CN117070360A - Organoid culture method and production system - Google Patents
Organoid culture method and production system Download PDFInfo
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- CN117070360A CN117070360A CN202311042189.6A CN202311042189A CN117070360A CN 117070360 A CN117070360 A CN 117070360A CN 202311042189 A CN202311042189 A CN 202311042189A CN 117070360 A CN117070360 A CN 117070360A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
Abstract
The invention provides a method for culturing an organoid and a production system. Wherein, the above-mentioned organoid culture method includes the primary establishment and subculture that go on sequentially, wherein, the primary establishment includes: performing glue adding and plating, complete culture and primary establishment detection on a sample to be established by using a production system to obtain organoid primary cells; subculture includes: and (3) digesting, blowing, sucking, resuspension, glue adding and plating, complete culture and organoid establishment detection are carried out on the organoid primary cells by using a production system, so that organoids are obtained. Can solve the problem of large organoid difference prepared by manual culture in the prior art, and is suitable for the organoid culture field.
Description
Technical Field
The invention relates to the field of organoid culture, in particular to a method and a production system for organoid culture.
Background
The organoids can be used as a research tool and have great value in the fields of developmental biology, disease pathology, cytobiology, regeneration mechanism, accurate medical treatment, drug toxicity, drug efficacy test and the like. By means of organoids, it is possible to more efficiently and safely help humans overcome many biological and medical research problems. The primary organoid establishment refers to a test technological process from organoid sample through a series of experimental operations to organoid culture generation, and the process comprises the steps of carrying out technological operations such as sample pretreatment, matrix gel mixing, inoculation and plating, incubation and culture, observation and analysis on normal or cancerous cell tissues obtained from human organ tissues, and finally forming organoid culture with organ key characteristics. The organoid subculture refers to a test technological process from a primary established organoid sample to the generation of a new-generation organoid culture through a series of experimental operations, wherein the process comprises the steps of recovering, digesting, counting, shunting, freezing, inoculating, plating, incubating, culturing, observing and analyzing and the like the primary established organoid sample, and finally forming the new-generation organoid culture.
The primary establishment and subculture of the organoids are necessary processes for organoid preparation, the primary establishment and subculture of the organoids are complex in process, severe in environmental requirements, and have a large amount of repeated labor during manual operation, and the organoids prepared by manual operation are large in difference and low in flux. However, at present, the primary establishment and subculture of organoids still remain in the manual experimental stage, and the main pain point of preventing industrialization is that the batch experiment is difficult to be homogenized.
Disclosure of Invention
The invention mainly aims to provide a production system of a culture method of a organoid, which aims to solve the problem of large organoid difference prepared by manual culture in the prior art.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a method for culturing an organoid, the method comprising sequentially performing primary establishment and subculture, wherein the primary establishment comprises: performing glue adding and plating, complete culture and primary establishment detection on a sample to be established by using a production system to obtain organoid primary cells; subculture includes: and (3) digesting, blowing, sucking, resuspension, glue adding and plating, complete culture and organoid establishment detection are carried out on the organoid primary cells by using a production system, so that organoids are obtained.
Further, the rubberized plating comprises a first rubberized plating in primary establishment and a second rubberized plating in subculture; complete culture includes a first complete culture in primary establishment and a second complete culture in subculture;
the primary establishment includes: a1 Performing first glue adding and paving on the sample to be built, wherein the first glue adding and paving comprises the steps of adding matrigel into the sample to be built, blowing, sucking and mixing uniformly, sub-packaging the matrigel into a plurality of holes of a first pore plate, and solidifying the matrigel to obtain a first colloid; a2 Adding a complete culture medium into the first colloid to obtain a first complete culture system; performing first complete culture and primary establishment detection on a first complete culture system, and if the first complete culture system passes the primary establishment detection, completing primary establishment to obtain organoid primary cells;
subculture includes: b1 Adding digestive juice into the primary cells of the organoids which are established in the primary stage, digesting, stopping digestion, centrifuging, and sucking and removing the supernatant to obtain a digested sample; b2 Blowing and sucking the resuspension digested sample to obtain organoid cell suspension; combining organoid cell suspensions in a plurality of wells of a first well plate to obtain a combined sample; b3 Performing second glue adding and paving on the combined sample, wherein the second glue adding and paving comprises the steps of adding matrigel into the combined sample, blowing and sucking the matrigel into a plurality of holes of a second pore plate after uniform mixing, and solidifying the matrigel to obtain a second colloid; b4 Adding a complete culture medium into the second colloid to obtain a second complete culture system; performing second complete culture and organoid establishment detection on the second complete culture system, and if the second complete culture system passes organoid establishment detection, completing subculture to obtain organoids; if the second complete culture system does not pass the organoid establishment test, continuing the second complete culture and re-performing the organoid establishment test.
Further, a 2) includes: adding a complete culture medium into the first colloid to obtain a first complete culture system, performing primary establishment detection, and starting to perform first complete culture; when the first complete culture is carried out for 36-48 hours, carrying out primary establishment detection, judging whether bacteria are infected, and if the first complete culture system is not infected, continuing the first complete culture; when the first complete culture is carried out for 96-120 hours, carrying out primary establishment detection, judging whether organoids appear, if organoids appear in the first complete culture system, carrying out inclined liquid discarding on the first complete culture system, adding a complete culture medium again, and continuing the first complete culture; and when 144-168 hours are carried out in the first complete culture, carrying out primary establishment detection, judging whether passage is needed, if the passage is needed, obliquely discarding liquid to obtain the primary organoid cells, carrying out subsequent passage culture operation on the primary organoid cells, and if the passage is not needed, continuing culturing the primary organoid cells.
