CN112877279B

CN112877279B - Light source controllable cell culture box for rat embryonic stem cells

Info

Publication number: CN112877279B
Application number: CN201911267985.3A
Authority: CN
Inventors: 谌兵来
Original assignee: Shanghai Houchao Biotechnology Co ltd
Current assignee: Shanghai Houchao Biotechnology Co ltd
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2023-03-28
Anticipated expiration: 2039-12-11
Also published as: CN112877279A

Abstract

The invention discloses a culture solution for rat embryonic stem cells, which comprises a culture medium, a light protectant and a culture additive; the light protectant is mixture of fructus Lycii extract, radix astragali extract, and tea polyphenols. The invention also discloses a formula method and an incubator based on the culture solution. The invention promotes the culture of the rat embryonic stem cells in a lighting mode, does not influence the functions of the embryonic stem cells and has positive effect.

Description

Light source controllable cell culture box for rat embryonic stem cells

Technical Field

The invention belongs to the technical field of cell culture. In particular to a light source controllable cell culture box for rat embryonic stem cells.

Background

Chinese patent CN103031272A discloses a culture medium for rat embryonic stem cells, which indicates that differentiation of rat embryonic stem cells can be effectively inhibited by adding a differentiation inhibitor, an embryonic stem cell strain of a rat capable of reproductive passage is established, and the cultured rat stem cells can have stable karyotype after targeting.

The applicant also found through experiments that there is a certain inhibitory effect on the culture of rat embryonic stem cells after adding the differentiation inhibitor, so that it is necessary to provide a new solution to this problem.

Disclosure of Invention

The invention aims to provide a culture solution and a culture method for culturing rat embryonic stem cells, and a culture device developed based on the culture method.

In a first aspect of the invention, there is provided a culture solution for rat embryonic stem cells, the culture solution comprising a culture medium, a culture additive;

the culture medium is prepared by mixing a DMEM/F12 culture medium and a nerve cell basic culture medium;

the culture supplement comprises a differentiation inhibitor and pluriptin, and the differentiation inhibitor comprises a GSK inhibitor, a FGFR inhibitor and a MAPK inhibitor, and the supplement further comprises: beta-mercaptoethanol; l-glutamine;

the technicians in the field can also configure the components and the contents of the culture medium and the culture additives disclosed in the Chinese patent CN 103031272A;

for those skilled in the art, the difference is that a photoprotection agent is also added into the culture solution, and the photoprotection agent can be a mixture of medlar extract, astragalus extract and tea polyphenol; all the substances have better radiation resistance; the reason for adding the light protective agent is that the light method is adopted to promote the growth of the rat embryonic stem cells, and in order to avoid the adverse effect of the light on the cells, a proper amount of light protective agent is added, so that the influence on the rat embryonic stem cells is reduced, the survival rate of the rat embryonic stem cells is improved, and the karyotype stability is improved;

the traditional Chinese medicine extract is selected as the light protectant, so that the anti-irradiation capacity can be improved, meanwhile, certain nutrition can be provided for the culture of rat embryonic stem cells, and the dual effects are achieved;

the added mass of the light protective agent is 1 to 20 percent of the mass of the whole culture solution, and the preferred mass of the light protective agent accounts for 10 percent of the mass of the whole culture medium.

In a second aspect of the present invention, there is provided a method for culturing rat embryonic stem cells, which comprises culturing rat embryonic stem cells in the presence of the culture solution, and irradiating light during the culturing process.

The essence of illumination is to generate a certain frequency, which further promotes the growth of cells, and the illumination that can be used is visible light.

In a third aspect of the present invention, there is provided a light source controllable cell incubator, which is mainly based on the problems of the existing illumination incubator, and comprises: the space in the incubator is limited, the illumination intensity range which can be changed by changing the distance from the LED lamp to the culture dish is very small, and the requirement of cell culture cannot be met at all; secondly, the environment of above-mentioned equipment incubator is unanimous when using, can't do the contrast test group, needs to set up a plurality of equipment and carries out the contrast test, has increased the experimental cost.

