Human pluripotent stem cell single cell cloning method and culture medium thereof

Abstract

The invention provides a human pluripotent stem cell single cell cloning method and a culture medium thereof, and belongs to stem cell culture. A single cell is inoculated into a 96-hole or 384-hole culture plate by utilizing a limited dilution technology, and meanwhile, the proliferation process of the single cell is tracked and recorded in real time by virtue of a living cell analysis system, so that the obtained clone is ensured to be derived from the single cell. According to the method provided by the invention, a large number of clone groups derived from single cells can be simply, conveniently, quickly and easily obtained, and a solid foundation is laid for future large-scale production and industrial technical platforms.

Description

Technical Field

[0001] The invention belongs to stem cell culture, and specifically relates to a human pluripotent stem cell single cell cloning method and a culture medium thereof. Background Art

[0002] Human pluripotent stem cells (PSCs), with their unlimited proliferation potential and unique ability to differentiate into various functional cells of the body, are regarded as "seed" cells with great research and application value. In many medical research fields such as cell therapy, drug screening, and disease model construction, human pluripotent stem cells have shown broad application prospects, heralding a profound change in the medical field. However, how to safely and stably expand and maintain single-cell-derived human pluripotent stem cells in vitro is a key scientific problem that needs to be solved urgently.

[0003] In an in vitro culture environment, human pluripotent stem cells usually grow in the form of monolayer, flat clones, and the cells in the clones are all derived from the same starting cell. By picking and amplifying these monoclones, cell lines of a single cell source can be obtained. The cells in these cell lines are highly homogeneous, providing valuable experimental materials for clinical treatment and basic scientific research. However, the current method of picking monoclones mainly relies on physical and mechanical methods, that is, using fine tools such as glass needles under a microscope to mechanically separate the clones, and inoculating the separated cell clusters onto feeder cells for subsequent amplification. This method is not only technically demanding and complex to operate, but also inefficient, the size of the clusters is difficult to unify, and the cell clusters are prone to floating, thereby increasing the risk of cross-"contamination". In addition, the use of feeder cells may also introduce additional safety hazards. Especially when a large number of single-cell clones (such as more than 50) need to be picked and amplified, such as when conducting gene editing or drug screening research, physical and mechanical methods are particularly time-consuming, labor-intensive and inefficient.

[0004] Although there are a variety of single-cell printing devices on the market that are designed to achieve single-cell cloning, these devices generally face the problems of high cost, complex operation and low efficiency. More importantly, these devices were originally designed mainly for suspended single cells, and their applicability is significantly limited for cell types such as human pluripotent stem cells that have clonal growth characteristics. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for single-cell cloning of human pluripotent stem cells, which utilizes limiting dilution and a living cell analysis system to efficiently obtain human pluripotent stem cell lines derived from a single cell, thereby meeting the needs of large-scale production.

[0006] The present invention provides an application of a caspase inhibitor Emricasan combined with Chroman-1 in the establishment of a single cell cloning system of human pluripotent stem cells.

[0007] The present invention provides a culture medium for single-cell cloning of human pluripotent stem cells, comprising a single-cell culture medium for human pluripotent stem cells and a culture medium for expanding human pluripotent stem cells;

[0008] The human pluripotent stem cell expansion medium is TeSR with a volume ratio of 1:19 TM -AOF 20XSupplement and TeSR TM - A mixture of AOF Basal Medium;

[0009] The human pluripotent stem cell single cell culture medium is based on the human pluripotent stem cell expansion culture medium and further comprises the following components: 10 μM Y-27632, 4-6 μM caspase inhibitor Emricasan and 0.04-0.06 μM Chroman-1.

[0010] The invention provides application of the culture medium in establishing a single cell cloning system of human pluripotent stem cells.

[0011] The present invention provides a method for single-cell cloning of human pluripotent stem cells, comprising the following steps:

[0012] The digested human pluripotent stem cell single cell is prepared into a single cell suspension using the human pluripotent stem cell single cell culture medium described in the above technical solution;

[0013] Inoculating the single cell suspension in a cell culture plate coated with recombinant human laminin 521, ensuring that the number of cells inoculated per well does not exceed 0.5 on average;

[0014] The cell culture plate inoculated with a single cell is supplemented with human pluripotent stem cell expansion culture medium after culturing for 1 day, and then the fresh human pluripotent stem cell expansion culture medium described in the above technical solution is replaced by half-medium replacement every 4 days. During the culture period, regular observation and / or photography are performed. When the clonal growth of human pluripotent stem cells is observed and / or photographed, and the process of a single cell dividing into two cells and two cells dividing into four cells is observed and / or photographed in the early stage of culture, a cloned human pluripotent stem cell single cell is obtained.

