CN114480274A - Method for directionally differentiating Pan cells in intestinal organoid - Google Patents
Method for directionally differentiating Pan cells in intestinal organoid Download PDFInfo
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- CN114480274A CN114480274A CN202111654672.0A CN202111654672A CN114480274A CN 114480274 A CN114480274 A CN 114480274A CN 202111654672 A CN202111654672 A CN 202111654672A CN 114480274 A CN114480274 A CN 114480274A
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/23—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the gastro-intestinal tract
Abstract
The invention provides a method for directionally differentiating Pan cells in intestinal organoids, which comprises the following steps: obtaining an intestinal organoid; placing the intestinal organoid in an organoid culture medium; adding DAPT and CHIR99021 to the organoid culture and stimulating the intestinal organoids in the organoid culture medium for a predetermined time. According to the method for directionally differentiating the Pan cells in the intestinal organoids, disclosed by the invention, a large amount of high-purity Pan cells can be obtained in a short time by adding DAPT and CHIR99021 into the culture medium.
Description
Technical Field
The invention relates to the field of animal organoid culture, in particular to a method for directionally differentiating Pan cells in intestinal organoids.
Background
Pan cells are unique epithelial cells located at the bottom of crypts of small intestine and near colon, and although the number of Pan cells is small, the Pan cells secrete defensins, lysozyme and the like to resist pathogenic microorganisms and play an important role in regulating host immune function. In addition, other important functions of Pan cells are proved by research, for example, Pan cells provide necessary habitat signals for Lgr5 positive stem cells, and are closely related to the occurrence and development of intestinal tumors. There are many areas of Pan cell function that have yet to be explored, and there is a need to find a way to obtain large quantities of Pan cells in vitro for relevant studies.
Organoid technology has become one of the most popular leading technologies in the biological and medical fields in recent years. The intestinal organoid is a three-dimensional intestinal cell culture system, can utilize intestinal stem cells of human or animals to proliferate and differentiate into an organ-specific cell set in a laboratory, and can differentiate by a cell sequencing and space-limiting system in a manner similar to that of the human or animal in vivo, so that self-renewal and self-construction are completed. The method is mainly applied to the establishment of disease models, the screening and detection of medicines and the research in the aspect of regenerative medicine. However, organoid culture techniques also have limitations, and the differentiation of various types of intestinal cells into organoids is complicated due to the uncontrollable differentiation of intestinal organoids cultured in vitro, which is a technical problem of obtaining a large amount of Pan cells in a targeted manner.
Disclosure of Invention
To achieve the above and other related objects, the present invention provides a method for directional differentiation of Pangolian cells in intestinal organoids, comprising the steps of: obtaining an intestinal organoid; placing the intestinal organoid in an organoid culture medium; adding DAPT and CHIR99021 to the organoid culture and stimulating the intestinal organoids in the organoid culture medium for a predetermined time.
Optionally, the preset time is 5-10 days.
Optionally, the acquiring the intestinal analog device comprises the following steps:
taking small intestine, washing the intestinal canal with cold PBS for several times, and removing intestinal contents;
cutting the intestinal canal longitudinally, and cutting into intestinal sections with preset lengths;
transferring the section of intestine into a test tube filled with PBS;
shaking the test tube for cleaning, separating out a first supernatant, and removing the first supernatant;
repeating the previous step for a plurality of times until the first supernatant is clear;
discarding the first supernatant obtained last;
adding crypt separation liquid into the test tube, and shaking gently to separate out a second supernatant;
removing the second supernatant;
adding PBS into the test tube, shaking the test tube to separate out a third supernatant, observing whether an intestinal tract unit exists or not, and collecting the third supernatant;
repeating the steps until a preset volume of the third supernatant is collected;
centrifuging the third supernatant to obtain intestinal cells;
resuspending the intestinal tract cells by using a washing solution, and centrifuging the resuspended intestinal tract cells to obtain the intestinal tract organoid;
and repeating the previous step.