Consequently controllable type cell culture case of light source after this application improves, including box, moving mechanism, interval mechanism and lighting mechanism, moving mechanism installs in the inboard bottom of box, interval mechanism installs the upper end at moving mechanism, lighting mechanism installs the upper end at interval mechanism, the inside of box is equipped with environment monitoring device, the both sides of box are equipped with intake pipe and outlet duct respectively, the intake pipe with the outlet duct all communicates with the box is inside, be equipped with the camera on the inside wall of box, the camera sets up towards interval mechanism, the front end of box is equipped with the chamber door, the lower extreme of box is equipped with the cabinet, the side of box is equipped with the display.

Further, the moving mechanism comprises a moving plate, pulleys are arranged on two sides of the moving plate, slide rails are arranged on two sides of the interior of the box body and are in sliding fit with the pulleys on two sides of the moving plate respectively, two grooves are formed in the upper end of the moving plate, a culture box is arranged above the moving plate, two convex edges are arranged at the lower end of the culture box and are in sliding fit with the grooves, the length of each convex edge is half of the length of each groove, and a pull block is arranged at the front end of the culture box.

Further, the spacing mechanism includes fixed frame and a plurality of baffle, a plurality of the baffle is all installed at the middle part of fixed frame, a plurality of the baffle equally divides into a plurality of culture tank with the space in the fixed frame, the bottom of cultivateing the box is equipped with the inserting groove, the lower extreme of fixed frame is pegged graft with the inserting groove and is cooperated.

Further, the interval mechanism includes the culture block, the culture block is installed in the bottom in the box, the lower extreme of culture block is equipped with two cultivation chambeies that are the symmetry and set up, every it all is provided with the cultivation drawer to cultivate the intracavity, cultivate the drawer with cultivate chamber sliding fit, every it all is equipped with the cultivation room on the drawer to cultivate, every the one end of cultivating the drawer all is equipped with the movable block.

Further, lighting mechanism includes fixed plate, light, cup joints frame and installing frame, the top at the box inboard is installed to the fixed plate, the light is equipped with a plurality of and all installs the lower extreme at the fixed plate, cup joint and set up the cover and establish the upper end at cultivateing the box, cup joint and be equipped with a plurality of mounting groove on the frame, a plurality of mounting groove and a plurality of cultivation groove one-to-one, equal joint has an installing frame on every mounting groove, all installs a light-passing board in every installing frame, all is equipped with the lug on every installing frame.

Further, lighting mechanism includes that fixed plate, light, a plurality of take out the groove and a plurality of take out the board, the top at the box inboard is installed to the fixed plate, the light is provided with a plurality of and all installs the lower extreme at the fixed plate, the upper portion of culture block is provided with a plurality of and takes out the groove, a plurality of take out the groove and fall into two sets of upper ends of cultivateing the chamber respectively, and the equal top-down in groove of every group is equidistant setting, a plurality of take out the board and install respectively a plurality of take out the inslot, every take out and all be equipped with two logical grooves, every on the board all be equipped with the light-passing board in leading to the inslot.

Furthermore, a heating plate and a heat insulation layer are arranged in the side wall of the box body, and a handle is arranged outside the box door.

Further, double-layer glass is arranged below the illuminating lamp, and the middle of the double-layer glass is a vacuum layer.

Furthermore, an oxygen concentration sensor, a CO2 concentration sensor, an illumination sensor, a humidity sensor and a temperature sensor are arranged on the environment monitoring device.

Further, the intake pipe with all be equipped with the control valve on the outlet duct, be equipped with the cabinet that disinfects in the cabinet, the inside top of the cabinet that disinfects is provided with a plurality of ultraviolet lamp.

Compared with the existing illumination incubator, the beneficial effects of the application lie in:

firstly, the upper end of the culture box is provided with the plurality of light-transmitting plates, the illumination intensity in the culture tank corresponding to the lower part of each light-transmitting plate is different by changing the transmittance of the light-transmitting plates, meanwhile, a plurality of groups of contrast tests can be carried out in one culture box, the technical problems that the regulation and control range of the illumination intensity in the culture box is small and a plurality of groups of contrast tests cannot be made in the prior art are solved, and the test cost can be reduced.

Secondly, the fixing frame can be divided into different numbers of culture tanks by using different numbers of partition plates, so that the fixing frame can be arranged according to test requirements, and the test operation is convenient.