[0015] Preferably, the coating concentration ratio of the recombinant human laminin 521 to the calcium and magnesium-containing DPBS is 1:(10-50).

[0016] Preferably, the solvent for digesting the human pluripotent stem cell single cell includes Accutase digestion solution;

[0017] The digestion time of the Accutase digestion solution is 4 to 6 minutes.

[0018] Preferably, the method for ensuring that the average number of cells inoculated per well does not exceed 0.5 is that when inoculated in a 384-well cell culture plate, the inoculation volume of the single cell suspension is 20 μl / well, and the cell density of the single cell suspension is <25 cells / mL;

[0019] When inoculated in a 96-well cell culture plate, the inoculation volume of the single cell suspension is 40 μl / well, and the cell density of the single cell suspension is <25 cells / mL.

[0020] Preferably, the amount of supplementary liquid of the human pluripotent stem cell expansion culture medium is 1.5 to 2.5 times the volume of the culture medium in the culture well.

[0021] Preferably, the interval between two consecutive timed observations and / or photographs varies according to the culture period:

[0022] After inoculating a single human pluripotent stem cell, observe and / or take photos after 30 minutes of static culture;

[0023] When the culture period is 0 to 72 h, the interval between observation and / or photography is 0.5 to 2 h;

[0024] When the culture time is more than 72 hours and less than 120 hours, the interval between observation and / or photography is 4 to 10 hours;

[0025] When the culture period exceeds 120 h, the interval between observations and / or photographs is 10 to 12 h.

[0026] Preferably, the culture time is 10 to 14 days.

[0027] The present invention provides an application of a caspase inhibitor Emricasan combined with Chroman-1 in the cloning and establishment of human pluripotent stem cell single cell lines. In view of the problem of low cloning efficiency, the present invention combines Emricasan and Chroman-1 for the cloning culture of human pluripotent stem cell single cells. The addition of the two molecules not only reduces the bubbles generated during the inoculation process, but also ensures that the human pluripotent stem cell single cells maintain a round morphology, and significantly enhances the cell's adhesion ability to the coated matrix gel. This positive change can be seen in a short time after cell dissociation. Compared with the traditional method, the technical solution of the present invention significantly improves the adhesion efficiency and the number of clones of human pluripotent stem cell single cells, which is conducive to the large-scale preparation of human pluripotent stem cell single cells.

[0028] The present invention provides a method for cloning a single cell of a human pluripotent stem cell. On the basis of Emricasan and Chroman-1 being used in combination for cloning and culturing a single cell of a human pluripotent stem cell, the method also skillfully combines the limiting dilution technique of a single cell suspension with timed observation and / or photographing, thereby realizing the safe, stable and efficient expansion of human pluripotent stem cells of a monoclonal origin in vitro. In the early stage, by accurately configuring the concentration of the cell suspension, it is ensured that the number of cells inoculated in each culture unit (such as a specific area or culture well of a culture dish) does not exceed 0.5 cells on average, thereby greatly simplifying the selection process of clones. Subsequently, the cells are monitored in real time and / or images are collected, thereby accurately grasping the key dynamic processes such as cell growth, division and clone formation, and providing strong technical support for the screening and expansion of clones. The method provided by the present invention is not only simple to operate and has high work efficiency, but also has excellent scalability. More importantly, the single cell clones obtained by this method have high purity, which provides a solid foundation for the subsequent development of cell lines, the construction of disease models and the application in the field of regenerative medicine. Compared with existing single-cell cloning methods, the present invention has shown significant advantages in terms of cost, efficiency, purity and wide application, and has opened up a new path for the research and application of human pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the half-fluid exchange strategy adopted by the present invention;

[0030] Figure 2 A diagram of a human pluripotent stem cell dividing into two single cells;

[0031] Figure 3 Results of screening and generating stable single cell clones using a live cell analysis system;

[0032] Figure 4 This is a typical morphology of human pluripotent stem cells;

[0033] Figure 5 This is the result of immunofluorescence staining of human pluripotent stem cells;

[0034] Figure 6 The results of flow cytometry detection of human pluripotent stem cells;

[0035] Figure 7 The results are the statistical results of the clone numbers of single cells of human pluripotent stem cells in different treatment groups. DETAILED DESCRIPTION

[0036] The present invention provides an application of a caspase inhibitor Emricasan combined with Chroman-1 in the establishment of a single cell cloning system of human pluripotent stem cells.

