CN116240094B - Separation device and SVF preparation method - Google Patents
Separation device and SVF preparation method Download PDFInfo
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- CN116240094B CN116240094B CN202310508070.7A CN202310508070A CN116240094B CN 116240094 B CN116240094 B CN 116240094B CN 202310508070 A CN202310508070 A CN 202310508070A CN 116240094 B CN116240094 B CN 116240094B
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/10—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by centrifugation ; Cyclones
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0653—Adipocytes; Adipose tissue
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0661—Smooth muscle cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention relates to a separation device and a preparation method of SVF, wherein the separation device comprises: a transducer and at least two syringes. The converter comprises a conversion pipe and an adjusting piece, wherein the conversion pipe is provided with an adjusting section, the adjusting section is positioned between a first end of the conversion pipe and a second end of the conversion pipe, the adjusting piece is connected with the adjusting section, the adjusting piece is movable relative to the adjusting section so as to change the circulation gap of the adjusting section, and two injectors are respectively connected with the first end of the conversion pipe and the second end of the conversion pipe. The separation device provided by the embodiment of the invention has excellent separation effect and can improve the yield and the activity of SVF cells.
Description
Technical Field
The invention relates to the technical field of medical equipment, in particular to a separation device and a SVF preparation method.
Background
Adipose tissue is used as a source of adult stem cells and can be used in plastic surgery and regenerative medicine. The vascular matrix component (SVF) isolated from adipose tissue is a heterogeneous cell population, and the important components contained in the matrix vascular component (SVF) are mainly adipose mesenchymal stem cells, endothelial progenitor cells, endothelial cells, regulatory T cells, macrophages, smooth muscle cells, pericytes, preadipocytes and the like. Among the main components of SVF described above, adipose mesenchymal stem cells (ADSCs) have self-replicative capacity and multipotency, and have chemotaxis, homing, migration and low antigenicity, and can differentiate into bone cells, cartilage cells, muscle cells, hematopoietic stromal cells, nerve cells, and the like.
In the related art, the preparation process of SVF cells mainly includes an enzymatic digestion method and a mechanical emulsification method. Enzymatic digestion separates the contents into two distinct phases by digesting the fat portion of the fat aspirate with collagenase: mature adipocyte fraction and target cell fraction, and then obtaining SVF cells by centrifugation. However, the enzyme digestion method has problems of excessive treatment of tissue cells and enzyme residue clinically, and the enzyme digestion time is too long, which results in complicated operation steps and high cost and has a safety hazard.
In the mechanical emulsification method, the SVF cell yield prepared by using the Closed tube equipment of Tiryaki company is 1.34×10 6 cell/mL, SVF cell viability was 85.82%.
As an example, the Fatstem kit apparatus from CORIOS Soc.Coop, which produces SVF cells with a yield of 3X 10 4 cell/mL, SVF cell viability was 52.00%.
Taking myStem equipment from myStem LLC as an example, SVF cells were prepared with a yield of 8X 10 3 cell/mL, SVF cell viability was 43.00%.
Among them, most of the fat converters in the mechanical emulsification method are designed with hollow blades or fine wire mesh structures, and the extracted fat extract and fat cells are usually pushed back and forth by means of a syringe and broken, thinned and emulsified by passing through the hollow blades or fine wire mesh holes of the fat converters. However, while adipose tissues and adipocytes are crushed, refined and emulsified, SVF cells entrapped within the adipose particles and dispersed among the adipocytes are also chopped or crushed together, thus resulting in low SVF cell yield and poor cell viability.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent.
Therefore, the embodiment of the invention provides a separation device which can improve the yield and the activity of SVF cells and has excellent separation effect.
The embodiment of the invention provides a preparation method of SVF.
The separation device of the embodiment of the invention comprises: a converter comprising a conversion tube having an adjustment section between a first end of the conversion tube and a second end of the conversion tube, and an adjustment member connected to the adjustment section, the adjustment member being movable relative to the adjustment section to vary a flow gap of the adjustment section; at least two syringes, two said syringes are connected to the first end of the transfer tube and the second end of the transfer tube, respectively.
According to the separating device provided by the embodiment of the invention, the fat extract to be separated can be repeatedly pushed through the converter through the two injectors, and the adjusting piece can move relative to the adjusting section to change the circulation gap of the adjusting section, so that the circulation gap of the adjusting section can be adjusted to a proper position for pushing, the crushing, thinning and emulsifying of adipose tissues and fat cells can be realized, and SVF cells can smoothly pass through the circulation gap and are not damaged. Therefore, the separation device of the embodiment of the invention can greatly improve the yield and the cell viability of SVF cells, so that the separation effect of the separation device is excellent.
In some embodiments, the adjustment member is located outside the adjustment section, the adjustment section is resilient, and the adjustment member can press against an outer wall of the adjustment section to change the flow gap of the adjustment section.
In some embodiments, the converter further comprises a housing, the conversion tube is disposed through the housing, the adjusting member is connected to the housing, and the adjusting member is movable relative to the housing.
In some embodiments, the adjusting member includes a main body portion, a clamping portion, a first extrusion portion and a second extrusion portion connected to each other, the conversion tube is disposed through the main body portion, the adjusting section is disposed between the first extrusion portion and the second extrusion portion, and the clamping portion can be clamped with different positions of the main body portion, so that the first extrusion portion and the second extrusion portion are close to and far away from each other.
