CN111139218A - Method for rapidly and efficiently preparing embryoid body by inducing pluripotent stem cell or embryonic stem cell - Google Patents
Method for rapidly and efficiently preparing embryoid body by inducing pluripotent stem cell or embryonic stem cell Download PDFInfo
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Abstract
The invention discloses a method for preparing an embryoid body by induced pluripotent stem cells or embryonic stem cells quickly and efficiently, which digests induced pluripotent stem cells iPS/embryonic stem cells ES into small cell masses or single cells; inoculating the culture medium with proper concentration; culturing under a horizontal shaking condition to prepare a polymeric sphere; separating the polymerized cell spheres and continuing culturing to form embryoid bodies. The method forms the cell polymeric sphere under the conditions of proper cell concentration and proper horizontal rotation and shaking, and then performs differentiation culture on the cell sphere to form the embryoid body.
Description
Technical Field
The invention belongs to the field of biotechnology and cell biology, and particularly relates to a method for quickly and efficiently preparing an embryoid body by using induced pluripotent stem cells or embryonic stem cells, so as to quickly, efficiently and high-flux form the embryoid body.
Background
Embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are cells with self-renewal and totipotency, have the differentiation capacity of three germ layers and the capacity of differentiating all types of cells, and have great potential values in the aspects of cell replacement therapy, the research of pathogenesis, new drug screening, the treatment of clinical diseases such as nervous system diseases, cardiovascular diseases and the like. The ES/iPS can form a spherical structure with three germ layers of inner, middle and outer under certain culture conditions, which has high similarity with the early embryonic development stage of mammals in morphology and is called an Embryoid Bodies (EBs). At present, many differentiation systems are established based on an embryoid body differentiation system, such as hematopoietic stem cells, natural killer cells, neural stem cells, cardiac muscle cells and the like, and the embryoid body and the preparation technology thereof have wide application in the fields of embryonic development research, regenerative medicine, cell therapy and the like.
The traditional preparation method of the embryoid body comprises a hanging drop method (hanging drops) and a centrifugal method. The pendant drop method has complex operation and low forming efficiency of the pseudo-embryo body, and is difficult to prepare on a large scale. The centrifugation method is that a 96-well plate or a commercial microporous structure plate is used for evenly distributing cells, and the cells are pressed together by using centrifugal force, but the method is also complicated to operate, the centrifuged cells are stacked in a sheet shape and cannot directly form a spherical structure, the formed embryoid body has a loose structure, and the embryoid body is easy to disintegrate into single cells after being transferred to a differentiation medium, so that the subsequent differentiation development is influenced.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for quickly and efficiently preparing an embryoid body by using induced pluripotent stem cells or embryonic stem cells, which has low cost, simple and convenient operation and high speed and efficiency.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for preparing embryoid bodies rapidly and efficiently by using induced pluripotent stem cells or embryonic stem cells comprises the following steps:
1) digesting the induced pluripotent stem cell iPS/embryonic stem cell ES into small cell masses or single cells;
2) inoculating the culture medium with proper concentration;
3) culturing under a horizontal shaking condition to prepare a polymeric sphere;
4) separating the polymerized cell spheres and continuing culturing to form embryoid bodies.
The method specifically comprises the following steps:
1) digesting the iPS/ES cells into small cell masses or single cells using appropriate digestion reagents;
2) the iPS/ES cells were packed at 1X 105From ml to 2X 106Inoculating the culture medium at a density of individual cells/ml and transferring to a non-tissue-processing culture vessel;
3) placing the culture container in a carbon dioxide incubator, and horizontally shaking and culturing for 8-120 hours at 20 rpm-200 rpm;
4) filtering the culture by using a double-sided filter screen, removing free and dead cells and cell debris, collecting polymerized cell spheres, and re-suspending the aggregated cell spheres in a differentiation medium;
5) the polymerized cell spheres are continuously cultured to form embryoid bodies.
Preferably, the concentration of cells seeded is 5X 105/mL。
The culture medium is all suitable for maintaining dryness or differentiation of iPS/ES cells.
Preferably, the speed of the horizontal rotation culture is 70 rpm.
