CN117070444A - Method for establishing human pluripotent stem cell monoclonal selected and transferred strain - Google Patents
Method for establishing human pluripotent stem cell monoclonal selected and transferred strain Download PDFInfo
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Abstract
The application relates to a method for establishing a plant by monoclonal picking and transferring of human pluripotent stem cells, which comprises the steps of observing human pluripotent stem cell clones meeting preset conditions by a visual method, and photographing and marking to obtain clones to be picked; after clone digestion is carried out by ReLeSR digestive juice, carrying out picking transfer by utilizing prefabricated human pluripotent stem cell subculture liquid, picking a plurality of monoclonal antibodies, and respectively inoculating the monoclonal antibodies into a culture container which is prepared in advance and is coated with recombinant human Laminin521 (Laminin 521); and (3) continuously culturing the monoclonal by using fresh human pluripotent stem cell subculture liquid to obtain a cell strain of a monoclonal source. The method and the device effectively solve the problems of the physical and mechanical picking monoclonal technology, and are simpler to operate and higher in efficiency. The method not only can digest and obtain uniform cell 'clusters', but also can avoid cross 'pollution' between the selected and transmitted clones caused by floating of the cell clusters without adding culture solution to resuspend the cells after digestion is completed.
Description
Technical Field
The application relates to the field of stem cells and regenerative medicine, in particular to a method for establishing a monoclonal transmission strain of a human pluripotent stem cell.
Background
The pluripotent stem cells have unlimited proliferation capability, can differentiate to generate all types of functional cells of an organism, are very ideal seed cells, and have wide application prospects in the research fields of cell treatment, drug screening, disease models and the like, and the application of the pluripotent stem cells is expected to bring a new medical revolution.
In early research stages, the culture of human pluripotent stem cells is very challenging, requires passaging by physical and mechanical operations, and has complex operation steps and low working efficiency. Although human pluripotent stem cells can be digested into single cells using trypsin or collagenase, it leads to a decrease in cell viability, to the appearance of karyotype abnormalities, or to spontaneous differentiation. Subsequent studies have found that the use of neutral protease (dispase) or cell digestive juice (accutase), especially in combination with ROCK inhibitors, can significantly enhance the survival of human pluripotent stem cells. In addition, several non-enzymatic digests are widely used, including EDTA-based modified cell dissociation solutions and ReLeSR products. It is emphasized that non-enzymatic digests are generally more akin to physical mechanical manipulation passaging, i.e., the dissociation of cells into clumps of only a few cells for passaging. Whereas enzymatic digestion methods, such as cell digests (Accutase) or trypsin (TrypLE), can digest human pluripotent stem cells into single cells for passaging. Although these methods make in vitro culture of human pluripotent stem cells more stable and simple, the need for monoclonal selection of established strains of human pluripotent stem cells has not been met.
When the human pluripotent stem cells are cultured in vitro, the cells in the clone grow in a single-layer, flat-like clone, and are derived from common initial cells. Through picking and amplifying the monoclonal, a cell strain with single clone source can be obtained, and cells in the cell strain have good homogeneity, thereby being more beneficial to clinical application and basic scientific research. The current method of picking up a monoclonal is mainly a physical mechanical method, namely, mechanically dividing the clone under a microscope by using a glass needle, and then inoculating the divided cell mass into feeder cells for subsequent expansion culture. The method has higher technical requirements on operators, has complex operation steps and low working efficiency, is difficult to homogenize the size of the agglomerate, and is easy to float up cell agglomerates so as to cause the risk of cross pollution of the picking and transferring clone. In addition, the need for feeder cells presents a safety risk. It has been shown that when more than 50 single cell clones need to be picked up, for example when the cells are genetically modified, the method of physical mechanical picking is very labor intensive and inefficient.
Therefore, the method for constructing the monoclonal transmission strain of the human pluripotent stem cells efficiently, rapidly and simply solves the problem of cross pollution of the transmission clone caused by floating of cell masses, and promotes the development of the human pluripotent stem cells in clinical application and basic scientific research, which is a problem to be solved urgently at present.
Disclosure of Invention
In view of the above, the present application provides a method for constructing a monoclonal strain of a pluripotent stem cell of a human, which solves the above-mentioned problems.