In some embodiments, the adjustment member is threadably engaged with the housing; or, the regulating piece is provided with a plurality of clamping positions along the length direction of the regulating piece, the shell is provided with a matching part, and different clamping positions can be clamped with the matching part so as to change the circulation gap of the regulating section.
In some embodiments, the adjusting member includes a connecting member, a first extrusion block and a second extrusion block, the connecting member being connected to the housing and the first extrusion block, the adjusting section being disposed between the first extrusion block and the second extrusion block, the connecting member being movable relative to the housing so as to move the first extrusion block and the second extrusion block toward and away from each other.
In some embodiments, the shell comprises two half shells, the two half shells are detachably connected, the connecting piece is connected with one of the two half shells, the second extrusion block is arranged on the other of the two half shells, the half shells are respectively provided with a first pipe groove and a second pipe groove along the length direction of the conversion pipe, the first pipe groove is abutted against the outer edge of the first end of the conversion pipe, and the second pipe groove is abutted against the outer edge of the second end of the conversion pipe; and/or the converter comprises two first connectors, the two first connectors are respectively arranged at two ends of the conversion pipe and detachably connected with the injector, the half shell is respectively provided with a first matching groove and a second matching groove along the length direction of the conversion pipe, the first matching groove is abutted against the outer edge of one of the first connectors, and the second matching groove is abutted against the outer edge of the other of the first connectors; and/or one of the two half shells is provided with a positioning part, the other half shell is provided with a positioning groove, and the positioning part is matched in the positioning groove.
In some embodiments, the plurality of adjusting sections are arranged at intervals along the length direction of the conversion pipe, the plurality of adjusting pieces are arranged, and the plurality of adjusting pieces are in one-to-one correspondence with the plurality of adjusting sections; or, the converter includes the casing, the regulating part is the regulation hole, the regulation hole is located in the casing, the regulation hole extends along predetermineeing the direction, the regulation hole is followed predetermineeing the clearance of direction and diminishing gradually, the transfer pipe wears to locate the regulation hole, the regulation section with the inner wall elasticity butt in regulation hole, the transfer pipe is followed predetermineeing the direction is movable, in order to change the circulation clearance of regulation section.
In some embodiments, the syringe includes a syringe barrel having an end detachably connected to an end of the transition tube, an injection rod slidably disposed within the syringe barrel, and a piston detachably connected to the injection rod.
According to another embodiment of the present invention, a method for preparing SVF using the separation device according to any one of the embodiments of the present invention includes the steps of:
(a) Centrifuging the mixture of fat and swelling liquid for one time through a centrifugal separator to obtain a fat layering substance;
(b) Extracting the fat layering material by using the injector to obtain an interlayer mixture;
(c) Connecting the syringes with the converter, and injecting the two syringes to perform fat emulsification;
(d) And performing secondary centrifugation on the emulsified fat through the centrifugal separator to obtain SVF cell sap.
According to the preparation method of the SVF, the fat extract to be separated can be pushed through the converter repeatedly through the two injectors, and the adjusting piece can move relative to the adjusting section to change the circulation gap of the adjusting section, so that the circulation gap of the adjusting section can be adjusted to a proper position for pushing, the crushing, thinning and emulsifying of adipose tissues and fat cells can be realized, and the SVF cells can smoothly pass through the circulation gap and are not damaged. Therefore, the separation device of the embodiment of the invention can greatly improve the yield and the cell viability of SVF cells, so that the separation effect of the separation device is excellent.
In some embodiments, in step (d), the SVF cellular fluid is prepared by steps comprising: extracting the cell liquid after secondary centrifugation and the upper milky white cell matrix layer thereof by using the injector, adding physiological saline, mixing and re-suspending to obtain a matrix cell suspension mixture; and centrifuging the mixture for three times through the centrifugal separator to obtain the SVF cell sap.
In some embodiments, the centrifugal force of the three centrifugation is 500-2000 Xg and the centrifugation time of the three centrifugation is 1-15min.
Drawings
Fig. 1 is a front view of a separation device according to a first embodiment of the present invention.
Fig. 2 is a schematic diagram of a converter of a separation device according to a first embodiment of the invention.
Fig. 3 is a schematic view showing the installation of a transfer pipe and a first joint of a separation device according to a first embodiment of the present invention.
Fig. 4 is a cross-sectional view of a transducer of a separation device according to a first embodiment of the invention.
Fig. 5 is a schematic view of one of the half shells of the separation device of the first embodiment of the present invention.
Fig. 6 is a schematic view of another half-shell of the separation device of the first embodiment of the invention.
Fig. 7 is a front view of one of the half shells of the separating apparatus of the first embodiment of the present invention.
Fig. 8 is a front view of another half-shell of the separation device of the first embodiment of the present invention.
Fig. 9 is a schematic view of the connection member of the separation device according to the first embodiment of the present invention.
Fig. 10 is a schematic view of a first extrusion block of a separation device according to a first embodiment of the present invention.
Fig. 11 is a schematic view of a syringe of the separation device of the first embodiment of the present invention.
Fig. 12 is a cross-sectional view of a syringe of the separation device of the first embodiment of the present invention.
Fig. 13 is a schematic view of a syringe and piston of a separation device according to a first embodiment of the present invention.
Fig. 14 is a front view of a separation device according to a second embodiment of the present invention.
Fig. 15 is an isometric view of a separator device according to a second embodiment of the present invention.
Fig. 16 is a front view of a separating apparatus of a third embodiment of the invention.
Fig. 17 is an isometric view of a separator device according to a third embodiment of the present invention.
