CN111979175A - Method for removing residual pluripotent stem cells in stem cell differentiation system - Google Patents
Method for removing residual pluripotent stem cells in stem cell differentiation system Download PDFInfo
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- CN111979175A CN111979175A CN202010889268.0A CN202010889268A CN111979175A CN 111979175 A CN111979175 A CN 111979175A CN 202010889268 A CN202010889268 A CN 202010889268A CN 111979175 A CN111979175 A CN 111979175A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0081—Purging biological preparations of unwanted cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses a method for removing residual pluripotent stem cells in a stem cell differentiation system, which comprises the steps of washing and soaking, water bath oscillation, primary centrifugation, primary extraction, division inhibition, secondary centrifugation, secondary extraction and purity detection. Compared with the traditional method, the method has the advantages of higher efficiency and better clearing effect, can effectively limit the division and differentiation of the pluripotent stem cells, and improves the purity of the separated cells; the differential centrifugation method is adopted to separate the cells, and the stem cells are separated according to the adherence of the stem cells, so that the accuracy of cell separation is improved. Solves the problem that the residual pluripotent stem cells in the stem cell differentiation system can not be effectively removed in the prior art, thereby influencing the purity of the cells.
Description
Technical Field
The invention belongs to the field of stem cells, and particularly relates to a method for removing residual pluripotent stem cells in a stem cell differentiation system.
Background
Stem cells are a class of cells that have unlimited or immortal self-renewal capacity, capable of producing at least one type of highly differentiated progeny cells. The research of stem cells is widely concerned by scientists and generations and has the necessity, and the stem cells have extremely wide application prospect in the fields of cell repair, developmental biology, pharmacology and the like of life science. Among them, pluripotent stem cells are the hot spot and focus of current stem cell research.
Pluripotent stem cells have the potential to differentiate into various cell tissues, but lose the ability to develop into complete individuals, and the development potential is limited to a certain extent. Can differentiate into all cells in the body, and thus form all tissues and organs of the body. Therefore, the research of the pluripotent stem cells not only has important theoretical significance, but also has great application value in the aspects of organ regeneration, repair and disease treatment.
In the actual cell culture process, part of pluripotent stem cells are always remained after the differentiation of the stem cells is finished, and in order to ensure the purity of the differentiated pluripotent stem cells, the existing clearing mode is complicated and complex, and the clearing effect is not obvious, so that the method for simply, conveniently and effectively clearing the residual pluripotent stem cells in the stem cell differentiation system is designed to meet the actual requirement.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for removing residual pluripotent stem cells in a stem cell differentiation system, and solves the problem that the residual pluripotent stem cells in the stem cell differentiation system in the prior art cannot be effectively removed, so that the cell purity is influenced.
The purpose of the invention can be realized by the following technical scheme:
a method for removing residual pluripotent stem cells in a stem cell differentiation system comprises the following steps:
s1, washing and soaking
Preferably selecting a stem cell differentiation product, placing the stem cell differentiation product on a sterile tray, repeatedly and slowly washing the stem cell differentiation product by using PBS (phosphate buffer solution), and then soaking the stem cell differentiation product in sterile physiological saline;
s2 oscillating in water bath
Taking out the treated stem cell differentiation product, placing the stem cell differentiation product in a culture medium, adding CAS and EDTA into the culture medium, and oscillating in a water bath;
s3, primary centrifugation
After water bath oscillation, placing stem cell differentiation products in a centrifuge for centrifugal separation;
s4, extracting for the first time
After the primary centrifugation is finished, extracting supernatant liquor;
s5, inhibiting splitting
Transferring the large particles on the lower layer left after the primary extraction into a test tube, adding the mixed solution into the test tube, slowly stirring by using a sterile glass rod, and standing;
s6, secondary centrifugation
Pouring the lower layer large particles into the centrifuge again for centrifugal separation;
s7, secondary extraction
After secondary centrifugation, taking out supernatant, and collecting large particles at the lowest layer;
s8, purity detection
And (4) carrying out purity detection on the supernatant in the primary extraction and the large-particle substances in the secondary extraction.
