Device for separating and enriching bone marrow stem cells in vitro

Abstract

The utility model discloses an in-vitro separation and enrichment device for bone marrow stem cells, and relates to the technical field of in-vitro separation and enrichment of stem cells. The utility model provides an in-vitro separation and enrichment device for bone marrow stem cells, which comprises a plurality of liquid storage pipes which are connected with one another and are internally provided with nylon net filter membranes with different pore diameters; the enriching structure is connected with the filtering structure, and a filter plate which is used for bearing an enriching material and is provided with holes is arranged in the enriching structure. The filter structure comprises a first liquid storage pipe, a second liquid storage pipe and a third liquid storage pipe which are detachably connected, a first filter membrane is arranged in the first liquid storage pipe, and a second filter membrane is arranged in the second liquid storage pipe; by designing the detachably connected enrichment tube, after stem cells flow into the enrichment tube, the enrichment material adsorbed with bone marrow stem cells can be conveniently taken out, and the operation is convenient.

Description

Technical Field

[0001] The utility model relates to the technical field of in vitro separation and enrichment of stem cells, in particular to a device for in vitro separation and enrichment of bone marrow stem cells. Background Art

[0002] Stem cells are primitive cells with the potential for self-renewal and multidirectional differentiation, which can differentiate into one or more cells that constitute human tissues or organs under certain conditions. Self-renewal means that stem cells can maintain the size of the stem cell population through cell division; multidirectional differentiation means that stem cells have plasticity. The in vitro separation and enrichment technology of stem cells is an important research direction in the biomedical field, and is of great significance for the further use and research of stem cells.

[0003] At present, due to the plasticity of stem cells, people often extract various stem cells for pairing in order to maintain the normal function of various tissue cells in the human body. They are often used to treat blood system diseases and tissue and organ transplantation. In the process of stem cell extraction, a stem cell separation device is often required to separate and re-extract the stem cells in the plasma. Traditional stem cell separation and enrichment methods usually have problems such as incomplete separation, poor enrichment effect, and cumbersome operation. It is often impossible to completely separate stem cells from other cells, resulting in poor enrichment effect. The extracted stem cells are not pure enough and it is difficult to meet the needs of clinical treatment and research. Some methods require multiple steps, which are cumbersome, not conducive to batch processing, and not easy to promote.

[0004] After searching, the Chinese patent with the announcement number CN213835299U proposes a stem cell filtration separator, including a base, the upper surface of the base is rotatably connected with a first liquid storage tube, the outer wall of the first liquid storage tube is fixedly connected with a first bevel gear, the upper surface of the base is fixedly connected with a motor, the output shaft of the motor is fixedly connected with a second bevel gear, the second bevel gear is meshed with the first bevel gear, the upper end of the first liquid storage tube is rotatably connected with a sealing cover, the end surface of the sealing cover is respectively penetrated with a first sampling tube and a second sampling tube, the first sampling tube and the second sampling tube are respectively movably connected with the sealing cover, and the first liquid collection tube and the second liquid collection tube are arranged on the sealing cover, so that the first liquid collection tube and the second liquid collection tube can slide up and down, so that after the blood is centrifuged and layered in the first liquid storage tube, the positions of the first liquid collection tube and the second liquid collection tube can be adjusted to absorb liquids of different layers, thereby simplifying the operation. However, the device is relatively complicated, and centrifugation is required when removing plasma and red blood cells, which is inconvenient to operate. Utility Model Content

[0005] In view of the above-mentioned problems, the utility model aims to provide a device for in vitro separation and enrichment of bone marrow stem cells, which can accurately separate stem cells from other cells to make the obtained stem cells relatively pure; at the same time, it has a simple structure, is easy to operate, and is easy to promote.

[0006] The main idea of ​​the technical solution adopted by the utility model is as follows: by designing a three-section series-connected filtering structure, the blood to be separated passes through a double-layer filter membrane, and tissues of different diameters are respectively located in different structures; an enrichment structure is connected to the second filtering structure, which can adsorb and enrich bone marrow stem cells for subsequent extraction or direct use; by designing a detachably connected enrichment tube, after the stem cells flow into the enrichment tube, the enrichment material adsorbed with the bone marrow stem cells can be easily taken out, and the operation is convenient, which can improve efficiency.