Thirdly, the double-layer glass is arranged, the temperature of the upper end and the temperature of the lower end of the double-layer glass can be isolated through the vacuum arrangement between the double-layer glass, and the phenomenon that the heat generated by the illuminating lamp affects cells in the culture tank below the double-layer glass is avoided, so that the experimental result is inaccurate.

Fourthly, the cells can be cultured in the culture chambers in the plurality of culture drawers, and the cells cultured in the culture drawers can be taken out by drawing the culture drawers, so that the test operation is convenient, and the structure is simple.

Fifth, in the invention, the drawing plate is inserted in the drawing groove, and the intensity of light entering the culture chamber is changed by drawing the light-transmitting plates on the drawing plate, so that the illumination intensity in each culture chamber is changed, and a comparison test is convenient to carry out.

Drawings

FIG. 1 is a photograph of rat embryonic stem cells after 30% irradiation with visible light;

FIG. 2 is a photograph of rat embryonic stem cells after irradiation with 60% visible light;

FIG. 3 is a photograph of rat embryonic stem cells after irradiation with 100% visible light;

FIG. 4 is a schematic perspective view of an incubator according to the present application;

FIG. 5 is a partial top view of an incubator of the present application;

FIG. 6 isbase:Sub>A cross-sectional view taken along line A-A of FIG. 5;

FIG. 7 is a schematic view showing the use state of the moving mechanism of the incubator of the present application;

FIG. 8 is a partially exploded view of an incubator of the present application;

FIG. 9 is an enlarged view at B in FIG. 0;

FIG. 10 is a partially exploded perspective view of example 8 in an incubator of the present application.

Reference numerals:

the device comprises a box body 1, a moving mechanism 2, a moving plate 2a, pulleys 2b, sliding rails 2c, a groove 2d, a culture box 2e, ribs 2f, a pull block 2g, a spacing mechanism 3, a fixed frame 3a, partition plates 3b, a culture tank 3c, an insertion groove 3d, a culture block 3e, a culture cavity 3f, a culture drawer 3g, a moving block 3h, a lighting mechanism 4, a fixed plate 4a, a lighting lamp 4b, a sleeving frame 4c, a mounting frame 4d, a mounting groove 4e, a light-transmitting plate 4g, a protruding block 4h, a drawing groove 4j, a drawing plate 4k, an environment monitoring device 5, an air inlet pipe 6, an air outlet pipe 7, a camera 8, a box door 9, a storage cabinet 10, a display 11, a heating plate 12, a heat insulation layer 13, double-layer glass 14, a control valve 15, a handle 16, a sterilization cabinet 17 and an ultraviolet lamp 18.

Detailed Description

The preparation method and the culture method of the cell culture solution are basically consistent with the technical scheme disclosed in the Chinese patent CN103031272A, and the difference is that a light protection agent is also added into the prepared culture solution.

Example 1

Preparation of culture solution

Adding 10mM CHIR99021, 10mM PD0325901 and 10mM Pluriptin (SC 1) in turn into 100ml of N2B27 mixed medium so that the final concentration of CHIR99021 is 3. Mu. Mol/L; PD0325901 with a final concentration of 1. Mu. Mol/L; the final concentration of Pluriptin (SC 1) is 1. Mu. Mol/L; weighing fructus Lycii extract, radix et rhizoma Rhei extract and tea polyphenols mixture (all commercially available) at 10% of the total mass of the above culture medium, adding into culture solution to obtain the preferred culture solution, storing at 4 deg.C for 1 month, and preparing into multiple parts for use.

Wherein, the N2B27 mixed culture medium is prepared according to the following formula:

100ml of DMEN/F12-N12 culture medium;

nerve cell basal medium/B27 medium 100ml;

200. Mu.l of 0.1M beta-mercaptoethanol.

The DMEN/F12-N12 culture medium is prepared according to the following formula:

100ml of DMEN/F12 culture medium;

1ml of N2 additive.

The nerve cell basic culture medium/B27 culture medium is prepared according to the following formula:

100ml of nerve cell basic culture medium;

the nerve cell culture supplement factor B27 ml;

200mM L-glutamine 0.5ml.

Example 2

Primary culture of rat embryonic stem cells

Fibroblasts treated with MMC, which lose mitotic activity, were planted in 96-well plates to make feeder layers.

