KR20170019049A

KR20170019049A - Time lapse incubator

Info

Publication number: KR20170019049A
Application number: KR1020150112899A
Authority: KR
Inventors: 최규완; 서호철; 전진현; 양윤석; 서용수
Original assignee: 을지대학교 산학협력단; 주식회사 씨엔씨바이오텍
Priority date: 2015-08-11
Filing date: 2015-08-11
Publication date: 2017-02-21
Also published as: KR101772151B1

Abstract

Provided is a time lapse cell incubator comprising: an incubation room providing incubation environments suitable for an incubate; an incubation chamber accommodating an incubation container in the incubation room; an imaging system photographing the incubate in the incubation room; a temperature adjusting device for adjusting the temperature in the incubation room; a gas mixing system including at least two gas mixing chambers to adjust the concentration of injected gas of the incubation room; and a computer for controlling the operation of the imaging system, the temperature adjusting device and the gas mixing system.

Description

Time lapse incubator < RTI ID = 0.0 >

The present invention relates to time-lapse cell incubators.

At present, one out of seven couples in Korea is infertile. Of these, 95% are depressed due to stress. As infertility is recognized as a social problem, efforts to cope with infertility are being carried out in various ways including medical care, policy, . In general, infertility can be overcome by artificial procedures such as in vitro fertilization (IVF) and artificial insemination, but implanting a large number of embryos to improve pregnancy rate leads to multiple pregnancies, resulting in increased social costs and ethical problems due to artificial abortion . Therefore, it is important to select the most healthy embryos. In general, selection of transplanted embryos is performed in the order of pronucleus, blastocere stage, embryo fragmentation, blastocyst, And the shape of the blastocyst. However, there is still a limitation of the selection of embryos that can not be observed with the naked eye, such as when the time for blast is progressing too fast or the fragment is disappeared and the expansion of the blastocyst is increased.

Korean Patent Laid-Open Publication No. 2009-0020614 discloses a culture observation system which makes it possible to observe the culture condition of a culture with a microscopic image.

However, in the case of the above-mentioned prior art, only the real-time observation of the culturing condition is taken into consideration and the environmental conditions suitable for culturing are not considered.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a time-lapse cell incubator in which the environment suitable for cell culture is controlled. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a culture chamber for providing a suitable culture environment of a culture; A culture chamber for accommodating a culture container inside the culture chamber; An imaging system for imaging a culture inside the culture chamber; A temperature adjusting device for adjusting a temperature inside the culture chamber; A gas mixing system including at least two gas mixing chambers for regulating the concentration of the gas to be injected into the culture chamber; And a computer for controlling the operation of the imaging system, the temperature controller, and the gas mixing system.

According to one embodiment of the present invention as described above, an optimal embryo sorting effect can be realized through a suitable culture environment and real-time embryo observation. Of course, the scope of the present invention is not limited by these effects.

1 is a perspective view illustrating a time-lapse cell incubator 100 according to an embodiment of the present invention.
2 is a perspective view of a time-lapse cell incubator 100 according to an embodiment of the present invention.
3 is a perspective view showing the internal structure of the culture chamber 140 constituting the culture chamber 130 of the time-lapse cell culture apparatus.
FIG. 4 is a schematic block diagram showing an A-type time lapse photography apparatus 180 constituting the inside of a culture room 130 of a time-lapse cell culture apparatus 100 according to an embodiment of the present invention.
FIG. 5 is a schematic diagram showing a B-type time lapse photographing apparatus 180 'constituting the inside of a culture room 130 of a time-lapse cell incubator 100 according to an embodiment of the present invention.
6 is a perspective view of a gas mixing system 160 that supplies gas to a culture chamber of a time-lapse cell incubator 100. FIG.
7 is an exploded view schematically illustrating a process in which a gas is mixed by a gas mixing system 160 and injected through a filter.

According to one aspect of the present invention, there is provided a culture chamber for providing a suitable culture environment of a culture; A culture chamber for accommodating a culture container inside the culture chamber; An imaging system for imaging a culture inside the culture chamber; A temperature adjusting device for adjusting a temperature inside the culture chamber; A gas mixing system including at least two gas mixing chambers for regulating the concentration of the gas to be injected into the culture chamber; And a computer for controlling the operation of the imaging system, the temperature controller and the gas mixing system.