Preferably, the culture time is 24-48 hours by horizontal rotation.
The invention has the beneficial effects that: the forming rate is high, the formed cell aggregate is connected tightly, and is not easy to be disintegrated in subsequent culture; the operation is simple, and the inoculation is performed according to a proper density and then the horizontal shaking culture is performed, so that the labor cost is greatly saved; high and flexible flux, 106The basal area of each cell is 10cm2In the culture hole200-300 cell aggregates are formed and developed into embryoid bodies, and the cell amount and the bottom area of the culture container can be increased according to requirements to improve the yield of the embryoid bodies; the cost is low, and commercial micro-pore plates or 96-pore plates do not need to be purchased.
Drawings
FIG. 1 is a graph (40-fold magnification) of cell aggregates formed after 24 hours of horizontal shaking culture in example 1 of the present invention.
FIG. 2 is a graph (100-fold magnification) of cell aggregates formed after 24 hours of horizontal shaking culture in example 1 of the present invention.
FIG. 3 is a diagram (100-fold magnification) of embryoid bodies formed after transferring the cell aggregates of the present invention into an APEL medium and culturing in suspension for 11 days.
FIG. 4 is a morphological diagram (200 times magnification) of the embryoid bodies of the present invention after adherent culture for 2 days.
Detailed Description
The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustration:
the present invention is not to be limited in scope by the embodiments of the present invention. The implementation conditions used in the examples can be adjusted according to the conditions of the specific manufacturer, and the implementation conditions not specified are generally the conditions in routine experiments.
The method for rapidly and efficiently preparing the embryoid body by using the induced pluripotent stem cell or the embryonic stem cell comprises the following steps:
1) digesting the induced pluripotent stem cell iPS/embryonic stem cell ES into small cell masses or single cells;
2) inoculating the culture medium with proper concentration;
3) culturing under a horizontal shaking condition to prepare a polymeric sphere;
4) separating the polymerized cell spheres and continuing culturing to form embryoid bodies.
The method specifically comprises the following steps:
1) digesting the iPS/ES cells into small cell masses or single cells using appropriate digestion reagents;
2) the iPS/ES cells were packed at 1X 105From ml to 2X 106The density of each cell/ml is inoculated into the cultureNutrient medium and transferring to a non-tissue-treated culture vessel;
3) placing the culture container in a carbon dioxide incubator, and horizontally shaking and culturing for 8-120 hours at 20 rpm-200 rpm;
4) filtering the culture by using a double-sided filter screen, removing free and dead cells and cell debris, collecting polymerized cell spheres, and re-suspending the aggregated cell spheres in a differentiation medium;
5) the polymerized cell spheres are continuously cultured to form embryoid bodies.
Preferably, the concentration of cells seeded is 5X 105/mL。
The culture medium is all suitable for maintaining dryness or differentiation of iPS/ES cells.
Preferably, the speed of the horizontal rotation culture is 70 rpm.
Preferably, the culture time is 24-48 hours by horizontal rotation.
The invention is suitable for human and animal embryonic stem cells, induced pluripotent stem cells and other pluripotent stem cells.
The principle of the EB preparation method which is established by the invention and is efficient and simple lies in that a liquid culture medium forms a micro vortex by utilizing horizontal shaking, cells are gathered to the center of the vortex and are fully contacted with each other, the cells are aggregated and connected into uniform and regular spherical cell clusters, the formed cell clusters are tightly connected, the cells are not easily dispersed into single cells in subsequent culture, and the formation rate of a pseudoembryo is high.
The invention has the main application range of formation and application of the embryoid body, and provides a new technical means for developmental research, in-vitro cell differentiation, organoid formation, disease model construction, cell/tissue/organ alternative treatment, cell treatment and gene treatment.
The invention changes the traditional hanging drop or centrifugal method, utilizes the horizontal shaking with proper rotating speed and proper cell concentration to ensure that cells are fully contacted with each other in the culture process to form stable and compact spherical aggregates, and continues to culture to form embryoid bodies.
Example 1
In a 1.6-well plate, Metrgel is used as a matrix, mTeSR1 is used as a culture medium to culture iPS cells until the coverage of the plate bottom reaches 70% -80%.