1-3 the feeder layer prepared as described above was used. The supernatant of the 96-well plate was aspirated off, and 150. Mu.l of the culture solution prepared in example 1 was added to each well to form 4 samples.

Sucking white rat blastocysts of day 4.5 with a mouth control tube, adding one blastocyst per well, and placing in an incubator disclosed in the application; the culture was carried out for 3 days without exposure to light, with 30% intensity of visible light, with 60% intensity of visible light, and with 100% intensity of visible light, respectively, at the same time, during which the culture medium was not replaced.

By day 4, the culture results were as follows:

subsequent experiments with D were abandoned and rat embryonic stem cells from a to C were labeled as first generation.

Example 3

Passage and analysis

Passage was performed within 1-3 days according to the size of the rat embryonic stem cell clone masses in A to C. After the rat embryonic stem cells are cultured in the culture solution prepared in the example 1, most of the rat embryonic stem cells can form good clone clusters, and the positive clone rate can reach 60-90%. And more than 98% of the cells are rat embryonic stem cells through detection, which shows that the illumination does not generate obvious side effect on the rat embryonic stem cells, and better rat embryonic stem cells can be cultured while the culture solution can effectively inhibit the differentiation of the rat stem cells.

The specific steps are disclosed in chinese patent CN103031272a, and are not described herein again.

Example 4

Reproductive potential test

The rat embryonic stem cells obtained in example 3 are used for detecting the reproductive potential, and specific steps are disclosed in Chinese patent CN103031272A, so that the expected effect is finally obtained, which indicates that the light irradiation does not have obvious influence on the reproductive potential

Example 5

Target test

The specific steps are disclosed in Chinese patent CN103031272A, and the target practice test also achieves the expected effect.

Example 6

Stem cell culture and analysis

The light irradiation has positive effect on the culture of the rat embryonic stem cells, but the light irradiation is limited, and the excessive light irradiation can be reflected, so the corresponding light irradiation intensity is selected according to the actual situation.

Example 7

Specific improvements and uses of incubators

Referring to fig. 4 to 10, the cell culture box with the controllable light source comprises a box body 1, a moving mechanism 2, a spacing mechanism 3 and a lighting mechanism 4, wherein the moving mechanism 2 is installed at the bottom of the inner side of the box body 1, the spacing mechanism 3 is installed at the upper end of the moving mechanism 2, the lighting mechanism 4 is installed at the upper end of the spacing mechanism 3, an environment monitoring device 5 is arranged inside the box body 1, an air inlet pipe 6 and an air outlet pipe 7 are respectively arranged at two sides of the box body 1, the air inlet pipe 6 and the air outlet pipe 7 are both communicated with the inside of the box body 1, a camera 8 is arranged on the inner side wall of the box body 1, the camera 8 is arranged towards the spacing mechanism 3, a box door 9 is arranged at the front end of the box body 1, a storage cabinet 10 is arranged at the lower end of the box body 1, and a display 11 is arranged at the side of the box body 1; separate into a plurality of space with 2 tops of moving mechanism through spacing mechanism 3, in order to carry out the multiunit contrast experiment simultaneously, lighting mechanism 4 through the top can provide illumination, and can make the illumination intensity inequality in a plurality of space through lighting mechanism 4, thereby carry out the contrast experiment under the different illumination intensity, can adjust the air composition in the box 1 through intake pipe 6 and outlet duct 7, can real time monitoring box 1 interior cell culture's the condition through camera 8, and can show the picture through display 11, thereby be convenient for the experimenter to observe, can real time monitoring box interior cell culture environment through environment monitoring device 5, in order to ensure that the environment of incubator is under the control, storage cabinet 10 is used for depositing standby equipment.

As shown in fig. 7 and 8, the moving mechanism 2 includes a moving plate 2a, pulleys 2b are disposed on two sides of the moving plate 2a, slide rails 2c are disposed on two sides of the interior of the box 1, the two slide rails 2c are respectively in sliding fit with the pulleys 2b on two sides of the moving plate 2a, two grooves 2d are disposed at the upper end of the moving plate 2a, a culture box 2e is disposed above the moving plate 2a, two ribs 2f are disposed at the lower end of the culture box 2e, the ribs 2f are in sliding fit with the grooves 2d, the length of the ribs 2f is half of the length of the grooves 2d, and a pull block 2g is disposed at the front end of the culture box 2 e; before an experiment, the culture box 2e is pulled by the pulling block 2g, the culture box 2e moves through the sliding fit of the convex edge 2f and the groove 2d, and because the length of the convex edge 2f is half of the length of the groove 2d, when the culture box 2e moves out for a half, the other half of the culture box 2e moves out through the sliding fit of the sliding rail 2c and the pulley 2b, the culture box 2e moves out of the box body 1 completely, and meanwhile, the lower end of the culture box 2e is supported by the half of the moving plate 2a, so that the culture box 2e can be kept stable when moving out of the box body 1.