Further, the stem cell differentiation product in S1 is soaked in sterile physiological saline for 25-40 min.
Further, the stem cell differentiation product in S2 is shaken in water bath at 35-40 ℃ for 25-40 min under the environment.
Further, in the step S3, the rotation speed of the centrifuge is controlled to be changed to 30-60 rpm/S, so that the rotation speed is stabilized at 500-600 rpm, and the centrifuge is kept for 15-25 min; then the speed is controlled to be changed to 30-50 rpm/s again, so that the rotating speed is 700-840 rpm, and the centrifuge is kept for 20-30 min.
Further, the mixed solution in S5 is at least one of cyclophosphamide, busulfan, methotrexate, and astaxanthin.
Further, in the step S6, the rotation speed of the centrifuge is controlled to be changed to 30-50 rpm/S, so that the rotation speed is stabilized at 300-450 rpm, and the centrifuge is kept for 10-15 min; then the speed is controlled to be changed to 50-60 rpm/s again, so that the speed is 600-800 rpm, and the centrifuge is kept for 20-30 min.
The invention has the beneficial effects that:
1. compared with the traditional method, the method has the advantages of higher efficiency and better clearing effect, can effectively limit the division and differentiation of the pluripotent stem cells, and improves the purity of the separated cells;
2. the invention adopts a differential centrifugation method to separate the cells and separates the stem cells according to the adherence of the stem cells, thereby improving the accuracy of cell separation.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a schematic overall flow diagram of an embodiment of the present invention.
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.
In the description of the present invention, it is to be understood that the terms "opening," "upper," "lower," "thickness," "top," "middle," "length," "inner," "peripheral," and the like are used in an orientation or positional relationship that is merely for convenience in describing and simplifying the description, and do not indicate or imply that the referenced component or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be considered as limiting the present invention.
As shown in fig. 1, a method for removing residual pluripotent stem cells from a stem cell differentiation system comprises the following steps: washing and soaking, water bath oscillation, primary centrifugation, primary extraction, fission inhibition, secondary centrifugation, secondary extraction and purity detection.
The present application is further described below by reference to several examples.
Example 1
S1, washing and soaking
Selecting 10ml of stem cell differentiation products, placing the stem cell differentiation products on an aseptic tray, repeatedly and slowly washing the stem cell differentiation products for five times by using 1000ml of PBS (phosphate buffered saline), then placing the stem cell differentiation products in aseptic physiological saline with the concentration of 0.68 percent, and soaking the stem cell differentiation products for 30 min;
s2 oscillating in water bath
Taking out the treated stem cell differentiation product, placing the stem cell differentiation product in a culture medium, adding 5ml of CAS (cell separation fluid) and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 2:3 into the culture medium after 120min, and oscillating in water bath at 37 ℃ for 30 min;
s3, primary centrifugation
After water bath oscillation, placing a stem cell differentiation product in a centrifuge, controlling the speed to change to 50rpm/s until the speed is 500rpm, stopping acceleration, keeping the speed stable, centrifuging for 20min, controlling the speed to change to 40rpm/s again until the speed is 700rpm, stopping acceleration, keeping the speed stable, centrifuging for 25min, and placing the multipotent stem cells at the lowest layer and attaching to the inner wall of the centrifuge;
s4, extracting for the first time
After the primary centrifugation is finished, extracting supernatant liquor;
s5, inhibiting splitting
Transferring the large particles at the lower layer left after the primary extraction into a test tube, adding a mixed solution of 0.2ml of cyclophosphamide, 0.3ml of busulfan and 0.1ml of astaxanthin into the test tube, slowly stirring for 1min by using a sterile glass rod, and standing for 25 min;
s6, secondary centrifugation
Pouring the lower-layer large particles into the centrifuge again, controlling the rotation speed to change to 30rpm/s until the rotation speed is 300rpm, stopping acceleration, keeping the rotation speed stable, centrifuging for 10min, controlling the rotation speed to change to 50rpm/s again until the rotation speed is 600rpm, stopping acceleration, keeping the rotation speed stable, and centrifuging for 20 min;
s7, secondary extraction
After secondary centrifugation, taking out supernatant, and collecting large particles at the lowest layer;
s8, purity detection
And (4) carrying out purity detection on the supernatant in the primary extraction and the large-particle substances in the secondary extraction.