[0007] In order to achieve the above purpose, the technical solution adopted by the utility model is as follows:

[0008] A device for separating and enriching bone marrow stem cells in vitro, comprising:

[0009] The filtering structure comprises a plurality of liquid storage tubes, the liquid storage tubes are connected to each other and nylon mesh filter membranes with different pore sizes are arranged in the liquid storage tubes;

[0010] The enrichment structure is connected to the filtering structure and is internally provided with a filter plate with holes for carrying the enrichment material.

[0011] Through the above technical solution, further: the filtering structure includes a first liquid storage tube, a second liquid storage tube and a third liquid storage tube that are detachably connected, a first filter membrane is arranged in the first liquid storage tube, and a second filter membrane is arranged in the second liquid storage tube.

[0012] Through the above technical solution, further: the pore size of the first filter membrane is 20 μm, and the pore size of the second filter membrane is 5 μm.

[0013] Through the above technical solution, further: the enrichment structure is detachably connected to the second liquid storage tube, and a valve three is provided at the connection.

[0014] Through the above technical solution, further: a piston 1 is slidably connected in the third liquid storage tube, and an operating rod 1 is fixedly connected to the piston 1.

[0015] The beneficial effects of the utility model are:

[0016] 1. By designing a three-section filtration structure in series, the blood to be separated passes through the double-layer filter membrane in sequence, and blood components of different diameters are located in different structures;

[0017] 2. By designing a fourth liquid storage tube for enrichment connected to the second filter structure, bone marrow stem cells can be adsorbed and enriched, which is convenient for subsequent extraction or direct use;

[0018] 3. By designing a detachable enrichment tube, after the stem cells flow into the enrichment tube, the enrichment material adsorbed with bone marrow stem cells can be easily taken out, and the operation is convenient;

[0019] 4. This device can simultaneously complete the separation and enrichment of stem cells, which is convenient for subsequent extraction, and has a simple structure, making it easy to extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the three-dimensional structure of a device for in vitro separation and enrichment of bone marrow stem cells according to the utility model;

[0021] Figure 2 It is a three-dimensional structural schematic diagram of the base and the support frame of the utility model;

[0022] Figure 3 It is a front view schematic diagram of the first liquid storage tube of the utility model;

[0023] Figure 4 This is a front view structural diagram of the second liquid storage tube of the utility model;

[0024] Figure 5 This is a schematic diagram of the front view structure of the third liquid storage tube of the utility model;

[0025] Figure 6 This is a front view structural diagram of the fourth liquid storage tube of the utility model;

[0026] Figure 7 This is a front view structural diagram of the fourth liquid storage tube and the second liquid storage tube of the utility model after installation;

[0027] Wherein: 1. first liquid storage tube; 101. liquid inlet tube 1; 102. liquid outlet tube 1; 103. first filter membrane; 104. valve 1; 105. filter tube 1; 2. second liquid storage tube; 201. liquid inlet tube 2; 202. liquid outlet tube 2; 203. second filter membrane; 204. valve 2; 205. filter tube 2; 3. third liquid storage tube; 301. liquid inlet tube 3; 302. piston 1; 303. operating rod 1; 304. filter tube 3; 4. fourth liquid storage tube; 401. liquid inlet tube 4; 402. connecting tube; 403. enrichment tube; 404. operating tube; 405. valve 3; 406. piston 2; 407. operating rod 2; 408. filter plate; 5. heparin cap; 6. base; 7. support frame; 701. telescopic rod; 702. arc support plate. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0029] The inventors have found that, at present, due to the plasticity of stem cells, people often extract various stem cells for pairing in order to maintain the normal function of various tissue cells in the human body, which is often used to treat blood system diseases and tissue organ transplantation. In the process of stem cell extraction, a stem cell separation device is often required to separate and re-extract the stem cells in the plasma. Traditional stem cell separation and enrichment methods usually have problems such as incomplete separation, poor enrichment effect, and cumbersome operation. It is often impossible to completely separate stem cells from other cells, resulting in poor enrichment effect. The extracted stem cells are not pure enough and it is difficult to meet the needs of clinical treatment and research. Some methods require multiple steps of operation, which are cumbersome, not conducive to batch processing, and not easy to promote.

