KR20120005886A

KR20120005886A - Incuvator for culturing cell

Info

Publication number: KR20120005886A
Application number: KR1020100066573A
Authority: KR
Inventors: 김영래
Original assignee: 주식회사 엔바이오텍
Priority date: 2010-07-09
Filing date: 2010-07-09
Publication date: 2012-01-17
Also published as: KR101192324B1

Abstract

PURPOSE: A cell culture incubator is provided to completely sterilize fungi and virus in a chamber and to ensure optimal cell culture condition. CONSTITUTION: A cell culture incubator comprises: a chamber(10) having a culture room(13) inside for culturing cells; a heater mounted in the chamber to heat the culture room inside the chamber; a temperature sensor for sensing temperature in the culture room; and a plasma generator for sterilizing the inside the chamber by generating plasma in the culture room. The heater is mounted at the all sides of the chamber and comprises a first heater used during cell culture and a second heater for sterilizing the inside of the chamber. The incubator is selectively operated between a culture mode and a clean mode.

Description

Incubator for culturing cell

The present invention relates to an incubator for cell culture for artificially culturing cells, and more particularly, to an incubator for cell culture that can be sterilized so that the cell culture space is always kept clean.

Cell culture is a culture of cells isolated from tissues of an organism under artificial conditions, and the cells to be cultured are very diverse, such as animal cells, sperm, eggs, anaerobic cells, microorganisms, and the like.

Since the cells are sensitive to the surrounding environment, the cell is cultured in an incubator that provides optimal conditions for cell growth, such as temperature, humidity, and the concentration of carbon dioxide.

Conventional cell culture incubator is provided with a shelf in which cells are placed in the chamber, the chamber is provided with a heater so that the space inside the chamber is maintained at a temperature and humidity suitable for culturing the cells. In addition, depending on the type of cells, the concentration of carbon dioxide should be kept constant, so that the carbon dioxide gas is constantly supplied and discharged to the chamber.

On the other hand, the inside of the chamber of the incubator should always be kept clean and sterilized, the incubator should be sterilized at regular intervals. In the conventional incubator, since the system for sterilizing the inside of the chamber is not equipped, cleaning is performed by disinfecting the inside of the chamber with alcohol or the like or disinfecting the shelf in a separate sterilizer while the power of the incubator is turned off.

Recently, in order to solve this problem, a method of attaching an ultraviolet lamp to the inside of the chamber and irradiating ultraviolet rays into the chamber at regular intervals or introducing hydrogen peroxide gas into the chamber has been developed.

However, only the method of irradiating ultraviolet rays or using hydrogen peroxide had a limitation in maintaining the sterile state inside the chamber. There is a risk that the inside of the chamber is contaminated and causes abnormalities in cell culture.

On the other hand, in the conventional incubator, the heater is disposed on the bottom or ceiling of the chamber. Since the heater is biased on one side of the chamber, the temperature of the entire chamber is not uniformly maintained, and a temperature gradient occurs in each chamber region. There was a problem that the condensation occurs, the disruption in the cell culture.

The present invention is to solve the above problems, excellent sterilization and disinfection function so that the inside of the chamber is always maintained aseptic state, the cell culture for improved structure to maintain the temperature inside the chamber evenly over the entire area The purpose is to provide an incubator.

Incubator for cell culture according to the present invention for achieving the above object is a chamber in which a culture chamber is formed to cultivate cells, a heater installed in the chamber to heat the culture chamber inside the chamber, of the culture chamber inside the chamber And a plasma generator for sterilizing the interior of the chamber by generating a plasma in the chamber and a temperature sensor for detecting a temperature.

In one embodiment of the invention the chamber is made of a polyhedron shape, the heater is installed on all sides of the chamber.

In addition, in one embodiment of the present invention, the heater is provided with a first heater used at the time of cell culture, and a second heater used to sterilize the interior of the chamber, the first heater is the best inside the chamber It can be maintained at 60 ℃, the second heater can be maintained at up to 90 ℃.

In addition, in one embodiment of the present invention is selectively operated between the culture mode for culturing the cells in the chamber, and the clean mode for sterilizing the interior of the chamber after removing the culture vessel containing the cells in culture. In the clean mode, at least one or both of the second heater and the plasma generator may be operated.