Fig. 18 is a front view of a separating apparatus of a fourth embodiment of the invention.
Fig. 19 is a front view of a separating apparatus of a fifth embodiment of the invention.
Fig. 20 is an isometric view of a separator device according to a fifth embodiment of the invention.
FIG. 21 is a schematic diagram showing an enlarged view of SVF cells isolated by the method for preparing SVF according to the sixth embodiment of the present invention under a microscope of 100X.
FIG. 22 is a schematic diagram showing an enlarged view of SVF cells isolated by the method for preparing SVF according to the seventh embodiment of the present invention under a microscope of 100X.
FIG. 23 is an enlarged view of SVF cells isolated by the method for preparing SVF according to the seventh embodiment of the present invention under a microscope of 400X.
Reference numerals:
1. a converter;
11. a switching tube; 111. a regulating section;
12. an adjusting member; 121. a connecting piece; 1211. a clamping table; 122. a first extrusion block; 1221. a clamping groove; 123. a second extrusion block; 124. a main body portion; 125. a clamping part; 126. a first pressing part; 127. a second pressing part; 128. an adjustment aperture;
13. a housing; 131. a half shell; 1311. a first pipe groove; 1312. a second pipe groove; 1313. a first mating groove; 1314. a second mating groove; 1315. a positioning groove; 1316. a positioning part; 132. a visual window;
14. a first joint;
2. a syringe; 21. a syringe; 22. an injection rod; 23. a piston; 24. and a second joint.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The separation apparatus and the method of preparing SVF according to the embodiment of the present invention are described below with reference to fig. 1 to 23.
As shown in fig. 1 to 4, a separation apparatus according to a first embodiment of the present invention includes: a transducer 1 and at least two syringes 2. The converter 1 comprises a conversion tube 11 and an adjustment member 12, the conversion tube 11 having an adjustment section 111, the adjustment section 111 being located between a first end of the conversion tube 11 (e.g. the left end of the conversion tube in fig. 3) and a second end of the conversion tube 11 (e.g. the right end of the conversion tube in fig. 3), the adjustment member 12 being connected to the adjustment section 111, the adjustment member 12 being movable relative to the adjustment section 111 to change the flow gap of the adjustment section 111. Two syringes 2 are connected to a first end of the transfer tube 11 and a second end of the transfer tube 11, respectively.
It will be appreciated that as shown in fig. 1 and 2, the cross-sectional flow area of the first end of the transition tube 11 and the cross-sectional flow area of the second end of the transition tube 11 are both smaller than the cross-sectional flow area of the adjustment section 111. The regulator 12 may adjust the flow gap of the regulator segment 111 to be less than the adipocyte diameter (e.g., 200 um) and greater than the SVF cell population diameter (e.g., 2um to 70 um). The two injectors 2 may cyclically push the fat extract so that the fat extract may be repeatedly pushed through the adjustment section 111.
According to the separation device of the embodiment of the invention, the fat extract to be separated can be pushed through the converter 1 repeatedly through the two injectors 2, and the circulation gap of the adjusting section 111 can be adjusted to a proper position for pushing due to the adjustable circulation gap of the adjusting section 111, so that the crushing, thinning and emulsifying of adipose tissues and fat cells can be realized, and SVF cells can smoothly pass through the circulation gap without being damaged. Therefore, the separation device of the embodiment of the invention can greatly improve the yield and the cell viability of SVF cells, so that the separation effect of the separation device is excellent.
Alternatively, the converter 1 may employ multi-gear adjustment. For example, the flow gap of the adjustment section 111 is different for each stage of gear. It will be appreciated that during the initial pushing, a gear with a larger flow gap may be selected, and then the distance to the flow gap is sequentially reduced to increase the yield and viability of the SVF cells. For another example, the converter 1 can be adjusted steplessly, i.e. the flow gap can be adjusted to an arbitrary distance. The adjustment mode of the converter 1 is not particularly limited in the present application.
Alternatively, as shown in fig. 1, 2 and 4, the adjusting member 12 is located outside the adjusting section 111, the adjusting section 111 has elasticity, and the adjusting member 12 may press the outer wall of the adjusting section 111 to change the circulation gap of the adjusting section 111. It will be appreciated that the adjustment member 12 is mounted externally of the adjustment section 111, the adjustment member 12 changing the size of the flow gap by squeezing the adjustment section 111. For example, the conversion tube 11 is a silicone tube, and the conversion tube 11 can be elastically deformed by external force. Because the conversion tube 11 is a silica gel hose, the damage to SVF cells during the pushing of the fat extract can be further avoided, the yield and the activity of the SVF cells are improved, and the device has the advantages of simple structure, convenient operation and convenient processing and manufacturing.
In the second embodiment, as shown in fig. 14 and 15, the adjusting member 12 includes a main body 124, a clamping portion 125, a first pressing portion 126 and a second pressing portion 127 connected to each other, the conversion tube 11 is disposed through the main body 124, the adjusting section 111 is disposed between the first pressing portion 126 and the second pressing portion 127, and the clamping portion 125 can be clamped to make the first pressing portion 126 and the second pressing portion 127 approach to and separate from each other. For example, the engagement position of the engagement portion 125 and the main body portion 124 is adjustable. When the distance of the circulation gap needs to be adjusted, the clamping portion 125 may be fixed at different positions of the main body portion 124, so that the first pressing portion 126 and the second pressing portion 127 have different pitches.
