阅读说明：本技术 葡聚糖凝胶色谱柱及脐血间充质干细胞外泌体的分离方法 (Glucan gel chromatographic column and method for separating umbilical cord blood mesenchymal stem cell exosome ) 是由 石宏 翟平生 宁眺 于 2021-07-26 设计创作，主要内容包括：本发明公开了一种葡聚糖凝胶色谱柱及脐血间充质干细胞外泌体的分离方法,所述葡聚糖凝胶色谱柱包括U型柱管、脱脂棉球和收集管；所述U型柱管内部设置有葡聚糖凝胶,并且底部设置有收集口,所述收集口上设置有开关；所述脱脂棉球设置于所述收集口内；所述收集管位于所述收集口的下方。本发明葡聚糖凝胶色谱柱够快速分离得到外泌体,仅仅通过自身重力实现体积超过300ml的外泌体分离,无需真空抽离,操作简单,效率高,此外,该葡聚糖凝胶可以重复使用,降低了分离制备成本。(The invention discloses a sephadex chromatographic column and a method for separating umbilical cord blood mesenchymal stem cell exosomes, wherein the sephadex chromatographic column comprises a U-shaped column tube, a degreasing cotton ball and a collecting tube; the inner part of the U-shaped column tube is provided with glucan gel, the bottom of the U-shaped column tube is provided with a collecting port, and a switch is arranged on the collecting port; the absorbent cotton ball is arranged in the collecting port; the collecting pipe is positioned below the collecting opening. The sephadex chromatographic column can be used for quickly separating to obtain exosomes, the exosomes with the volume of more than 300ml can be separated only through self gravity, vacuum extraction is not needed, the operation is simple, the efficiency is high, and in addition, the sephadex can be repeatedly used, so that the separation and preparation cost is reduced.)

1. A sephadex chromatographic column is characterized by comprising a U-shaped column tube, a degreasing cotton ball and a collecting tube;

the inner part of the U-shaped column tube is provided with glucan gel, the bottom of the U-shaped column tube is provided with a collecting port, and a switch is arranged on the collecting port;

the absorbent cotton ball is arranged in the collecting port;

the collecting pipe is positioned below the collecting opening.

2. The sephadex chromatography column of claim 1, further comprising a fixation bracket, a collar, a linkage, and a fixation screw;

the ring sleeve is arranged on the outer wall of the U-shaped column pipe;

one end of the connecting rod is connected with the ring sleeve, and the other end of the connecting rod is fixed on the fixed support through the fixed screw.

3. The sephadex chromatographic column of claim 1 or 2, wherein the sephadex accounts for 0.15 to 0.3 times the volume of the U-shaped column tube, and the volume of the U-shaped column tube is 800 to 1200 ml.

4. A method for separating umbilical cord blood mesenchymal stem cell exosomes by using the sephadex chromatographic column as claimed in any one of claims 1 to 3, which is characterized by comprising the following steps:

(a) swelling the sephadex, and then placing the sephadex into the U-shaped column tube;

(b) adding sterilized deionized water into the U-shaped column tube for stirring, then opening the collection port switch for draining liquid, and repeating for 2-4 times;

(c) centrifuging the umbilical cord blood stem cell culture solution at a high speed, and collecting supernatant;

(d) slowly adding the supernatant into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch, collecting the discharged liquid by using the collection tube, when the liquid level is reduced to 0-0.5 cm away from the surface of the sephadex, closing the collection port switch, slowly adding the physiological saline into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch, and continuously collecting the discharged liquid, wherein the discharged liquid is collected according to 5-7 ml per tube;

(e) measuring the discharge liquid of each tube by adopting a full-wavelength ultraviolet spectrophotometer, observing by using a high-power microscope, and determining a collecting tube containing the umbilical cord blood mesenchymal stem cell exosome according to the measuring and observing results;

(f) and removing the first tube and the last tube of the collecting tube containing the umbilical cord blood mesenchymal stem cell exosomes according to the collecting time, and combining the collecting tubes in the middle to obtain the umbilical cord blood mesenchymal stem cell exosomes.

5. The method according to claim 4, wherein in the step (c), the centrifugal force rcf of the high-speed centrifugation is not less than 12000 g.

6. The method of claim 4, wherein in step (d), the volume of supernatant added is 0.3 to 0.5 times the volume of the sephadex.

