

The above-mentioned microwave is an electromagnetic wave having a very high frequency and has a wavelength of 1 mm to 1 m, a frequency of 300 MHz to 300 GHz, and a photon energy of 1.24 meV to 1.24 μeV.

The present invention relates to a method for culturing a cell culture medium at a target temperature, for example, 37 ° C to 38 ° C, in particular, using a microwave, A method of warming the culture at a temperature of 37 ° C and using a microwave-heated medium for cell culture is presented. As shown in the examples of the present invention, heating the medium using microwaves reduces the contamination probability of the medium by eliminating contact with water, thereby preventing the cleaning and contamination of the medium and shortening the time required for heating the medium Can be obtained.

The frequency of the microwave is 2.4 GHz to 2.5 GHz and the wavelength is 0.11 m to 0.12 m, but the present invention is not limited thereto. And the target temperature of the culture medium by microwave is not limited to 37 ° C to 38 ° C.

The microwaves are generated by a magnetron. When microwaves are used, the temperature of the medium can be increased without using any water other than the medium, and contamination of the medium caused by water can be prevented. The microwave passes through most of the material in addition to the metal. When it encounters a resonant substance by its frequency, it vibrates the molecule as it is absorbed by the substance. Representative substances are water, followed by fat, carbohydrate, and protein. Therefore, the portion containing a lot of water and fat is first heated, and the heat is transferred to another portion.

More specifically, a microwave having a very short wavelength penetrates deeply into a material, and heat is generated due to molecular vibrations inside the material, so that the material can be evenly heated to a deep portion in a short time. The magnetron has a power consumption of about 2 kW and generates a high frequency output of about 1.2 kW. The present invention discloses a medium heating method using a heating principle of a domestic microwave oven.

3 to 6, an embodiment 100 of a culture medium heating apparatus for cell culture according to the present invention includes a main body 110 for accommodating a culture tube 1 and a magnetron 130 for generating a microwave . A cavity 111 is formed in the main body 110 to radiate microwaves and the medium tube 1 is placed in a tube holder 120 for supporting the medium tube, 111).

As the medium heating method, the principle of a general domestic microwave oven can be applied as described above. Despite the concern that when the medium is heated using a domestic microwave oven, the medium composition may be lost due to high temperature, the medium may lose its function as a medium. According to the experimental results of the present invention, It can be confirmed that it can be implemented. According to the embodiments and the experimental examples of the present invention, since the heating time of the medium is short, a timer which is cut off in a short time, for example a timer, can be applied to the medium heating apparatus.

The medium heating method and the cell culture method according to the present invention are effective in shortening the time or removing germs by using the principle of microwave oven, that is, microwaves. Referring to FIG. 6, . &Lt; / RTI >

(a) First, 50 mL of the medium is placed in a tube (tube for exhausting), the tube is loaded in the cavity of the domestic microwave oven, and the tube is heated for 10 seconds using microwave.

(b) After removing the existing medium from the flask containing the cells, divide the cells into a new flask, and add 5 mL of the microwave-heated medium to the cells distributed in the new flask. Namely, the culture medium for cell culture heated to 37 DEG C by the microwave is put into a culture container (flask) containing the cells.

(c) Next, the flask containing the cells and the fresh medium is placed in an incubator with a temperature of 37 ° C and a carbon dioxide concentration of 5%, and the cells are cultured.

In the conventional method, it takes 60 minutes (t ₁ = 3600 s) using a constant temperature water bath to heat the medium, so that it takes a long time to prepare the medium However, in the present invention, when the amount of the medium was equally set to 50 mL and heated for 10 seconds (t ₂ = 10 s), a value of 37 ° C, which is the temperature obtained in the constant temperature water tank, was obtained.

In addition, since the medium is heated by using microwaves, it is possible to provide a clean medium deviating from molds which are not necessary for cell propagation and which may occur in a constant temperature water bath. The medium heating apparatus used in the experiment of the present invention is a household microwave oven (Samsung Electronics RE-C21AB, rated voltage 220 V, 60 Hz, oscillation frequency 2450 MHz).