[0030] Based on the above findings, the present application proposes a device for in vitro separation and enrichment of bone marrow stem cells. By designing a three-section series-connected filtering structure, the blood to be separated can be filtered through a double layer, and cell tissues of different diameters are located in different structures respectively; an enrichment structure is connected to the second-section filtering structure, which can adsorb and enrich bone marrow stem cells for subsequent extraction or direct use; by designing a detachable enrichment tube, after the stem cells flow into the enrichment tube, the enrichment material adsorbed with bone marrow stem cells can be easily removed, and the operation is convenient. Embodiment 1

[0031] See also Figure 1-Figure 5 The present application discloses a device for in vitro separation and enrichment of bone marrow stem cells, including a first liquid storage tube 1, the first liquid storage tube 1 includes a filter tube 105 with a hollow lumen, and a liquid inlet tube 101 and a liquid outlet tube 102 are arranged at both ends. The liquid inlet tube 101 is provided with an internal thread, and the liquid inlet tube 101 is threadedly connected with a heparin cap 5. When injecting blood to be separated, a syringe is used to penetrate the rubber head of the heparin cap 5, and the syringe piston is pushed to inject the blood into the first liquid storage tube 1, which can prevent the liquid to be separated from contacting with the air, causing contamination and affecting the quality of stem cell separation. In addition, the heparin cap 5 can be punctured multiple times and repeatedly, and it only needs to be disinfected before each operation.

[0032] The second liquid storage tube 2 includes a filter tube 205 with a hollow lumen, with a liquid inlet tube 201 and a liquid outlet tube 202 provided at both ends. The liquid inlet tube 201 is provided with an internal thread, and the liquid outlet tube 102 of the first liquid storage tube 1 is provided with an external thread on the outside, which is threadedly connected to the liquid inlet tube 201, so that the first liquid storage tube 1 is connected to the second liquid storage tube 2, and the substance in the first liquid storage tube 1 can move to the second liquid storage tube 2.

[0033] The third liquid storage tube 3 includes a filter tube 3 304 with a hollow lumen, one end of which is provided with a liquid inlet tube 3 301, the liquid inlet tube 3 301 is provided with an internal thread, and the liquid outlet tube 2 202 of the second liquid storage tube 2 is provided with an external thread on the outside, which is threadedly connected with the liquid inlet tube 3 301, so that the second liquid storage tube 2 is connected to the third liquid storage tube 3, and the material in the second liquid storage tube 2 can move to the third liquid storage tube 3. The other end of the filter tube 3 304 is slidably connected with a piston 1 302, the piston 1 302 is located inside the filter tube 3 304, and the piston 1 302 is fixedly connected with an operating rod 1 303. By pulling the operating rod 1 303, the position of the piston 1 302 can be adjusted, and the air pressure inside the filter tube 3 304 can be adjusted at the same time. When the first liquid storage tube 1, the second liquid storage tube 2 and the third liquid storage tube 3 are all kept in a connected state, the air pressure in the entire tube cavity can be adjusted by pulling the operating rod 303, causing the material in the first liquid storage tube 1 to flow to the second liquid storage tube 2, and the material in the second liquid storage tube 2 to flow to the third liquid storage tube 3.

[0034] In order to facilitate the use of the device, a base 6 and three support frames 7 are designed, and the liquid storage tubes are placed on the support frames 7 respectively. The support frames 7 are fixedly connected to the base 6. The support frames 7 include a telescopic rod 701 and an arc-shaped support plate 702. A rubber layer is provided inside the arc-shaped support plate 702 to increase friction and prevent the liquid storage tubes from detaching from the support frame 7. In order to facilitate observation, the first liquid storage tube 1, the second liquid storage tube 2 and the third liquid storage tube 3 are all made of transparent material. Embodiment 2

[0035] See also Figure 3-Figure 4 The first filter membrane 103 is disposed inside the outlet pipe 102 of the first liquid storage tube 1. The first filter membrane 103 is a nylon mesh filter membrane with a pore size of 20 μm. Cells and other tissues larger than 20 μm cannot pass through, and cells and other tissues smaller than 20 μm flow toward the second liquid storage tube 2 and undergo the next round of filtration. A valve 104 is also disposed inside the outlet pipe 102 of the first liquid storage tube 1. By adjusting the switch of the valve 104, it is possible to adjust whether the substance in the first liquid storage tube 1 flows out, thereby operating each step according to the experimental requirements.