Optionally, the number of times the intestinal tube is flushed with the cold PBS is 3-4 times; the predetermined length is 0.2 cm-0.6 cm; adding the crypt separation solution into the test tube, and then shaking gently at 3-6 ℃ for 15-35 min; the preset volume is 40 ml-60 ml.
Optionally, in the process of centrifuging the third supernatant, the rotation speed of a centrifuge is 300 × g, the time of the centrifugation is 3min to 7min, and the temperature of the centrifugation is 2 ℃ to 6 ℃; in the process of carrying out centrifugal treatment on the resuspended intestinal cells, the rotating speed of a centrifugal machine is 300 Xg, the centrifugal treatment time is 3 min-7 min, and the centrifugal treatment temperature is 2-6 ℃.
Optionally, the wash solution comprises DMEM, BSA, and a double antibody. .
Optionally, said placing said intestinal organoid in organoid culture medium comprises:
resuspending the intestinal organoid with an organoid culture medium and matrigel according to a ratio to obtain an organoid suspension;
suspending the organoid suspension and dripping the organoid suspension into the culture hole.
As described above, the method for directionally differentiating Pan cells in intestinal organoids of the present invention has the following beneficial effects: according to the method for directionally differentiating the Pan cells in the intestinal organoids, disclosed by the invention, a large amount of high-purity Pan cells can be obtained in a short time by adding DAPT and CHIR99021 into the culture medium.
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FIG. 1 is a flow chart of the method of the present invention for the directional differentiation of Pangolian cells in intestinal organoids.
FIG. 2 is a comparison graph of immunofluorescence staining of intestinal organoids cultured by the method for directional differentiation of Pangolian cells in intestinal organoids of the present invention with intestinal organoids cultured in a control group without DAPT and CHIR 99021; wherein, the picture (a) in figure 2 is the immunofluorescence staining pattern of the intestinal organoid obtained by the culture of the control group without adding DAPT and CHIR99021, and the picture (b) in figure 2 is the immunofluorescence staining pattern of the intestinal organoid obtained by the culture of the method for directionally differentiating Pangolian cells in the intestinal organoid of the invention.
FIG. 3 is a comparison of Lyz1 gene of intestinal organoid obtained by culturing the method of differentiating Pangolian cells directionally in intestinal organoid of the present invention and Lyz1 gene of intestinal organoid obtained by culturing the control group without DAPT and CHIR 99021.
FIG. 4 is a comparison graph of gene expression of Lyz1 gene of intestinal organoid obtained by culturing the method for directional differentiation of Pangolin cells from intestinal organoid of the present invention and Lyz1 gene of intestinal organoid obtained by culturing the control group without DAPT and CHIR 99021.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art. The basic principles of the invention, as defined in the following description, may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the invention.
The method is mainly applied to the establishment of disease models, the screening and detection of medicines and the research in the aspect of regenerative medicine. However, organoid culture techniques also have limitations, and the differentiation of various types of intestinal cells into organoids is complicated due to the uncontrollable differentiation of intestinal organoids cultured in vitro, which is a technical problem of obtaining a large amount of Pan cells in a targeted manner.
Referring to fig. 1, the present invention provides a method for directionally differentiating Pangolian cells in intestinal organoids, which comprises the following steps:
s1: obtaining an intestinal organoid;
s2: placing the intestinal organoid in an organoid culture medium;
s3: adding DAPT (gamma-secretase inhibitor) and CHIR99021(GSK3 inhibitor) to the organoid culture, and stimulating the intestinal organoids in the organoid culture medium for a predetermined time.
According to the method for directionally differentiating the Pan cells in the intestinal organoids, disclosed by the invention, a large amount of high-purity Pan cells can be obtained in a short time by adding DAPT and CHIR99021 into the culture medium.
As an example, the preset time is 5-10 days.
Specifically, the preset time may be 6 days, 7 days, 8 days, 9 days, or the like, and preferably, in this embodiment, the preset time is 7 days.
