Extraction method for primary tissue cell

Abstract

Disclosed in the present invention is an extraction method for a primary tissue cell, comprising the following steps: acquiring an organ tissue; then using mutual rotation motion of two grinding planes each having a rectangular pyramid-like surface microstructure to shear the organ tissue; using a digestive enzyme solution to digest the sheared organ tissue; then adding fetal bovine serum to terminate digestion; carrying out centrifugation for the first time to remove a supernatant; and adding a red blood cell lysis buffer, carrying out dispersion by blowing and carrying out uniform mixing, and then carrying out centrifugation again to remove a supernatant to obtain an extracted primary cell. According to the present invention, the mutual rotation motion of two grinding planes each having a prismatic microstructure is used to shear the organ tissue, and the tissue is sheared by a shearing force generated when the prismatic microstructures of the two grinding planes are staggered, so that the extrusion damage effect on tissue cells when a soft tissue is cut can be reduced; moreover, continuous shearing is carried out by means of a plurality of prismatic microstructures in an annular array, so that the shearing effect of a tissue is improved, more favorable conditions are created for cell extraction, the extracted cells are high in viability and good in activity, and the extraction amount is high.

Description

A method for extracting primary tissue cells Technical Field

[0001] The present invention relates to the technical field of tissue cell extraction, and in particular to a method for extracting primary tissue cells. Background Art

[0002] Driven by the current trend of precision medicine, personalized treatment is gradually becoming the trend of precision treatment. However, extracting specific primary tissue cells from patient organs and tissues has always been a difficult problem.

[0003] Existing primary tissue cell extraction methods use most rotors to centrifuge and disrupt tissues, which results in a high degree of cell damage and low primary cell extraction efficiency. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a method for extracting primary tissue cells to solve the problem of low efficiency of primary cell extraction in the prior art.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A method for extracting primary tissue cells comprises the following steps: obtaining organ tissue, then using two grinding planes with prismatic microstructures to rotate relative to each other to shear the organ tissue, digesting the sheared organ tissue with a digestive enzyme solution, then adding fetal bovine serum to terminate the digestion, performing a first centrifugation to remove the supernatant, then adding red blood cell lysis solution, blowing and mixing, and then centrifuging again to remove the supernatant to obtain extracted primary cells.

[0007] As a further improvement of the above technical solution, the two grinding planes respectively include a rotating bottom surface and a rotating top surface, and the rotating bottom surface and the rotating top surface are rotatably connected via an axial hole connection portion. The rotating bottom surface is provided with a first shearing block having a triangular prism structure, and the rotating top surface is provided with a second shearing block having a quadrangular pyramid structure. When the rotating bottom surface and the rotating top surface rotate relative to each other, the first shearing block and the second shearing block are staggered to have a shearing force, and the organ tissue is sheared by the shearing force.

[0008] As a further improvement of the above technical solution, there are multiple first shear blocks on a radius of the rotating bottom surface, and the multiple first shear blocks are respectively arranged in a circular array with the center of the rotating bottom surface; there are multiple second shear blocks on a radius of the rotating top surface, and the multiple second shear blocks are respectively arranged in a circular array with the center of the rotating top surface.

[0009] As a further improvement of the above technical solution, the rotating bottom surface is divided into multiple annular shearing areas according to the radius size, the number of first shearing block annular arrays in different shearing areas is different, and the number of first shearing block annular arrays in the same shearing area is different; the rotating top surface is also divided into multiple annular shearing areas according to the radius size, the number of second shearing block annular arrays in different shearing areas is different, and the number of second shearing block annular arrays in the same shearing area is different.

[0010] As a further improvement of the above technical solution, the size of the first shearing block in the same shearing area on the rotating bottom surface gradually increases along the direction of increasing radius; the size of the second shearing block in the same shearing area on the rotating top surface gradually increases along the direction of increasing radius.

[0011] As a further improvement of the above technical solution, the digestive enzyme solution comprises DMEMF12, Collagenase I, Collagenase IV, Dispase and DNase.