According to one aspect of the present application, there is provided a method for monoclonal selection and propagation of human pluripotent stem cells, comprising the steps of:
observing the clone of the human pluripotent stem cells meeting preset conditions by a visual method, and photographing and marking to obtain the clone to be picked;
after clone digestion is carried out on the to-be-picked clone by ReLeSR digestive juice, carrying out picking transfer by utilizing prefabricated human pluripotent stem cell subculture liquid, sequentially picking a plurality of monoclonal antibodies, and respectively inoculating the monoclonal antibodies into a culture container which is prepared in advance and is coated with the Laimin 521 protein;
and continuously culturing the monoclonal by using fresh human pluripotent stem cell subculture liquid to obtain a cell strain of a monoclonal source.
In one possible implementation manner, after the obtaining the clone of the human pluripotent stem cell meeting the preset condition, photographing and marking, obtaining the clone to be picked, further includes:
and washing the clone to be picked by using a washing liquid to wash away cell fragments and dead cells.
In one possible implementation, the formulation of the human pluripotent stem cell subculture solution is as follows:
in one possible implementation, when preparing a culture container coated with the Laminin521 protein in advance, it includes:
obtaining a Laminin521 protein and thawing;
diluting the thawed Laimin 521 protein according to a preset proportion by using a Du's phosphate buffer (D-becco' phosphate-buffer);
and placing the diluted Laminin521 protein into a culture container, and coating under a preset coating condition.
In one possible implementation, the digestion of the clone to be picked by the ReLeSR digest includes:
after digestion of the clones to be picked for 2 minutes at room temperature, the ReLeSR digest was aspirated and digestion was continued for 3 minutes in an incubator at 37 ℃.
In one possible implementation, the picking using the pre-prepared human pluripotent stem cell subculture solution comprises:
collecting 20 mu L of the human pluripotent stem cell subculture liquid, transferring to 500 mu L of the human pluripotent stem cell subculture liquid, and blowing and dispersing into small blocks by using a 200 mu L pipetting gun.
In one possible implementation, the number of monoclonal antibodies includes 10-15.
In one possible implementation manner, before the continuous culture of the monoclonal by using the new human pluripotent stem cell subculture solution to obtain a cell strain of a monoclonal source, the method further comprises:
the culture vessel inoculated with the monoclonal was placed at 37℃and saturated humidity, and the culture was continued in a 5% carbon dioxide incubator.
The application has the beneficial effects that:
the application dissociates human pluripotent stem cells by using a ReleSR digestive juice, namely a non-enzymatic digestive juice, absorbs dissociated single clones one by one under a microscope, and inoculates the single clones into a culture container coated by a Laminin521 protein to prepare a cell strain from a monoclonal source. The method specifically comprises the following steps: observing the clone of the human pluripotent stem cells meeting preset conditions by a visual method, and obtaining the clone to be picked after photographing and marking; after clone digestion is carried out by ReLeSR digestive juice, carrying out picking transfer by utilizing prefabricated human pluripotent stem cell subculture liquid, picking a plurality of monoclonal antibodies, and inoculating the monoclonal antibodies into a culture container which is prepared in advance and is coated with the Laminin521 protein; and (3) continuously culturing the monoclonal by using fresh human pluripotent stem cell subculture liquid to obtain a cell strain of a monoclonal source. The problems of the physical and mechanical picking monoclonal technology are effectively solved, the operation is simpler, and the efficiency is higher. The method not only can digest and obtain uniform cell 'clusters', but also can avoid cross 'pollution' between the selected and transmitted clones caused by floating of the cell clusters without adding culture solution to resuspend the cells after digestion is completed. Furthermore, the application does not need the support of feeder cells, does not have animal origin, is more beneficial to future clinical application, can obtain a large number of cell strains with monoclonal origin by a simple method, and can be used for a large-scale and industrialized technical platform in the future.
Other features and aspects of the present application will become apparent from the following detailed description of exemplary embodiments, which proceeds with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments, features and aspects of the application and together with the description, serve to explain the principles of the application.
FIG. 1 is a schematic flow chart of a method for monoclonal cross-strain establishment of human pluripotent stem cells according to an embodiment of the application;
FIG. 2 is a diagram showing a representative clone of human pluripotent stem cells according to the method for monoclonal selection and propagation of human pluripotent stem cells according to the embodiment of the application;
FIG. 3 shows a flowchart of digestion and picking of clones of human pluripotent stem cells in a method for monoclonal picking and establishing a strain of human pluripotent stem cells according to an embodiment of the application;
FIG. 4 shows human pluripotent stem cell clones prior to digestion in a monoclonal pick-and-strain method of human pluripotent stem cells according to an embodiment of the application;
FIG. 5 shows clones of human pluripotent stem cells after digestion in the monoclonal selected and transferred strain method of human pluripotent stem cells according to the embodiment of the application;
FIG. 6 is a graph showing the cloning trace left in situ after cloning and picking in the method for constructing a monoclonal picking plant of a human pluripotent stem cell according to the embodiment of the application;
FIG. 7 is a diagram showing a typical monoclonal morphology of human pluripotent stem cells in the method for monoclonal cross-strain construction of human pluripotent stem cells according to the embodiment of the application.