[0037] In the present invention, the concentration of the caspase inhibitor Emricasan is preferably 4 to 6 μM, and can be 5 μM. The concentration of Chroman-1 is preferably 0.04 to 0.06 μM, and can be 0.05 μM. Adding Emricasan and Chroman-1 to the culture medium during the process of single-cell cloning and establishment of human pluripotent stem cells is beneficial to increasing the number of clones of human pluripotent stem cells, while enhancing the adhesion ability of cells to the coated matrix gel, thereby improving the adhesion efficiency of human pluripotent stem cells; in addition, the round morphology of human pluripotent stem cells can be maintained, and bubbles caused during the inoculation process can be reduced. The present invention has no special restrictions on the sources of Emricasan and Chroman-1, and they can be obtained by commercial channels known in the art. In the embodiment of the present invention, the Emricasan is purchased from MCE Company, and the Chroman-1 is purchased from MCE Company.

[0038] In one embodiment of the present invention, TeSR TM -AOF 20X Supplement and TeSR TM -AOF BasalMedium as the basal culture medium, a human pluripotent stem cell single cell culture medium formed by Emricasan, Chroman-1 and Y-27632, and a basal culture medium containing only Y-27632 for human pluripotent stem cell single cell cloning culture (control culture medium). The results showed that the culture medium containing Emricasan and Chroman-1 was significantly superior to the control culture medium in terms of single cell clone number, adhesion efficiency and cell morphology.

[0039] The present invention provides a culture medium for single-cell cloning of human pluripotent stem cells, comprising a single-cell culture medium for human pluripotent stem cells and a culture medium for expanding human pluripotent stem cells. The single-cell culture medium for human pluripotent stem cells is used for culture on day 0 to 1; the culture medium for expanding human pluripotent stem cells is used on day 2 to 14.

[0040] In the present invention, the human pluripotent stem cell expansion medium is TeSR with a volume ratio of 1:19. TM -AOF 20XSupplement and TeSR TM -AOF Basal Medium. The human pluripotent stem cell single cell culture medium is based on the human pluripotent stem cell expansion medium, and also includes the following components: 10 μM Y-27632, 4-6 μM caspase inhibitor Emricasan and 0.04-0.06 μM Chroman-1. The present invention has no special restrictions on the configuration method of the two culture media, and the configuration method of the cell culture medium known in the art can be used.

[0041] The invention provides application of the culture medium in establishing a single cell cloning system of human pluripotent stem cells.

[0042] In the present invention, the method for establishing a human pluripotent stem cell single cell cloning system comprises obtaining a large number of clone populations derived from single cells.

[0043] The present invention provides a method for single-cell cloning of human pluripotent stem cells, comprising the following steps:

[0044] The digested human pluripotent stem cell single cell is prepared into a single cell suspension using the human pluripotent stem cell single cell culture medium described in the above technical solution;

[0045] Inoculating the single cell suspension in a cell culture plate coated with recombinant human laminin 521, ensuring that the number of cells inoculated per well does not exceed 0.5 on average;

[0046] The cell culture plate inoculated with a single cell is supplemented with human pluripotent stem cell expansion culture medium after culturing for 1 day, and then the fresh human pluripotent stem cell expansion culture medium described in the above technical solution is replaced by half-medium replacement every 4 days. During the culture period, regular observation and / or photography are performed. When the clonal growth of human pluripotent stem cells is observed and / or photographed, and the process of a single cell dividing into two cells and two cells dividing into four cells is observed and / or photographed in the early stage of culture, a cloned human pluripotent stem cell single cell is obtained.

[0047] The present invention uses the human pluripotent stem cell single cell culture medium described in the above technical solution to prepare a single cell suspension of the digested human pluripotent stem cell single cell.