[0012] As a further improvement of the above technical solution, the concentration of Collagenase I in the digestive enzyme solution is 2 mg / ml, the concentration of Collagenase IV is 2 mg / ml, the concentration of Dispase is 2 mg / ml, and the concentration of DNase is 1500 units / ml.

[0013] As a further improvement of the above technical solution, the chopped organ tissue is digested in a digestive enzyme solution on a shaking platform at 37° C. for 15 minutes, and then fetal bovine serum is added to terminate the digestion.

[0014] As a further improvement of the above technical solution, the parameters for centrifugal removal of the supernatant are: speed 1000 rpm, time 5 min.

[0015] As a further improvement of the above technical solution, the volume of the added red blood cell lysis solution is 3 times the volume of the remaining precipitate after removing the supernatant.

[0016] The beneficial effects of the present invention are: organ tissue is sheared by the mutual rotation of two grinding planes with prismatic microstructures, and the tissue is sheared by the shear force generated when the prismatic microstructures in the two grinding planes intersect, which can reduce the squeezing and damaging effects on tissue cells when cutting soft tissue. At the same time, the continuous shearing of multiple prismatic microstructures in the annular array improves the shearing effect of the tissue, creating more favorable conditions for cell extraction. Therefore, the present invention has a high extraction rate for primary cells extracted from organ tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below with reference to the accompanying drawings and examples.

[0018] FIG1 is a schematic diagram of the assembly of two grinding planes in the present invention;

[0019] FIG2 is a cross-sectional view of the structure of FIG1;

[0020] 3-5 are respectively a perspective view, a top view and a cross-sectional view of the rotating top surface of the present invention;

[0021] 6-8 are respectively a perspective view, a top view and a cross-sectional view of the rotating bottom surface of the present invention;

[0022] FIG. 9 is a schematic diagram showing the positions of the first shear block and the second shear block in the present invention.

[0023] FIG10 is a fluorescent image of tissue extracted live and dead tissues according to Example 1 of the present invention;

[0024] FIG11 is a diagram showing the primary tissue cell extraction test results of Example 1 of the present invention;

[0025] FIG12 is a fluorescent image of tissue extraction of dead or alive tissue according to Example 2 of the present invention;

[0026] FIG13 is a diagram showing the primary tissue cell extraction and detection results of Example 2 of the present invention.

[0027] Reference numerals: 10, rotating bottom surface; 11, first shearing block; 12, rotating hole; 20, rotating top surface; 21, second shearing block; 22, rotating axis. Implementation Method

[0028] The following will clearly and completely describe the concept, specific structure and technical effects of the present invention in combination with the embodiments and drawings, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In addition, all the connection / connection relationships involved in the patent do not refer to the direct connection of components, but refer to the fact that a better connection structure can be formed by adding or reducing connection accessories according to the specific implementation situation. The various technical features in the invention can be combined interactively without conflicting with each other.

[0029] A method for extracting primary tissue cells comprises the following steps: obtaining organ tissue, then using two grinding surfaces with prismatic microstructures to rotate relative to each other to shear the organ tissue, digesting the sheared organ tissue with a digestive enzyme solution on a shaker at 37°C for 15 minutes, then adding fetal bovine serum (FBS) to terminate the digestion, performing a first centrifugation at a speed of 1000 rpm for 5 minutes to remove the supernatant, then adding a red blood cell lysis solution with a volume three times that of the remaining sediment volume, blowing the mixture to disperse and mix, and then centrifuging again at a speed of 1000 rpm for 5 minutes to remove the supernatant, thereby obtaining extracted primary cells.

[0030] The components of the digestive enzyme solution are: DMEMF12 cell culture medium, Collagenase I (collagenase I) at a concentration of 2 mg / ml, Collagenase IV (collagenase IV), Dispase (dispersase) and DNase (deoxyribonuclease) at a concentration of 1500 units / ml.