Further, the blowing and sucking resuspension includes: for each digestion sample, blowing and sucking 5 positions of the digestion sample respectively, wherein each position is blown and sucked for 4 times with the distance of 1.1-1.3mm from the hole bottom, and the total volume of liquid in each blowing and sucking hole is 40% -60%; preferably, the 5 positions include: a middle position A at the center of the hole, and a position B, a position C, a position D and a position E which are uniformly arranged on the circumference with the middle position A as the center and the radius of 0.85-1.15mm respectively.
Further, b 4) includes: adding a complete culture medium into the second colloid to obtain a second complete culture system, then performing organoid establishment detection, and starting to perform second complete culture; and (3) when the second complete culture is carried out for 48-72 hours, carrying out organoid establishment detection, judging whether subculture is completed, and if the second complete culture system passes the organoid establishment detection, carrying out inclined liquid discarding on the second complete culture system, and then supplementing and adding a complete culture medium to obtain organoid primary cells.
Further, the tilting waste liquid includes: tilting the system by 13-17 degrees, sucking liquid from the edge by adhering to the wall of the hole at a distance of 0.5mm from the bottom of the hole, and discarding; preferably, 450-550. Mu.L of liquid is aspirated from the rim.
Further, b 1) includes: adding digestive juice into primary organoid cells, performing digestion at 30-40 ℃, performing blowing and sucking operation by using a gun head every 2min during digestion, blowing and sucking operation for 2 times each time, wherein the total volume of liquid in each blowing and punching is 35-45%, and the distance between the gun head and the hole bottom is 1.2mm; adding a basic culture medium after digestion is finished, blowing and sucking uniformly, and stopping digestion; sealing the membrane, centrifuging, tearing the membrane, and absorbing and discarding the supernatant to obtain a digestion sample.
Further, the merging in b 2) includes: the first well plate was tilted 13-17 °, and 1000 μl of organoid cell suspension was aspirated from each well edge, 0.5mm from the well bottom, to obtain pooled samples.
In order to achieve the above object, according to a second aspect of the present invention, there is provided a production system of an organoid which performs the above-described organoid culture method, the production system comprising: the functional module is used for realizing sample preparation and comprises a storage culture module, a pipetting module, a culture module, a temperature control module, a centrifugation module, an identification module, a sealing film and tearing film module and an imaging module; the serial module is used for physically connecting the functional modules and comprises a guide rail and/or a mechanical arm; and a control module electrically connected to the functional module and the serial module, respectively; the storage culture module is used for temporarily storing, culturing or preserving samples, wherein the samples comprise samples to be established for the primary, organoid primary cells and organoids; the pipetting module is used for adding, sucking and oscillating liquid; the temperature control module is used for heating or cooling the sample or the matrigel; the centrifugal module is used for centrifuging the sample; the identification module is used for identifying the label and the identification information of the sample; the film sealing and tearing module is used for sealing and tearing the film of the container of the sample, so that the loss and pollution of the sample are prevented; the imaging module is used for detecting the sample; the serial module is used for transferring samples between the functional modules; the control module is used for controlling the sample to be transferred between the functional modules through the serial modules according to the identification information of the sample, and controlling the functional modules to process the sample, and finally obtaining the organoids.
Further, the production system further comprises a tilting module; the tilting module is used for tilting the sample and is used for realizing tilting and liquid suction together with the liquid-transferring module.
In order to achieve the above object, according to a third aspect of the present application, there is provided a computer-readable storage medium including a stored program, wherein the program executes the above-described incubation method.
In order to achieve the above object, according to a fourth aspect of the present application, there is provided an automated processing system for samples, comprising a memory having a computer program stored therein and a processor configured to execute the above incubation method by the computer program.
By applying the technical scheme of the application, the organoid culture method is utilized, the organoid culture is carried out by using the production system with the sample to be established as the starting point, the problem of large difference between different samples of manually cultured organoids is solved, and organoids with good uniformity can be obtained.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows a layout of an organoid production system according to an embodiment of the application
Fig. 2 shows a perspective view of an organoid production system according to an embodiment of the application.
Fig. 3 shows a side view of an organoid production system according to an embodiment of the application.
FIG. 4 shows a flow chart of a method of organoid culture according to example 1 of the application.
Fig. 5 shows schematic diagrams of the blowing and sucking resuspension positions according to embodiments 1 and 5 of the present application.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.
Term interpretation:
primary establishment: refers to the process of forming organoid primary cells having key features of an organ from a sample to be primary established (including normal or cancerous cells obtained from human organ tissue) through a series of experimental procedures.
Subculture: refers to the process from the primary establishment of finished organoid primary cells through a series of experimental procedures to the generation of a new generation of organoids.
Primary establishment detection: mainly for judging whether pollution exists in the sample. The primary set-up detection includes: 1) Judging whether to dye bacteria or not by judging whether the color of the culture medium turns yellow, turbid and the like; 2) Judging whether the background is clean or not and whether the background is polluted by mixed bacteria or not; 3) Judging the growth state of the cells according to the cell morphology, and judging whether the cells are apoptotic or not.
Organoid establishment detection: judging whether the organoid needs to be passaged or not according to the organoid duty ratio, and when the organoid duty ratio reaches a fixed threshold value, indicating that the organoid needs to be passaged, preventing the occurrence of phenomena such as contact inhibition and the like from affecting the proliferation and activity of the organoid.
Adding glue and paving: liquid matrigel is added into cells related to organoid culture, and the matrigel is solidified to obtain cell-containing colloid, wherein the matrigel is given to a three-dimensional supporting structure of the cells, so that aggregation, differentiation and functional expression of organoid cells can be promoted.
As mentioned in the background art, in the existing organoid preparation method, the method is completely manually operated, has low flux, high cost, poor consistency, unstable product quality and biological environment risk. However, organoids prepared using machines are less uniform and it is difficult to control product uniformity in batch preparations. Thus, the inventors have tried to develop a new organoid culture method in the present application, and have thus proposed a series of protection schemes of the present application.