The cell incubator may be a cell incubator having a tabletop shape.

In the cell incubator, the temperature regulating device may be composed of a heating device, a cooling device, and a temperature sensor, and the heating device may be mounted on a cover of the culture chamber.

In the cell incubator, the imaging system may transmit a light source irradiated from a light source device to a culture container, and photograph the transmitted image with a camera lens and output to a display connected to the computer. The light source may be an LED light source, a mercury lamp, an optical fiber, bright field illumination, dark field illumination, phase contrast, hoffman modulation contrast, differential interference contrast, And may be one or more selected from the group consisting of The display may be a CRT monitor, an LCD monitor, an LED monitor, a PDP or a tablet PC, and the lens may be an objective lens or a magnification changing lens, and the camera may be a ccd camera, a cmos camera, or a nmos camera.

In the cell incubator, the gas mixing system may be constituted by a plurality of gas mixing chambers, and a timer is provided in the gas mixing chamber. During the initial incubation, the mixed gas of the first chamber is injected into the culture chamber, The mixed gas of the second chamber can be injected. The timer measures the time from the time when the culture container is placed. When the set time has elapsed, the gas phase can be regulated and injected. The gas phase can contain 3 to 7% of CO ₂ , 2 to 5% of O ₂ and N ₂ 75 to 90%.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The embodiments of the present invention are described in order to more fully explain the present invention to those skilled in the art, and the following embodiments may be modified into various other forms, The present invention is not limited to the embodiment. Rather, these embodiments are provided so that this disclosure will be more thorough and complete, and will fully convey the concept of the invention to those skilled in the art. In the drawings, the thickness and size of each layer are exaggerated for convenience and clarity of explanation.

It is to be understood that throughout the specification, when an element such as a film, region or substrate is referred to as being "on", "connected to", "laminated" or "coupled to" another element, It will be appreciated that elements may be directly "on", "connected", "laminated" or "coupled" to another element, or there may be other elements intervening therebetween. On the other hand, when one element is referred to as being "directly on", "directly connected", or "directly coupled" to another element, it is interpreted that there are no other components intervening therebetween do. A uniform code refers to a uniform element. As used herein, the term "and / or" includes any and all combinations of one or more of the listed items.

Although the terms first, second, etc. are used herein to describe various elements, components, regions, layers and / or portions, these members, components, regions, layers and / It is obvious that no. These terms are only used to distinguish one member, component, region, layer or section from another region, layer or section. Thus, a first member, component, region, layer or section described below may refer to a second member, component, region, layer or section without departing from the teachings of the present invention.

Also, relative terms such as "top" or "above" and "under" or "below" can be used herein to describe the relationship of certain elements to other elements as illustrated in the Figures. Relative terms are intended to include different orientations of the device in addition to those depicted in the Figures. For example, in the figures the elements are turned over so that the elements depicted as being on the top surface of the other elements are oriented on the bottom surface of the other elements. Thus, the example "top" may include both "under" and "top" directions depending on the particular orientation of the figure. If the elements are oriented in different directions (rotated 90 degrees with respect to the other direction), the relative descriptions used herein can be interpreted accordingly.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" include singular forms unless the context clearly dictates otherwise. Also, " comprise "and / or" comprising "when used herein should be interpreted as specifying the presence of stated shapes, numbers, steps, operations, elements, elements, and / And does not preclude the presence or addition of one or more other features, integers, operations, elements, elements, and / or groups.

Hereinafter, embodiments of the present invention will be described with reference to the drawings schematically showing ideal embodiments of the present invention. In the figures, for example, variations in the shape shown may be expected, depending on manufacturing techniques and / or tolerances. Accordingly, the embodiments of the present invention should not be construed as limited to the particular shapes of the regions illustrated herein, but should include, for example, changes in shape resulting from manufacturing.