In the clean mode, after the temperature inside the chamber is heated to a first temperature, the temperature inside the chamber is maintained at the first temperature for a predetermined time, the operation of the second heater is stopped, and the temperature of the chamber is reduced. The temperature is controlled to be lowered to 2 temperatures, and when the clean mode ends, the cells are switched to the culture mode to place cells in the chamber, and the temperature of the culture chamber of the chamber is maintained at the second temperature while the first heater is operated.

In the cell culture incubator according to the present invention can be sterilized by plasma ions and sterilization by high temperature to completely sterilize fungi, viruses, etc. in the chamber, thereby providing an optimal environment for cell culture. There is an advantage that it is.

In addition, in an embodiment of the present invention, by placing a heater on all six surfaces of the chamber, it is possible to maintain a constant temperature of the culture chamber inside the chamber, and to maintain the temperature evenly in all regions inside the culture chamber.

In addition, in an embodiment of the present invention, the first heater for low temperature heating and the second heater for high temperature heating are separately provided, thereby increasing the thermal efficiency of the incubator and increasing durability.

1 is a schematic perspective view of an incubator for cell culture according to an embodiment of the present invention.
2 is a view showing the arrangement of the first heater and the second heater disposed on each side of the chamber of the cell culture incubator shown in FIG.
3 is a flow chart showing an operating state of the cell culture incubator shown in FIG.
4 is a graph showing an operating temperature in an operating state illustrated in FIG. 3.
5 is a graph showing the virus removal rate by the plasma generator.
6 is a graph showing the fungal bacteria removal rate by the plasma generator.
7 is a graph comparing the difference between the sterilization effect by the plasma and the sterilization effect by the ultraviolet lamp.

Hereinafter, with reference to the accompanying drawings will be described in more detail for the cell culture incubator according to an embodiment of the present invention.

1 is a schematic perspective view of a cell culture incubator according to an embodiment of the present invention, Figure 2 is a layout of the first heater and the second heater disposed on each side of the chamber of the cell culture incubator shown in FIG. The figure showing.

1 and 2, the cell culture incubator 100 according to an embodiment of the present invention includes a chamber 10, a first heater 21, a second heater 22, and a plasma generator.

The chamber 10 provides a space in which cells can be cultured, and includes a main body 11 and an outer door 12. In the present embodiment, the main body 11 has a hexahedron shape and the front surface is opened. The outer door 12 is coupled to the opened front surface of the main body 11. The outer door 12 is rotatably coupled to the main body 11 to open and close the inside of the main body 11. When the outer door 12 closes the main body 11, the culture chamber 13 closed by the main body 11 and the outer door 12 is formed. The chamber 10 is generally hexahedral as in this embodiment, but in other embodiments, may be a polyhedron of other shapes. And the inner wall 18 of the chamber 10 has a corner (c) is formed in a curved surface, unlike the angular corner is easy to clean, foreign matter is attached to the corner can solve the problem of contaminating the chamber 10.

In addition, in this embodiment, the inner door 14 is provided between the main body 11 and the outer door 12. The inner door 14 is made of a transparent material such as glass, and the inside of the culture chamber 13 can be observed while only the outer door 12 is opened.

In the culture chamber 13, a plurality of shelves 15 are stacked at regular intervals along the vertical direction. The shelf 15 has a culture vessel (not shown) in which cells are contained, and a plurality of through holes 15a penetrating between an upper surface and a lower surface are formed. Accordingly, the heated air may flow through the through holes 15a of the shelf 15.

On the other hand, a tray 16 containing water is disposed at the bottom of the culture chamber 13. As the water contained in the tray 16 is evaporated, the humidity in the culture chamber 13 is maintained. In addition, a thermometer (not shown) and a hygrometer (not shown) for detecting temperature and humidity inside the culture chamber 13 are installed, and the thermometer and hygrometer are electrically connected to the controller 70 of the cell culture incubator 100. It is connected. When the temperature and humidity in the culture chamber 13 is out of the set range, the thermometer and the hygrometer detect this and send a signal to the controller 70 so that the controller 70 adjusts the first heater 21 to be described later to adjust the temperature and humidity. Adjust The thermometer and the hygrometer may be provided separately, but in the present embodiment, a thermohygrometer type that senses temperature and humidity at the same time is used.