For example, the adjustment member 12 is an integral piece. In other words, as shown in fig. 14, the main body portion 124, the clamping portion 125, the first pressing portion 126, and the second pressing portion 127 are integrally formed, so that the manufacturing of the converter 1 can be facilitated, the mounting and the dismounting are convenient, and the cost is low.
In other embodiments, as shown in fig. 1 to 8, the converter 1 further includes a housing 13, the conversion tube 11 is disposed through the housing 13, the adjusting member 12 is connected to the housing 13, and the adjusting member 12 is movable relative to the housing 13. It will be appreciated that the housing 13 may fix the transition tube 11 and that the adjustment member 12 may be moved in a direction towards or away from the adjustment section 111 to vary the size of the flow gap.
Alternatively, as shown in fig. 2 and 4, the adjusting member 12 includes a connecting member 121, a first pressing block 122 and a second pressing block 123, the connecting member 121 is connected to the housing 13 and the first pressing block 122, the adjusting section 111 is disposed between the first pressing block 122 and the second pressing block 123, and the connecting member 121 is movable relative to the housing 13 to bring the first pressing block 122 and the second pressing block 123 toward and away from each other. It is understood that the connecting member 121 may drive the first extrusion 122 to move in a direction toward or away from the second extrusion 123 such that the first extrusion 122 and the second extrusion 123 compress the adjustment segment 111. By arranging the adjusting member 12 in the above structure, the separating device of the embodiment of the invention can improve the adjusting precision of the adjusting member 12, so that the separating effect of the fat extract is better.
Alternatively, as shown in fig. 2 and 4, the connecting member 121 is screw-fitted with the housing 13. It will be appreciated that the housing 13 is provided with a threaded hole into which the connector 121 is screwed, and that the connector 121 may be screwed in a direction towards the adjustment section 111 such that the connector 121 presses the adjustment section 111, thereby reducing the distance of the flow gap. Likewise, the connecting member 121 may be threaded in a direction away from the adjustment section 111 such that the connecting member 121 is spaced away from the adjustment section 111, thereby increasing the distance of the flow gap.
In other embodiments, the connector 121 is provided with a plurality of engaging portions (not shown) along its length, and the housing 13 is provided with a mating portion (not shown) with which different engaging portions can be engaged to change the circulation gap of the adjusting section 111. It will be appreciated that the connection 121 may be adjusted in multiple gears. Different clamping positions are matched with the matching parts, so that the connecting piece 121 can have different gears, and the circulation gaps of the adjusting sections 111 of each stage of gears are different. The separating device of the embodiment of the invention can facilitate the gear adjustment of the converter 1 by arranging the connecting piece 121 and the shell 13 in the structure, and has simple structural design and convenient operation.
Alternatively, as shown in fig. 9 and 10, the connecting piece 121 is provided with a clamping table 1211, the first extrusion block 122 is provided with a clamping groove 1221, and the clamping table 1211 is elastically clamped in the clamping groove 1221, so that the fixing of the connecting piece 121 and the first extrusion block 122 is completed, and the connecting piece is convenient to install and simple to process and manufacture.
In some embodiments, as shown in fig. 5 to 8, the housing 13 includes two half-shells 131, the two half-shells 131 are detachably connected, the connecting member 121 is connected to one of the two half-shells 131, and the second extrusion block 123 is provided on the other of the two half-shells 131. For example, the two half-shells 131 are connected by screws, thereby facilitating the installation and removal of the housing 13. When the conversion tube 11 needs to be installed, the two half-shells 131 can be separated, then the conversion tube 11 is installed between the two half-shells 131, and the two half-shells 131 are fixed through the screw, so that the installation work of the conversion tube 11 is completed.
For example, as shown in fig. 5 and 6, the half shell 131 is provided with a first tube groove 1311 and a second tube groove 1312 along the length direction of the converting tube 11, the first tube groove 1311 abuts against the outer edge of the first end of the converting tube 11, and the second tube groove 1312 abuts against the outer edge of the second end of the converting tube 11, and it is understood that the first tube groove 1311 and the second tube groove 1312 are both semicircular hole structures. Taking the first pipe slot 1311 as an example, the first pipe slots 1311 of the two half-shells 131 may be spliced into a round hole, so as to fix the end of the conversion pipe 11, so as to avoid the conversion pipe 11 from moving relative to the shell 13, and improve the reliability of installation of the conversion pipe 11.
Further, as shown in fig. 5 and 6, the converter 1 includes two first connectors 14, the two first connectors 14 are respectively disposed at two ends of the conversion tube 11 and detachably connected with the syringe 2, the half shell 131 is respectively provided with a first mating groove 1313 and a second mating groove 1314 along the length direction of the conversion tube 11, the first mating groove 1313 abuts against an outer edge of one of the first connectors 14, and the second mating groove 1314 abuts against an outer edge of the other first connector 14. For example, the first fitting 14 is a luer fitting. The first mating groove 1313 and the second mating groove 1314 are both half-quincuncial hole structures, so that the first connector 14 can be compressed, and the installation firmness of the first connector 14 is improved.
Alternatively, as shown in fig. 7 and 8, one of the two half-shells 131 is provided with a positioning portion 1316, and the other is provided with a positioning groove 1315, and the positioning portion 1316 is fitted in the positioning groove 1315, and it is understood that the positioning portion 1316 may be mounted in the positioning groove 1315 when the half-shells 131 are mounted, so that the housing 13 is pre-fixed, so that the screw member smoothly passes through the two half-shells 131, and the convenience of mounting the housing 13 is improved.