As shown in fig. 7, 8 and 9, the partition mechanism 3 includes a fixed frame 3a and a plurality of partition plates 3b, the plurality of partition plates 3b are all installed in the middle of the fixed frame 3a, the plurality of partition plates 3b equally divide the space in the fixed frame 3a into a plurality of culture tanks 3c, the bottom of the culture box 2e is provided with an insertion groove 3d, and the lower end of the fixed frame 3a is inserted and matched with the insertion groove 3 d; in the invention, the partition plate 3b and the partition plate 3b as well as the partition plate 3b and the fixed frame 3a are elastically connected, so that the partition plate 3b and the partition plate 3b as well as the partition plate 3b and the fixed frame 3a can rotate, and when the fixed frame 3a and the partition plate 3b are not used, the partition plate can be folded to reduce the storage space.

As shown in fig. 6, 7 and 8, the illuminating mechanism 4 comprises a fixing plate 4a, an illuminating lamp 4b, a sleeving frame 4c and a mounting frame 4d, the fixing plate 4a is mounted at the top of the inner side of the box body 1, the illuminating lamp 4b is provided with a plurality of lamps which are all mounted at the lower end of the fixing plate 4a, the sleeving frame 4c is sleeved at the upper end of the culture box 2e, the sleeving frame 4c is provided with a plurality of mounting grooves 4e, the mounting grooves 4e are in one-to-one correspondence with the culture grooves 3c, a mounting frame 4d is clamped on each mounting groove 4e, a light-transmitting plate 4g is mounted in each mounting frame 4d, and each mounting frame 4d is provided with a convex block 4h; provide illumination through light 4b, light 4b uses the electricity-saving lamp in order to reduce energy consumption, in addition, the transmittance of light-passing board 4g on every mounting groove 4e is all inequality, control light 4b through light-passing board 4g and provide illumination and get into the intensity in culture tank 3c, thereby make the illumination intensity in every culture tank 3c all inequality, thereby be convenient for experiment simultaneously at a plurality of culture tanks 3c, be convenient for contrast difference between the cell culture under the different illumination intensity, the setting up of installing frame 4d makes can change different installing frame 4d, make the transmittance of light-passing board 4g different, can make the illumination intensity in the culture tank 3c be the illumination intensity of experimental needs fast.

As shown in fig. 6, a heating plate 12 and a heat insulating layer 13 are arranged in the side wall of the box body 1, wherein the heating plate 12 is used for heating the environment in the incubator so as to control the temperature environment of cell culture, the heat insulating layer 13 is used for isolating the environment in the incubator from the external environment so as to reduce heat conduction and reduce heat loss in the incubator so as to reduce energy consumption, and a handle 16 is arranged outside the box door 9; the handle 16 is provided to facilitate quick opening of the door 9.

Double-layer glass 14 is arranged below the illuminating lamp 4b, and a vacuum layer is arranged between the double-layer glass 14; the temperature at both ends about double glazing 14 can be completely cut off in the vacuum setting between double glazing 14, and the heat of avoiding light 4b to produce influences the cell in the culture tank 3c of below, leads to the experimental result inaccurate.

The environment monitoring device 5 is provided with an oxygen concentration sensor, a CO2 concentration sensor, an illumination sensor, a humidity sensor and a temperature sensor; oxygen concentration, CO2 concentration, light intensity, humidity and temperature conditions in the incubator are respectively monitored through an oxygen concentration sensor, a CO2 concentration sensor, a light sensor, a humidity sensor and a temperature sensor, so that the environment in the incubator is timely controlled.