In this embodiment, the carbon dioxide gas is supplied into the culture chamber 13 through a supply line (not shown) connected from the external carbon dioxide source to the inside of the chamber 10. In addition, an impeller (not shown) is provided in the ceiling of the culture chamber 13 to promote the flow of air in the culture chamber 13.

In addition, in the present embodiment, an ultraviolet lamp u is installed at one side of the culture chamber 13 to sterilize the inside of the chamber 10. However, in the present invention, sterilization by the ultraviolet lamp u is secondary and sterilization is performed by the plasma generator and the second heater 22, which will be described later.

In the cell culture incubator 100 according to the present embodiment, the first heater 21 and the second heater 22 are provided. The first heater 21 is for heating the culture chamber 13 when the cells are cultured in the chamber 10, and the second heater 22 has the chamber 10 removed from the culture chamber 13. It is for heat sterilization of the inside.

The first heater 21 and the second heater 22 may be used in various forms, but in this embodiment, as shown in FIG. 2, an infrared heater method for heating an object while radiating infrared rays from a hot wire is employed. The first and second heaters 21 and 22 are installed between the outer wall 17 and the inner wall 18 of the chamber 10, and are insulated between the first and second heaters 21 and 22 and the outer wall of the chamber 10. The heat generated from the first and second heaters 21 and 22 is interposed and is transmitted to the culture chamber 13 through the inner wall 18 of the chamber 10 without being transferred to the outside of the chamber 10. As the inner wall 18 of the chamber 10, stainless steel (SUS) having excellent heat transfer rate is used, thereby improving thermal efficiency of the first and second heaters 21 and 22.

The first heater 21 is used at the time of cell culture, and has a performance capable of maintaining the temperature of the culture chamber 13 at most 60 ° C. or less. The optimum temperature at the time of cell culture varies from cell to cell, but generally 37 ° C., so that the temperature of the culture chamber can be optimally maintained by the first heater 21 used in the present embodiment.

The first heater 21 is installed on all six surfaces of the hexahedral chamber 10. That is, it is provided between five surfaces except the front surface of the main body 10 and the outer wall and the inner wall of the outer door 12. When the first heater 21 is installed on all six surfaces of the chamber 10 as described above, since the entire region of the culture chamber 13 is heated evenly, the temperature may be maintained evenly for each region in the culture chamber 13. In the conventional incubator, the heater is installed only at the bottom, so that the temperature does not appear evenly for each region inside the culture chamber, and in some cases, condensation occurs in the culture chamber, but in this embodiment, six surfaces of the chamber 10 are provided. All of these problems are solved by installing the first heater.

The first heaters 21 installed on the six surfaces of the chamber 10 may be installed separately, but in this embodiment, the heating wires installed on the six surfaces are all connected to one.

The second heater 22 is used to sterilize the inside of the chamber 10, and may heat the culture chamber 13 to a temperature of up to 90 ° C. When a certain time is heated in the chamber 10 to a temperature of 90 ℃ a number of bacteria die.

Like the first heater 21, the second heater 22 may be installed on all six surfaces of the chamber 10. However, in the present exemplary embodiment, the second heater 22 except the upper surface of the chamber 10 and the outer door 12 may be installed. It is installed only on four sides of the. Even if only four surfaces are installed, not only can the temperature of the culture chamber 13 be maintained at 90 ° C., but there is no effect on sterilization even if a slight temperature gradient occurs in the culture chamber 13 unlike when the cells are cultured. Because.

The second heaters 22 installed on the four surfaces of the chamber 10 may be separately installed, but in this embodiment, like the first heater 21, the heating wires installed on the four surfaces are all connected in one. .

In the cell culture incubator 100, which will be described later, since the cells are incubated for a predetermined time and then sterilized inside the chamber, and the cells are cultured to some extent, the first heater 21 and the second heater 22 together. Not used, only the second heater 22 is used for sterilization and only the first heater 21 is used for cell culture. Therefore, only the second heater 22 that can be heated to a high temperature of 90 ℃ can be used for both cell culture and sterilization, but if the heating wire is repeatedly used in the high temperature region of 90 ℃ and low temperature region of about 37 ℃ There is a problem that the durability of the heater is sharply lowered and the service life of the incubator is shortened. Therefore, in the present invention, the durability of the heater is increased by separately providing and using the first heater 21 and the second heater 22.