2. The medium was aspirated, 1mL DPBS was washed per well, DPBS was aspirated, 1mL GCDR was added, the cell culture chamber was placed for 6-8 minutes, GCDR was aspirated, 1mL mTeSR 3D inoculum medium (containing 10 μ M Y27632) was added per well, the bottom of the well was flushed with a 1mL pipette, the cells were exfoliated, and gently whipped several times to disperse the cells into clumps containing 20-30 cells.
3. According to the following steps: 1 or 1: 2 ratio the cell suspension in each well was transferred to a non-tissue treated 6-well plate, supplemented with mTeSR 3D inoculum medium (containing 10. mu. M Y27632) to 2ml, placed on a horizontal shaker in a carbon dioxide incubator and incubated for 24 hours with horizontal shaking at 70 rpm.
4. Observing the formation of cell spheres, and selecting direct inoculation or continuing to culture for 1-4 days according to the size of the cell spheres, and supplementing 224 mu L mTeSR 3D feeding culture medium to each hole every day.
A5.50 mL centrifuge tube was fitted with a 37 μ M pore size double-sided cell strainer, and the cultured cultures were filtered through the strainer and the culture wells were rinsed with 1mL DPBS and the rinse solution was filtered through the strainer.
6. The double-sided screen was inverted onto a new 50mL centrifuge tube and the screen was back-washed thoroughly with APEL medium to allow complete washing of the cell pellet into APEL medium.
7. According to the following steps: supplementing APEL to the corresponding volume according to the proportion of 6, inoculating the mixture into a non-tissue-treated T75 culture bottle or a 6-hole culture plate, standing and suspending for culture, replacing a fresh culture medium every 5 days, differentiating to form an embryoid body, and changing to adherent culture on the 11 th day.
Example 2
In a 1.6-well plate, Metrgel is used as a matrix, mTeSR1 is used as a culture medium to culture iPS cells until the coverage of the plate bottom reaches 70% -80%.
2. The medium was aspirated, the wells were washed with 1mL DPBS, DPBS was aspirated, 0.5mL ACCUTASE was added, the cells were placed in a cell incubator for 5-7 minutes, 1.5mL DPBS was added to each well to stop the digestion reaction, and the cells were dispersed into single cells by gentle pipetting several times using a 1mL pipette.
3. The cells were transferred to a 15ml centrifuge tube, centrifuged at 150g for 10 minutes and the supernatant discarded, and 1ml of mTeSR 3D inoculum medium containing 10. mu. M Y27632 was added to blow the cell pellet and the cells were counted.
4. The non-tissue-treated T75 flask was inoculated with 10mL of mTeSR 3D containing 10. mu. M Y27632 and inoculated with 7.5X 10 cells6Step 2 cells were filled to 15mL and placed on a horizontal shaker in a carbon dioxide incubator with 50rpm horizontal shaking for 24 hours.
5. Observing cell spheroid formation, and selecting direct inoculation or continuing to culture for 1-4 days according to the size of the cell spheroid, and supplementing 1350 mu L mTeSR 3D feeding culture medium every day.
A6.50 mL centrifuge tube was placed with a 37 μ M pore size double-sided cell strainer, and the cultured cultures were filtered through the strainer, and the flasks were rinsed with 5mL DPBS and the rinse was filtered through the strainer.
7. The double-sided screen was inverted onto a new 50mL centrifuge tube and the screen was back-washed thoroughly with APEL medium to allow complete washing of the cell pellet into APEL medium.
8. According to the following steps: 6, the culture medium is inoculated into a non-tissue-treated T75 culture bottle, the culture is kept still and suspended, the fresh culture medium is replaced every 5 days, the culture medium is differentiated to form an embryoid body, and the culture medium is changed to adherent culture on the 11 th day.
Example 3
In a 1.6-well plate, Metrgel is used as a matrix, mTeSR1 is used as a culture medium to culture iPS cells until the coverage of the plate bottom reaches 70% -80%.
