CN110420353B - Device and method for extracting fat-derived matrix vascular components - Google Patents
Device and method for extracting fat-derived matrix vascular components Download PDFInfo
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- CN110420353B CN110420353B CN201910688031.3A CN201910688031A CN110420353B CN 110420353 B CN110420353 B CN 110420353B CN 201910688031 A CN201910688031 A CN 201910688031A CN 110420353 B CN110420353 B CN 110420353B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
Abstract
The invention discloses an extraction device and an extraction method of fat-derived matrix vascular components, wherein the extraction device is used for extracting the matrix vascular components in adipose tissues and can realize centrifugal motion, vortex motion, oscillation motion, centrifugal oscillation motion and vortex oscillation motion of in vitro fat masses; the blood vessel matrix component contains various cell components which can be widely used in the multidirectional fields of fat transplantation, cartilage repair and the like; the matrix vascular component obtained by vortex oscillation adopted in the extraction method has high viable cell number, strong proliferation capacity, simple extraction method, low cost, safety, high efficiency and easy operation and implementation.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to an extraction device and an extraction method for fat-derived matrix vascular components.
Background
Adipose tissue is an important regenerative repair resource in the field of soft tissue repair in recent years, can be easily obtained through liposuction surgery, and has low surgery risk and small damage to patients. Some clinical researches find that a Stromal Vascular Fraction (SVF) from fat has obvious anti-inflammatory and early vascularization effects, and the adipose tissue transplantation effect of the added SVF is obviously superior to that of a simple adipose tissue, so that the SVF has wide prospects in the fields of tissue repair and regeneration.
At present, SVF is isolated and extracted from adipose tissue mainly by enzymatic digestion or high speed centrifugation. Among them, the enzymatic digestion requires high aseptic conditions, requires introduction of exogenous biological enzymes, is time-consuming and has high potential risks, is mostly used for preparing SVF under laboratory conditions, and is not approved for clinical application. The high-speed centrifugation method is a mechanical preparation method, is relatively safe, but the activity of SVF extracted by the high-speed centrifugation method is poor, and the improvement effect on the fat transplantation operation is still to be improved.
Therefore, how to prepare high-activity SVF efficiently and safely without introducing exogenous biological agents is a problem to be solved in clinical urgent need.
Disclosure of Invention
In order to solve the problems, the invention provides an extraction device and an extraction method for matrix vascular components from fat, the extraction device is used for extracting the matrix vascular components from adipose tissues, and can realize centrifugal motion, vortex motion, oscillation motion, centrifugal oscillation motion and vortex oscillation motion of in vitro fat masses. The blood vessel matrix component contains various cell components which can be widely used in the multidirectional fields of fat transplantation, cartilage repair and the like; the matrix vascular component obtained by vortex oscillation adopted in the extraction method has high viable cell number, strong proliferation capacity, simple extraction method, low cost, safety, high efficiency and easy operation and implementation.
In order to achieve the above object, the present invention adopts the following technical solutions.
(A) an apparatus for extracting blood vessel components from adipose derived substrates, comprising: the test tube rack comprises a bottom plate, a mounting plate, a tray, a test tube rack, a first driving device, a second driving device and a third driving device; the upper end of the bottom plate is fixedly provided with a first limiting pipe, the bottom end of the mounting plate is fixedly provided with a second limiting pipe matched with the first limiting pipe, and the first limiting pipe is sleeved in the second limiting pipe in a sliding manner; the mounting plate is driven to move up and down on the bottom plate through a first driving device; the upper end of the middle part of the mounting plate drives the tray to horizontally rotate through a second driving device; the upper end of the tray drives the test tube rack to horizontally rotate through a third driving device; the test tube rack comprises a connecting rod and a support frame, and the lower end of the connecting rod is fixedly connected with an output shaft of the third driving device; a support frame is detachably fixed at the upper end of the connecting rod, a plurality of clamping rings are circumferentially and symmetrically arranged on the support frame, and the clamping rings are used for clamping test tubes; the upper end bilateral symmetry of mounting panel is fixed with first support, be fixed with the riding wheel on the first support, the bottom of tray be provided with riding wheel assorted slide rail.