7. The method according to claim 4, wherein step (e) further comprises labeling the time and order of collection tubes containing umbilical cord blood mesenchymal stem cell exosomes as a reference for subsequent processing.

8. The method of claim 4, wherein in step (a), swelling comprises:

and (3) placing the sephadex into deionized water, heating and boiling for 3-5 h.

9. The method of claim 4, further comprising:

after the sephadex is used for 3-4 times, the sephadex is placed in deionized water to be boiled at high temperature for 4-5 hours, and then the sephadex is continuously filled in a column for use.

Technical Field

The invention relates to the technical field of separation and preparation of cell exosomes, and particularly relates to a glucan gel chromatographic column and a separation method of umbilical cord blood mesenchymal stem cell exosomes.

Background

At present, the preparation technology of the umbilical cord blood mesenchymal stem cell exosome is basically obtained by separating and precipitating from a cell culture solution by ultracentrifugation, and in the process, a plurality of impurities with approximate molecular weights are precipitated along with the precipitation, so that a mixture with exosome is obtained. The exosomes obtained by the methods are not very high in purity, and the application is limited. The exosome is a saccular corpuscle with the size of about dozens of nanometers and is larger than general protein, and the exosome can be obtained by selectively separating the molecular size of a sample by using gel columns with different intercepted relative molecular Masses (MWCO). The gel column filtration method is simple and efficient, does not influence the biological activity of the exosome, and is a new method for extracting the cell exosome. The grain size of the exosome is larger than that of protein and lipid substances, and the exosome can quickly pass through the separation column; and the interaction force between the protein and lipid substances and the filler is large, and the speed of the protein and lipid substances passing through the chromatographic column is low, so that the efficient separation of the exosome and the impurities is realized, and the exosome with high purity is obtained. The technology has the advantages of simple operation, convenient use and high enrichment efficiency. However, conventional columns have stringent requirements for loading, typically within 0.5mL, otherwise the separation efficiency of exosomes is reduced. Furthermore, due to severe contamination with proteins and lipids, the column cannot be reused many times (<3 times), limiting the application of the product.

Disclosure of Invention

The invention aims to provide a sephadex chromatographic column and a separation method of an exosome of umbilical cord blood mesenchymal stem cells, the sephadex chromatographic column can be used for quickly separating the exosome, the efficiency is high, and the sephadex can be reused, so that the separation and preparation cost is reduced.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a sephadex chromatographic column, which comprises a U-shaped column tube, a cotton wool ball and a collecting tube;

the inner part of the U-shaped column tube is provided with glucan gel, the bottom of the U-shaped column tube is provided with a collecting port, and a switch is arranged on the collecting port;

the absorbent cotton ball is arranged in the collecting port;

the collecting pipe is positioned below the collecting opening.

Preferably, the sephadex chromatographic column further comprises a fixing bracket, a ring sleeve, a connecting rod and a fixing screw;

the ring sleeve is arranged on the outer wall of the U-shaped column pipe;

one end of the connecting rod is connected with the ring sleeve, and the other end of the connecting rod is fixed on the fixed support through the fixed screw.

Preferably, the volume of the sephadex accounts for 0.15-0.3 times of that of the U-shaped column tube, and the volume of the U-shaped column tube is 800-1200 ml.

The second aspect of the invention provides a method for separating the umbilical blood mesenchymal stem cell exosomes by using the sephadex chromatographic column, which comprises the following steps:

(a) swelling the sephadex, and then placing the sephadex into the U-shaped column tube;

(b) adding sterilized deionized water into the U-shaped column tube for stirring, then opening the collection port switch for draining liquid, and repeating for 2-4 times;

(c) centrifuging the umbilical cord blood stem cell culture solution at a high speed, and collecting supernatant;

(d) slowly adding the supernatant into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch, collecting the discharged liquid by using the collection tube, when the liquid level is reduced to 0-0.5 cm away from the surface of the sephadex, closing the collection port switch, slowly adding the physiological saline into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch, and continuously collecting the discharged liquid, wherein the discharged liquid is collected according to 5-7 ml per tube;

(e) measuring the discharge liquid of each tube by adopting a full-wavelength ultraviolet spectrophotometer, observing by using a high-power microscope, and determining a collecting tube containing the umbilical cord blood mesenchymal stem cell exosome according to the measuring and observing results;

(f) and removing the first tube and the last tube of the collecting tube containing the umbilical cord blood mesenchymal stem cell exosomes according to the collecting time, and combining the collecting tubes in the middle to obtain the umbilical cord blood mesenchymal stem cell exosomes.