In a first exemplary embodiment of the present application, there is provided a method for culturing an organoid comprising sequentially performing primary establishment and subculture, wherein the primary establishment comprises: performing glue adding and plating, complete culture and primary establishment detection on a sample to be established by using a production system to obtain organoid primary cells; subculture includes: and (3) digesting, blowing, sucking, resuspension, glue adding and plating, complete culture and organoid establishment detection are carried out on the organoid primary cells by using a production system, so that organoids are obtained.
In the culture method, the production system is used for replacing manpower, so that the operation flow of organoid culture can be realized, the flux is improved, the cost is reduced, the product quality is improved, the product consistency is improved, and the biological environment risk of manual operation is reduced. In the culture method, a sample to be established as a primary is used as a starting point for culture, first organoid primary cells are obtained, and after the organoid primary cells are detected to be qualified, the organoid is further cultured by using the organoid primary cells as the starting point, so that a target product organoid is obtained.
Compared with artificial culture of organoids, the culture method has the advantages of high quality, high throughput, low cost and the like.
High quality: the traditional manual operation experiment mode has the difference in operation methods and habits among different people at different times, and is easy to cause the deviation of experiment results. The automatic operation replaces manual operation, so that experimental result deviation caused by human factors can be avoided, and the quality is improved as a whole.
High flux: because the experimental process flow is responsible, the traditional manual operation experimental mode has low efficiency and long time consumption. Especially when large batches of samples need to be processed, a lot of manpower and time are required. The automatic operation replaces manual operation, the all-weather uninterrupted operation can be realized, unattended operation or one person on duty can be realized, the per-person yield is greatly improved, and the yield per unit time is greatly improved.
Low cost: reagent consumable waste caused by manual operation errors can be avoided by replacing manual operation by automation, the reagent utilization rate is improved by micro-operation, and the unit cost is reduced as the average yield and the unit time yield are improved.
The production system is an automated device having functions such as pipetting, cell culturing, and enzyme-labeled detection, and the culture method can be realized by using the device.
In a preferred embodiment, the rubberized plating comprises a first rubberized plating in a primary setup and a second rubberized plating in a subculture; complete culture includes a first complete culture in primary establishment and a second complete culture in subculture; the primary establishment includes: a1 Performing first glue adding and paving on the sample to be built, wherein the first glue adding and paving comprises the steps of adding matrigel into the sample to be built, blowing, sucking and mixing uniformly, sub-packaging the matrigel into a plurality of holes of a first pore plate, and solidifying the matrigel to obtain a first colloid; a2 Adding a complete culture medium into the first colloid to obtain a first complete culture system; performing first complete culture and primary establishment detection on a first complete culture system, and if the first complete culture system passes the primary establishment detection, completing primary establishment to obtain organoid primary cells; subculture includes: b1 Adding digestive juice into the primary cells of the organoids which are established in the primary stage, digesting, stopping digestion, centrifuging, and sucking and removing the supernatant to obtain a digested sample; b2 Blowing and sucking the resuspension digested sample to obtain organoid cell suspension; combining organoid cell suspensions in a plurality of wells of a first well plate to obtain a combined sample; b3 Performing second glue adding and paving on the combined sample, wherein the second glue adding and paving comprises the steps of adding matrigel into the combined sample, blowing and sucking the matrigel into a plurality of holes of a second pore plate after uniform mixing, and solidifying the matrigel to obtain a second colloid; b4 Adding a complete culture medium into the second colloid to obtain a second complete culture system; performing second complete culture and organoid establishment detection on the second complete culture system, and if the second complete culture system passes organoid establishment detection, completing subculture to obtain organoids; if the second complete culture system does not pass the organoid establishment test, continuing the second complete culture and re-performing the organoid establishment test.
The reagents used in the present application, including but not limited to matrigel, complete media, digestive juice, basal media, etc., are all commonly used in the prior art for the manual preparation of organoids.
The matrigel used in the application is Growth Factor Reduced (GFR) matrigel of corning company, and the product number is 356230; the digestive juice is TrypLE Express Enzyme (1X) of Thermofish company, no phenol red, and the product number is 12604013; the basic culture medium is DMEM/F12 of HyClone company, and the product number is SH30023.01B; the complete culture medium is added with different nutritional factors according to different cultured organoids, and the complete culture medium has mature formula and preparation method in the prior art. In the above-described culture method, the composition of the complete medium used in primary establishment and subculture may be the same for the same organoids, or may be flexibly adjusted, and the first complete culture and the second complete culture may be performed using different complete media, respectively.
The reagent used in the application is not limited to the reagent with the specific brand or model, and the reagent in the prior art can be flexibly selected in the actual culture method, so that the corresponding function can be realized. In a preferred embodiment, a 2) comprises: adding a complete culture medium into the first colloid to obtain a first complete culture system, performing primary establishment detection, and starting to perform first complete culture; when the first complete culture is carried out for 36-48 hours, carrying out primary establishment detection, judging whether bacteria are infected, and if the first complete culture system is not infected, continuing the first complete culture; when the first complete culture is carried out for 96-120 hours, carrying out primary establishment detection, judging whether organoids appear, if organoids appear in the first complete culture system, carrying out inclined liquid discarding on the first complete culture system, adding a complete culture medium again, and continuing the first complete culture; and when 144-168 hours are carried out in the first complete culture, carrying out primary establishment detection, judging whether passage is needed, if the passage is needed, obliquely discarding liquid to obtain the primary organoid cells, carrying out subsequent passage culture operation on the primary organoid cells, and if the passage is not needed, continuing culturing the primary organoid cells.