According to the fat source matrix blood vessel component extraction device, the second limiting pipe on the mounting plate can slide up and down relatively in the first limiting pipe on the bottom plate, and the first driving device drives the mounting plate to vibrate up and down, so that the test tube rack connected with the mounting plate is driven to vibrate up and down. The clamping rings on the test tube rack are used for placing a plurality of test tubes at one time, and the test tubes are used for placing an extract to be extracted (the extract to be extracted in the application is in-vitro fat mass), so that the working efficiency of the extract to be extracted is improved; the connecting rod can be dismantled with the support frame and be connected, can change different support frames according to actual demand. The riding wheels on the first supports on the two sides of the mounting plate can slide on the sliding rails on the tray, the riding wheels on the first supports are used for supporting the tray to rotate horizontally, the second driving device is used for driving the tray to rotate horizontally, and the rotation of the extract to be extracted around the output shaft of the second driving device is realized. The third driving device is used for driving the test tube rack to rotate horizontally, so that the extract to be extracted can rotate around the output shaft of the third driving device, and further the centrifugal motion of the extract to be extracted can be realized. The device for extracting the blood vessel components of the fat source matrix realizes the vortex motion of the extract to be extracted by combining the rotation and the revolution of the extract to be extracted; the first driving device realizes the oscillating motion of the extract to be extracted, and the third driving device realizes the centrifugal motion of the extract to be extracted.
Preferably, the support frame is hinged or welded with the clamping ring.
According to the fat source matrix blood vessel component extraction device, the support frame is divided into two types, wherein one type is that the support frame is hinged with the clamping ring, and the other type is welded with the clamping ring into a whole. When the extract is needed to be centrifuged, the support frame hinged with the clamping ring is selected, the test tube on the clamping ring is thrown away under the action of the third driving device, and the centrifugation of the extract is realized according to the difference of the molecular weight of the substance. When the extract to be treated needs to do vortex motion, the support frame welded with the clamping ring is selected, and the vortex motion of the extract to be treated is realized under the action of the second driving device and the third driving device.
Preferably, the top end of the connecting rod is provided with a screw hole, the center of the support frame is provided with a through hole, a hand screw is assembled in the screw hole and the through hole, and the hand screw penetrates through the through hole and is screwed into the screw hole to fixedly connect the support frame and the connecting rod.
According to the fat source matrix blood vessel component extraction device, the screw rod of the hand screw penetrates through the support frame and is screwed into the screw hole of the connecting rod, so that the connecting rod is detachably connected with the support frame, and the support frame can be used as a support frame for vortex motion or centrifugal motion according to actual requirements.
Preferably, the first driving means comprises a first motor, a cam and a roller; the middle part of the bottom plate is fixedly provided with a first motor through a second support, an output shaft of the first motor is fixedly provided with a cam, and a roller is arranged at a position, opposite to the cam, on the mounting plate.
According to the device for extracting the fat-derived matrix blood vessel components, the first motor is fixed on the bottom plate through the second bracket, the output shaft of the first motor drives the cam to do circular motion, and the cam drives the roller to move up and down, so that the up-and-down oscillating motion to be extracted is realized. In addition, the cam is provided with two symmetrical convex parts, and the cam can vibrate the extract to be extracted twice in the process of one-time circular motion, so that the extraction efficiency of the extract to be extracted is improved.
Preferably, the second driving device comprises a second motor, the second motor is fixed in the middle of the mounting plate, and an output shaft of the second motor is fixedly connected with the tray.
According to the device for extracting the fat-derived stromal vascular fraction, the output shaft of the second motor is fixedly connected with the tray, the tray is driven to horizontally rotate by the second motor, and the test tube rack on the tray can do revolution motion around the output shaft of the second motor.
Preferably, the third driving device comprises a third motor, the third motor has a mounting seat, a centrifugal groove is formed in the center of the tray, and a plurality of vortex grooves are uniformly formed in the tray along the radial direction or/and the circumferential direction; the centrifugal groove and the vortex groove are respectively detachably connected with the mounting seat; and an output shaft of the third motor is connected with the connecting rod through a coupler.
According to the device for extracting the fat-derived stromal vascular fraction, the mounting seat at the bottom of the third motor is detachably connected with the centrifugal groove or the vortex groove on the tray, and the third motor can be fixed at different positions on the tray according to actual requirements. When the third motor is fixed on the centrifugal groove, the second motor is started, and the centrifugal motion of the extract to be extracted can be realized; when the third motor is fixed on the vortex groove, the second motor and the third motor are started, and the vortex motion of the extract to be extracted can be realized. In addition, when the third motor is fixed on the centrifugal groove, the first motor and the second motor are started, and the centrifugal oscillation motion of the extract to be extracted can be realized; when the third motor is fixed on the vortex groove, the first motor, the second motor and the third motor are started, and vortex oscillation motion of the extract can be realized. The tray is evenly provided with a plurality of vortex grooves along the radial direction, the distance between the third motor and the output shaft of the second motor can be adjusted, and the extract to be treated can do vortex motion along different radiuses. A plurality of vortex grooves are uniformly formed in the circumferential direction of the tray, a plurality of samples to be extracted can be processed at the same time, and the processing efficiency is high.