FIG. 7 is a graph showing a temperature change with time when 50 mL of water is heated using a microwave. FIG. 7 (a) shows distilled water and FIG. 7 (b) shows a general water tank . Referring to FIG. 7, it can be seen that the time required to heat the distilled water and the water to 37 ° C. is about 10 seconds or less. Since the medium contains water as a basic component as food or nutrients of cells including water and protein, it can be confirmed that it can be heated to 37 ° C in a short time by microwave.

Next, FIG. 8 is a graph showing changes in the volume of the object to be heated by heating the water and the medium at 37 DEG C using microwaves, respectively. As the amount (volume) of the object to be heated increases, Is increased. Fig. 8 (a) is for water, and Fig. 8 (b) is for medium. It is confirmed that the slope of water and medium is similar in the range of 25 to 50 ml and 50 to 75 ml .

9 is a photograph (HeLa Cell) of a HeLa cell (scale bar = 100 mu m) taken after cell culture by an experimental group (medium heated by microwave) and a control group (medium heated by a constant temperature water bath) This is the image of the last cell.

More specifically, FIG. 9 (a) is a bright field image of a cell using a heated medium using a constant temperature water bath, and FIG. 9 (b) is a cell viability assay (Cell viability measurement) image. FIG. 9 (c) is a bright field photograph of a cell using a microwave heated medium, and FIG. 9 (d) is a cell viability assay photograph of a cell using a microwave heated medium.

10 (a) is a photograph of the third day of the cell using a medium (experimental group) heated with microwave, and FIG. 10 is a photograph showing the viability of cells by the experimental group and the control group. (b) is a photograph of the third day of the cell using a medium (control group) heated in a constant temperature water bath, FIG. 10 (c) is a photograph of the seventh day of the cell using a medium heated by microwave, ) Is a photograph of the 7th day of the cell using a medium heated in a constant temperature bath.

According to the cell culture experiment using the medium heated to 37 ° C using a microwave and a constant temperature water bath, the total cell number and cell viability by date in the cell culture by the experimental group and the control group are shown in the following table 1].

division Experimental group (microwave) Control group (constant temperature bath) Day 3
Total cell number 3.5 X 10E6 cells / mL 3.9 X 10E6 cells / mL Survival rate 90% 93% Day 7
Total cell number 2.7 X 10E6 cells / mL 2.0 X 10E6 cells / mL Survival rate 87% 82%

Referring to [Table 1], the cell survival rate on day 3 in the cell culture by the medium heated with microwave and the medium heated by the constant temperature water bath is slightly lower, but the cell survival rate on the 7th day is rather superior .

Meanwhile, FIG. 11 is a graph showing changes in cell confluence by the experimental group and the control group, showing the confluence change in the flask for one week. In Fig. 11, the red graph is the density of cells using a medium heated in a microwave (experimental group), and the blue graph is the density of cells using a medium (control group) heated in a constant temperature water bath.

According to the above-mentioned experimental results, there is no significant difference in the results of the cell culture using the experimental group (medium heated by microwave) and the control group (medium heated by the constant temperature bath), and the experimental group provides an excellent culture environment It can be seen that there is a more useful technical value in that it can rather shorten the time required for heating the medium and reduces the risk of contamination of the medium.

It will be apparent to those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or scope of the invention as defined in the appended claims. .

Therefore, the above-described embodiments are to be considered as illustrative rather than restrictive, and thus the present invention is not limited to the above description, but may be modified within the scope of the appended claims and equivalents thereof.

1: medium tube 100: medium medium heater
110: main body 111: cavity
120: tube holder 130: magnetron

Claims

A cell culture medium heating method for heating a cell culture medium comprising:
And heating the cell culture medium to a target temperature by irradiating microwaves to the cell culture medium.

The method according to claim 1,
Wherein the frequency of the microwave is 2.4 GHz to 2.5 GHz and the wavelength is 0.11 m to 0.12 m.

3. The method according to claim 1 or 2,
Wherein the target temperature is 37 占 폚 to 38 占 폚.

(A) heating the cell culture medium to a target temperature by irradiating the cell culture medium with microwave; And
And (b) introducing the culture medium for cell culture into a culture container containing the cells.