KR20030049190A - Storage method of eukaryote cell on transfer - Google Patents

Abstract

PURPOSE: A method of storing eukaryotic cells in a vessel filled with carbon dioxide is provided, thereby safely transferring and maintaining eukaryotic cells and tissues, improving the survival rate thereof and inhibiting cell differentiation. CONSTITUTION: Eukaryotic cells are stored in a vessel filled with 5 to 30% by volume of carbon dioxide while transferring the same. The eukaryotic cells are cells cultured in human beings and animals, cancer cells, immortal cell lines, artificial tissue, transgenic cells or tissue.

Description

STORAGE METHOD OF EUKARYOTE CELL ON TRANSFER}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for storing eukaryotic cells during transport, and more particularly, to a method capable of transporting eukaryotic cells by maintaining eukaryotic cells in a state in which 5 to 30% by volume of carbon dioxide is filled. It is about.

[Private Technology]

Cells or tissues are essential for protein production, tissue transplantation, toxicity testing, reagent testing, and molecular biological research. Devices and methods have been developed that allow long-term storage of cells. Commonly used cell storage method is cryopreservation method using cryogenic (-196 ℃) of liquid nitrogen, various methods for the storage and culture of other cells have been developed and widely used in cell line banks (ATCC, KCTC), etc. .

On the contrary, in the case of prolonged transport of cells, most cryopreserved cells are stored in dry ice and moved. Dry ice does not provide a stable cell transfer method because it provides a temperature of minus 20 degrees. When the cells are transported using dry ice, the physiological activity of the cells may be reduced, and when transported for a long time, the dry ice melts and it is difficult to maintain the best activity.

Another method of storage and transfer of prokaryotic cells is lyophilization, and in the case of live anaerobic prokaryotic cells, a method of storing them in a container filled with a carbon dioxide gas at an appropriate concentration according to the type of cells is used. However, eukaryotic cells such as tissues are more difficult to culture and transport than prokaryotic cells, and are difficult to maintain cellular activity.

An object of the present invention is to provide a eukaryotic cell storage method that can easily transport eukaryotic cells and tissues.

It is another object of the present invention to provide a eukaryotic cell storage method capable of safely transporting eukaryotic cells and tissues.

It is another object of the present invention to provide a method for safely maintaining eukaryotic cells and tissues.

1 is a micrograph observing keratinocytes stored in the carbon dioxide porch and keratinocytes left in the air,

Figure 2 is a graph measuring the cell survival rate of keratinocytes stored in the carbon dioxide porch and keratinocytes left in the air,

3 is a micrograph of the fibroblasts stored in the carbon dioxide porch and the fibroblasts left in the air.

Figure 4 is a graph measuring the cell viability of the fibroblasts stored in the carbon dioxide porch and the fibroblasts left in the atmosphere.

In order to achieve the above object, the present invention provides a eukaryotic cell storage method for storing eukaryotic cells in a container filled with carbon dioxide gas of 5 to 30% by volume during transfer of eukaryotic cells.

Hereinafter, the present invention will be described in detail.

The present inventors have completed the present invention by confirming that carbon dioxide gas forms a condition that can impart safety to the storage and transport of eukaryotic cells. In general, the concentration of carbon dioxide in vivo is maintained at about 5%. Therefore, this carbon dioxide concentration was applied to the storage and transport of eukaryotic cells to maintain the activity of eukaryotic cells.

Eukaryotic cell storage method of the present invention is to place the eukaryotic cells in a container filled with 5 to 30% by volume carbon dioxide gas. Eukaryotic cells referred to in the present invention include cells, cancer cells, immortalized cell lines, artificial tissues, transformants or tissues cultured in humans and animals.

If the filling amount of the carbon dioxide gas is less than 5% by volume it is difficult to maintain the physiological activity of the eukaryotic cells, when the excess of 30% by volume may cause a problem that the cells die due to excessively high carbon dioxide concentration. The most preferable filling amount of carbon dioxide is 5-10% by volume.

The container of the present invention may use a kind of material capable of maintaining the concentration of carbon dioxide, and may be a container, a carbon dioxide pouch, a bag, or the like, used for culturing or transporting anaerobic bacteria.

In order to fill the bag with carbon dioxide gas, a carbon dioxide gas-releasing solution capable of releasing 5 to 30% by volume of carbon dioxide gas is charged or filled with carbon dioxide gas and then stored at a temperature of about 30 degrees. A container filled with carbon dioxide is maintained at a concentration of carbon dioxide that can hold cells and tissue for about five days.

In addition, the container filled with the carbon dioxide gas preferably further includes a device capable of maintaining a constant concentration of the carbon dioxide gas in the container during cell storage. The device detects the concentration of carbon dioxide gas to produce carbon dioxide gas or discharge carbon dioxide gas at a constant rate per hour. Such devices are necessary for long-term storage and transport of more than 5 days.

Cells used in the above eukaryotic cell storage method is preferably stored in a container cultured with 30 to 70% confluence in consideration of cell proliferation during storage and transport, but the degree of culture of the cells is not limited thereto. Preferably, cells cultured with 50% confluency are immersed in the culture solution and stored in a container. The culture medium is preferably a conventional cell culture medium, it is more preferable to use a suitable medium according to the characteristics and types of cells.