Preferably, the bottom end of the mounting seat is provided with an external thread, and the centrifugal groove and the vortex groove are respectively provided with screw holes matched with the external thread.
According to the fat-derived stromal vascular fraction extraction device, the mounting seat is detachably connected with the centrifugal groove and the vortex groove through the external threads matched with the internal threads on the screw hole, so that the position of the third motor can be conveniently adjusted and fixed.
Preferably, the device further comprises a spring, and two ends of the spring are fixedly connected with the bottom plate and the mounting plate respectively.
According to the device for extracting the fat-derived stromal vascular fraction, the elastic action of the spring is used for supporting the mounting plate, so that the pressure of the cam and the roller is reduced.
Preferably, the device further comprises a protective cover, wherein the protective cover is used for covering the whole fat-derived stromal vascular fraction extracting device.
According to the fat source matrix blood vessel component extraction device, the protective cover is used for providing a sterile and temperature-controlled environment, so that the extract to be extracted is extracted or stored at the temperature of 4-37 ℃, and the pollution of the environment to the extract to be extracted is reduced.
(II) the extraction method of the fat-derived stromal vascular fraction comprises the following steps:
and 4, filtering the centrifugate to obtain the stromal vascular fraction.
Preferably, in the step 2, the frequency of the vortex oscillation is 5-50 Hz, and the time of the vortex oscillation is 2-25 min.
Further preferably, in step 2, the frequency of the vortex oscillation is 30Hz, and the time of the vortex oscillation is 15 min.
Preferably, in the step 3, the rotation speed of the centrifugation is 500-1500 rpm, and the time of the centrifugation is 1-5 min.
Further preferably, in step 3, the rotation speed of the centrifugation is 1000rpm, and the time of the centrifugation is 2 min.
Drawings
The invention is described in further detail below with reference to the figures and specific embodiments.
FIG. 1 is a schematic view of the structure of a cellulite mass at rest;
FIG. 2 is a schematic structural diagram of a fat mass in a high-speed centrifugation state in the prior art;
FIG. 3 is a schematic view of the structure of the fat mass in vortex oscillation state according to the present application;
FIG. 4 is a schematic view showing the construction of the fat-derived stromal vascular fraction extracting apparatus of the present invention;
fig. 5 is a schematic view of a first configuration of the test tube rack of fig. 4;
FIG. 6 is a top view of FIG. 5;
fig. 7 is a schematic view of a second configuration of the test tube rack of fig. 4;
FIG. 8 is a top view of the support bracket of FIG. 7;
FIG. 9 shows Trypan blue staining results of SVF suspensions extracted under different treatment methods; wherein the ordinate is the number of living cells (cell counts) in the unit of × 105(ii) a The abscissa is different processing methods, namely low-frequency vortex oscillation (vibration), enzyme digestion (enzyme digestion), high-speed centrifugation (centrifugation) and nano emulsification (nano emulsification);
FIG. 10 shows the results of a proliferation potency test of SVF suspensions extracted under different treatment methods; wherein the abscissa is the number of Days (Days) and the ordinate is the Absorbance value at 450nm (Absorbance of 450 OD);
FIG. 11 shows the results of B-mode ultrasonography for SVF suspensions extracted under different treatment methods; wherein, the abscissa is time, and the unit is week (week); the ordinate is the volume maintenance (survival rate) in%;
FIG. 12 shows the results of an oil-Red-O staining test after 6 months of implantation of SVF suspensions extracted under different treatment methods; wherein, the upper part in the figure is respectively an A-concussion group and a B-enzyme digestion group from left to right; the lower part of the figure is respectively provided with a C-centrifugation group and a D-emulsification group from left to right;
FIG. 13 shows the results of HE staining tests after 6 months of implantation of SVF suspensions extracted under different treatment methods; wherein, the upper part in the figure is respectively an A-concussion group and a B-enzyme digestion group from left to right; the lower part of the figure is respectively a C-centrifugation group and a D-emulsification group from left to right.
In the above figures: 1, a bottom plate; 101 a first limiting pipe; 102 a second bracket; 2, mounting a plate; 201 a second limiting tube; 202 a first support; 203, a riding wheel; 3, a tray; 301 a centrifugal tank; 302 a spiral groove; 4, a test tube rack; 410 a connecting rod; 420, a support frame; 421 a snap ring; 422 through holes; 430 test tubes; 440 hand-screw; 5 a first motor; 501 a cam; 502 a roller; 6 a second motor; 7 a third motor; 701, a mounting seat; 8 springs.