2. The medium was aspirated off, 1mL of DPBS was washed per well, DPBS was aspirated off, 1mL of GCDR was added, the cell culture chamber was placed and treated for 6-8 minutes, GCDR was aspirated off, 1mL of APEL medium (containing 10. mu. M Y27632) was added per well, the bottom of the well was flushed with a 1mL pipette, the cells were exfoliated, and gently tapped several times to disperse the cells into clumps containing 20-30 cells.
2. The cell suspension in each well was transferred to a non-tissue treated 6-well plate, supplemented with 1mL of APEL medium, and placed on a horizontal shaker in a carbon dioxide incubator for 24 hours with horizontal shaking at 70 rpm.
A3.50 mL centrifuge tube was fitted with a 37 μ M pore size double-sided cell strainer, and the cultured cultures were filtered through the strainer and the culture wells were rinsed with 1mL DPBS and the rinse solution was filtered through the strainer.
4. The double-sided screen was inverted onto a new 50mL centrifuge tube and the screen was back-washed thoroughly with APEL medium to allow complete washing of the cell pellet into APEL medium.
6. According to the following steps: supplementing APEL to the corresponding volume according to the proportion of 6, inoculating the mixture into a non-tissue-treated T75 culture bottle or a 6-hole culture plate, standing and suspending for culture, replacing a fresh culture medium every 5 days, differentiating to form an embryoid body, and changing to adherent culture on the 11 th day.
Example 4
In a 1.6-well plate, Metrgel is used as a matrix, mTeSR1 is used as a culture medium to culture iPS cells until the coverage of the plate bottom reaches 70% -80%.
2. And (3) sucking and removing the culture medium, cleaning each hole by using 1mL of DPBS, sucking and removing the DPBS, adding 1mL of 50uM EDTA solution, placing the mixture into a cell culture box for treatment for 5-7 minutes, sucking and removing the EDTA solution, adding 1mL of mTeSR1 culture medium (containing 10 mu M Y27632 and 0.05% PVA) into each hole, blowing and flushing the bottom of each hole by using a 1mL pipette to remove the cells, and gently blowing and beating the cells for a plurality of times to disperse the cells into lumps containing 20-30 cells.
3. According to the following steps: 1 ratio the cell suspension in each well was transferred to a non-tissue treated 6-well plate, supplemented with mTeSR1 medium (containing 10. mu. M Y27632, 0.05% PVA) to 2mL, placed on a horizontal shaker in a carbon dioxide incubator, and incubated for 24 hours with horizontal shaking at 70 rpm.
4. Supplemented with 1m TeSR1 medium (containing 0.05% PVA) was placed on a horizontal shaker in a carbon dioxide incubator and incubated for 24 hours with horizontal shaking at 70 rpm.
A5.50 mL centrifuge tube was fitted with a 37 μ M pore size double-sided cell strainer, and the cultured cultures were filtered through the strainer and the culture wells were rinsed with 1mL DPBS and the rinse solution was filtered through the strainer.
6. The double-sided screen was inverted onto a new 50mL centrifuge tube and the screen was back-washed thoroughly with APEL medium to allow complete washing of the cell pellet into APEL medium.
7. According to the following steps: supplementing APEL to the corresponding volume according to the proportion of 6, inoculating the mixture into a non-tissue-treated T75 culture bottle or a 6-hole culture plate, standing and suspending for culture, replacing a fresh culture medium every 5 days, differentiating to form an embryoid body, and changing to adherent culture on the 11 th day.
Example 5
In a 1.6-well plate, Metrgel is used as a matrix, mTeSR1 is used as a culture medium to culture iPS cells until the coverage of the plate bottom reaches 70% -80%.
2. Removing the culture medium by aspiration, washing each well with 1mL of DPBS, removing DPBS by aspiration, adding 0.5mL of ACCUTASE to digest the cells, placing the cells in a cell incubator to treat for 5-7 minutes, adding 1.5mL of DPBS to each well to terminate the digestion reaction, and gently pipetting the cells several times by using a 1mL pipette to disperse the cells into single cells.