All be equipped with control valve 15 on intake pipe 6 and the outlet duct 7, the setting of control valve 15 is used for controlling the switch of intake pipe 6 and outlet duct 7, be convenient for let in different cultivation gas in to the incubator, with the air in changing the incubator according to the experiment demand, be equipped with the cabinet 17 that disinfects in the cabinet 10, the inside top of cabinet 17 that disinfects is provided with a plurality of ultraviolet lamp 18, cabinet 10 is used for placing other experimental facilities, like the light-passing board 4g of different transmittances, experimental facilities can be placed in cabinet 17 that disinfects before the use, shine through ultraviolet lamp 18 and disinfect it, the bacterium that prevents on the experimental facilities can cause the influence to the cell culture.

The working principle of the embodiment is as follows: the cell culture experiment device has the advantages that the upper part of the moving mechanism 2 is divided into the plurality of culture tanks 3c through the spacing mechanism 3, so that a plurality of groups of comparison experiments can be carried out at the same time, the illumination can be provided through the illuminating lamps 4b above the culture tanks 3c, the illumination environment in the corresponding culture tanks 3c is changed through the light-transmitting plates 4g at the upper ends of the culture tanks 3c, so that the comparison experiments under different illumination intensities can be carried out, the air composition in the box body 1 can be adjusted through the air inlet pipe 6 and the air outlet pipe 7, the cell culture condition in the box body 1 can be monitored in real time through the camera 8, pictures can be displayed through the display 11, so that an experimenter can conveniently observe the cell culture environment in the culture can be monitored in real time through the environment monitoring device 5.

Example 8

Referring to fig. 10, the spacing mechanism 3 includes a culture block 3e, the culture block 3e is installed at the bottom in the box body 1, two culture chambers 3f are symmetrically arranged at the lower end of the culture block 3e, a culture drawer 3g is arranged in each culture chamber 3f, the culture drawers 3g are in sliding fit with the culture chambers 3f, a culture chamber is arranged on each culture drawer 3g, and a moving block 3h is arranged at one end of each culture drawer 3 g; cultivate the cell through the culture room in a plurality of cultivation drawer 3g, can cultivate drawer 3g through the extraction and take out the cell of wherein cultivateing, be convenient for carry out test operation, and simple structure.

Comparing the spacing mechanism 3 in embodiment 8 with the spacing mechanism 3 in embodiment 7, the spacing mechanism 3 in embodiment 7 can divide a plurality of culture tanks 3c by the mounting frame 4d and the plurality of partition plates 3b, and different numbers of partition plates 3b can be used according to experiment requirements, so that the culture tanks 3c with the number required by the experiment can be divided, the mounting mode is simple, and the mounting frame 4d and the partition plates 3b can be folded when being stored, thereby reducing the occupation of storage space. Embodiment 8 has simple structure's characteristics, and its using-way is simpler, only need when using will cultivate drawer 3g and take out through movable block 3h, culture the cell in the culture room can.

Illuminating mechanism 4 includes fixed plate 4a, light 4b, a plurality of is taken out groove 4j and a plurality of and is taken out board 4k, the top at box 1 inboard is installed to fixed plate 4a, light 4b is provided with a plurality of and all installs the lower extreme at fixed plate 4a, the upper portion of culture block 3e is provided with a plurality of and takes out groove 4j, a plurality of is taken out groove 4j and is divided into two sets of upper ends that set up at two cultivation chambeies 3f respectively, the equal top-down in every group takes out groove 4j is equidistant setting, a plurality of is taken out board 4k and is installed respectively in a plurality of takes out groove 4j, all be equipped with two logical grooves on every takes out board 4k, it all is equipped with light-passing board 4g to lead to the inslot every.