Detailed Description
FIG. 1 is a schematic view of the structure of a cellulite mass at rest; as can be seen from FIG. 1, adipocytes and SVF cells were uniformly distributed in vitro.
FIG. 2 is a schematic structural diagram of a fat mass in a high-speed centrifugation state in the prior art; as is clear from FIG. 2, in the high-speed centrifugation state (at a centrifugation speed of 1500 rpm), the fat cells are crushed and squeezed together, SVF passes through the narrow gaps between the fat cells and sinks toward the tube bottom, and SVF is crushed and deformed, and the activity is affected. From this, it was found that it was difficult to separate adipocytes from SVF cells under high-speed centrifugation, and the activity of the extracted SVF cells was weak.
FIG. 3 is a schematic view of the structure of the fat mass in vortex oscillation state according to the present application; as can be seen from FIG. 3, in the vortex oscillation state, the space between the fat cells is expanded or contracted rhythmically with the oscillation motion, and at the enlarged cell space, SVF cells smoothly pass through the space and sink toward the bottom of the tube. Therefore, vortex oscillation is beneficial to separation of fat cells and SVF cells, extraction of the SVF cells is facilitated, and activity of the extracted SVF cells is strong.
Referring to fig. 4 to 6, an apparatus for extracting a vascular component of adipose-derived matrix according to an embodiment of the present disclosure includes: the test tube rack comprises a bottom plate 1, a mounting plate 2, a tray 3, a test tube rack 4, a first driving device, a second driving device and a third driving device; a first limiting pipe 101 is fixed at the upper end of the bottom plate 1, a second limiting pipe 201 matched with the first limiting pipe 101 is fixed at the bottom end of the mounting plate 2, and the first limiting pipe 101 is sleeved in the second limiting pipe 201 in a sliding mode; the mounting plate 2 is driven to move up and down on the bottom plate 1 through a first driving device; the upper end of the middle part of the mounting plate 2 drives the tray 3 to horizontally rotate through a second driving device; the upper end of the tray 3 drives the test tube rack 4 to horizontally rotate through a third driving device; the test tube rack 4 comprises a connecting rod 410 and a supporting frame 420, and the lower end of the connecting rod 410 is fixedly connected with an output shaft of the third driving device; a support frame 420 is detachably fixed at the upper end of the connecting rod 410, a plurality of clamping rings 421 are symmetrically arranged on the support frame 420 in the circumferential direction, and the clamping rings 421 are used for clamping the test tubes 430; the upper end bilateral symmetry of mounting panel 2 is fixed with first support 202, be fixed with riding wheel 203 on the first support 202, the bottom of tray 3 be provided with riding wheel 203 assorted slide rail.
In the above embodiment, the second limiting tube 201 on the mounting plate 2 can slide up and down relatively in the first limiting tube 101 on the bottom plate 1, and the first driving device drives the mounting plate 2 to vibrate up and down, so as to drive the test tube rack 4 connected with the mounting plate 2 to vibrate up and down. The plurality of snap rings 421 on the test tube rack 4 are used for placing a plurality of test tubes 430 at one time, and the test tubes 430 are used for placing an extract to be extracted (the extract to be extracted in the application is an in vitro fat mass), so that the work efficiency of the extract to be extracted is improved; the connecting rod 410 is detachably connected with the supporting frame 420, and different supporting frames 420 can be used according to actual requirements. The riding wheels 203 on the first brackets 202 on two sides of the mounting plate 2 can slide on the sliding rails on the tray 3, the riding wheels 203 on the first brackets 202 are used for supporting the tray 3 to horizontally rotate, and the second driving device is used for driving the tray 3 to horizontally rotate so as to realize the revolution of the extract to be extracted around the output shaft of the second driving device. The third driving device is used for driving the test tube rack 4 to rotate horizontally, so that the extract to be extracted can rotate around the output shaft of the third driving device, and further the centrifugal motion of the extract to be extracted can be realized. The device for extracting the blood vessel components of the fat source matrix realizes the vortex motion of the extract to be extracted by combining the rotation and the revolution of the extract to be extracted; the first driving device realizes the oscillating motion of the extract to be extracted, and the third driving device realizes the centrifugal motion of the extract to be extracted.