Meanwhile, in the present invention, a plasma generator (not shown) is provided to sterilize the chamber 10. The plasma generator is a known device that activates ambient air or gas in an excited ion state, and is disposed on the ceiling of the chamber 10. Ions activated by the plasma generator sterilize bacteria, fungi, and the like in the chamber 10.

Sterilization effect by plasma is shown in FIGS. 5 and 6. 5 is a graph showing the virus removal rate by the plasma generator, Figure 6 is a graph showing the fungal bacteria removal rate by the plasma generator.

In order to test the virus sterilization effect by plasma, the virus was suspended in a box of 1 m ³ and the residual rate was examined for 10 minutes. Referring to FIG. 5, almost all of the virus floating in the air remained in the natural state without plasma ions in the box after 10 minutes. However, when plasma ions were maintained in the box at 7,000 / Cm ³ , 99% of the virus was removed after 10 minutes. In addition, when the plasma ions were maintained at a high concentration of 50,000 / Cm ³ , 99.9% of the virus was removed.

In addition, we tested the ability to remove suspended fungi. That is, in a laboratory of 13.2 m ² area, plasma ions were released at a concentration of 3,000 / Cm ³ and 200 minutes later, the residual rate of fungi was measured by an air sampler. Referring to FIG. 6, fungi were naturally reduced slightly after 200 minutes in the absence of plasma ions, but most remained. On the other hand, almost all fungi were present after 200 minutes when plasma ions were released at a concentration of 3,000 / Cm ³ . It was confirmed that it was removed.

That is, when plasma ions are generated in the chamber 10 through the plasma generator, it is confirmed that viruses and bacteria in the chamber 10 can be almost completely removed. In the conventional cell culture incubator, the ultraviolet lamp or hydrogen peroxide was supplied to sterilize the inside of the chamber, but in the present invention, the sterilization rate of the inside of the chamber 10 was dramatically increased by introducing plasma ions.

The operation of the incubator 100 for cell culture consisting of the above configuration will be described. 3 is a flow chart showing the operating state of the cell culture incubator shown in Figure 1, Figure 4 is a graph showing the operating temperature in the operating state shown in FIG.

3 and 4, the cell culture incubator 100 is operated in a clean mode and a culture mode. Based on 24 hours, 16 hours is operated in a culture mode for culturing cells, and 8 hours is operated in a clean mode for sterilizing the interior of the chamber 10.

The clean mode is further divided into a heating section, a sustaining section, and a cooling section. The power is supplied to the second heater 22 to set the temperature inside the chamber 10 for 50 minutes to the first temperature set up to 90 ° C in this embodiment. Raise. Afterwards, while maintaining the temperature inside the chamber 10 at about 90 ± 5 ° C. while operating the second heater 22, the inside of the chamber 10 is heat sterilized.

Plasma ions are generated inside the chamber 10 by operating the plasma generator intermittently for about 5 minutes in this heating section and the continuous section. The plasma starts to operate approximately when the temperature of the culture chamber approaches 70 ° C. When the plasma generator is continuously operated, excessive ozone ions are generated inside the chamber, and thus, it is preferable to operate intermittently while stopping for 5 minutes and 55 minutes as in the present embodiment.

When the sterilization by heating and the sterilization by plasma ions are performed in the heating section, the inside of the chamber 10 is completely sterilized. In the cooling section, the second heater 22 is turned off and the temperature of the culture chamber 13 is increased by natural cooling. The second temperature, which is suitable for cell culture, is lowered to 37 ° C in this example.

When the cooling section is completed, the culture vessel containing the cells is placed on a shelf, and the first heater 21 is supplied with power while supplying carbon dioxide, and the temperature of the culture chamber 13 is fixed to the second temperature for 16 hours. Keep it. During the incubation period, the temperature and humidity in the culture chamber 13 are continuously detected by a thermo-hygrometer, and the concentration of carbon dioxide is continuously detected by a sensor (not shown). The controller 70 receives the signals of the thermo-hygrometer and the carbon dioxide concentration sensor so as to adjust the calorific value of the first heater 21 so that the conditions of temperature, humidity, and carbon dioxide concentration inside the culture chamber 13 are optimally maintained. After 16 hours of incubation, the process returns to the clean mode and sterilizes the inside of the chamber 10.