Optionally, the half shell 131 may be made of medical stainless steel (1 Cr18Ni 9T), 316L stainless steel, 317L stainless steel, TC4 titanium alloy, medical plastic (PC, PP, ABS), and the like.
The material of the first joint 14 may be medical stainless steel (1 Cr18Ni9T, 316L or 317L), TC4 titanium alloy, medical plastic (PC, PP, ABS), or the like.
The connecting piece 121, the first extrusion block 122 and the second extrusion block 123 may be made of medical stainless steel (1 Cr18Ni 9T), 316L stainless steel, 317L stainless steel, TC4 titanium alloy, etc.
In the third embodiment, as shown in fig. 16 and 17, the plurality of adjustment sections 111 are provided, the plurality of adjustment sections 111 are arranged at intervals along the length direction of the conversion pipe 11, the plurality of adjustment members 12 are provided, and the plurality of adjustment members 12 are in one-to-one correspondence with the plurality of adjustment sections 111. For example, the adjusting members 12 are arranged on the housing 13 at intervals along the length direction of the converting tube 11, and the plurality of adjusting members 12 correspond to different adjusting sections 111, so that the effect of separating the SVF cells from the fat extract can be better. The specific number of adjustment members 12 is not specifically limited in this application.
In the fourth embodiment, as shown in fig. 18, the adjusting member 12 is an adjusting hole 128, the adjusting hole 128 is provided in the housing 13, and the adjusting hole 128 extends along a predetermined direction. For example, the extending direction of the adjustment hole 128 is orthogonal to the longitudinal direction of the conversion tube 11. The gap of the adjusting hole 128 along the preset direction gradually decreases, the converting tube 11 penetrates through the adjusting hole 128, and the adjusting section 111 elastically abuts against the inner wall of the adjusting hole 128. The transfer tube 11 can be moved in a preset direction such that the pressing force of the adjustment holes 128 against the adjustment section 111 is gradually increased such that the flow gap of the adjustment section 111 is adjustable.
It should be understood that the specific structure of the adjusting member 12 of the present application includes, but is not limited to, the above-mentioned adjusting structure (such as the threaded connection structure, the clamping structure, or the adjusting hole 128 structure), but may be other forms of adjusting structure already disclosed in the prior art, which is not limited in this application.
In some embodiments, as shown in fig. 2 and 17, a visible window 132 is provided on the housing 13 to observe the emulsification process in the conversion tube 11, so that an operator can determine whether separation is completed according to the emulsification condition of fat. For example, the viewing windows 132 are arranged on both sides of the housing 13 in the front-rear direction of the housing 13, and the front-rear direction of the housing 13, the length direction of the switching tube 11, and the up-down direction of the housing 13 are orthogonal to each other.
In an example, as shown in fig. 2, the housing 13 includes two half-shells 131, the two half-shells 131 are detachably connected in the up-down direction, and part of the visible windows 132 are provided on the two half-shells 131, respectively. When the two half-shells 131 are assembled, a complete visual window 132 can be spliced.
In a fifth embodiment, as shown in fig. 19 and 20, the outer wall of the housing 13 adopts a closed arrangement. In other words, the two side walls of the housing 13 in the front-rear direction are not provided with windows, so that the exposure of the adjustment section 111 to the outside can be avoided, and the probability of damage of the adjustment section 111 due to an outside collision is reduced.
Alternatively, as shown in fig. 11 to 13, the syringe 2 includes a syringe barrel 21, a syringe rod 22, and a piston 23, and an end of the syringe barrel 21 is detachably connected to an end of the transfer tube 11, for example, an end of the syringe barrel 21 is fitted with a second connector 24, and the second connector 24 is a luer connector. The piston 23 is slidably disposed within the syringe 21, and the injection rod 22 is detachably connected to the piston 23. For example, injection rod 22 is threadably connected to piston 23.
It will be appreciated that since the injection rod 22 is detachably connected to the plunger 23, the syringe 21 and the plunger 23 may form a centrifuge tube. That is, after the syringe 21 removes the injection rod 22, a stopper may be provided at the end of the syringe 21, and then the syringe 21 may be transferred to a centrifugal separator for separation.
In a preferred embodiment, the injector 2 is a centrifugal separator. It will be appreciated that the injector 2 of the present embodiment may be used as an injection device or as a centrifugal separator, thereby simplifying the operation steps of the SVF production method and providing higher SVF yield and activity. According to another embodiment of the present invention, a method for preparing SVF using the separation apparatus of the present invention comprises the steps of:
(a) Injecting swelling liquid into the body in the process of fat extraction, so that fat extraction is facilitated, the fat and the swelling liquid are extracted from the body together, and the extracted liquid is a mixture of the fat and the swelling liquid;
(b) Centrifuging the mixture of fat and swelling liquid for one time through a centrifugal separator to obtain a fat layering substance;
(c) Extracting the fat layering material by using a syringe 2 to obtain an intermediate layer mixture;
(d) Connecting the syringes with the converter 1, injecting the two syringes 2, and emulsifying the fat cells;
(e) And (3) performing secondary centrifugation on the emulsified fat cells through a centrifugal separator to obtain SVF cell sap.