[0036] In order to remove red blood cells in the blood, ACK red blood cell lysis solution is injected into the first storage tube 1 through the heparin cap 5, the volume ratio of blood to ACK red blood cell lysis solution is 1:3, and the blood and ACK lysis buffer are incubated at room temperature for 3-5 minutes. During this incubation process, red blood cell lysis should be clearly visible, and the red blood cells in the blood can be lysed to less than 5μm.

[0037] A second filter membrane 203 is arranged inside the second outlet tube 202 of the second liquid storage tube 2. The second filter membrane 203 is made of a nylon mesh filter membrane with a pore size of 5μm. Cells and other tissues larger than 5μm cannot pass through, and cells and other tissues smaller than 5μm flow toward the third liquid storage tube 3 and undergo the next round of filtration. A valve 204 is also arranged inside the second outlet tube 202 of the second liquid storage tube 2. By adjusting the switch of the valve 204, it is possible to adjust whether the substances in the second liquid storage tube 2 flow out, so as to operate each step according to the experimental requirements. The plasma, white blood cells and lysed red blood cells in the second liquid storage tube 2 can pass through the second filter membrane 203 and flow to the third liquid storage tube 3, and the stem cells are retained in the second liquid storage tube 2.

[0038] Through double-layer filtration, cells larger than 20 μm and smaller than 5 μm and other blood components are isolated into the first liquid storage tube 1 and the third liquid storage tube 3 respectively. Since the diameter of bone marrow stem cells is in the range of 5 μm-20 μm, bone marrow stem cells remain in the second liquid storage tube 2, and the bone marrow stem cells are completely separated from other cells, thereby improving the separation efficiency. Embodiment 3

[0039] refer to Figure 6-Figure 7 The outer surface of the second liquid storage tube 2 is provided with a threaded hole, which is threadedly connected with the liquid inlet tube 401 of the fourth liquid storage tube 4. A valve 3 405 is arranged inside the liquid inlet tube 401. By adjusting the switch of the valve 3 405, it is possible to adjust whether the material in the fourth liquid storage tube 4 flows in or not. For easy observation, the fourth liquid storage tube 4 is made of transparent material.

[0040] The fourth liquid storage tube 4 also includes a connecting tube 402, an enrichment tube 403 and an operating tube 404, all of which are hollow tubular structures. The connecting tube 402 is fixedly connected to the liquid inlet tube 401, and the connecting tube 402 has a larger diameter. The end of the connecting tube 402 away from the liquid inlet tube 401 is provided with a thread, which is threadedly connected to the enrichment tube 403. The other end of the enrichment tube 403 is threadedly connected to the operating tube 404. The operating tube 404 is slidably connected with a piston 2 406, which is located inside the tube cavity of the third liquid storage tube 3. The piston 2 406 is fixedly connected with an operating rod 2 407. By pulling and pulling the operating rod 2 407, the position of the piston 2 406 can be adjusted, and the air pressure inside the fourth liquid storage tube 4 can be adjusted at the same time.

[0041] A filter plate 408 with holes is provided in the enrichment tube 403, and the pore size is 5 microns, which can filter substances below 5 μm. Substances smaller than 5 μm in the second liquid storage tube 2 but not entering the third liquid storage tube 3 can be filtered into the operation tube 404 through the filter plate 408, so that the enriched bone marrow stem cells are purer. The filter plate 408 is fixedly connected to the enrichment tube 403, and a DBM enrichment material for enriching bone marrow stem cells is placed on the filter plate 408. DBM is Demineralized Bone Matrix, and this material is used to enrich bone marrow stem cells in this embodiment. In addition, DBM can also be replaced with other materials that can absorb stem cells, such as gelatin sponge, hydrogel or other porous nano-biomaterials.

[0042] After the valve 403 is opened, the stem cell suspension retained in the second liquid storage tube 2 enters the fourth liquid storage tube 4, the bone marrow stem cells remain on the enrichment material in the enrichment tube 403, and other substances enter the operating tube 404 through the filter plate 408 and are discharged along the stem cell outlet tube. By pulling the second operating rod 407, the position of the second piston 406 can be adjusted, and the air pressure inside the fourth liquid storage tube 4 can be adjusted at the same time, so that the residual substances in the second liquid storage tube 2 enter the fourth liquid storage tube 4 under the action of pressure.