The cell culture incubator 100 operated as described above and the sterilization effect of the incubator operated by the ultraviolet lamp was prepared.

The test conditions are as follows. 100 μl of Aspergillus niger fungi were inoculated into a PDA (potato dextrose agar) on a 60 mm diameter dish and inoculated into 5 (n = 5) culture dishes, each with an incubator with a plasma generator and an ultraviolet lamp. After treatment for 6 hours, 12 hours, 18 hours, and 24 hours in the attached incubator, respectively, the result of incubation for 52 hours in an incubator at a temperature of 30 ° C. and a carbon dioxide concentration of 5% was analyzed through NIH ImageJ. The analysis area was 23.75 cm ² .

The results are shown in FIG. Referring to FIG. 7, in the first six hours, the mold growth area was small when temporarily treated with an ultraviolet lamp, but the growth of mold was suppressed when plasma ion treatment was performed over time. After the last 24 hours, the fungus growth area was only 13% in the case of plasma sterilization, but the fungus growth area was almost 4 times higher in the case of sterilization by UV lamp.

In other words, it was confirmed that the efficiency of the plasma sterilization treatment was much higher than that of the ultraviolet lamp sterilization treatment. In particular, the Aspergillus Niger fungus used in the experiment can be seen that almost all viruses and fungi can be removed in the cell culture incubator according to the present invention is a very difficult species sterilization.

As described above, in the cell culture incubator 100 according to the present invention can be sterilized by plasma ions and sterilization by high temperature to completely sterilize fungi, viruses, etc. in the chamber, thereby culturing the cells Can provide an optimal environment.

Although the present invention has been described with reference to one embodiment shown in the accompanying drawings, this is merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent other embodiments are possible. Could be. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.

100 ... incubator for cell culture 10 ... chamber
11 ... main unit 12 ... external door
13 ... Culture Room 21 ... First Heater
22 ... second heater 70 ... controller

Claims

A chamber in which a culture chamber is formed so as to culture the cells;
A heater installed in the chamber to heat the culture chamber inside the chamber;
A temperature sensor detecting a temperature of the culture chamber inside the chamber; And
And a plasma generator for sterilizing the interior of the chamber by generating a plasma in the culture chamber inside the chamber.

The method of claim 1,
The chamber is made of a polyhedron shape,
The heater is an incubator for cell culture, characterized in that installed on all sides of the chamber.

The method according to claim 1 or 2,
The heater is an incubator for cell culture, characterized in that the first heater used for cell culture and the second heater used to sterilize the interior of the chamber are provided separately.

The method of claim 3,
The second heater is a cell culture incubator, characterized in that for heating the interior of the chamber to 90 ℃ to sterilize the chamber.

The method of claim 3,
It is selectively operated between the culture mode for culturing the cells in the chamber and the clean mode for sterilizing the interior of the chamber after removing the culture vessel containing the cells in culture from the chamber,
The incubator for cell culture, characterized in that at least one of the second heater and the plasma generator is operated in the clean mode.

The method of claim 5,
In the clean mode, after the temperature inside the chamber is heated to the first temperature, the temperature inside the chamber is maintained at the first temperature for a predetermined time, the operation of the second heater is stopped, and the temperature of the chamber is set to the second temperature. Incubator for cell culture, characterized in that the control to lower to a temperature.

The method of claim 6,
When the clean mode is finished, the cell culture incubator to switch to the culture mode to place the cells in the chamber, the first heater is operated so that the temperature of the chamber of the chamber is maintained at a second temperature.

The method of claim 1,
The plasma generator incubator for cell culture, characterized in that the intermittent operation.

The method of claim 1,
The culture chamber may be placed in the culture vessel containing the cells, a plurality of shelves are formed with a plurality of through holes are spaced apart at regular intervals along the vertical direction,
The inner wall of the chamber is an incubator for cell culture, characterized in that the corner region is formed in a curved surface.