[0048] In the present invention, the solvent for digestion of the human pluripotent stem cell single cell includes Accutase digestion solution. The Accutase digestion solution is preferably warmed before digestion. The temperature of the warm bath is preferably 36-38°C, which can be 37°C, which is conducive to improving the enzyme activity of the Accutase digestion solution. The amount of the Accutase digestion solution added is preferably 0.5mL (12-well plate) or 1mL (6-well plate). The digestion time of the Accutase digestion solution is preferably 4-6min, which can be 5min. After digestion, it is preferably centrifuged by the digestion solution, collecting the precipitated cells, resuspending them with human pluripotent stem cell single cell culture fluid, filtering, and preparing a single cell suspension. The speed of the centrifugation is preferably 400g, which can be 300-600g. The centrifugation time is preferably 3-5min, which can be 3min. The filtration is preferably carried out using a cell sieve with a pore size of 40μm, and the filtrate collected is the culture fluid containing single cells.

[0049] In the present invention, the method for preparing the single cell suspension is preferably to perform gradient dilution using the limiting dilution method (10-fold dilution), and the cell density after dilution is calculated according to the well plate specifications and the volume of culture medium added. Taking a 384-well cell culture plate as an example, if the inoculation volume is 20 μL, the corresponding cell density after dilution is <25 cells / mL; if it is a 96-well cell culture plate, if the inoculation volume of each well is 40 μL, the corresponding cell density after dilution is <25 cells / mL.

[0050] After obtaining the single cell suspension, the present invention inoculates the single cell suspension into a cell culture plate coated with recombinant human laminin 521 to ensure that the number of cells inoculated per well does not exceed 0.5 on average.

[0051] In the present invention, the coating concentration ratio of the recombinant human laminin 521 to the calcium and magnesium-containing DPBS is preferably 1:19, and can be 1:10 to 45. The coating of the recombinant human laminin 521 is beneficial to supporting the growth of stem cells, promoting the attachment and survival of newly inoculated stem cells, improving the proliferation ability of stem cells, maintaining the potential for cell differentiation, and providing a simulated niche environment for human stem cells.

[0052] In the present invention, the method for ensuring that the average number of cells inoculated per well does not exceed 0.5 is preferably that when inoculated in a 384-well cell culture plate, the inoculation volume of the single cell suspension is 20 μl / well, and the cell density of the single cell suspension is <25 cells / mL; when inoculated in a 96-well cell culture plate, the inoculation volume of the single cell suspension is 40 μl / well, and the cell density of the single cell suspension is <25 cells / mL.

[0053] In order to efficiently and stably obtain clones derived from single cells and avoid the problems of low efficiency of manual picking and easy "cross contamination", the present invention proposes an efficient single-cell inoculation method based on limiting dilution. This method ensures that the number of cells inoculated in each well does not exceed 0.5 cells by accurately calculating the specifications of the well plate, the volume of culture medium added, and the cell density after dilution, which not only meets the experimental needs, but also meets the requirements of later clinical applications. In an embodiment of the present invention, a gradient dilution method is used to dilute the single-cell suspension of human pluripotent stem cells to accurately control the cell density within a range of less than 25 cells / ml. Subsequently, the prepared cell suspension is thoroughly mixed and inoculated into a 384-well plate using a spray gun, with 20 μL of single-cell suspension added to each well.

[0054] After the inoculation is completed, the present invention adds human pluripotent stem cell expansion culture medium to the cell culture plate inoculated with the single cell after culturing for 1 day, and then replaces the fresh human pluripotent stem cell expansion culture medium described in the above technical solution by half-replacement every 4 days. During the culture period, regular observation and / or photography are performed. When the clonal growth of human pluripotent stem cells is observed and / or photographed, and the process of single cell division into two cells and two cells division into four cells is observed and / or photographed in the early stage of culture, the cloned human pluripotent stem cell single cell is obtained.

[0055] In the present invention, the amount of supplemented human pluripotent stem cell expansion culture medium is preferably 1.5 to 2.5 times the volume of the original culture medium in the culture wells. For example, the volume of supplemented human pluripotent stem cell expansion culture medium for a 384-well plate is preferably 50 μL / well, and the volume of supplemented human pluripotent stem cell expansion culture medium for a 96-well plate is preferably 60 μL / well.