[0043] Application Cases

[0044] The utility model can be used for in vitro separation and enrichment of bone marrow stem cells in blood, and its specific use process is as follows:

[0045] (1) Close valve 104 and valve 3 405, penetrate the rubber head of the heparin cap 5 with a syringe, and push the syringe piston to inject blood and ACK red blood cell lysis solution into the first liquid storage tube 1 in a ratio of 1:3.

[0046] (2) Incubate the blood sample with ACK lysis buffer at room temperature for 3-5 minutes. During this incubation process, red blood cell lysis should be clearly visible. In order to improve the lysis efficiency, the first storage tube 1 can be manually shaken to allow the red blood cell lysis solution to fully contact the blood. The red blood cell lysis solution lyses the red blood cells in the blood into cells smaller than 5μm.

[0047] (3) Open valve 104 to allow the substance in the first liquid storage tube 1 to flow into the second liquid storage tube 2 through the first filter membrane 103. Cells and other tissues larger than 20 μm in the first liquid storage tube 1 are retained, while the others flow into the second liquid storage tube 2. The cells in the second liquid storage tube 2 are all smaller than 20 μm. When the flow speed is too slow, the internal air pressure can be adjusted by pulling out the operating rod 303 to promote the substance in the first liquid storage tube 1 to flow into the second liquid storage tube 2.

[0048] (4) Continue to pull the operating rod 303 outward, and the material in the second liquid storage tube 2 flows into the third liquid storage tube 3 through the second filter membrane 203. The cells and other tissues larger than 5 μm in the second liquid storage tube 2 are retained, and the cells that flow into the second liquid storage tube 2 are all smaller than 5 μm. The cells with a diameter between 5 μm and 20 μm are retained in the second liquid storage tube 2. The diameter of bone marrow stem cells is within this range, and the bone marrow stem cells are completely separated from other cells, thereby improving the separation efficiency.

[0049] (5) Close valve 104 and valve 204, open valve 3 405, and the bone marrow stem cells in the second liquid storage tube 2 are enriched through the fourth liquid storage tube 4 at the bottom. The fourth liquid storage tube 4 is initially in a closed state. After the filtration is completed, open valve 3 405 to allow the residual cells in the second liquid storage tube 2 to flow into the fourth liquid storage tube 4 through the liquid inlet tube 4 401 and through the enrichment tube 403. The bone marrow stem cells are adsorbed on the enrichment material through the enrichment effect, and other components flow into the operation tube 404 through the small holes in the enrichment tube 403.

[0050] (6) The operation tube 404 and the enrichment tube 403 are separated from the device in turn, and the enriched material can be taken out from the enrichment tube 403, and then bone marrow stem cells can be extracted from the enriched material. Alternatively, the bone marrow stem cell-enriched material can be removed from the enrichment device and directly used for local implantation.

[0051] The above shows and describes the basic principle, main features and advantages of the utility model. Those skilled in the art should understand that the utility model is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principle of the utility model. Without departing from the spirit and scope of the utility model, the utility model may have various changes and improvements, which fall within the scope of the utility model to be protected. The scope of protection of the utility model is defined by the attached claims and their equivalents.

Claims

1. A device for separating and enriching bone marrow stem cells in vitro, characterized in that: include: The filtering structure comprises a plurality of liquid storage tubes, the liquid storage tubes are connected to each other and filter membranes with different pore sizes are arranged in the liquid storage tubes; An enrichment structure connected to the filtration structure and having a filter plate (408) with holes therein for carrying the enrichment material; The filtering structure comprises a first liquid storage tube (1), a second liquid storage tube (2) and a third liquid storage tube (3) which are detachably connected, a first filter membrane (103) being arranged in the first liquid storage tube (1), and a second filter membrane (203) being arranged in the second liquid storage tube (2).

2. The device for in vitro separation and enrichment of bone marrow stem cells according to claim 1, characterized in that: The pore size of the first filter membrane (103) is 20 μm, and the pore size of the second filter membrane (203) is 5 μm.

3. The device for in vitro separation and enrichment of bone marrow stem cells according to claim 2, characterized in that: The enrichment structure is detachably connected to the second liquid storage tube (2), and a third valve (405) is provided at the connection.

4. The device for in vitro separation and enrichment of bone marrow stem cells according to claim 3, characterized in that: A piston 1 (302) is slidably connected inside the third liquid storage tube (3), and an operating rod 1 (303) is fixedly connected to the piston 1 (302).

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