1 shows a schematic diagram of a time-lapse cell incubator 100 according to some embodiments of the present invention. The time-lapse cell incubator 100 of the present invention is equipped with a tabletop type body model which is easy to move and operate so that related work can be carried out easily in cell culture. 1, the cell incubator main body 110 includes a door 120 so as to be opened and closed in the form of a cabinet, a culture chamber 130 formed at an upper end of the cell culture chamber 130, a culture chamber 130, Is composed of six culture chambers 140 in total. A computer keyboard 150 is installed at the center of the upper part of the main body, and a monitor 112 is installed at the upper right of the main body to input the culture conditions and monitor the condition of the culture space in real time. For example, an image photographed through a time-lapse camera (not shown) installed in the culture room 130 is displayed in real time on a display 112 that is collected in a computer (not shown) and connected to the computer. The display 112 provides information for confirming the operation state of the cell incubator 100 according to an embodiment of the present invention and displays various variables such as the culture conditions of the cell incubator 100 and the photographing interval of the camera, The keyboard 120 can be omitted if the user can recognize the touch or drag of the finger and input the variable directly on the screen. The display 112 may be a CRT monitor, an LCD monitor, an LED monitor, an OLED monitor, or a PDP monitor, and may be an integrated PC or a tablet PC configured integrally with the computer main body. In this case, It is possible.

FIG. 2 is a perspective view showing an internal configuration of the time-lapse cell incubator 100. As shown in FIG. 2, when the door 120 of the main body 110 is opened, the inside of the main body 110 is composed of an upper portion and a lower portion. At the lower end, there is a storage space A in which recording documents related to culture can be stored. And a gas mixing system 160 is installed at the upper left corner. A VOC filter (165) is installed on the right side of the gas mixing system (160). A gas supply port (not shown) is provided on the rear surface to communicate with the inside of the culture chamber 130, and a gas supply pipe (not shown) is connected to the gas supply port. (CO ₂ , O _2, and N ₂ ) flowing from the outside of the gas mixing system 160 are mixed according to the preset concentration, and the mixed gas is supplied from the first gas hose installed at the rear portion of the chamber through the VOC filter 165, (Not shown) in the culture chamber 130 to the culture chamber. At this time, the supply of the gas may be performed by mixing the gas, and optionally, only one gas may be selectively supplied. Meanwhile, a computer 155 is installed on the upper right side of the body, and the concentration of the gas and the injection time can be adjusted by adjusting the value of the gas mixing system 160 using the computer keyboard 150. Of course, the setting conditions may be inputted through the display 112. [

The computer 155 also stores images taken by a time lapse camera installed in the culture room 130 at a photographing date (photographing time), temperature data (incubator temperature, stage temperature) output from the incubator control device, (Not shown) of the computer 155 in correspondence with the information on the culture environment such as the concentration (CO ₂ , O ₂ and N ₂ gas concentration). The auxiliary storage device may be a hard disk, a CD-RW, a CD-R, a DVD-RW, a DVD-R, a DVD-RAM, a flash memory, or a solid state drive (SSD). Information about the camera image, the shooting date, and the culture environment saved in the auxiliary memory device is displayed in association with each camera image on the cell observation system display. It is possible to appropriately confirm the culture information by displaying it together with the information about the photographed camera image culture environment, so that reliability as observation data can be achieved.

FIG. 3 is a perspective view of a culture chamber 140 constituting a culture chamber 130 at an upper end of the body of the time-lapse cell culture apparatus 100. Since the culture chamber is composed of six chambers in the culture chamber 130 as described above, a maximum of 6 samples can be separately cultured, and when the sample is a human germ cell, embryos of up to 6 patients are cultured A maximum of 72 embryos per 12 chambers can be cultured at one time, so that the capacity of the culture sample can be increased and the number of the culture chambers 140 can be decreased or increased as needed.

When the incubation chamber 140 is placed in the chamber 140, the chamber lower part 144 and the rectangular chamber body 142, which are formed with grooves according to the shape and size of the container, And a chamber cover 146. The culture container may be constituted of a container, for example, glass or resin, at least the bottom surface and the top surface being transparent to light. An inlet (not shown) for injecting the gas and the temperature supplied from the culture chamber 130 is provided in the chamber lower part 144 to enable an environment suitable for culturing to be maintained.

In the culture environment of the cell, major stress is caused by temperature change, change of gas concentration, non-optimization of pH, and introduction of volatile organic solvents (VOCs). These problems are caused by opening and closing of the door The air will enter the inside and the gas inside will leak out. Since the incubation chamber 140 is constituted by a minimum space for culturing, it is possible to minimize the problems of changes in temperature and gas concentration due to opening and closing occurring in a conventional cell incubator when a sample is added or removed. For example, after the door is opened and closed, the CO ₂ concentration can be restored to 5 minutes, the concentration of 0 ₂ can be recovered within 15 minutes, and the chamber cover 146 is provided with a heating system. Do.