Detailed Description
Various exemplary embodiments, features and aspects of the application will be described in detail below with reference to the drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. Although various aspects of the embodiments are illustrated in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated.
It should be understood, however, that the terms "center," "longitudinal," "transverse," "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counter-clockwise," "axial," "radial," "circumferential," and the like indicate or are based on the orientation or positional relationship shown in the drawings, and are merely for convenience of describing the application or simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore should not be construed as limiting the application.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the application. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In some instances, well known methods, procedures, components, and circuits have not been described in detail so as not to obscure the present application.
As shown in FIG. 1, the method for monoclonal cross-linking and establishment of the human pluripotent stem cells comprises the following steps:
s100, visually observing the clone of the human pluripotent stem cells meeting preset conditions, and photographing and marking to obtain the clone to be picked;
s200, after clone digestion is performed on the to-be-picked clones through ReLeSR digestive juice, picking and transferring are performed by utilizing prefabricated human pluripotent stem cell subculture liquid, a plurality of monoclonal antibodies are picked, and the monoclonal antibodies are respectively inoculated into a culture container which is prepared in advance and is coated with the Laimin 521 protein;
s300, continuously culturing the monoclonal by using fresh human pluripotent stem cell subculture liquid to obtain a cell strain of a monoclonal source.
In the embodiment, a high-efficiency, rapid and feeder-independent method for monoclonal propagation and establishment of human pluripotent stem cells is provided, the human pluripotent stem cells are dissociated by using a ReleSR digestive fluid, namely a non-enzymatic digestive fluid, the dissociated individual clones are sucked one by one under a microscope, and the individual clones are inoculated into a culture container coated with a Lamin 521 protein, so that a cell strain of a monoclonal source is prepared. The problems of the physical and mechanical picking monoclonal technology are effectively solved, the operation is simpler, and the efficiency is higher. The method not only can digest and obtain uniform cell 'clusters', but also can avoid cross 'pollution' between the selected and transmitted clones caused by floating of the cell clusters without adding culture solution to resuspend the cells after digestion is completed. Furthermore, the application does not need the support of feeder cells, does not have animal origin, is more beneficial to future clinical application, can obtain a large number of cell strains with monoclonal origin by a simple method, and can be used for a large-scale and industrialized technical platform in the future.
Specifically, the step S100 is performed by observing the clone of the human pluripotent stem cells meeting preset conditions by a visual method, and photographing and marking are performed to obtain the clone to be picked. Here, under a microscope, a clone with large area, clear edge and compactness is selected, and the cell in the clone is full in shape and high in nuclear mass ratio, and has no obvious differentiation; clones which are far away from other clones are selected as far as possible, so that the picking operation is convenient. Wherein prior to selecting the human pluripotent stem cell clone, the microscope/stereoscopic mirror is placed in a biosafety cabinet in advance, and irradiated with ultraviolet light for at least 30 minutes. Further, after selecting clones, the clones of the human pluripotent stem cells to be picked up are circled at the bottom of the culture container with a Mark pen, and the clones to be picked up are photographed and numbered.
And then, after the clone to be picked is digested by ReLeSR digestive juice, picking and transferring by using prefabricated human pluripotent stem cell subculture liquid, picking a plurality of monoclonal antibodies, and respectively inoculating the monoclonal antibodies into a culture container which is prepared in advance and is coated with the Laimin 521 protein.
In one possible implementation manner, after the obtaining the clone of the human pluripotent stem cell meeting the preset condition, photographing and marking, obtaining the clone to be picked, further includes: and washing the clone to be picked by using a washing liquid to wash away cell fragments and dead cells. Specifically, a proper volume of washing liquid (for example, 1 mL is added to each hole of a 6-hole plate, and other culture containers are added with pluripotent stem cell subculture liquid) is added into the culture plate, and the culture plate is gently shaken to wash away cell fragments and dead cells.
In one possible implementation, the formulation of the human pluripotent stem cell subculture solution is as follows:
in this example, when preparing a human pluripotent stem cell subculture solution, a commercially available human pluripotent stem cell culture solution such as mTESR plus culture solution of Stem Cell Technology company or the like was used, Y27632 was added at a final concentration of 10. Mu.M, and the culture solution was required to be returned to room temperature (18-26 ℃) before the experiment.