As an example, the acquiring the intestinal analog comprises the following steps:
s101: taking small intestine, washing the intestinal canal with cold PBS (phosphate buffered saline) for several times, and removing intestinal contents;
s102: cutting the intestinal canal longitudinally, and cutting into intestinal sections with preset lengths;
s103: moving the section of intestine into a test tube filled with PBS;
s104: shaking the test tube for cleaning, separating out a first supernatant, and removing the first supernatant;
s105: repeating the previous step for a plurality of times until the first supernatant is clear;
s106: discarding the first supernatant obtained last;
s107: adding crypt separation liquid into the test tube, and shaking gently to separate out a second supernatant;
s108: removing the second supernatant;
s109: adding PBS into the test tube, shaking the test tube to separate out a third supernatant, observing whether an intestinal tract unit exists or not, and collecting the third supernatant;
s110: repeating the steps until a preset volume of the third supernatant is collected;
s111: centrifuging the third supernatant to obtain intestinal cells;
s112: resuspending the intestinal tract cells by using a washing solution, and centrifuging the resuspended intestinal tract cells to obtain the intestinal tract organoid;
s113: and repeating the previous step.
Specifically, in step S101, a small intestine, which is an animal small intestine, is taken, and preferably, in this embodiment, a small intestine of a mouse is taken as an example.
The cold PBS refers to PBS below room temperature.
As an example, the number of times the intestinal tube is flushed with the cold PBS is 3-4 times; the predetermined length is 0.2 cm-0.6 cm; adding the crypt separation solution into the test tube, and then shaking gently at 3-6 ℃ for 15-35 min; the preset volume is 40 ml-60 ml.
Specifically, in step S102, the predetermined length may be 0.3cm, 0.4cm, 0.5cm, or the like, and preferably, the predetermined length is 0.4cm in this embodiment.
Specifically, in step S103, in this example, the section of intestine is transferred to a 50ml tube containing 20ml of PBS.
Specifically, in step S107, in this example, 30ml of the crypt-separated liquid was added to the mixture to contain 10mm EDTA (Chinese name ethylenediamine tetraacetic acid C)10H16N2O8) The PBS (1). Specifically, the tube was shaken gently at 4 ℃ for 30min to precipitate a second supernatant.
Specifically, in step S109, the volume of PBS added to the test tube is 20ml, and the presence or absence of the intestinal unit can be observed under a microscope.
Specifically, in step S110, the volume of the collected third supernatant is 50 ml.
As an example, in the process of centrifuging the third supernatant, the rotation speed of a centrifuge is 300 × g, the centrifuging time is 3min to 7min, and the centrifuging temperature is 2 ℃ to 6 ℃; in the process of carrying out centrifugal treatment on the resuspended intestinal cells, the rotating speed of a centrifugal machine is 300 Xg, the centrifugal treatment time is 3 min-7 min, and the centrifugal treatment temperature is 2-6 ℃.
Specifically, in step S111, the time for centrifuging the third supernatant may be 4min, 5min, or 6min, and the like, and preferably, in this embodiment, the time for centrifuging is 5 min; the temperature of the centrifugation treatment may be 3 ℃, 4 ℃, or 5 ℃ or the like, and preferably, in the present embodiment, the time of the centrifugation treatment is 4 minutes.
Further, in step S112, the volume of the washing solution is 10 ml. The time for performing centrifugal treatment on the resuspended intestinal tract cells can be 4min, 5min or 6min, etc., preferably, in this embodiment, the time for performing centrifugal treatment is 5 min; the temperature of the centrifugation treatment may be 3 ℃, 4 ℃, or 5 ℃ or the like, and preferably, in the present embodiment, the time of the centrifugation treatment is 4 minutes.
By way of example, the washing solution includes DMEM (a medium containing various amino acids and glucose), BSA, and a double antibody (streptomycin mixture).
As an example, said placing said intestinal organoid in organoid culture medium comprises:
resuspending the intestinal organoid with an organoid culture medium and matrigel according to a ratio to obtain an organoid suspension;
suspending the organoid suspension and dripping the organoid suspension into the culture hole.