[0056] In the present invention, the half-replacement method replaces the human pluripotent stem cell expansion culture medium in the above technical solution with fresh culture medium, and only part of the culture medium is replaced each time. For example, taking the inoculation in a 96-well cell culture plate as an example, each culture well contains 100 μL in total, and 40 μL of culture medium in each culture well is removed on the 5th day of culture, and 40 μL of fresh human pluripotent stem cell expansion culture medium is added to continue culture. The culture conditions are preferably 36-38°C, which can be 37°C. The culture time is preferably 10d. The present invention is beneficial to replenish the nutrients required for cell growth by performing a rehydration operation after the first day of inoculation and culture, and the half-replacement method can effectively reduce the disturbance and loss of cells. Although the addition of Rock pathway inhibitor Y-27632 during the inoculation process plays a vital role in preventing apoptosis of human pluripotent stem cells and improving cell adhesion survival rate. However, it is worth noting that the continuous use of Rock pathway inhibitors for a long time may induce differentiation of human pluripotent stem cells. Therefore, after the second day of inoculation, it is necessary to promptly switch to a culture medium without Rock pathway inhibitors to avoid adverse effects on cells. By replenishing and half-replacing the fluid, the survival and proliferation of single human pluripotent stem cells are improved, the stability and activity of the cells are maintained to the maximum extent, and the accuracy and reliability of the results are ensured.

[0057] In the present invention, the interval between two consecutive timed observations and / or photographs is preferably different according to different culture periods: after inoculating a single human pluripotent stem cell, observe and / or photograph it after static culture for 30 minutes;

[0058] During the culturing period of 0 to 72 h, the interval between observation and / or photography was 2 h;

[0059] When the culture time exceeds 72 hours and is less than 120 hours, the interval between observation and / or photography is 8 hours;

[0060] When the culture lasts for more than 120 h, the interval between observations and / or photographs is 12 h.

[0061] In the present invention, in order to more conveniently monitor and / or collect cell clone images in real time, the culture is preferably performed in a living cell analysis system.

[0062] The method provided by the present invention is to inoculate a single cell into a 96-well or 384-well culture plate through a limiting dilution strategy. Subsequently, the proliferation process of a single cell from one to two, and then two to four, can be tracked and recorded in real time with the help of a living cell analysis system, thereby ensuring that the resulting clones are derived from a single cell. It can be seen that the present invention provides a simpler method than conventional methods, which can easily obtain a large number of clonal populations derived from single cells, which is crucial for constructing a stable human pluripotent stem cell single-cell cloning system. This method is not only efficient and accurate, but also lays a solid foundation for future large-scale production and industrialization technology platforms.

[0063] The following is a detailed description of a method for single-cell cloning of human pluripotent stem cells provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] A method for single-cell cloning of human pluripotent stem cells

[0066] 1. Before single-cell cloning of human pluripotent stem cells, two specific culture media need to be prepared. These two culture media are optimized for different stages of cell cloning to ensure healthy growth and effective expansion of cells. The first is the formula of human pluripotent stem cell single-cell culture media, see Table 1, used on days 0-1; the second is the formula of human pluripotent stem cell expansion culture media, see Table 2, used on days 2-14.

[0067] Table 1 Human pluripotent stem cell single cell culture medium

[0068]

[0069]

[0070] Table 2 Human pluripotent stem cell expansion culture medium

[0071] Solution name concentration 500mL added volume <![CDATA[TeSR TM -AOFBasalMedium]]> - 475mL <![CDATA[TeSR TM -AOF20XSupplement]]> 20× 25mL

[0072] 2. Prepare a 96-well plate coated with Lamninin 521 (recombinant human laminin 521) protein.

[0073] DPBS (Dulbecco's phosphate-buffered saline) containing calcium ions and magnesium ions was mixed with Lamninin 521, wherein the volume ratio of DPBS to Lamninin 521 (100 μg / mL) was 19:1; 90 μl was added to each well of a 96-well plate and incubated in a 37° C. incubator overnight.

[0074] 3. Digestion to Single Cells

[0075] Preheat the Accutase digestion solution at 37°C in advance, add 0.5 mL to each well of the 12-well plate, and place in a 37°C incubator for digestion for 5 minutes. Centrifuge the obtained human pluripotent stem cell single cell, resuspend it in human pluripotent stem cell single cell culture medium, filter it with a 40μm cell sieve, collect the filtrate, and obtain single cells.