In one possible implementation, when preparing a culture container coated with the Laminin521 protein in advance, it includes: obtaining a Laminin521 protein and thawing; diluting the thawed Laimin 521 protein according to a preset proportion by using a Du's phosphate buffer (D-becco' phosphate-buffer); and placing the diluted Laminin521 protein into a culture container, and coating under a preset coating condition. That is, when preparing a culture container coated with Laminin521, after thawing Laminin521 protein, the Laminin521 protein is diluted with DPBS of calcium and magnesium according to a ratio of 1:40, and a proper volume of diluted Laminin521 is sucked into the culture container, and incubated in a culture box at 37 ℃ for 2 hours or coated overnight at 4 ℃.
In one possible implementation, the digestion of the clone to be picked by the ReLeSR digest includes: after digestion of the clones to be picked for 2 minutes at room temperature, the ReLeSR digest was aspirated and digestion was continued for 3 minutes in an incubator at 37 ℃. The digestion time needs to be strictly controlled to prevent the cells from being excessively digested.
In one possible implementation, the picking using the pre-prepared human pluripotent stem cell subculture solution comprises: collecting 20 mu L of the human pluripotent stem cell subculture liquid, transferring to 500 mu L of the human pluripotent stem cell subculture liquid, and blowing and dispersing into small blocks by using a 200 mu L pipetting gun. Wherein, when 20. Mu.L was used for collection, a part of the liquid was retained, ensuring that the clone could be aspirated. If the culture containers to be picked up are more, the culture containers can be digested and picked up in sequence, so that the solution is prevented from being dried off, and the survival of cells is prevented from being influenced.
In one possible implementation, the number of monoclonal antibodies includes 10-15. In this embodiment, preferably 10-15 single clones are picked up, and if the total number of clones before picking up is less than 10, all single clones are picked up and are in one-to-one correspondence according to the number. When a plurality of monoclonal antibodies were inoculated into a culture vessel coated with a Laminin521 protein prepared in advance, the liquid in the Laminin521 protein coating plate was sucked, the picked monoclonal antibodies were inoculated into corresponding wells, and the culture vessel was uniformly mixed, and information such as cell name, lot number, and generation number was marked on the lid of the culture vessel.
Further, in one possible implementation manner, the method further includes, before the step of continuously culturing the monoclonal with the new human pluripotent stem cell subculture solution to obtain a cell strain of monoclonal origin: the culture vessel inoculated with the monoclonal was placed at 37℃and saturated humidity, and the culture was continued in a 5% carbon dioxide incubator. And then continuously culturing the monoclonal by using fresh human pluripotent stem cell subculture liquid through a step S300 to obtain a cell strain of a monoclonal source. Specifically, the next day fresh human pluripotent stem cell expansion culture medium is replaced, the cloning growth condition is observed and recorded, and the cloning cell state and growth density are recorded.
The following will specifically describe the process of observing and labeling, digesting and picking up clones of human pluripotent stem cells and culturing established cell lines by picking up clones.
1. Human pluripotent stem cell stem monoclonal observation and labeling
Under a microscope, selecting clones with large area, compactness and clear edges; the cells within the clones were full in morphology, high in nuclear mass ratio, and not significantly differentiated (as shown in fig. 2 as typical human pluripotent stem cell clones, indicated by white arrows). Clones which are far away from other clones are selected as far as possible, so that the picking operation is convenient, and cross contamination between different clones is prevented. And (3) circling the clone of the human pluripotent stem cells to be picked up at the bottom of the culture container by using a Mark pen, and photographing and numbering each clone to be picked up.
2. Human pluripotent stem cell stem monoclonal digestion and picking transfer
After the clone labeling is completed, the clone digestion and the picking of the human pluripotent stem cells are sequentially completed (as shown in fig. 3 and 4). In the biosafety cabinet, the culture solution is sucked, and a proper amount of DPBS is added to clean the cells. Adding a proper amount of digestion solution ReLeSR, firstly digesting for 2 minutes at room temperature, and sucking the digestion solution; digestion was then continued for 3 minutes at 37℃in an incubator. It was observed that the cells within the clone were detached, but not completely dispersed, and still maintained the clone morphology (as shown in fig. 5). mu.L of human pluripotent stem cell subculture solution was aspirated using a pipette, observed under a microscope, and individual clones were blow-aspirated and transferred into a culture vessel previously coated with the Laminin521 protein. After completion of the picking, the free clone trace was observed in situ (as shown in FIG. 6). And (5) according to the requirements, picking and transmitting corresponding numbers of monoclone and marking. The following day, cells were observed for cell attachment and replaced with fresh human pluripotent stem cell expansion medium.