FIGS. 4 to 5 show a schematic configuration diagram of a time lapse apparatus 180 installed in the culture chamber 130 in the time-lapse cell incubator 100. FIG. There is a difference in the position of the light source device and the number of mirrors in the classification of the time-lapse device, and the configuration of other time-lapse device is the same.

4, the photographing using the time-lapse apparatus 180 of the type A is performed when the light source irradiated from the light source device 183 provided on the rear surface of the culture chamber 130 is illuminated by the first mirror 181 ) Is transmitted to the culture container and the transmitted image is incident on the second mirror (182) installed under the culture chamber (130). An image of the cell is acquired by the lens and the image is captured by the camera and displayed as a display. The light source may utilize LED illumination, mercury lamp, optical fiber, bright field illumination, dark field illumination, phase contrast, hoffman modulation contrast, differential interference contrast, or fluorescence . An infrared or near-infrared light source may also be used to reduce phototoxicity and improve the contrast ratio between the cell membrane and the interior portions of the cell. The lens may be an objective lens or a magnification varying lens, and the camera may be a camera using a CCD, CMOS or NMOS as an image pickup element. The camera body 185 coupled with the lens 184 and a ball screw device 187 for driving the camera body 185 are installed in the image pickup device and are movable in the horizontal direction by driving the motor. Therefore, it is possible to capture images according to the date and time set sequentially from the first chamber to the sixth chamber constituting the incubation chamber, and it is possible to take images in the order of the set chambers. Between the culture chamber 130 and the photographing apparatuses 182, 184, 155 and 187 is partitioned by a partition 186 and the air and the temperature in the culture chamber are sealed so as not to enter the photographing apparatus.

The time-lapse device 180 is connected to a computer through a communication line for data communication (not shown), and coordinate movement, irradiation of a light source, and image capturing by a camera are controlled and output to a computer according to computer settings.

It is assumed that the total amount of light received by the imaging system corresponds to, for example, about 24 minutes of continuous low-level light exposure in the case of imaging for 5 days. The light intensity for a time-lapse imaging system is typically used in assisted reproductive microscopy due to the low power of the LED (e.g., using a 1W LED compared to a typical 100W halogen bulb) and the high sensitivity of the camera sensor It is much lower than the brightness. Thus, the total amount of light energy received by an embryo when using a time-lapse imaging system is comparable or less than the amount of energy received during routine handling in an IVF clinic. In addition, the exposure time can be significantly shortened to reduce the total amount of light exposure to the embryo / stem cells. In the case of two day imaging, the image is captured every five minutes at 0.5 second light exposure per image, and the total amount of low level light exposure is less than 5 minutes.

5, the photographing using the B-type time lapse device 180 'is performed such that the light source device 183 is positioned on the upper surface of the culture chamber 140, and the light source, which has passed through the culture container, It is a structure that enters directly. The configuration of the other photographing apparatus is the same as that of the A type time lapse apparatus.

The time-lapse photographing is a function of automatically photographing by setting a predetermined time interval, recording the photographed camera image in a memory of a computer, and is suitable for observing a temporal change of a living cell, It is possible to set a plurality of shooting times or a plurality of shooting times for each shooting point. Especially, in the multi-point time-lapse where a plurality of shooting points are set at the same time, the stage moving time after the shooting, the shooting time, and the camera exposure time are influenced, so that it can be set in consideration of the time-lapse interval time. The interval time can be adjusted in units of seconds, minutes and hours, and is preferably 1 minute to 30 minutes in order to capture a meaningful morphological event. Also, the time interval between images taken can vary depending on the amount of cellular activity. For example, an image can be frequently photographed every few seconds or every minute during an activity period, whereas during an inactivity period an image can be taken every 10 or 15 minutes or at longer intervals. Real-time image analysis on the captured image can be used to detect when and how to change the time interval.