In a preferred embodiment, the blow-suction resuspension comprises: for each digestion sample, blowing and sucking 5 positions of the digestion sample respectively, wherein each position is blown and sucked for 4 times with the distance of 1.1-1.3mm from the hole bottom, and the total volume of liquid in each blowing and sucking hole is 40% -60%; preferably, the 5 positions include: a middle position A at the center of the hole, and a position B, a position C, a position D and a position E which are uniformly arranged on the circumference with the middle position A as the center and the radius of 0.85-1.15mm respectively. Preferably, the line connecting position B and position D is perpendicular to the line connecting position C and position E.
The organoid primary cells obtained after the primary establishment are in a state of layered distribution of colloid and culture medium, the proliferated cells are mainly distributed in the colloid, and after digestion is completed in the stage of subculture, the digested samples are required to be uniformly mixed, so that the subsequent separation of the cells is facilitated. Compared with manual operation, the gun head position, blowing and sucking times, blowing and sucking volume and other parameters can be flexibly adjusted when the gun is blown and sucked for heavy suspension, whether the mixing is uniform or not is judged visually, and when an automatic production system is used for the operation, the uniform dispersion of organoids in each hole is difficult to ensure. And the blind increase of the blowing and sucking strength (including but not limited to the blowing and sucking times and the blowing and sucking volume) can also influence the activity of cells, even lead to the breakage and death of the cells, and influence the successful proceeding of the subsequent organoid culture.
After digestion, the effect of the blow-suction resuspension is to completely dissociate the matrigel and disperse the organoids to obtain organoid cell suspensions. In the application, the inventor finds out a blowing-sucking resuspension method suitable for a production system through a large number of experiments, and the distances between other 4 positions compared with the middle position are controlled through respectively blowing-sucking 5 positions of the digestion sample, so that a better resuspension effect is achieved. The blowing-sucking resuspension performed by the method has low residual degree of colloid at the bottom of the hole, high uniformity degree of organoid cell suspension and no influence on the survival state and activity of cells.
The blowing-sucking resuspension parameters are tested by using 24 pore plates, if the selected pore plates are different, the height from the bottom of the pore is not required to be adjusted, the relative volume of blowing-sucking is also not required to be adjusted, but the actual volume of blowing-sucking is required to be adjusted, the blowing-sucking volume in the 24 pore plates is 400-600 mu L each time, for example, the blowing-sucking volume is reduced by half compared with the 24 pore plates by using 48 pore plates.
In a preferred embodiment, b 4) comprises: adding a complete culture medium into the second colloid to obtain a second complete culture system, then performing organoid establishment detection, and starting to perform second complete culture; and (3) when the second complete culture is carried out for 48-72 hours, carrying out organoid establishment detection, judging whether subculture is completed, and if the second complete culture system passes the organoid establishment detection, carrying out inclined liquid discarding on the second complete culture system, and then supplementing and adding a complete culture medium to obtain organoid primary cells.
In a preferred embodiment, the tilting waste comprises: tilting the system by 13-17 degrees, and discarding the system from the edge, wherein the attaching distance is 0.5mm from the hole bottom; preferably, 450-550. Mu.L of liquid is aspirated from the rim.
In the above culture method, since the culture system in the partial culture stage is in a state of layered distribution of colloid and culture medium, and the proliferated organoids are distributed in the colloid, when the supernatant is sucked and discarded for such a system, not only the thorough suction of the supernatant is ensured, but also the proliferated organoids are ensured not to be affected by pipetting. Unlike manual operations, which can flexibly adjust the position and the suction volume of the gun head, it is difficult to thoroughly suck the supernatant and prevent the gun head from adversely affecting the colloid, particularly the proliferation cells, when an automated production system is used for the operation.
In the present application, the inventors have found out a method of obtaining the above-mentioned oblique discard solution through a large number of experiments. The method can ensure the liquid discarding efficiency of the supernatant, ensure the normal form of the dome of the organoid, ensure the normal function of the organoid obtained by subsequent preparation, and prevent sample pollution caused by liquid overflow due to overlarge inclination angle. The angles at which the above systems are inclined include, but are not limited to, 13 °, 14 °, 15 °, 16 °, or 17 °.
In a preferred embodiment, b 1) comprises: adding digestive juice into primary organoid cells, performing digestion at 30-40deg.C, performing blowing and sucking operation with a gun head every 2min during digestion, blowing and sucking for 2 times, wherein 35-45% of total volume of liquid in each blowing and punching (350-450 μl for 24-well plate), and the distance between gun head and bottom of well is 1.2mm; adding a basic culture medium after digestion is finished, blowing and sucking uniformly, and stopping digestion; sealing the membrane, centrifuging, tearing the membrane, and absorbing and discarding the supernatant to obtain a digestion sample.
Digestion in the above culture methods can dissociate matrigel and dissociate large organoids into small organoids or cells for subsequent passage.
In a preferred embodiment, the combining in b 2) comprises: the first well plate was tilted 13-17 °, and 1000 μl of organoid cell suspension was aspirated from each well edge, 0.5mm from the well bottom, to obtain pooled samples.
The tilt angles used in the above combinations include, but are not limited to, 13 °, 14 °, 15 °, 16 °, or 17 °.
In a second exemplary embodiment of the present application, there is provided a production system of an organoid which performs the above-described organoid culture method, the production system comprising: the functional module is used for realizing sample preparation and comprises a pipetting module, a storage culture module, a temperature control module, a centrifugation module, an identification module, a sealing film and tearing film module and an imaging module; the serial module is used for physically connecting the functional modules and comprises a guide rail and/or a mechanical arm; and a control module electrically connected to the functional module and the serial module, respectively; the storage culture module is used for temporarily storing, culturing or preserving samples, wherein the samples comprise samples to be established for the primary, organoid primary cells and organoids; the pipetting module is used for adding, sucking and oscillating liquid; the temperature control module is used for heating or cooling the sample or the matrigel; the centrifugal module is used for centrifuging the sample; the identification module is used for identifying the label and the identification information of the sample; the film sealing and tearing module is used for sealing and tearing the film of the container of the sample, so that the loss and pollution of the sample are prevented; the imaging module is used for detecting the sample; the serial module is used for transferring samples between the functional modules; the control module is used for controlling the sample to be transferred between the functional modules through the serial modules according to the identification information of the sample, and controlling the functional modules to process the sample, and finally obtaining the organoids.