Specifically, the volume of the organoid suspension dropped into the culture well is 50 μ l, and the culture well is a 24-well plate.
Further, after the organoid suspension is suspended and dropped into the culture hole, the organoid suspension is quickly placed into an incubator and continuously cultured for 15 min.
As an example, the culture medium may be replaced every two days during the above-described culture process.
Specifically, the intestinal organoids can be cultured in the organoid culture medium obtained in step S2 to serve as a control group, i.e., the culture medium in the control group is not added with DAPT and CHIR99021 to stimulate the culture. The culture medium can be replaced every two days in the culture process.
The control group and the protocol of the present application are cultured for a period of time (e.g., 7 days), and then intestinal organoids are collected for gene detection and immunofluorescent staining, respectively, as shown in fig. 2 to 4, and as can be seen from fig. 2 to 4, compared to the control group, the present application can obtain a large amount of Pan cells with high purity in a short time by adding DAPT and CHIR99021 to the culture medium.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. A method for directionally differentiating Pan cells in intestinal organoids is characterized by comprising the following steps:
obtaining an intestinal organoid;
placing the intestinal organoid in an organoid culture medium;
adding DAPT and CHIR99021 to the organoid culture and stimulating the intestinal organoids in the organoid culture medium for a predetermined time.
2. The method of gut organoid directed differentiation of Pangolian cells according to claim 1, wherein: the preset time is 5-10 days.
3. The method of gut organoid directed differentiation of Pangolian cells according to claim 1, wherein: the intestinal tract collector obtaining method comprises the following steps:
taking small intestine, washing the intestinal canal with cold PBS for several times, and removing intestinal contents;
cutting the intestinal canal longitudinally, and cutting into intestinal sections with preset lengths;
transferring the section of intestine into a test tube filled with PBS;
shaking the test tube for cleaning, separating out a first supernatant, and removing the first supernatant;
repeating the previous step for a plurality of times until the first supernatant is clear;
discarding the first supernatant obtained last;
adding crypt separation liquid into the test tube, and shaking gently to separate out a second supernatant;
removing the second supernatant;
adding PBS into the test tube, shaking the test tube to separate out a third supernatant, observing whether an intestinal tract unit exists or not, and collecting the third supernatant;
repeating the steps until a preset volume of the third supernatant is collected;
centrifuging the third supernatant to obtain intestinal cells;
resuspending the intestinal tract cells by using a washing solution, and centrifuging the resuspended intestinal tract cells to obtain the intestinal tract organoid;
and repeating the previous step.
4. A method of gut organoid directed differentiation of pangolin cells according to claim 3, wherein: the number of times of flushing the intestinal canal with the cold PBS is 3-4; the predetermined length is 0.2 cm-0.6 cm; adding the crypt separation solution into the test tube, and then shaking gently at 3-6 ℃ for 15-35 min; the preset volume is 40 ml-60 ml.
5. A method of gut organoid directed differentiation of pangolin cells according to claim 3, wherein: in the process of centrifuging the third supernatant, the rotating speed of a centrifuge is 300 Xg, the centrifuging time is 3-7 min, and the centrifuging temperature is 2-6 ℃; in the process of carrying out centrifugal treatment on the resuspended intestinal cells, the rotating speed of a centrifugal machine is 300 Xg, the centrifugal treatment time is 3 min-7 min, and the centrifugal treatment temperature is 2-6 ℃.
6. A method of gut organoid directed differentiation of pangolin cells according to claim 3, wherein: the washing solution comprises DMEM, BSA and double antibody.
7. The method of gut organoid directed differentiation of Pangolian cells according to any of claims 1 to 6, wherein: said placing said intestinal organoid in organoid culture medium comprises:
resuspending the intestinal organoid with an organoid culture medium and matrigel according to a ratio to obtain an organoid suspension;
suspending the organoid suspension and dripping the organoid suspension into the culture hole.
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