3. Culture of built-up cell strain by picking and transferring clone
The next day after the transfer is completed, the fresh human pluripotent stem cell expansion culture solution (such as mTESR plus and the like) is replaced, and the culture is continued for 4 days. After the clone grows up, digestion is carried out by using ReLeSR under the condition of room temperature digestion for 2 minutes, and digestive juice is sucked; digestion was then continued for 3 minutes at 37℃in an incubator. After digestion was completed, an appropriate amount of human pluripotent stem cell culture medium containing 10 μ M Y27632 was added to the culture vessel, dissociated human pluripotent stem cells were gently blown from the bottom of the culture vessel using a pipette, and then the cell suspension was inoculated into the Laminin521 coated culture vessel at an appropriate density. The next day, the culture medium is replaced by fresh human pluripotent stem cell culture medium, and the culture is continued for 3 days, so that cloning of a typical human pluripotent stem cell form (shown in fig. 7) can be observed, namely, the monoclonal strain establishment is successful.
In conclusion, compared with the traditional physical and mechanical picking monoclonal technology, the method is simpler to operate and high in efficiency, and uniform cell 'clusters' can be obtained through digestion. After digestion is completed, the cells do not need to be resuspended by adding culture solution, so that cross pollution between the selected and transmitted clones caused by floating of cell masses is avoided; in addition, the technology does not need the support of feeder cells, does not have animal origin, and is more beneficial to future clinical application.
The foregoing description of embodiments of the application has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
Claims (8)
1. The method for constructing the human pluripotent stem cell monoclonal selected and transferred strain is characterized by comprising the following steps of:
observing the clone of the human pluripotent stem cells meeting preset conditions by a visual method, and photographing and marking to obtain the clone to be picked;
after clone digestion is carried out on the to-be-picked clone by ReLeSR digestive juice, carrying out picking transfer by utilizing prefabricated human pluripotent stem cell subculture liquid in sequence, picking a plurality of monoclonal antibodies, and respectively inoculating the monoclonal antibodies into a culture container which is prepared in advance and is coated with the Laimin 521 protein;
and continuously culturing the monoclonal by using fresh human pluripotent stem cell subculture liquid to obtain a cell strain of a monoclonal source.
2. The method for constructing a plant by monoclonal picking up and transferring of human pluripotent stem cells according to claim 1, wherein the steps of obtaining the clone of the human pluripotent stem cells meeting the preset conditions, photographing and marking the clone to be picked up, and then:
and washing the clone to be picked by using a washing liquid to wash away cell fragments and dead cells.
3. The method for monoclonal cross-breeding and establishment of human pluripotent stem cells according to claim 1, wherein the formula of the human pluripotent stem cell subculture solution is as follows:
。
4. the method for constructing a monoclonal stem cell line of human pluripotent stem cells according to claim 1, wherein when preparing a culture vessel coated with Laminin521 protein in advance, comprising:
obtaining a Laminin521 protein and thawing;
diluting the thawed Laimin 521 protein according to a preset proportion by using D-becco' spline-bufferedsaline containing calcium and magnesium;
and placing the diluted Laminin521 protein into a culture container, and coating under a preset coating condition.
5. The method for monoclonal cross-strain establishment of human pluripotent stem cells according to claim 1, wherein the digestion of the clone to be selected by slesr digest comprises:
after digestion of the clones to be picked for 2 minutes at room temperature, the ReLeSR digest was aspirated and digestion was continued for 3 minutes in an incubator at 37 ℃.
6. The method for constructing a plant by monoclonal picking and transferring of the human pluripotent stem cells according to claim 1, wherein the picking and transferring by using the prefabricated human pluripotent stem cell subculture solution comprises the following steps:
collecting 20 mu L of the human pluripotent stem cell subculture liquid, transferring to 500 mu L of the human pluripotent stem cell subculture liquid, and blowing and dispersing into small blocks by using a 200 mu L pipetting gun.
7. The method for monoclonal cross-propagation of human pluripotent stem cells according to claim 1, wherein the number of the monoclonal cells comprises 10 to 15.
8. The method for constructing a monoclonal transmission line of a human pluripotent stem cell according to claim 1, wherein the method further comprises, before the step of continuously culturing the monoclonal cell by using fresh human pluripotent stem cell subculture solution to obtain a cell line of monoclonal origin:
the culture vessel inoculated with the monoclonal was placed at 37℃and saturated humidity, and the culture was continued in a 5% carbon dioxide incubator.
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