In the organoid production system, the functional module is responsible for preparing a sample, and comprises a pipetting module, a storage culture module, a temperature control module, a centrifugation module, an identification module, a sealing film and tearing film module and an imaging module. The functional modules are respectively and independently arranged, and the number of devices corresponding to the modules can be flexibly adjusted according to the requirements of the system. The pipetting module comprises, but is not limited to, an automated pipetting workstation, and can be used for adding, sucking, discarding, vibrating and the like of various liquids. The identification module includes, but is not limited to, a code scanner, which can identify label information on a sample consumable, such as a 24-well plate, a 96-well plate, or the like. The sealing film and tearing film module can realize sealing film treatment before entering the centrifugal module and other modules and tearing film treatment after exiting the centrifugal module and other modules for consumable materials containing samples. The storage culture module includes, but is not limited to, an incubator capable of effecting culture of sample cells, or a temporary storage rack, a middle index, etc. for transferring, temporarily storing samples in a production system, or a refrigerator for preserving samples. The temperature control module is used for heating or cooling the sample, the matrigel and the like. The imaging module is capable of taking a photograph or other detection of the sample, including but not limited to detection of absorbance, cell count, etc., using ultraviolet, visible light, etc. The imaging module includes, but is not limited to, a microplate reader having a photographing function.
The functional modules are connected by means of a serial module comprising a rail and/or a robot arm, said connection comprising a substantial connection by means of the rail and a connection by means of the robot arm enabling a sample transfer between the serial modules.
The control module is respectively and electrically connected with the functional module and the serial module, controls the serial module to transmit the sample according to the sample information identified by the identification module, controls the processing condition of the functional module to the sample, and updates the sample information so as to control the sample processing to be correctly performed in the next step of processing.
In a preferred embodiment, the production system further comprises a tilting module; the tilting module is used for tilting the sample and is used for realizing tilting and liquid suction together with the liquid-transferring module.
The organoid production system used in the present application is shown in figures 1, 2 and 3. In FIG. 1, (1) pipetting station, (2) film tearing machine, (3) centrifuge, (4) film sealing machine, (5) temporary storage rack, (6) indexing, (7) enzyme-labeled imager, (8) linear guide rail, (9) cooperation mechanical arm, carriage, and (d) incubator,Code reader, < >>Spin-dump board station, < >>Work ofA platform. The tilting module and the temperature control module are integrated in the pipetting workstation. Fig. 2 and 3 show a perspective view and a side view, respectively, of the organoid production system.
The pipetting workstation (1) is the pipetting module, (2) a film tearing machine, (4) a film sealing machine is the film sealing and tearing module, (3) a centrifuge is the centrifugal module, (5) a temporary storage rack, (6) a medium-shifting and medium-culturing box, a medium-culturing box and a medium-culturing box,The rotary unloading plate station is the storage temporary storage module, (7) the enzyme-labeled imager is the imaging module, (8) the linear guide rail, (9) the cooperation mechanical arm is the serial module, and the cartridge is provided with the serial module>The code reader is the identification module. The above (1) ->Are all carried in->And on the working platform, a complete production system is formed together.
In a third exemplary embodiment of the present application, a computer-readable storage medium is provided, the computer-readable storage medium including a stored program, wherein the program when run performs the above-described incubation method.
In a fourth exemplary embodiment of the application, an automated sample processing system is provided, comprising a memory having a computer program stored therein and a processor configured to perform the above-described incubation method by means of the computer program.
It should be noted that, for simplicity of description, the foregoing method embodiments are all described as a series of acts, but it should be understood by those skilled in the art that the present application is not limited by the order of acts described, as some steps may be performed in other orders or concurrently in accordance with the present application. Further, those skilled in the art will appreciate that the embodiments described in the specification are presently preferred embodiments, and that the acts are not necessarily required for the present application.
From the above description of the embodiments, it will be clear to those skilled in the art that the present application may be implemented by means of hardware devices such as software and detection devices. Based on such understanding, portions of the data processing in the present application may be embodied in the form of a software product, which may be stored in a storage medium, such as a ROM/RAM, magnetic disk, optical disk, etc., including instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to perform the methods of various embodiments or portions of embodiments of the application.
The application is operational with numerous general purpose or special purpose computing system environments or configurations. For example: personal computers, server computers, hand-held or portable devices, tablet devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.
It will be apparent to those skilled in the art that some of the modules or steps of the application described above may be implemented in a general purpose computing device, they may be concentrated on a single computing device, or distributed across a network of computing devices, or they may alternatively be implemented in program code executable by a computing device, so that they may be stored in a memory device for execution by the computing device, or they may be separately fabricated into individual integrated circuit modules, or multiple modules or steps of them may be fabricated into a single integrated circuit module. Thus, the present application is not limited to any specific combination of hardware and software.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
Example 1
The above-described organoid culture methods, as shown in FIG. 4, include, but are not limited to:
sealing film: samples (cell suspensions) to be primary established are stored on 24-well centrifugation plates, requiring a first sealing membrane.
Centrifuging and tearing the film: and (5) centrifuging and tearing the membrane by using a 24-hole centrifugal plate.
Discarding the supernatant: 3mL of the supernatant was aspirated and discarded.
Adding matrigel: placing the gel in a low-temperature module in advance, pre-cooling to 0 ℃, adding 160 mu L into the cell sediment, and blowing, sucking and mixing uniformly.