According to the preparation method of the SVF, according to the embodiment of the invention, the fat cell liquid to be separated can be pushed through the converter repeatedly through the two injectors, and the adjusting piece can move relative to the adjusting section to change the circulation gap of the adjusting section, so that the circulation gap of the adjusting section can be adjusted to a proper position for pushing, the crushing, thinning and emulsifying of fat tissues and fat cells can be realized, and the SVF cells can smoothly pass through the circulation gap and are not damaged. Therefore, the separation device of the embodiment of the invention can greatly improve the yield and the cell activity of SVF cells, which are higher than those of SVF cells separated by the related separation devices listed in the background art, so that the separation effect of the separation device is excellent.
Specifically, in step e, "the emulsified adipocytes are subjected to secondary centrifugation by a centrifugal separator to obtain an SVF cell sap" includes the steps of:
extracting the cell liquid after secondary centrifugation and the upper layer of the milky white cell matrix layer by using a syringe, adding physiological saline, mixing and re-suspending to obtain a matrix cell suspension mixture;
physiological saline is injected into the stromal cell suspension mixture, and the stromal cell suspension mixture is centrifuged through a centrifugal separator for the third time to separate out SVF cell sap.
It can be appreciated that the preparation method of SVF according to the embodiment of the invention can further improve the enrichment degree of SVF cells by centrifuging the separated fat cell liquid for three times through a centrifugal separator, so that the yield and the output of SVF cells are higher.
The inventors of the present application found through experimental studies that when the SVF cells prepared by three centrifugation are used in the preparation method of the SVF of the examples of the present invention, the yield and viability of the obtained SVF cells are significantly higher than those of the SVF cells isolated by the related isolation apparatuses listed in the background art. Therefore, the separation device and the SVF preparation method can greatly improve the yield and the activity of SVF cells, and have excellent separation effect.
Alternatively, the centrifugal force of the three centrifugation is 500-2000 Xg, and the centrifugal time of the three centrifugation is 1-15min. The inventors of the present application found through experimental studies that when the centrifugal force and the centrifugal time of the triple centrifugation use the above parameters, the separation effect of SVF cells can be further improved.
In some embodiments, the method of preparing SVF comprises the following specific steps:
s1: selecting the abdomen fat or leg fat of a receiver as a SVF cell source, and preferentially selecting the abdomen fat;
s2: sterilizing the fat extraction area by using medical gauze and iodophor;
s3: preparing a swelling liquid;
s4: cutting two small openings in the sterilized liposuction region by using a surgical knife blade, and injecting swelling liquid;
s5: extracting 100-300ml of fat (mixed with swelling liquid) by using a liposuction needle;
s6: standing for layering, removing lower blood and swelling solution, and remaining fat mixture;
s7: transferring the fat mixture into a centrifugal separator through a luer connector, screwing down an injection rod, and performing centrifugal separation, wherein the centrifugal force is 500-2000 Xg, the centrifugal time is 1-15min, and the centrifugal force is 600-800 Xg and the centrifugal time is 1-5min as a preferred embodiment. Centrifuging the fat extract into three layers, wherein the lowest layer is hematoedema distention liquid, part of flocculent extracellular matrix (ECM) and fascia layer, the middle layer is fat layering substance, and the uppermost layer is fat layer;
S8: the injection rod of the injector is screwed on, and the bottom swelling liquid, blood water, part flocculent ECM and fascia are slowly and stably pushed out downwards;
s9: extracting middle-layer fat layering matters by using an injector, connecting the injector to two sides of a conversion pipe, adjusting connecting pieces step by step to change the circulation gap of an adjusting section, repeatedly pushing and pulling the injector to treat the fat layering matters so as to crush fat cells, and separating SVF cell components from fat matrixes;
the circulation gap of the adjusting section is provided with a plurality of stages of gear adjustment, and the adjustment range is 5mm to 70 mu m. As a preferred embodiment, the flow gap of the adjustment section 111 is provided with three-stage gear adjustment: the first grade gear is set at a distance of 4-5mm, and the injector is repeatedly pushed for more than or equal to 10 times to preliminarily emulsify the fat extract. The secondary gear is arranged at a distance of 2-3mm, and the injector is repeatedly pushed for more than or equal to 10 times until the injector is pushed smoothly, and no obvious resistance is felt. Setting a space of 50-70 mu m between three stages of gears, basically attaching the tube walls of the adjusting sections, separating extract cells from matrixes by elastic extrusion of the conversion tube, and repeatedly pushing the injector for 20 times at the space;
then, performing secondary centrifugal separation on the fat tissue treated by the separation device, wherein the centrifugal force is 500-2500 Xg, the centrifugal time is 1-25min, and as a preferential embodiment, the centrifugal force is 2000-2500 Xg, the centrifugal time is 10-15min, and the fat extract is centrifuged into three layers, wherein the upper part of the bottom layer is a milky white layer and the lower part of the bottom layer is a SVF cell liquid layer;
S10: and (5) extracting the SVF cell liquid layer by using a syringe to obtain the required SVF cell liquid.
S11: as another embodiment, SVF cell sap is washed and purified 3-4 times with physiological saline or PBS.
S12: as another embodiment, the milky white layer and the SVF cell liquid layer in step S9 are directly extracted by syringe, and an appropriate amount of physiological saline, preferably 1:1, repeatedly shaking and uniformly mixing; the mixture is placed in a centrifugal separator and centrifuged three times for a centrifugal force of 500-2000 Xg and a centrifugal time of 1-15min, and as a preferred example, for a centrifugal force of 600-800 Xg and a centrifugal time of 1-5min. So that the fat extract is centrifuged into two layers, wherein the lower layer is enriched purified SVF cell sap.