3. Transferring the cells into a 15mL centrifuge tube, centrifuging at 150g for 10 min, discarding the supernatant, adding 1mL APEL medium containing 10 μ M Y27632, blowing and mixing the cell precipitate, counting the cells, and diluting the cell suspension to 10% using APEL medium containing 10 μ M Y276325one/mL.
In 4.96 well round bottom low adhesion culture plates, 100. mu.L of cell suspension per well was added and incubated on a horizontal shaker in a carbon dioxide incubator for 24 hours at 150rpm with horizontal shaking.
5. Cell spheroid formation was observed, the culture was switched to static culture, 50. mu.L of upper medium was aspirated away at 6 th, and 50. mu.L of fresh APEL medium was supplemented.
6. On day 11, the embryoid bodies were aspirated from each well and transferred to tissue culture treated plates for culture in adherent cells.
As shown in FIGS. 1 to 4, FIG. 1 shows the aggregation of cells formed after 24 hours of horizontal shaking culture in example 1 of the present invention (magnification: 40 times), which shows that a large number of regular round cell spheres are formed. FIG. 2 shows the cell aggregates formed in example 1 after 24 hours of horizontal shaking culture (magnification 100 times), and it can be seen that the cell aggregates have a compact outer structure, a rough surface, and a clear boundary. FIG. 3 shows that the embryoid bodies (amplified by 100 times) formed after the cell aggregates are transferred into an APEL medium for suspension culture for 11 days lose regular round appearance and have smooth surfaces. FIG. 4 shows the morphology (200 times magnification) of embryoid bodies after 2 days of adherent culture, wherein the embryoid bodies grow adherently, differentiated fusiform and polygonal cells climb out around the adherent position, and the differentiated cells continuously proliferate and grow outwards.
In summary, the disclosure of the present invention is not limited to the above-mentioned embodiments, and persons skilled in the art can easily set forth other embodiments within the technical teaching of the present invention, but such embodiments are included in the scope of the present invention.
Claims (6)
1. A method for preparing an embryoid body by inducing pluripotent stem cells or embryonic stem cells quickly and efficiently is characterized by comprising the following steps:
1) digesting the induced pluripotent stem cell iPS/embryonic stem cell ES into small cell masses or single cells;
2) inoculating the culture medium with proper concentration;
3) culturing under a horizontal shaking condition to prepare a polymeric sphere;
4) separating the polymerized cell spheres and continuing culturing to form embryoid bodies.
2. The method for rapidly and efficiently preparing an embryoid body by using the induced pluripotent stem cell or the embryonic stem cell according to claim 1, which comprises the following steps:
1) digesting the iPS/ES cells into small cell masses or single cells using appropriate digestion reagents;
2) the iPS/ES cells were packed at 1X 105From ml to 2X 106Inoculating the culture medium at a density of individual cells/ml and transferring to a non-tissue-processing culture vessel;
3) placing the culture container in a carbon dioxide incubator, and horizontally shaking and culturing for 8-120 hours at 20 rpm-200 rpm;
4) filtering the culture by using a double-sided filter screen, removing free and dead cells and cell debris, collecting polymerized cell spheres, and re-suspending the aggregated cell spheres in a differentiation medium;
5) the polymerized cell spheres are continuously cultured to form embryoid bodies.
3. The method for the rapid and efficient production of embryoid bodies using induced pluripotent stem cells or embryonic stem cells according to claim 2, wherein the inoculated cells are present in a concentration of 5X 105/mL。
4. The method for rapidly and efficiently preparing embryoid bodies from induced pluripotent stem cells or embryonic stem cells according to claim 1 or 2, wherein the culture medium is any culture medium suitable for the maintenance of dryness or differentiation of iPS/ES cells.
5. The method for rapidly and efficiently preparing an embryoid body from induced pluripotent stem cells or embryonic stem cells according to claim 2, wherein the horizontal rotation culture speed is 70 rpm.