[0076] 4. Cell Counting

[0077] The cell density after dilution is calculated according to the well plate specifications and the volume of culture medium added. The number of cells inoculated per well should not exceed 0.5. Taking a 384-well plate as an example, if the volume added to each well is 20 μL, the corresponding cell density after dilution is <25 cells / mL.

[0078] 5. Dilute the single cell suspension to the corresponding cell density by gradient dilution according to the method in Table 3.

[0079] Table 3 Cell density of human pluripotent stem cells after gradient dilution

[0080]

[0081] 6. Inoculate the diluted single-cell suspension into a 384-well plate, ensuring that the number of cells per well does not exceed 0.5 and the volume of each well is 20 μL.

[0082] 7. Place the culture plate inoculated with single cells in a living cell analysis system and culture at 37°C. Place the living cell analysis system in a 37°C incubator and take pictures after standing for 30 minutes.

[0083] 8. From 0 to 72 hours, set the photo taking interval to every 2 hours.

[0084] 9. After 24 hours of culture, the culture plate was removed, and 50 μL / well of human pluripotent stem cell expansion culture medium was added to the 384-well plate, and the plate was placed in the living cell analysis system for continued culture and photography. Figure 1 Later, the half-medium replacement culture method was carried out;

[0085] 10. From 72 to 120 hours, set the photo taking interval to every 8 hours.

[0086] 11. After 120 hours of cultivation, set the photo taking interval to once every 12 hours.

[0087] 12. After 10 days of continuous collection, observe the size of the cell clones and decide whether to continue collection or subculture.

[0088] 13. On the 10th day, if there is human pluripotent stem cell clone growth in the well, trace back to see whether the process of a single cell dividing into two and two into four was captured in the early stage of collection; if so, the human pluripotent stem cell single cell cloning in this well is successful, and the culture is continued by subculture according to the above culture method. The human pluripotent stem cell cell line obtained by subculture is observed, and immunofluorescence detection and loss cytometry are used for detection.

[0089] The experimental results are as follows Figure 2 As shown in the figure, at the single-cell stage after inoculation, the cells have successfully divided into two cells (the left picture shows the cells before the medium is changed, and the arrows show the divided cells). In addition, within 22 hours before the medium is changed, the morphology and movement of these cells remained normal without abnormal changes. However, after the medium is changed, the phenomenon of cell disappearance is observed ( Figure 2 The middle right picture shows cell loss caused by the fluid replacement operation. No original cells can be observed at the position indicated by the arrow). This may be due to human operation or environmental factors during the fluid replacement process.

[0090] The live cell analysis system can continuously track single cells after inoculation. By setting a custom time interval for taking photos, the system can accurately record and analyze the cell division process, capturing the key moment when a single cell divides into two and then into four, thereby ensuring that the obtained monoclonal clones are derived from a single cell origin. With this powerful tool, clones that can stably produce single-cell sources can be screened to provide high-quality cell resources for subsequent experiments and applications ( Figure 3 ).

[0091] The results after passage are shown in Figure 4 The results showed that they can be rapidly expanded and maintain the typical pluripotent stem cell morphology. Figure 5 ) and flow cytometry ( Figure 6 ), indicating that the single cell clone established by the present invention has pluripotency.

[0092] Comparative Example 1

[0093] The human pluripotent stem cell single cell cloning and establishment were carried out according to the method of Example 1, except that the human pluripotent stem cell single cell culture medium was TeSR TM -AOF Basal Medium and TeSR TM-AOF 20X Supplement (19:1) is a basal medium, comprising 0.1% by weight of Y-27632 (denoted as Y-27632).

[0094] The number of single cell clones of human pluripotent stem cells in Example 1 and Comparative Example 1 was counted by direct observation under an inverted microscope. Figure 7 .