Preferably, in the step (c), the centrifugal force rcf of the high-speed centrifugation is not less than 12000 g.

Preferably, in the step (d), the volume of the added supernatant is 0.3 to 0.5 times of the volume of the sephadex gel.

Preferably, the step (e) further comprises marking the collection time and sequence of the collection tubes containing the umbilical cord blood mesenchymal stem cell exosomes as a reference for subsequent processing.

Preferably, in the step (a), the swelling comprises:

and (3) placing the sephadex into deionized water, heating and boiling for 3-5 h.

Preferably, the method further comprises:

after the sephadex is used for 3-4 times, the sephadex is placed in deionized water to be boiled at high temperature for 4-5 hours, and then the sephadex is continuously filled in a column for use.

Compared with the prior art, the invention has the beneficial effects that at least:

the sephadex chromatographic column can be used for quickly separating to obtain exosomes, the exosomes with the volume of more than 300ml can be separated only through self gravity, vacuum extraction is not needed, the operation is simple, the efficiency is high, and in addition, the sephadex can be repeatedly used, so that the separation and preparation cost is reduced.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a schematic diagram of a Sephadex chromatographic column provided by an embodiment of the invention;

in the drawings, there is shown in the drawings,

1-U-shaped column tube; 2-absorbent cotton balls; 3-a collection tube; 4-sephadex; 5-a collection port; 6-a switch; 7-fixing the bracket; 8-ring sleeve; 9-a connecting rod; 10-fixing screws.

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

Example 1

In this embodiment, the sephadex chromatographic column is shown in fig. 1, and comprises a U-shaped column tube 1, a cotton wool ball 2, a collection tube 3, a fixing bracket 7, a ring sleeve 8, a connecting rod 9 and a fixing screw 10;

the inner part of the U-shaped column tube 1 is provided with the sephadex 4, the bottom of the U-shaped column tube is provided with a collecting port 5, and the collecting port 5 is provided with a switch 6;

the absorbent cotton ball 2 is arranged in the collecting opening 5;

the collecting pipe 3 is positioned below the collecting port 5;

the ring sleeve 8 is arranged on the outer wall of the U-shaped column tube 1;

one end of the connecting rod 9 is connected with the ring sleeve 8, and the other end is fixed on the fixed support 7 through a fixed screw 10.

The sephadex chromatographic column can be used for quickly separating to obtain exosomes, the exosomes with the volume of more than 300ml can be separated only through self gravity, vacuum extraction is not needed, the operation is simple, the efficiency is high, and in addition, the sephadex can be repeatedly used, so that the separation and preparation cost is reduced.

In the invention, the addition amount of the sephadex 4 in the U-shaped column tube 1 is not strictly limited, and the sephadex can be added according to the actual requirement by a person skilled in the art; for example, in one embodiment, the addition amount of the sephadex 4 is any value of 0.15 to 0.3 in terms of the volume multiple of the U-shaped column tube 1; specifically, it may be 0.15, 0.2 or 0.3.

Example 2

This example is a method for separating umbilical cord blood mesenchymal stem cell exosomes by using the sephadex chromatographic column in example 1, which comprises the following steps:

(a) placing the sephadex into deionized water, heating and boiling for 5h, and placing into a U-shaped column tube;

(b) adding sterilized deionized water into the U-shaped column tube, stirring, opening a collection port switch to discharge liquid, and repeating the operation for 2 times;

(c) centrifuging the umbilical cord blood stem cell culture solution at a high speed, and collecting supernatant, wherein the centrifugal force rcf of the high-speed centrifugation is 12000 g; the granular substances and dead cells in the liquid can be effectively removed, and the impurity pollution is reduced;

(d) slowly adding the supernatant into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch, collecting the discharged liquid by using the collection tube, when the liquid level is reduced to 0.2cm away from the surface of the sephadex, closing the collection port switch, slowly adding physiological saline into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch again, and continuously collecting the discharged liquid, wherein the adding volume of the supernatant is 0.5 times of the volume of the sephadex, and the discharged liquid is collected according to 7ml per tube;