The eukaryotic cell storage method of the present invention maintains the activity of eukaryotic cells to improve survival rate and inhibit cell differentiation.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Carbon dioxide gas porch filled with 5% by volume to 10% by volume of carbon dioxide was purchased from BBL's Campy Pouch system (BBL, 260656) and used.

Example 1 Storage of Keratinocytes

Keratinocytes were cultured in KGM (Clonetics, CC-3101) medium until they had 70 percent confluency. The culture vessel in which the cultured keratinocytes were cultured was placed in a carbon dioxide gas porch and left to stand at room temperature after sealing.

Control

Keratinocytes were left at room temperature as a control.

Example 2: Measurement of Cell Activity According to Cell Storage

The keratinocytes of Example 1 and control were measured for cell activity over time while standing at room temperature.

At 1, 2, 3, and 6 days, keratinocytes were observed for morphological changes on an inverted microscope, and the degree of cell death was measured by MTT analysis.

(1) microscopic observation

As a result of microscopic observation, keratinocytes of the control group showed early differentiation of cells after 2-3 days of room temperature, and the experimental group left in a porch filled with carbon dioxide showed normal morphology until 6 days of standing.

Figure 1 is a micrograph observing the cell activity of keratinocytes after 3 days at room temperature, a is keratinocytes stored in the carbon dioxide porch, b is keratinocytes left in the atmosphere. When stored in a carbon dioxide porch, the number of cells was larger than that in the air, and the activity of the cells was also excellent. In addition, the control group left in the air showed a differentiation characteristics.

(2) MTT analysis

The keratinocytes of Example 1 and the control were placed in 96-well plates and incubated for 4 hours in a medium containing 0.5 mg / ml of MTT solution. 200 μl dimethylsulfoxide (DMSO) solution was added to the wells and dissolved, and the absorbance was measured at 540 nm using an ELISA reader. The cell viability was measured by converting the absorbance of each solution, shown in Table 1 and FIG.

D0 D1 D2 D3 D6 Control group (%) 100 + 4.6 115 + 1.9 108 + 3.9 113 + 4.1 122 + 5.6 Example 1 (%) 100 + 4.6 108 + 4.1 106 + 4.1 109 + 7.2 131 + 13.3

Cell viability of Example 1 using the carbon dioxide porch was superior to the control.

Therefore, cell storage using carbonic acid gas porch is an optimal method of cell storage during transport by improving cell viability and inhibiting the differentiation of cells.

Example 3: Fibroblasts

Fibroblasts were cultured in DMEM (Gibco, 12800-0580) media until they had about 90% confluency. The fibroblasts were placed in a carbon dioxide gas porch (Campy pouch system, BBL, 260656) to prepare an experimental group, and the control group was left in the air and left at room temperature.

(1) microscopic observation

The cell morphology of fibroblasts was observed under a microscope.

Figure 3 is a micrograph observing the cell activity of the fibroblasts, a is the fibroblasts stored in the carbon dioxide porch, b is the fibroblasts left in the atmosphere. Cells kept in the carbon dioxide porch retained cell numbers, while most of the fibroblasts left in the air were dead. Morphological changes were not observed in the cells of the experimental and control groups.

(2) MTT analysis

Cell viability of the experimental group and the control group was measured by MTT analysis. As shown in Table 2 and Figure 4, the cell survival rate of the experimental group and the control group showed a big difference. That is, the survival rate of the experimental group was significantly superior to the control group.

D0 D1 D2 D3 D6 Control group (%) 100 + 18.1 123 + 4.3 43 + 2.6 15 + 0.6 16 + 1.5 Experimental group (%) 100 + 18.1 109 + 4.2 99 + 9.5 94 + 1.9 118 + 2.0

Therefore, the cell storage method using carbon dioxide gas is a method that can transport the fibroblasts in an optimal state.

Example 4: Preservation of Tissue

Tissues are generally preserved at 4 ° C, but there is no standardized way to actually determine the state of tissues. In order to confirm the effectiveness of the carbon dioxide storage method, the skin tissues obtained for cell culture were stored in a carbon dioxide gas bag and then cell cultured. Viable cell number was measured by MTT assay after 24 hours and 48 hours incubation, the results are shown in Table 3.

D0 D1 D2 Control group (%) 100 + 7.5 52.2 + 15.9 9.3 + 2.4 Experimental group (%) 100 + 7.5 78.9 + 8.4 27.2 + 5.5

As a result of the experiment, the number of cultured cells was much higher than that of the tissues stored at room temperature, and the tissue activity was improved by storing the tissues in a carbon dioxide gas bag.

The present invention not only maintains eukaryotic activity by storing eukaryotic cells using carbon dioxide, but also maintains eukaryotic cells in their initial state.

Claims (2)

Eukaryotic cell storage method for storing the eukaryotic cells in a container filled with 5 to 30% by volume of carbon dioxide during the transport of eukaryotic cells. The method of claim 1, wherein the eukaryotic cells are cells, cancer cells, immortalized cell lines, artificial tissues, transformants or tissues cultured in humans and animals.