First glue adding and paving: each sample was dispensed into 6 wells, 25. Mu.L per well, to obtain a first gel.
Gel: placing the 24-hole culture plate on a temperature control module at 37 ℃, standing for 15min, and completely solidifying the matrigel to obtain a first colloid.
Adding a complete medium: 500. Mu.L of complete medium was added to each well to obtain a first complete culture system.
D0 (day 0 of first complete culture) photographs were taken: and (5) taking a first bright field picture, and recording the picture.
Culturing: placing the culture medium into an automatic incubator for culture to perform first complete culture.
D2 (day 2 of the first complete culture, 36-48 hours for the first complete culture) photographs were taken: and taking a photo in the open field on day 2, recording the photo, judging whether bacteria are infected, continuing the subsequent steps when the bacteria are not infected, and discarding the bacteria.
Culturing: placing the culture medium into an automatic incubator to continue the first complete culture.
D5 (day 5 of first complete culture, 96-120 hours for first complete culture) photographs were taken: and 5, taking a photo in the open field, recording the photo, judging whether organoids appear, if so, continuing the subsequent steps, and if not, discarding.
Oblique liquid discarding: put in a tilting module, after tilting, draw 500 μl of liquid from the rim and discard.
Adding a complete medium: 500. Mu.L of complete medium was added to each well.
Culturing: placing the culture medium into an automatic incubator to continue the first complete culture.
D8 (day 8 of first complete culture, 144-168 hours for first complete culture) photographs were taken: and (8) taking a photo in the open field on the 8 th day, recording the photo, and judging whether passage is needed. If necessary, the following procedure is continued for the obtained organoid primary cells. If not, the culture is continued.
Oblique liquid discarding: put in a tilting module, after tilting, draw 500 μl of liquid from the rim and discard.
Adding digestive juice: adding 0.45mL of digestive juice, blowing and sucking, and mixing.
Digestion: the 24-well centrifuge plate was placed on a heating module at 37℃and the time consumption was determined according to the type of sample.
Terminate digestion, suction resuspension and combining: adding 2.5mL of basic culture medium, blowing and sucking a heavy suspension digestion sample by using a 5mL gun head, blowing and sucking 5 positions at each hole for 4 times at a distance of 1mm from the middle position A, the middle position B, the middle position C, the middle position D and the middle position E respectively (a schematic diagram of 5 positions of blowing and sucking heavy suspension is shown in figure 5, wherein A represents the middle position, B represents the middle position B, C represents the middle position C, D represents the middle position D, E represents the middle position E and F represents the hole wall), and blowing and sucking for 4 times at a distance of 1.2mm from the hole bottom for each time, and blowing and sucking volume of 400 mu L each time to obtain organoid cell suspension. Organoid cell suspensions in multiple wells are pooled to obtain pooled samples.
Sealing, centrifuging and tearing: sealing the membrane by a 24-hole centrifugal plate, centrifuging and tearing the membrane.
Discarding the supernatant: aspirate 2.5mL supernatant and discard.
Adding matrigel: and adding 280 mu L of matrigel pre-cooled to 0 ℃ in a low-temperature module into the digested sample in advance, and blowing, sucking and uniformly mixing.
And (3) second glue adding and paving: each sample was dispensed into 12 wells, 25 μl per well.
Gel: placing the 24-hole culture plate on a temperature control module at 37 ℃, standing for 15min, and completely solidifying the matrigel to obtain a second colloid.
Adding a complete medium: 500. Mu.L of complete medium was added to each well to obtain a second complete culture system.
D0 (second full culture day 0) photograph: and (5) taking a first bright field picture, and recording the picture.
Culturing: and (5) placing the culture medium into an automatic incubator for second complete culture.
D3 (day 3 of the second complete culture, 48-72 hours of the second complete culture) photographs were taken: and (3) taking a photograph in the open field on the 3 rd day, recording the photograph, judging whether subculture is completed or not, and continuing the culture after the completion of the subculture.
Oblique liquid discarding: put in a tilting module, after tilting, draw 500 μl of liquid from the rim and discard.
Adding a complete medium: 500. Mu.L of complete medium was added to each well to obtain organoids.
In the above culture method, the culture vessel used includes, but is not limited to, various specifications of well plates, the addition and suction amounts of reagents in each step, and parameters such as culture time, digestion time, clotting time, etc. can be flexibly adjusted according to specific culture conditions, including, but not limited to, the values disclosed in the examples of the present application. The flow chart of the above-mentioned cultivation method is shown in FIG. 4.
Example 2
The operation steps of the inclined liquid discarding are as follows:
the recovery plate is placed in an inclined module and inclined by 15 degrees, 1000 mu L of gun head is used, the distance between the gun head and the bottom of the hole is 0.5mm, 550 mu L of culture solution is sucked, the volume of the culture solution is between 450 and 550 mu L, and the whole suction is ensured, and the gun head and the culture solution are discarded. The liquid discarding efficiency is 95%, the dome shape of the organoid is normal, and the gun head does not touch the side wall of the orifice plate.
Other procedure was as in example 1.
Example 3
Oblique liquid discarding: the difference compared to example 2 is the inclination angle of the recovery plate.
Organoid samples in different wells of the same batch were taken, each with an inclination angle of 0 °,5 °,10 °,20 °,25 ° for oblique fluid removal, and then the amount of remaining culture fluid was visually observed and the percentage of fluid removal to the total amount of stock culture fluid was estimated (as shown in table 1).
Table 1:
| sequence number | Inclination angle | Liquid discarding efficiency | Organoid dome morphology |
| 1 | 0° | 60％ | Normal state |
| 2 | 5° | 70％ | Normal state |
| 3 | 10° | 80％ | Normal state |
| 4 | 15° | 95％ | Normal state |
| 5 | 20° | 90％ | Dome displacement |
| 6 | 25° | 90％ | The dome is displaced and liquid overflows |
At an inclination of 15℃the drainage efficiency was highest and did not affect the morphology of organoid dome (dome) in the culture well.