Sixth embodiment:
the superficial abdominal fat of the subject was extracted, a swelling solution (40 ml of 2% lidocaine, 1:1000 epinephrine, 10ml of 5% sodium carbonate, and 1000ml of physiological saline) was prepared, and after sterilization and incision, 100ml of fat extract was extracted with a 1.8X200 liposuction needle.
Placing the fat extract in a centrifugal separator for centrifugal separation, wherein the centrifugal force is 700×g, the centrifugal time is 1min, the fat extract is centrifuged into three layers, the uppermost layer is a fat layer, and the lowermost layer is a bloodred swelling liquid and a blood water layer.
The injection rod of the injector is screwed on, the bottom swelling liquid and the blood water layer are slowly and stably pushed out downwards, the fat cell layer is extracted by the injector of the separating device, then the injector and the converter are screwed together, the connecting piece is continuously rotated so as to adjust the circulation gap of the adjusting section, the circulation gap is sequentially set to be 5mm, 2mm and 70 mu m, and simultaneously the injector is repeatedly pushed, so that fat continuously passes through the converting pipe in a reciprocating manner, the emulsification of the fat is realized, and the cell disruption is ensured while the SVF cells are completely reserved. The number of pushing is 10 times or more in the range of 5mm and 2mm, and 20 times in the range of 70 μm.
Injecting the emulsified fat into a centrifugal separator for secondary centrifugal separation, wherein the centrifugal force is 2060 Xg, the centrifugal time is 15min, the fat extract is centrifuged into three layers, the upper layer is a fat layer, the middle layer is a layer containing partial SVF cell fat, fragments and fat drops (the bottom of the middle layer is a milky light layer), the lowest layer is a SVF layer, and the lower SVF layer is extracted by a syringe, thus obtaining the required SVF cell sap. As shown in FIG. 21, the cell morphology was observed under a microscope of 100X and the count was 9.6X10 6 Cell viability was calculated to be > 90% by trypan blue staining per ml.
Seventh embodiment:
the superficial abdominal fat of the subject was extracted, a swelling solution (40 ml of 2% lidocaine, 1:1000 epinephrine, 10ml of 5% sodium carbonate, and 1000ml of physiological saline) was prepared, and after sterilization and incision, 100ml of fat extract was extracted with a 1.8X200 ton liposuction needle.
Placing the fat extract in a centrifugal separator for centrifugal separation, wherein the centrifugal force is 700×g, the centrifugal time is 1min, the fat extract is centrifuged into three layers, the uppermost layer is a fat layer, and the lowermost layer is a bloodred swelling liquid and a blood water layer.
The injection rod of the injector is screwed on, the bottom swelling liquid and the blood water layer are slowly and stably pushed out downwards, the fat cell layer is extracted by the injector of the separating device, then the injector and the converter are screwed together, the connecting piece is continuously rotated so as to adjust the circulation gap of the adjusting section, the circulation gap is sequentially set to be 5mm, 2mm and 5 mu m, and simultaneously the injector is repeatedly pushed, so that fat continuously passes through the converting pipe in a reciprocating manner, the emulsification of the fat is realized, and the cell disruption is ensured while the SVF cells are completely reserved. The number of pushing is 10 times or more in the range of 5mm and 2mm, and 20 times in the range of 5 μm.
Injecting the emulsified fat into a centrifugal separator for secondary centrifugal separation, wherein the centrifugal force is 2060 Xg, the centrifugal time is 15min, the fat extract is centrifuged into three layers, the upper layer is a fat layer, the middle layer is a fat layer containing part of SVF cells, fragments and fat droplets (the bottom of the middle layer is a milky light layer), and the lowest layer is a SVF layer.
The SVF suspension layer and the upper milky white layer were drawn up by syringe connection at 1:1 into proper amount of physiological saline for injection. The mixture was placed in a centrifugal separator and shaken well under shaking, the centrifugal force was selected to be 700 Xg, and the centrifugal time was selected to be 1min. After centrifugation, the extract was separated into two layers, the effective SVF cell sap was about 4 times the amount obtained by secondary centrifugation, and finally the lowest SVF-enriched layer was extracted. The morphology of SVF cells was observed under a microscope of 100X (as shown in FIG. 22) and under a microscope of 400X (as shown in FIG. 23), respectively.
As shown in FIG. 22, the cell morphology was observed under a microscope of 100X and the count was 3X 10 7 Cell viability was calculated to be > 90% by trypan blue staining per ml.
It can be seen that the SVF cell yield and viability obtained by the second centrifugation in the SVF preparation method according to the sixth embodiment of the present invention and the SVF cell yield and viability obtained by the third centrifugation in the SVF preparation method according to the seventh embodiment of the present invention are significantly higher than those obtained by the related separation apparatuses listed in the background art. Therefore, the separation device and the SVF preparation method can greatly improve the yield and the activity of SVF cells, and have excellent separation effect.
It can be appreciated that the SVF cells prepared by the SVF preparation method of the embodiment of the invention can be applied to a plurality of fields of treating osteoarthritis, tendon injury repair, cosmetology and plastic, chronic wound healing, diabetic foot ulcer, pediatric cerebral palsy, cerebral apoplexy, tissue regeneration repair and the like.