[0031] 1 and 2 , the two grinding surfaces in the above embodiment respectively include a rotating bottom surface 10 and a rotating top surface 20. The rotating bottom surface 10 and the rotating top surface 20 are rotatably connected via an axial-hole connecting portion. The axial-hole connecting portion includes a rotating hole 12 provided at the center of the rotating bottom surface 10 and a rotating shaft 22 provided at the center of the rotating top surface 20. When the rotating shaft 22 is located in the rotating hole 12 and rotates, the rotating top surface 20 and the rotating bottom surface 10 rotate relative to each other.

[0032] Specifically, referring to Figures 3 to 5, several first shear blocks 11 of triangular prism structure are provided on the rotating bottom surface 10, and there are multiple first shear blocks 11 on a radius of the rotating bottom surface 10, and the multiple first shear blocks 11 are respectively arranged in a circular array with the center of the rotating bottom surface 10; in addition, the rotating bottom surface 10 is divided into multiple circular shearing areas according to the radius size, and the number of circular arrays of first shear blocks 11 in different shearing areas is different, and the number of circular arrays of first shear blocks 11 in the same shearing area is different; further, the size of the first shear block 11 in the same shearing area on the rotating bottom surface 10 gradually increases along the direction of increasing radius.

[0033] 6-8 , the rotating top surface 20 is provided with a plurality of second shear blocks 21 of quadrangular pyramid structure, and there are multiple second shear blocks 21 on a radius of the rotating top surface 20, and the multiple second shear blocks 21 are respectively arranged in a circular array with the center of the rotating top surface 20; in addition, the rotating top surface 20 is also divided into multiple circular shearing areas according to the radius size, and the number of circular arrays of the second shear blocks 21 in different shearing areas is different, and the number of circular arrays of the second shear blocks 21 in the same shearing area is different; further, the size of the second shear blocks 21 in the same shearing area on the rotating top surface 20 gradually increases along the direction of increasing radius.

[0034] Referring to Figure 9 , the first shearing blocks 11 and the second shearing blocks 21 are interlaced. When the rotating bottom surface 10 and the rotating top surface 20 rotate relative to each other, the first shearing blocks 11 and the second shearing blocks 21 interlock and move, thereby generating a shearing force that shreds organ tissue. In this embodiment, by providing different shearing areas, numbers, and sizes of the first shearing blocks 11 and the second shearing blocks 21, the shearing effect of soft tissue can be further improved, thereby facilitating the extraction of primary tissue cells.

[0035] In Example 1, organ tissue (weighing 0.038 g) was prepared, and primary cells were extracted using the primary cell extraction method of the present invention. Then, calcein-AM and propidium iodide (PI) solutions were used to stain live cells and dead cells, respectively, at 37° C. for 15 minutes. The fluorescent image after staining is shown in FIG10 , and the extracted primary cell parameters are shown in FIG11 . Finally, the data of the extracted primary cells were measured as follows:

[0036] Total cell count: 3.36x10^5 / ml;

[0037] Viable cell count: 1.84x10^5 / ml;

[0038] Total cell concentration: 3.36*10^6 / ml;

[0039] Viable cell concentration: 1.84*10^6 / ml;

[0040] Cell viability: 54.80%;

[0041] Average diameter: 15.66 μm;

[0042] Average roundness: 0.70;

[0043] Agglomeration rate: 31.42%;

[0044] Live cell extraction rate: 1.84x10^5 / 0.038=4.842x10^6 unit / g.

[0045] In Example 2, organ tissue (weighing 0.036 g) was prepared, and primary cells were extracted using an existing method (wherein the tissue was broken by manual shearing or centrifugation, and the remaining steps were the same). Then, calcein-AM and propidium iodide (PI) solutions were used to stain live cells and dead cells, respectively, at 37° C. for 15 minutes. The fluorescent image after staining is shown in FIG12 , and the parameters of the extracted primary cells are shown in FIG13 . Finally, the data of the extracted primary cells were measured as follows:

[0046] Total cell count: 7.36x10^4 / ml;

[0047] Viable cell count: 5.17x10^4 / ml;

[0048] Total cell concentration: 7.36*10^5 / ml;

[0049] Viable cell concentration: 5.17*10^5 / ml;

[0050] Cell viability: 70.32%;

[0051] Average diameter: 15.39 μm;

[0052] Average roundness: 0.75;

[0053] Agglomeration rate: 19.79%;

[0054] Live cell extraction rate: 5.17x10^4 / 0.036=1.436x10^6 unit / g.