Example 4
Oblique liquid discarding: the difference compared to example 2 is the height of the gun head from the bottom of the hole.
Organoid samples from different wells of the same batch were taken, pipetting was performed using gun heads at heights of 0.25mm,1.0mm,1.5mm,2.0mm,2.5mm, respectively, and then visually observing the amount of remaining broth and estimating the percentage of broth to the total amount of broth (as shown in Table 2), indicating that the drainage efficiency was highest and did not touch the well plate side wall when the gun heads were at a height of 0.5mm from the bottom of the well.
Table 2:
example 5
The steps of blowing, sucking and resuspension are as follows:
the 5mL gun head is used for blowing and sucking the heavy suspension digestion sample, 5 positions are respectively a middle position A, a position B, a position C, a position D and a position E, the offset distance between the position B, the position C, the position D and the position E and the middle position is 1mm, the blowing and sucking distance between each position and the bottom of the hole is 1.2mm for 4 times, the blowing and sucking volume is 400 mu L each time, and the gun head is discarded after the blowing and sucking are finished. The residual degree of matrigel is lower, and the uniformity degree of suspension is better. A schematic diagram of 5 positions of blowing and sucking resuspension is shown in fig. 5, wherein A represents an intermediate position, B represents a position B, C represents a position C, D represents a position D, E represents a position E, and F represents a hole wall.
Example 6
Blowing and sucking heavy suspension: the difference compared with example 5 is the number and volume of blowing and sucking, and then the residual degree of matrigel at the bottom of the recovery plate hole and the uniformity degree of suspension were judged visually. As shown in Table 3, the residual degree of matrigel was higher than that of example 5 (No. 4) according to the number of times of blowing and sucking and the volume of blowing and sucking of numbers 1, 2, 3 and 5, and the uniformity of suspension after blowing and sucking resuspension was lower than that of example 1.
Table 3:
| sequence number | Number of blowing and sucking times | Volume of blowing and sucking | Residual degree of matrigel | Uniformity of suspension |
| 1 | 1 time | 1000μL | ++++ | + |
| 2 | 2 times | 800μL | +++ | ++ |
| 3 | 3 times | 600μL | ++ | +++ |
| 4 | 4 times | 400μL | + | ++++ |
| 5 | 5 times | 200μL | ++ | ++++ |
Example 7
Blowing and sucking heavy suspension: the difference compared with example 5 is the position of blowing and sucking, and then the residual degree of matrigel at the bottom of the recovery plate hole and the uniformity degree of suspension are judged by naked eyes. As shown in Table 4, the residual degree of matrigel was higher than that of example 5 (No. 4) at the blowing and sucking positions of Nos. 1, 2 and 3, and the uniformity of suspension after blowing and sucking resuspension was lower than that of example 5.
Table 4:
example 8
Blowing and sucking heavy suspension: the difference compared with example 5 is the offset distance between the blowing and sucking position and the middle, and then the residual degree of matrigel at the bottom of the recovery plate hole and the uniformity degree of suspension are judged by naked eyes. As shown in Table 5, the residual degree of matrigel was higher than that of example 1 and the uniformity of suspension after blowing and sucking resuspension was lower than that of example 5 according to the offset distances of numbers 1, 2, 3 and 5.
Table 5:
| sequence number | Distance of deviation | Residual degree of matrigel | Uniformity of suspension |
| 1 | 0.25mm | ++++ | + |
| 2 | 0.5mm | +++ | ++ |
| 3 | 0.75mm | ++ | +++ |
| 4 | 1mm | + | ++++ |
| 5 | 1.25mm | ++ | +++ |
From the above description, it can be seen that the above embodiments of the present application achieve the following technical effects: the method and the system can realize the effect of automatically preparing the organoids, and have the advantages of high quality, high flux, low cost and the like compared with the artificial organoids. And through a large number of experiments and researches, the automatic operation steps for automatically preparing the organoids are optimized, and compared with the method for carrying out vertical and single suction by using an automatic pipetting system, the method for culturing the organoids in the application optimizes and researches, ensures that the automatic culturing method can realize better organoid culturing effect and ensures that a production system can produce organoids with better uniformity and meeting requirements.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A method for culturing an organoid, characterized in that the method for culturing an organoid comprises primary establishment and subculture performed sequentially, wherein,
the primary establishing includes: sequentially carrying out glue adding and plating, complete culture and primary establishment detection on a sample to be established by using a production system to obtain organoid primary cells;
the subculture comprises: and sequentially performing digestion, blowing and sucking resuspension, glue adding and plating, complete culture and organoid establishment detection on the organoid primary cells by using the production system to obtain the organoid.
2. The method of claim 1, wherein the plating comprises a first plating in the primary establishment and a second plating in the subculture; the complete culture comprises a first complete culture in the primary establishment and a second complete culture in the subculture;
The primary establishing includes:
a1 Performing the first glue adding and paving on the sample to be built, wherein the first glue adding and paving comprises the steps of adding matrigel into the sample to be built, blowing, sucking and mixing uniformly, sub-packaging the matrigel into a plurality of holes of a first pore plate, and solidifying the matrigel to obtain a first colloid;
a2 Adding a complete culture medium into the first colloid to obtain a first complete culture system; performing the first complete culture and the primary establishment detection on the first complete culture system, and if the first complete culture system passes the primary establishment detection, completing the primary establishment to obtain the organoid primary cells;
the subculture comprises:
b1 Adding digestive juice to the primary cells of the organoids which are completed by the primary establishment, digesting, stopping digestion, centrifuging, and absorbing and discarding the supernatant to obtain a digested sample;
b2 Blowing and sucking to resuspend the digested sample to obtain organoid cell suspension; combining the organoid cell suspensions in a plurality of wells of the first well plate to obtain a combined sample;
b3 Performing the second glue adding and paving on the combined sample, wherein the second glue adding and paving comprises the steps of adding the matrigel into the combined sample, blowing, sucking and mixing uniformly, sub-packaging into a plurality of holes of a second pore plate, and solidifying the matrigel to obtain a second colloid;
b4 Adding the complete culture medium into the second colloid to obtain a second complete culture system; performing the second complete culture and the organoid establishment detection on the second complete culture system, and if the second complete culture system passes the organoid establishment detection, completing the subculture to obtain the organoid; if the second complete culture system does not pass the organoid build test, continuing the second complete culture and re-conducting the organoid build test.