Example 2
S1, washing and soaking
Selecting 20ml of stem cell differentiation products, placing the stem cell differentiation products on an aseptic tray, repeatedly and slowly washing the stem cell differentiation products for five times by using 1500ml of PBS (phosphate buffered saline), then placing the stem cell differentiation products in aseptic physiological saline with the concentration of 0.7%, and soaking the stem cell differentiation products for 25 min;
s2 oscillating in water bath
Taking out the treated stem cell differentiation product, placing the stem cell differentiation product in a culture medium, adding 8ml of CAS (cell separation liquid) and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 3:5 into the culture medium after 100min, and oscillating the stem cell differentiation product in a water bath at the temperature of 35 ℃ for 40 min;
s3, primary centrifugation
After water bath oscillation, placing a stem cell differentiation product in a centrifuge, controlling the rotation speed to change to 60rpm/s, stopping acceleration until the rotation speed is 600rpm, then keeping the rotation speed stable, centrifuging for 15min, then controlling the rotation speed to change to 45rpm/s again, stopping acceleration until the rotation speed is 780rpm, then keeping the rotation speed stable, centrifuging for 20min, and placing the multipotent stem cells on the lowest layer and attaching to the inner wall of the centrifuge;
s4, extracting for the first time
After the primary centrifugation is finished, extracting supernatant liquor;
s5, inhibiting splitting
Transferring the large lower-layer particles left after the primary extraction into a test tube, adding a mixed solution of 0.25ml of cyclophosphamide, 0.1ml of busulfan, 0.35ml of methotrexate and 0.2ml of astaxanthin into the test tube, slowly stirring for 1.5min by using a sterile glass rod, and then standing for 20 min;
s6, secondary centrifugation
Pouring the lower-layer large particles into the centrifuge again, controlling the rotation speed to be 40rpm/s, stopping acceleration until the rotation speed is 400rpm, keeping the rotation speed stable, centrifuging for 15min, controlling the rotation speed to be 60rpm/s again, stopping acceleration until the rotation speed is 760rpm, keeping the rotation speed stable, and centrifuging for 20 min;
s7, secondary extraction
After secondary centrifugation, taking out supernatant, and collecting large particles at the lowest layer;
s8, purity detection
And (4) carrying out purity detection on the supernatant in the primary extraction and the large-particle substances in the secondary extraction.
Example 3
S1, washing and soaking
Selecting 10ml of stem cell differentiation product, placing the stem cell differentiation product on a sterile tray, repeatedly and slowly washing the stem cell differentiation product for six times by using 1200ml of PBS (phosphate buffered saline), then placing the stem cell differentiation product in sterile physiological saline with the concentration of 0.68 percent, and soaking the stem cell differentiation product for 40 min;
s2 oscillating in water bath
Taking out the treated stem cell differentiation product, placing the stem cell differentiation product in a culture medium, adding 7ml of CAS (cell separation fluid) and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 3:4 into the culture medium after 150min, and oscillating in water bath at 40 ℃ for 25 min;
s3, primary centrifugation
After water bath oscillation, placing a stem cell differentiation product in a centrifuge, controlling the rotation speed to change to 60rpm/s, stopping acceleration until the rotation speed is 600rpm, then keeping the rotation speed stable, centrifuging for 20min, then controlling the rotation speed to change to 30rpm/s again, stopping acceleration until the rotation speed is 840rpm, then keeping the rotation speed stable, centrifuging for 20min, and then placing the multipotent stem cells on the lowest layer and attaching the multipotent stem cells to the inner wall of the centrifuge;
s4, extracting for the first time
After the primary centrifugation is finished, extracting supernatant liquor;
s5, inhibiting splitting
Transferring the large particles at the lower layer left after the primary extraction into a test tube, adding a mixed solution of 0.2ml of cyclophosphamide and 0.25ml of astaxanthin into the test tube, slowly stirring for 1min by using a sterile glass rod, and then standing for 20 min;
s6, secondary centrifugation
Pouring the lower-layer large particles into the centrifuge again, controlling the rotation speed to be 50rpm/s, stopping acceleration until the rotation speed is 400rpm, keeping the rotation speed stable, centrifuging for 15min, controlling the rotation speed to be 60rpm/s again, stopping acceleration until the rotation speed is 680rpm, keeping the rotation speed stable, and centrifuging for 30 min;
s7, secondary extraction
After secondary centrifugation, taking out supernatant, and collecting large particles at the lowest layer;
s8, purity detection
And (4) carrying out purity detection on the supernatant in the primary extraction and the large-particle substances in the secondary extraction.