6. The method for rapidly and efficiently preparing an embryoid body from induced pluripotent stem cells or embryonic stem cells according to claim 2, wherein the culture time is 24 to 48 hours by horizontal rotation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647552A (en) * | 2020-06-18 | 2020-09-11 | 中国人民解放军联勤保障部队第九二〇医院 | Method for rapidly and efficiently preparing embryoid bodies by inducing pluripotent stem cells |
| CN112410286A (en) * | 2020-11-30 | 2021-02-26 | 河南省生殖健康科学技术研究院(河南省出生缺陷干预工程技术研究中心) | Method for constructing pregnancy induced abortion drug screening model by using induced pluripotent stem cells and application |
| CN115873795A (en) * | 2023-02-02 | 2023-03-31 | 苏州血霁生物科技有限公司 | Method for differentiating hematopoietic stem/progenitor cells, culture medium and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009050694A1 (en) * | 2007-10-15 | 2009-04-23 | Technion Research & Development Foundation Ltd. | Methods of generating embryoid bodies and uses of same |
| CN102358892A (en) * | 2011-09-23 | 2012-02-22 | 安徽农业大学 | Manufacturing method for embryoid bodies of bovine induced pluripotent stem (iPS) cells |
| JP2016010330A (en) * | 2014-06-27 | 2016-01-21 | 国立大学法人 千葉大学 | METHOD FOR EXPRESSING A PLURALITY OF FOREIGN GENES IN EMBRYOID BODY CONSISTING OF INDUCED PLURIPOTENT STEM CELL (iPS CELL) |
-
2020
- 2020-01-16 CN CN202010048972.3A patent/CN111139218A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009050694A1 (en) * | 2007-10-15 | 2009-04-23 | Technion Research & Development Foundation Ltd. | Methods of generating embryoid bodies and uses of same |
| CN102358892A (en) * | 2011-09-23 | 2012-02-22 | 安徽农业大学 | Manufacturing method for embryoid bodies of bovine induced pluripotent stem (iPS) cells |
| JP2016010330A (en) * | 2014-06-27 | 2016-01-21 | 国立大学法人 千葉大学 | METHOD FOR EXPRESSING A PLURALITY OF FOREIGN GENES IN EMBRYOID BODY CONSISTING OF INDUCED PLURIPOTENT STEM CELL (iPS CELL) |
Non-Patent Citations (6)
| Title |
|---|
| STEMCELL TECHNOLOGIES: "Expansion of Human Pluripotent Stem Cells as Aggregates in Suspension Culture Using mTeSR™3D", 《EXPANSION OF HUMAN PLURIPOTENT STEM CELLS AS AGGREGATES IN SUSPENSION CULTURE USING MTESR™3D》 * |
| STEMCELL TECHNOLOGIES: "mTeSR™3D", 《MTESR™3D》 * |
| STEMCELL TECHNOLOGIES: "STEMdiff™ APEL™2 Medium", 《STEMDIFF™ APEL™2 MEDIUM》 * |
| 薛社普: "《医学细胞生物学》", 31 March 2019, 中国协和医科大学出版社 * |
| 袁毅君等: "不同方法从小鼠胚胎干细胞分化拟胚体", 《中兽医医药杂志》 * |
| 阮卫民等: "一种获得猪诱导性多能干细胞的新方法", 《中国科学:生命科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647552A (en) * | 2020-06-18 | 2020-09-11 | 中国人民解放军联勤保障部队第九二〇医院 | Method for rapidly and efficiently preparing embryoid bodies by inducing pluripotent stem cells |
| CN112410286A (en) * | 2020-11-30 | 2021-02-26 | 河南省生殖健康科学技术研究院(河南省出生缺陷干预工程技术研究中心) | Method for constructing pregnancy induced abortion drug screening model by using induced pluripotent stem cells and application |
| CN112410286B (en) * | 2020-11-30 | 2023-10-27 | 河南省生殖健康科学技术研究院(河南省出生缺陷干预工程技术研究中心) | Method for constructing abortion caused by gestation drug screening model by using induced pluripotent stem cells and application |
| CN115873795A (en) * | 2023-02-02 | 2023-03-31 | 苏州血霁生物科技有限公司 | Method for differentiating hematopoietic stem/progenitor cells, culture medium and application |
| CN115873795B (en) * | 2023-02-02 | 2023-09-01 | 苏州血霁生物科技有限公司 | Method for differentiating hematopoietic stem/progenitor cells, culture medium and application |
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