3. A method of organoid culture according to claim 2, wherein a 2) comprises:
after the complete culture medium is added into the first colloid to obtain the first complete culture system, the primary establishment detection is carried out, and the first complete culture is started;
when the first complete culture is carried out for 36-48 hours, carrying out primary establishment detection, judging whether bacteria are infected or not, and if the first complete culture system is not infected, continuing the first complete culture;
when the first complete culture is carried out for 96-120 hours, carrying out primary establishment detection, judging whether the organoids appear, if so, carrying out inclined liquid discarding on the first complete culture system, adding the complete culture medium again, and continuing to carry out the first complete culture;
And when 144-168 hours are carried out on the first complete culture, carrying out primary establishment detection, judging whether passage is needed, if the passage culture is needed, obliquely discarding liquid to obtain the organoid primary cells, carrying out subsequent operation of the passage culture on the organoid primary cells, and if the passage culture is not needed, continuing to culture the organoid primary cells.
4. The method of organoid culture according to claim 2, wherein said blow-suction resuspension comprises: for each digestion sample, blowing and sucking 5 positions of the digestion sample respectively, wherein each position is blown and sucked for 4 times by 1.1-1.3mm from the bottom of the hole, and the total volume of liquid in each blowing and sucking hole is 40% -60%;
preferably, the 5 positions include: a middle position A positioned in the center of the hole, and a position B, a position C, a position D and a position E which are uniformly arranged on the circumference with the middle position A as the center and the radius of 0.85-1.15mm respectively.
5. The method of organoid culture according to claim 2, wherein b 4) comprises:
after the complete culture medium is added into the second colloid to obtain a second complete culture system, performing organoid establishment detection, and starting to perform the second complete culture;
And when the second complete culture is carried out for 48-72 hours, carrying out organoid establishment detection, judging whether the subculture is finished, and if the second complete culture system passes through the organoid establishment detection, carrying out inclined liquid discarding on the second complete culture system, and then supplementing and adding the complete culture medium to obtain the organoid primary cells.
6. The method of claim 3 or 5, wherein the oblique reject comprises: tilting the system by 13-17 degrees, sucking liquid from the edge by adhering to the wall of the hole at a distance of 0.5mm from the bottom of the hole, and discarding;
preferably, 450-550. Mu.L of liquid is aspirated from the rim.
7. The method of organoid culture according to claim 2, wherein b 1) comprises:
adding the digestive juice into the primary organoid cells, performing digestion at 30-40 ℃, and performing blowing and sucking operation by using a gun head every 2min during the digestion, wherein the blowing and sucking operation is performed 2 times each time, 35-45% of the total volume of liquid in each blowing and punching is performed, and the distance between the gun head and the hole bottom is 1.2mm; adding a basic culture medium after digestion is finished, blowing and sucking uniformly, and stopping digestion;
sealing the membrane, centrifuging, tearing the membrane, and absorbing and discarding the supernatant to obtain the digestion sample.
8. The method of organoid culture according to claim 2, wherein said combining in b 2) comprises:
the first well plate was tilted 13-17 °, and 1000 μl of the organoid cell suspension was aspirated from each well edge, respectively, at a distance of 0.5mm from the well bottom, to obtain the pooled samples.
9. A production system of an organoid, characterized in that it performs the method of organoid culture according to any of claims 1 to 8, said production system comprising:
the functional module is used for realizing sample preparation and comprises a storage culture module, a pipetting module, a culture module, a temperature control module, a centrifugation module, an identification module, a sealing film and tearing film module and an imaging module;
a serial module for physically connecting the functional modules, the serial module comprising a rail and/or a robotic arm; and
the control module is electrically connected with the functional module and the serial module respectively;
the storage culture module is used for temporarily storing, culturing or preserving a sample, wherein the sample comprises the sample to be built for the primary generation, the organoid primary cells and the organoids;
the pipetting module is used for adding, sucking and oscillating liquid;
The temperature control module is used for heating or cooling the sample or the matrigel;
the centrifugation module is used for centrifuging the sample;
the identification module is used for identifying the label and the identification information of the sample;
the film sealing and tearing module is used for sealing and tearing the film of the container of the sample to prevent the sample from being lost and polluted;
the imaging module is used for detecting the sample;
the serial module is used for transferring the sample between the functional modules;
the control module is used for controlling the sample to be transferred between the functional modules through the serial modules according to the identification information of the sample, controlling the functional modules to process the sample, and finally obtaining the organoids.
10. The production system of claim 9, further comprising a tilt module;
the tilting module is used for tilting the sample and is used for realizing tilting and liquid suction through the combined action of the tilting module and the liquid suction module.
11. A computer-readable storage medium, characterized in that the computer-readable storage medium comprises a stored program, wherein the program when run performs the organoid culture method according to any of claims 1 to 8.
12. An automated processing system for samples, comprising a memory and a processor, wherein the memory has stored therein a computer program, the processor being arranged to execute the organoid culture method according to any of claims 1 to 8 by means of the computer program.
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