Example 4
S1, washing and soaking
Selecting 15ml of stem cell differentiation products, placing the stem cell differentiation products on an aseptic tray, repeatedly and slowly washing the stem cell differentiation products for five times by using 1000ml of PBS (phosphate buffered saline), then placing the stem cell differentiation products in aseptic physiological saline with the concentration of 0.69%, and soaking the stem cell differentiation products for 35 min;
s2 oscillating in water bath
Taking out the treated stem cell differentiation product, placing the stem cell differentiation product in a culture medium, adding 5ml of CAS (cell separation fluid) and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:4 into the culture medium after 120min, and oscillating in a water bath at 38 ℃ for 30 min;
s3, primary centrifugation
After water bath oscillation, placing a stem cell differentiation product in a centrifuge, controlling the rotation speed to change to 30rpm/s until the rotation speed is 600rpm, stopping acceleration, keeping the rotation speed stable, centrifuging for 25min, controlling the rotation speed to change to 50rpm/s again until the rotation speed is 750rpm, keeping the rotation speed stable, centrifuging for 30min, and placing the multipotent stem cells at the lowest layer and attaching to the inner wall of the centrifuge;
s4, extracting for the first time
After the primary centrifugation is finished, extracting supernatant liquor;
s5, inhibiting splitting
Transferring the large particles at the lower layer left after the primary extraction into a test tube, adding 0.2ml of astaxanthin into the test tube, slowly stirring for 1min by using a sterile glass rod, and standing for 30 min;
s6, secondary centrifugation
Pouring the lower layer large particles into the centrifuge again, controlling the rotation speed to be 45rpm/s, stopping acceleration until the rotation speed is 450rpm, keeping the rotation speed stable, centrifuging for 10min, controlling the rotation speed to be 50rpm/s again, stopping acceleration until the rotation speed is 800rpm, keeping the rotation speed stable, and centrifuging for 30 min;
s7, secondary extraction
After secondary centrifugation, taking out supernatant, and collecting large particles at the lowest layer;
s8, purity detection
And (4) carrying out purity detection on the supernatant in the primary extraction and the large-particle substances in the secondary extraction.
It should be noted that the supernatant separated in the first extraction is mostly differentiated cells in stem cell differentiation products, and the large granular substances separated in the second extraction are mostly pluripotent stem cells in stem cell differentiation products.