In the above-described time-lapse device, the cell may be an embryonic stem cell, which is undifferentiated stem cells (so-called ES cells) existing in an embryo, or an undifferentiated stem cell existing in each organ in a completed body, And includes cells used for regenerative medicine such as adult stem cells such as hematopoietic stem cells and neural tube cells as well as animals and human germ cells. The germ cells may be spermatocytes, sperms, oocytes, fertilized eggs, embryos, morula, blastocysts.

The function of the time-lapse incubator is to shoot the cultured state of the cultured cells and to output them as an image, thereby allowing the researcher to maximize the efficiency of culturing the cells by controlling the culturing conditions and selecting the cells according to the culturing conditions. The selection of the optimal embryo is very important for embryo transfer as described in the Background of the present invention, and the method of embryo selection is based on the correct timing, precise timing of confusion, precise timing of blastocyst stage, synchrony of division, Morphological or morphokinetic screening criteria such as the occurrence of multinucleation and the history of fragmentation have been considered. Traditionally, in IVF clinics, human embryonic viability has been assessed by monomorphological observations, for example, the presence of uniform sized mononucleate blastomere and degree of cell fragmentation (Rijinders PM et al., Hum Reprod, 13: 2869-73,1998). Recently, an embryo was cultured to blastocyst stage (5 days culture) and the chromosome status analysis through preimplantation genetic diagnosis (PGD) was also used to evaluate embryo quality (Milki A et al., Fertil Steril 73 : 126-9, 2000; Toukhy T et al., Hum Reprod, 6:20, 2009). However, there is also a potential risk in that these methods prolong the incubation period and destroy embryo integrity (Manipalviratn S et al., Fertil Steril, 91: 305-15, 2009) Several studies have shown that time-lapse imaging can be a useful tool for observing early embryonic development, and some methods have shown that after intracytoplasmic sperm injection (ICSI), human embryonic development (Nagy et al., Human Reproduction 9 (9): 1743-1748, 1994; Payne et al., Human Reproduction 12: 532-541, 1997).

FIG. 6 is a perspective view showing the appearance of a gas mixing system 160 installed inside the time-lapse cell incubator 100. The gases (CO ₂ , O _2, and N ₂ ) of the gas tank (not shown) separately mounted on the outside are introduced into the gas mixing chamber (shown in FIG. 7) contained in the gas mixing system 160, The volatile organic solvent is filtered through the VOC filter 165 installed on the right side of the cell culture container 110 and a gas delivery pipe (not shown) installed on the rear surface of the cell culture container 110 And is uniformly distributed into each culture chamber 140 through a distributor (not shown) installed in the culture chamber 130. [ In the gas mixing system 160, a temperature sensor (not shown) for detecting the gas temperature injected into the culture chamber is connected to the culture chamber control device, and the temperature inside the culture chamber and the culture chamber is uniformly maintained at the set temperature. Since the VOC filter 165 is installed outside the gas mixing system 160, the VOC filter 165 can be easily replaced after an appropriate period of use. The use period of the VOC filter 165 may be adjusted depending on the number of samples in the culture chamber 140 and the operation time of the gas mixing system 160, but is preferably 3 to 6 months.

FIG. 7 is a schematic diagram illustrating a gas mixing system 160 including a plurality of gas mixing chambers 168 and 169. As shown in FIG. Hereinafter, the gas mixing system will be described in detail. The gases (CO ₂ , O _2, and N ₂ ) introduced from the gas pipe installed on the rear surface of the gas mixing system 160 are first connected to the first chamber 168 under the inputted culture conditions using the computer keyboard 150 The gas concentration is controlled and mixed by the first gas concentration sensor 166. The concentration condition of the gas may be, for example, 3 to 7% of CO ₂ , 2 to 5% of O ₂ and 75 to 90% of N _{2 in} the case of culturing germ cells. The mixed gas in the first chamber 168 is filtered through the VOC filter 165 and the volatile organic solvent is filtered through the first gas hose pipe 195 connected to the culture chamber 130 from the rear side of the gas mixing system 160 130, respectively.