In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the device or element being referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the present invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
In the present invention, unless explicitly specified and limited otherwise, the terms "mounted," "connected," "secured," and the like are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally formed; may be mechanically connected, may be electrically connected or may be in communication with each other; either directly or indirectly, through intermediaries, or both, may be in communication with each other or in interaction with each other, unless expressly defined otherwise. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
In the present invention, unless expressly stated or limited otherwise, a first feature "up" or "down" a second feature may be the first and second features in direct contact, or the first and second features in indirect contact via an intervening medium. Moreover, a first feature being "above," "over" and "on" a second feature may be a first feature being directly above or obliquely above the second feature, or simply indicating that the first feature is level higher than the second feature. The first feature being "under", "below" and "beneath" the second feature may be the first feature being directly under or obliquely below the second feature, or simply indicating that the first feature is less level than the second feature.
For purposes of this disclosure, the terms "one embodiment," "some embodiments," "example," "a particular example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While the above embodiments have been shown and described, it should be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations of the above embodiments may be made by those of ordinary skill in the art without departing from the scope of the invention.
Claims (11)
1. A separation device, comprising:
a converter comprising a conversion tube having an adjustment section between a first end of the conversion tube and a second end of the conversion tube, and an adjustment member connected to the adjustment section, the adjustment member being movable relative to the adjustment section to vary a flow gap of the adjustment section;
At least two syringes connected to the first end of the transfer tube and the second end of the transfer tube, respectively;
the adjusting piece is located outside the adjusting section, the adjusting section is elastic, and the adjusting piece can squeeze the outer wall of the adjusting section so as to change the circulation gap of the adjusting section.
2. The separator device of claim 1, wherein the converter further comprises a housing, the conversion tube is disposed through the housing, the adjustment member is connected to the housing, and the adjustment member is movable relative to the housing.
3. The separator of claim 1, wherein the adjusting member includes a main body portion, a clamping portion, a first pressing portion, and a second pressing portion connected to each other, the switching tube is disposed through the main body portion, the adjusting section is disposed between the first pressing portion and the second pressing portion, and the clamping portion can be clamped to different positions of the main body portion so that the first pressing portion and the second pressing portion are close to and far from each other.
4. The separation device of claim 2, wherein the adjustment member is threadably engaged with the housing;
or, the regulating piece is provided with a plurality of clamping positions along the length direction of the regulating piece, the shell is provided with a matching part, and different clamping positions can be clamped with the matching part so as to change the circulation gap of the regulating section.
5. The separator of claim 2, wherein the adjustment member comprises a connector, a first squeeze block and a second squeeze block, the connector being coupled to the housing and the first squeeze block, the adjustment section being disposed between the first squeeze block and the second squeeze block, the connector being movable relative to the housing to move the first squeeze block and the second squeeze block toward and away from each other.
6. The separator device as claimed in claim 5, wherein said housing comprises two half-shells, said two half-shells being detachably connected, said connecting member being connected to one of said half-shells, said second pressing block being provided on the other of said half-shells,
the half shell is provided with a first pipe groove and a second pipe groove along the length direction of the conversion pipe respectively, the first pipe groove is abutted against the outer edge of the first end of the conversion pipe, and the second pipe groove is abutted against the outer edge of the second end of the conversion pipe;
and/or the converter comprises two first connectors, the two first connectors are respectively arranged at two ends of the conversion pipe and detachably connected with the injector, the half shell is respectively provided with a first matching groove and a second matching groove along the length direction of the conversion pipe, the first matching groove is abutted against the outer edge of one of the first connectors, and the second matching groove is abutted against the outer edge of the other of the first connectors;
And/or one of the two half shells is provided with a positioning part, the other half shell is provided with a positioning groove, and the positioning part is matched in the positioning groove.
7. The separator according to claim 1, wherein the plurality of the adjusting sections are arranged at intervals along the length direction of the transfer pipe, the plurality of the adjusting members are arranged in a plurality, and the plurality of the adjusting members are in one-to-one correspondence with the plurality of the adjusting sections;
or, the converter includes the casing, the regulating part is the regulation hole, the regulation hole is located in the casing, the regulation hole extends along predetermineeing the direction, the regulation hole is followed predetermineeing the clearance of direction and diminishing gradually, the transfer pipe wears to locate the regulation hole, the regulation section with the inner wall elasticity butt in regulation hole, the transfer pipe is followed predetermineeing the direction is movable, in order to change the circulation clearance of regulation section.
8. The separation device of any one of claims 1-7, wherein the syringe comprises a syringe barrel, an injection rod, and a piston, an end of the syringe barrel being removably connected to an end of the transition tube, the piston being slidably disposed within the syringe barrel, and the injection rod being removably connected to the piston.
9. A method for preparing SVF using the separation device of any one of claims 1 to 7, comprising the steps of:
(a) Centrifuging the mixture of fat and swelling liquid for one time through a centrifugal separator to obtain a fat layering substance;
(b) Extracting the fat layering material by using the injector to obtain an interlayer mixture;
(c) Connecting the syringes with the converter, and injecting the two syringes to perform fat emulsification;
(d) And performing secondary centrifugation on the emulsified fat through the centrifugal separator to obtain SVF cell sap.
10. The method of preparing SVF according to claim 9, wherein in step (d), the SVF cell sap is prepared by comprising the steps of:
extracting the cell liquid after secondary centrifugation and the upper milky white cell matrix layer thereof by using the injector, adding physiological saline, mixing and re-suspending to obtain a matrix cell suspension mixture;
and centrifuging the mixture for three times through the centrifugal separator to obtain the SVF cell sap.
11. The method for preparing SVF according to claim 10, wherein the centrifugal force of the three centrifugation is 500-2000 Xg, and the centrifugal time of the three centrifugation is 1-15min.
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