(e) measuring the discharge liquid of each tube by using a full-wavelength ultraviolet spectrophotometer, observing by using a high-power microscope, determining a collecting tube containing the exosome of the umbilical cord blood mesenchymal stem cells according to the measuring and observing results, and marking the collecting time and the sequence of the collecting tube as the reference of subsequent treatment;

(f) and removing the first tube and the last tube of the collecting tube containing the umbilical cord blood mesenchymal stem cell exosomes according to the collecting time, and combining the collecting tubes in the middle to obtain the umbilical cord blood mesenchymal stem cell exosomes.

Example 3

This example is a method for separating umbilical cord blood mesenchymal stem cell exosomes by using the sephadex chromatographic column in example 1, which comprises the following steps:

(a) placing the sephadex into deionized water, heating and boiling for 3h, and placing into a U-shaped column tube;

(b) adding sterilized deionized water into the U-shaped column tube, stirring, opening a collection port switch to discharge liquid, and repeating the operation for 4 times;

(c) centrifuging the umbilical cord blood stem cell culture solution at a high speed, and collecting supernatant, wherein the centrifugal force rcf of the high-speed centrifugation is 12000 g; the granular substances and dead cells in the liquid can be effectively removed, and the impurity pollution is reduced;

(d) slowly adding the supernatant into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch, collecting the discharged liquid by using the collection tube, when the liquid level is reduced to 0.5cm away from the surface of the sephadex, closing the collection port switch, slowly adding normal saline into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch again, and continuously collecting the discharged liquid, wherein the adding volume of the supernatant is 0.3 times of the volume of the sephadex, and the discharged liquid is collected according to 5ml per tube;

(e) measuring the discharge liquid of each tube by using a full-wavelength ultraviolet spectrophotometer, observing by using a high-power microscope, determining a collecting tube containing the exosome of the umbilical cord blood mesenchymal stem cells according to the measuring and observing results, and marking the collecting time and the sequence of the collecting tube as the reference of subsequent treatment;

(f) and removing the first tube and the last tube of the collecting tube containing the umbilical cord blood mesenchymal stem cell exosomes according to the collecting time, and combining the collecting tubes in the middle to obtain the umbilical cord blood mesenchymal stem cell exosomes.

Example 4

This example is a method for separating umbilical cord blood mesenchymal stem cell exosomes by using the sephadex chromatographic column in example 1, which comprises the following steps:

(a) placing the sephadex into deionized water, heating and boiling for 4h, and placing into a U-shaped column tube;

(b) adding sterilized deionized water into the U-shaped column tube, stirring, opening a collection port switch to discharge liquid, and repeating the operation for 3 times;

(c) centrifuging the umbilical cord blood stem cell culture solution at a high speed, and collecting supernatant, wherein the centrifugal force rcf of the high-speed centrifugation is 12000 g; the granular substances and dead cells in the liquid can be effectively removed, and the impurity pollution is reduced;

(d) slowly adding the supernatant into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch, collecting the discharged liquid by using the collection tube, when the liquid level is reduced to 0.4cm away from the surface of the sephadex, closing the collection port switch, slowly adding physiological saline into the U-shaped column tube, after the liquid level is balanced, opening the collection port switch again, and continuously collecting the discharged liquid, wherein the adding volume of the supernatant is 0.4 times of the volume of the sephadex, and the discharged liquid is collected according to 6ml per tube;

(e) measuring the discharge liquid of each tube by using a full-wavelength ultraviolet spectrophotometer, observing by using a high-power microscope, determining a collecting tube containing the exosome of the umbilical cord blood mesenchymal stem cells according to the measuring and observing results, and marking the collecting time and the sequence of the collecting tube as the reference of subsequent treatment;

(f) and removing the first tube and the last tube of the collecting tube containing the umbilical cord blood mesenchymal stem cell exosomes according to the collecting time, and combining the collecting tubes in the middle to obtain the umbilical cord blood mesenchymal stem cell exosomes.

The umbilical blood mesenchymal stem cell exosome obtained by the separation method has higher purity, can realize the separation of exosomes with the volume of more than 300ml, and has higher separation efficiency, simple operation and easy production.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.