The results of the comparison of the above examples with the prior art are shown in the following table.
| Example (A) | Clearing time | Cellular activity | Purity of |
| Example 1 | 6h | High strength | 99.5％ |
| Example 2 | 6h | High strength | 99.3％ |
| Example 3 | 7h | High strength | 99.6％ |
| Example 4 | 7h | High strength | 99.5％ |
| Prior Art | 8.5h | Is stronger | 94.3％ |
In conclusion, compared with the prior art, the method for removing the residual pluripotent stem cells in the stem cell differentiation system provided by the invention not only improves the removal efficiency, but also obviously improves the activity and purity of the removed cells.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (6)
1. A method for removing residual pluripotent stem cells from a stem cell differentiation system, comprising the steps of:
s1, washing and soaking
Preferably selecting a stem cell differentiation product, placing the stem cell differentiation product on a sterile tray, repeatedly and slowly washing the stem cell differentiation product by using PBS (phosphate buffer solution), and then soaking the stem cell differentiation product in sterile physiological saline;
s2 oscillating in water bath
Taking out the treated stem cell differentiation product, placing the stem cell differentiation product in a culture medium, adding CAS and EDTA into the culture medium, and oscillating in a water bath;
s3, primary centrifugation
After water bath oscillation, placing stem cell differentiation products in a centrifuge for centrifugal separation;
s4, extracting for the first time
After the primary centrifugation is finished, extracting supernatant liquor;
s5, inhibiting splitting
Transferring the large particles on the lower layer left after the primary extraction into a test tube, adding the mixed solution into the test tube, slowly stirring by using a sterile glass rod, and standing;
s6, secondary centrifugation
Pouring the lower layer large particles into the centrifuge again for centrifugal separation;
s7, secondary extraction
After secondary centrifugation, taking out supernatant, and collecting large particles at the lowest layer;
s8, purity detection
And (4) carrying out purity detection on the supernatant in the primary extraction and the large-particle substances in the secondary extraction.
2. The method for removing residual pluripotent stem cells from a stem cell differentiation system according to claim 1, wherein the stem cell differentiation product of S1 is soaked in sterile physiological saline for 25-40 min.
3. The method for removing residual pluripotent stem cells from a stem cell differentiation system according to claim 1, wherein the stem cell differentiation product of S2 is subjected to water bath shaking at 35-40 ℃ for 25-40 min.
4. The method according to claim 1, wherein the rotation speed of the centrifuge is controlled to be 30-60 rpm/S in step S3, so that the rotation speed is stabilized at 500-600 rpm, and the centrifuge is kept for 15-25 min; then the speed is controlled to be changed to 30-50 rpm/s again, so that the rotating speed is 700-840 rpm, and the centrifuge is kept for 20-30 min.
5. The method according to claim 1, wherein the mixture of S5 is at least one of cyclophosphamide, busulfan, methotrexate and astaxanthin.
6. The method for removing residual pluripotent stem cells from a stem cell differentiation system according to claim 1, wherein in S6, the centrifuge is first controlled to change the rotation speed to 30-50 rpm/S, so that the rotation speed is stabilized at 300-450 rpm, and the centrifuge is kept for 10-15 min; then the speed is controlled to be changed to 50-60 rpm/s again, so that the speed is 600-800 rpm, and the centrifuge is kept for 20-30 min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107075473A (en) * | 2014-11-07 | 2017-08-18 | 国立大学法人大阪大学 | The wherein cell mass of the removed induction of neoblast, its purposes and preparation method thereof |
| CN110050060A (en) * | 2016-10-17 | 2019-07-23 | 学校法人庆应义塾 | Undifferentiated stem cell remover and the method for removing undifferentiated stem cell |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107075473A (en) * | 2014-11-07 | 2017-08-18 | 国立大学法人大阪大学 | The wherein cell mass of the removed induction of neoblast, its purposes and preparation method thereof |
| CN110050060A (en) * | 2016-10-17 | 2019-07-23 | 学校法人庆应义塾 | Undifferentiated stem cell remover and the method for removing undifferentiated stem cell |
Non-Patent Citations (3)
| Title |
|---|
| HO‑CHANG JEONG 等: "Technical approaches to induce selective cell death of pluripotent stem cells", 《CELL. MOL. LIFE SCI.》 * |
| 左伟勇 等: "《细胞工程技术》", 30 September 2014 * |
| 曾庆坛 等: "降低/ 消除诱导性多能干细胞致瘤性的研究进展", 《血管与腔内血管外科杂志》 * |
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