[0055] It can be clearly seen from the comparison of the data of Example 1 and Example 2 that the extraction rate of primary tissue cells extracted by the method of the present invention is high.

[0056] The above is a specific description of the preferred implementation of the present invention, but the invention is not limited to the embodiments. Those skilled in the art can make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for extracting primary tissue cells, characterized in that: The following steps are involved: The organ tissue is obtained, and then two grinding planes with prismatic microstructures are used to rotate relative to each other to cut the organ tissue into pieces. The cut organ tissue is digested with a digestive enzyme solution, and then fetal bovine serum is added to terminate the digestion. The supernatant is removed by the first centrifugation, and then red blood cell lysis solution is added. After blowing and mixing, the supernatant is removed by centrifugation again to obtain the extracted primary cells.

2. The method for extracting primary tissue cells according to claim 1, characterized in that: The two grinding planes respectively include a rotating bottom surface and a rotating top surface, and the rotating bottom surface and the rotating top surface are rotatably connected via an axial hole connecting portion. The rotating bottom surface is provided with a plurality of first shearing blocks of triangular prism structures, and the rotating top surface is provided with a plurality of second shearing blocks of quadrangular pyramid structures. When the rotating bottom surface and the rotating top surface rotate relative to each other, the first shearing blocks and the second shearing blocks are staggered to have shearing force, and the organ tissue is sheared through the shearing force.

3. The method for extracting primary tissue cells according to claim 2, characterized in that: There are multiple first shear blocks on a radius of the rotating bottom surface, and the multiple first shear blocks are respectively arranged in a circular array with the center of the rotating bottom surface; there are multiple second shear blocks on a radius of the rotating top surface, and the multiple second shear blocks are respectively arranged in a circular array with the center of the rotating top surface.

4. The method for extracting primary tissue cells according to claim 3, characterized in that: The rotating bottom surface is divided into multiple annular shearing areas according to the radius size, and the number of first shearing block annular arrays in different shearing areas is different, and the number of first shearing block annular arrays in the same shearing area is different; the rotating top surface is also divided into multiple annular shearing areas according to the radius size, and the number of second shearing block annular arrays in different shearing areas is different, and the number of second shearing block annular arrays in the same shearing area is different.

5. The method for extracting primary tissue cells according to claim 4, characterized in that: The size of the first shearing block in the same shearing area on the rotating bottom surface gradually increases along the direction of increasing radius; the size of the second shearing block in the same shearing area on the rotating top surface gradually increases along the direction of increasing radius.

6. The method for extracting primary tissue cells according to claim 1, characterized in that: The digestive enzyme solution comprises DMEMF12, Collagenase I, Collagenase IV, Dispase and DNase.

7. The method for extracting primary tissue cells according to claim 6, characterized in that: In the digestive enzyme solution, the concentration of Collagenase I is 2 mg / ml, the concentration of Collagenase IV is 2 mg / ml, the concentration of Dispase is 2 mg / ml, and the concentration of DNase is 1500 unit / ml.

8. The method for extracting primary tissue cells according to claim 7, characterized in that: The minced organ tissues were digested in a digestive enzyme solution on a shaker at 37°C for 15 min, and then fetal bovine serum was added to terminate the digestion.

9. The method for extracting primary tissue cells according to claim 1, characterized in that: The parameters for centrifugation to remove the supernatant were: speed 1000 rpm, time 5 min.

10. The method for extracting primary tissue cells according to claim 1, characterized in that: The volume of red blood cell lysis buffer added was 3 times the volume of the remaining precipitate after removing the supernatant.

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