7, the gas mixing system 160 includes a second chamber 169, and a timer installed inside the chamber starts measurement from the time of placement of the culture container to the first chamber 168 ) Is automatically adjusted by the second gas concentration sensor 167 installed in the second chamber 169 and the VOC filter (gas phase) is regenerated by the gas phase The gas is delivered to the culture chamber 130 along the second gas hose pipe 198. [ The concentration of the reconditioned gas may be 3 to 7% of CO ₂ , 1 to 3% of O ₂ and 75 to 95% of N ₂ , for example in the case of germ cell culture. Here, the time for the resumption can be 48 to 96 hours and preferably 72 hours at the initial culturing time for confirming the fertilization for germ cells.

In addition, the gas mixture system 160 can be sequentially injected at a certain point of time during the incubation period by operating the first chamber 168 and the second chamber 169 as described above. However, The mixed gas of the first chamber 168 or the second chamber 169 may be selectively injected into the culture chamber 130 during the culture period depending on the cell and the operator so that the culture efficiency can be improved.

The present inventors have focused on metabolites consumed by embryos to overcome the limitations of conventional morphological or morphokinetic selection criteria in optimal embryo screening. Generally, when a sperm and an egg meet and become a fertilized egg, they stay in the fallopian tube until 3 days after fertilization and move to the uterus. When the environment of the ovary and the uterus is different, (nutriments) are different. That is, pyruvate or lactate is consumed as a main energy source from the pronucleus period to the 8th abortion in the oviduct, and glucose is consumed after 8 years of abortion. As the nutrient conditions change, so does the oxygen consumption. Unlike the uterus, the ovary is actually anaerobic. However, since the conventional cell culture apparatus supplies the same concentration of gas without considering these conditions, the initial culture condition is very important in the selection of the embryo, so that the present culture system is suboptimal. Accordingly, in the time-lapse cell culture apparatus according to an embodiment of the present invention, the gas phase of the embryo and the embryo after 3 days after the modification are different according to the operation of the timer installed in the mixing chamber, The present inventors have completed the present invention in order to provide an optimal culture system showing a high pregnancy rate through single embryo transfer by selecting the best healthy embryo in real time by confirming the cultivation situation in real time.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

100: Time-lapse cell incubator
130: Culture room
140: culture chamber
160: Gas mixing system
155: Computer
112: Display

Claims

A culture chamber for providing a suitable culture environment of the culture;
A culture chamber for accommodating a culture container inside the culture chamber;
An imaging system for imaging a culture inside the culture chamber;
A temperature adjusting device for adjusting a temperature inside the culture chamber;
A gas mixing system including at least two gas mixing chambers for regulating the concentration of the gas to be injected into the culture chamber; And
And a computer for controlling the operation of the imaging system, the temperature controller, and the gas mixing system.

The time-lapse cell incubator according to claim 1, wherein the cell incubator is in the form of a tabletop.

The method according to claim 1,
Wherein the temperature regulating device comprises a heating device, a cooling device, and a temperature sensor.

The method of claim 3,
Wherein the heating device is mounted on a cover of the culture chamber.

The method according to claim 1,
Wherein the imaging system is a time-lapse cell incubator in which a light source irradiated from a light source device is transmitted through a culture container, and the transmitted image is captured by a camera lens and output to a display connected to a computer.

6. The method of claim 5,
The light source may be an LED light source, a mercury lamp, an optical fiber, bright field illumination, dark field illumination, phase contrast, hoffman modulation contrast, differential interference contrast, And one or more species selected from the group consisting of time-lapse cell cultures.

6. The method of claim 5,
Wherein the display is a CRT monitor, an LCD monitor, an LED monitor, a PDP or a tablet PC.

6. The method of claim 5,
Wherein the lens is an objective lens or a magnification changing lens.

6. The method of claim 5,
Wherein the camera is a CCD camera, a CMOS camera, or an NMOS camera.

The method according to claim 1,
Wherein said gas mixing system comprises a plurality of gas mixing chambers.

11. The method of claim 10,
Wherein the gas mixing chamber is provided with a timer therein, and a mixed gas of the first chamber is injected into the culture chamber during the initial incubation, and a mixed gas of the second chamber is injected after a predetermined time has elapsed.

12. The method of claim 11,
The timer measures the time from the time when the culture container is placed, and the gas phase is adjusted and injected when the set time has passed.

12. The method of claim 11,
Characterized in that the gas phase is regulated to 3 to 7% of CO ₂ , 2 to 5% of O ₂ and 75 to 90% of N ₂ .