[0095] Depend on Figure 7 It can be seen that the results of two batches of human pluripotent stem cell cloning show that the number of single cell clones obtained by culturing with only the culture medium of Y-27632 is significantly lower than the number of single cells obtained by culturing with the culture medium of human pluripotent stem cells. Y-27632 is an efficient Rock pathway inhibitor, which significantly reduces the cell apoptosis rate during the passage of human pluripotent stem cells, but the culture medium containing only Y-27632 in the cloning of human pluripotent stem cells can promote the cloning of human pluripotent stem cells to a certain extent. The present invention not only effectively reduces the bubbles generated during the inoculation process by introducing two small molecules, Emricasan and Chroman-1, but also ensures that the human pluripotent stem cell single cells maintain their round morphology. More importantly, they significantly enhance the adhesion ability of cells to the coated matrix glue, and this positive change can be seen in a short time after cell dissociation. Emricasan, Chroman-1 and Y-27632 can increase the number of single cell clones and promote single cell adhesion.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Emricasan, a caspase inhibitor, combined with Chroman-1 in the establishment of single-cell cloning of human pluripotent stem cells.

2. A culture medium for single-cell cloning of human pluripotent stem cells, characterized in that: It includes a human pluripotent stem cell single cell culture medium and a human pluripotent stem cell expansion culture medium; The human pluripotent stem cell expansion medium is TeSR with a volume ratio of 1:19 TM -AOF 20XSupplement and TeSR TM - A mixture of AOF Basal Medium; The human pluripotent stem cell single cell culture medium is based on the human pluripotent stem cell expansion culture medium and further comprises the following components: 10 μM Y-27632, 4-6 μM caspase inhibitor Emricasan and 0.04-0.06 μM Chroman-1.

3. Use of the culture medium according to claim 2 in establishing single-cell cloning of human pluripotent stem cells.

4. A method for single-cell cloning of human pluripotent stem cells, characterized in that: The following steps are involved: The digested human pluripotent stem cell single cell is prepared into a single cell suspension using the human pluripotent stem cell single cell culture medium according to claim 2; Inoculating the single cell suspension in a cell culture plate coated with recombinant human laminin 521, ensuring that the number of cells inoculated per well does not exceed 0.5 on average; After 1 day of culture, the cell culture plate inoculated with the single cell is supplemented with the human pluripotent stem cell expansion culture medium of claim 2, and then the fresh human pluripotent stem cell expansion culture medium is replaced by half-medium replacement every 4 days. During the culture period, regular observation and / or photography are performed. When the clonal growth of the human pluripotent stem cells is observed and / or photographed, and the process of a single cell dividing into two cells and two cells dividing into four cells is observed and / or photographed in the early stage of culture, a cloned human pluripotent stem cell single cell is obtained.

5. The method for single-cell cloning of human pluripotent stem cells according to claim 4, characterized in that: The coating concentration ratio of the recombinant human laminin 521 to the calcium and magnesium-containing DPBS is 1:(10-50).

6. The method for single-cell cloning of human pluripotent stem cells according to claim 4, characterized in that: The solvent for digesting the human pluripotent stem cell single cell includes Accutase digestion solution; The digestion time of the Accutase digestion solution is 4 to 6 minutes.

7. The method for single-cell cloning of human pluripotent stem cells according to claim 4, characterized in that: The method for ensuring that the average number of cells inoculated per well does not exceed 0.5 is that when inoculated in a 384-well cell culture plate, the inoculation volume of the single cell suspension is 20 μl / well, and the cell density of the single cell suspension is <25 cells / mL; When inoculated in a 96-well cell culture plate, the inoculation volume of the single cell suspension is 40 μl / well, and the cell density of the single cell suspension is <25 cells / mL.

8. The method for single-cell cloning of human pluripotent stem cells according to claim 4, characterized in that: The amount of supplementary liquid of the human pluripotent stem cell expansion culture medium is 1.5 to 2.5 times the volume of the culture medium in the culture well.

9. The method for single-cell cloning of human pluripotent stem cells according to claim 4, characterized in that: The interval between two consecutive regular observations and / or photographs varies according to the culture period: After inoculating a single human pluripotent stem cell, observe and / or take photos after 30 minutes of static culture; When the culture period is 0 to 72 h, the interval between observation and / or photography is 0.5 to 2 h; When the culture time is more than 72 hours and less than 120 hours, the interval between observation and / or photography is 4 to 10 hours; When the culture period exceeds 120 h, the interval between observations and / or photographs is 10 to 12 h.

10. The method for single-cell cloning of human pluripotent stem cells according to any one of claims 4 to 9, characterized in that: The culture time is 10 to 14 days.

Images