Compare embodiment 8 with lighting mechanism 4 in embodiment 7, lighting mechanism 4 in embodiment 7 is when using, through install the installing frame 4d that has different light transmittance light-passing board 4g on cup jointing frame 4c to change the illumination intensity in the below culture tank 3c that corresponds, its using-way and structure are all very simple, and can satisfy the demand that the multiunit experiment goes on simultaneously. Embodiment 8 can use the illumination intensity in a plurality of light-transmitting plates 4g stack change corresponding cultivateing rooms simultaneously, and a plurality of light-transmitting plates 4g use simultaneously can further change the transmittance, and its use method is simple, directly take out or insert it can.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A light source controllable cell culture box for rat embryonic stem cells is characterized in that: the intelligent cabinet temperature monitoring device comprises a cabinet body, a moving mechanism, a spacing mechanism and a lighting mechanism, wherein the moving mechanism is installed at the bottom of the inner side of the cabinet body, the spacing mechanism is installed at the upper end of the moving mechanism, the lighting mechanism is installed at the upper end of the spacing mechanism, an environment monitoring device is arranged inside the cabinet body, an air inlet pipe and an air outlet pipe are respectively arranged on two sides of the cabinet body, the air inlet pipe and the air outlet pipe are both communicated with the inside of the cabinet body, a camera is arranged on the inner side wall of the cabinet body and faces the spacing mechanism, a cabinet door is arranged at the front end of the cabinet body, a storage cabinet is arranged at the lower end of the cabinet body, and a display is arranged on the side of the cabinet body;

the spacing mechanism comprises a fixed frame and a plurality of partition plates, the partition plates are all arranged in the middle of the fixed frame, the plurality of partition plates equally divide the space in the fixed frame into a plurality of culture tanks, insertion grooves are formed in the bottoms of the culture boxes, and the lower end of the fixed frame is in insertion fit with the insertion grooves;

the illumination mechanism comprises a fixed plate, an illuminating lamp, a sleeving frame and installation frames, wherein the fixed plate is installed at the top of the inner side of the box body, the illuminating lamp is provided with a plurality of installation grooves which are all installed at the lower end of the fixed plate, the sleeving frame is sleeved at the upper end of the culture box, the sleeving frame is provided with a plurality of installation grooves, the installation grooves are in one-to-one correspondence with the culture grooves, each installation groove is clamped with one installation frame, a light-transmitting plate is installed in each installation frame, and each installation frame is provided with a convex block;

the moving mechanism comprises a moving plate, pulleys are arranged on two sides of the moving plate, slide rails are arranged on two sides of the interior of the box body, the two slide rails are respectively in sliding fit with the pulleys on the two sides of the moving plate, two grooves are formed in the upper end of the moving plate, a culture box is arranged above the moving plate, two convex edges are arranged at the lower end of the culture box and are in sliding fit with the grooves, the length of each convex edge is half of that of each groove, and a pull block is arranged at the front end of the culture box;

the spacing mechanism comprises a culture block, the culture block is arranged at the bottom in the box body, two culture cavities which are symmetrically arranged are arranged at the lower end of the culture block, a culture drawer is arranged in each culture cavity, the culture drawers are in sliding fit with the culture cavities, a culture chamber is arranged on each culture drawer, and a moving block is arranged at one end of each culture drawer;

the illumination mechanism comprises a fixed plate, an illuminating lamp, a plurality of drawing grooves and a plurality of drawing plates, the fixed plate is arranged at the top of the inner side of the box body, the illuminating lamp is provided with a plurality of drawing grooves which are all arranged at the lower end of the fixed plate, the upper part of the culture block is provided with the plurality of drawing grooves, the plurality of drawing grooves are divided into two groups which are respectively arranged at the upper ends of the two culture cavities, each group of drawing grooves are arranged at equal intervals from top to bottom, the plurality of drawing plates are respectively arranged in the plurality of drawing grooves, each drawing plate is provided with two through grooves, and a light-passing plate is arranged in each through groove;

double-layer glass is arranged below the illuminating lamp, and a vacuum layer is arranged in the middle of the double-layer glass;

the culture method for culturing the rat embryonic stem cells by the light source controllable cell culture box comprises the following steps:

s1, culturing rat embryonic stem cells in the presence of a culture solution;

s2, in the culture process of the step S1, illumination is carried out;

wherein the culture solution comprises a culture medium, a light protection agent and a culture additive;

the light protectant is a mixture of fructus Lycii extract, radix astragali extract, and tea polyphenols;

the culture supplement comprises a differentiation inhibitor and pluriptin, and the differentiation inhibitor comprises a GSK inhibitor, a FGFR inhibitor and a MAPK inhibitor, and the supplement further comprises: beta-mercaptoethanol; l-glutamine.

2. A light source-controllable cell culture chamber for rat embryonic stem cells as claimed in claim 1, wherein: the light source of the illumination is visible light.