Referring to fig. 5-8, the support bracket 420 is hinged or welded to the snap ring 421 according to an embodiment of the present invention.
In the above embodiments, the supporting frame 420 of the present invention is divided into two types, one type is that the supporting frame 420 is hinged to the clamp ring 421 (as shown in fig. 5-6); and the other is that the support 420 and the snap ring are welded and integrally formed (as shown in fig. 7-8). When the extract needs to be centrifuged, the support 420 hinged with the clamp ring 421 is selected, the test tube 430 on the clamp ring 421 is thrown away under the action of the third driving device, and the centrifugation of the extract is realized according to the difference of molecular weight of the substance. When the extract to be extracted needs to do vortex motion, the support frame 420 welded with the clamp ring 421 is selected, and the vortex motion of the extract to be extracted is realized under the action of the second driving device and the third driving device.
Referring to fig. 4 to 8, according to an embodiment of the present invention, the top end of the connecting rod 410 has a screw hole, the center of the supporting frame 420 has a through hole 422, the screw hole and the through hole 422 are provided with a hand screw 440, and the hand screw 440 passes through the through hole 422 and is screwed into the screw hole to fixedly connect the supporting frame 420 and the connecting rod 410.
In the above embodiment, the screw rod of the hand screw 440 passes through the support frame 420 and is screwed into the screw hole of the connecting rod 410, so as to realize the detachable connection between the connecting rod 410 and the support frame 420, and further, the support frame 420 can be used as a support frame 420 for a vortex motion or a centrifugal motion according to actual requirements.
Referring to fig. 4, according to one embodiment of the present invention, the first driving means includes a first motor 5, a cam 501 and a roller 502; a first motor 5 is fixedly mounted in the middle of the bottom plate 1 through a second bracket 102, a cam 501 is fixed on an output shaft of the first motor 5, and a roller 502 is arranged on the mounting plate 2 at a position opposite to the cam 501.
In the above embodiment, the first motor 5 is fixed on the bottom plate 1 through the second bracket 102, the output shaft of the first motor 5 drives the cam 501 to make a circular motion, and the cam 501 drives the roller 502 to move up and down, so as to realize the up-and-down oscillating motion to be extracted. In addition, the cam 501 is provided with two symmetrical convex parts, and the cam 501 can make the extract to be extracted vibrate twice in the process of one circular motion, so that the extraction efficiency of the extract to be extracted is improved.
Referring to fig. 4, according to an embodiment of the present invention, the second driving device includes a second motor 6, the second motor 6 is fixed to a middle portion of the mounting plate 2, and an output shaft of the second motor 6 is fixedly connected to the tray 3.
In the above embodiment, the output shaft of the second motor 6 is fixedly connected to the tray 3, the tray 3 is driven by the second motor 6 to rotate horizontally, and the test tube rack 4 on the tray 3 can perform revolution motion around the output shaft of the second motor 6.
Referring to fig. 4, according to an embodiment of the present invention, the third driving device includes a third motor 7, the third motor 7 has a mounting seat 701, the center of the tray 3 is opened with a centrifugal groove 301, and the tray 3 is uniformly opened with a plurality of spiral grooves 302 along a radial direction or/and a circumferential direction; the centrifugal groove 301 and the vortex groove 302 are detachably connected with the mounting seat 701 respectively; the output shaft of the third motor 7 is connected with the connecting rod 410 through a coupler.
In the above embodiment, the mounting seat 701 at the bottom of the third motor 7 is detachably connected to the centrifugal groove 301 or the spiral groove 302 on the tray 3, and the third motor 7 can be fixed at different positions on the tray 3 according to actual requirements. When the third motor 7 is fixed on the centrifugal groove 301, the third motor 7 is started, and the centrifugal motion of the extract to be extracted can be realized; when the third motor 7 is fixed on the scroll groove 302, the second motor 6 and the third motor 7 are started, and the scroll motion of the extract to be extracted can be realized. In addition, when the third motor 7 is fixed on the centrifugal groove 301, the first motor 5 and the second motor 6 are started, so that the centrifugal oscillation motion of the extract to be extracted can be realized; when the third motor 7 is fixed on the vortex groove 302, the first motor 5, the second motor 6 and the third motor 7 are started, and vortex oscillation motion of the extract can be realized. The tray 3 is uniformly provided with a plurality of vortex grooves 302 along the radial direction, the distance between the third motor 7 and the output shaft of the second motor 6 can be adjusted, and the extract to be extracted can do vortex motion along different radiuses. The tray 3 is uniformly provided with a plurality of vortex grooves 302 along the circumferential direction, so that a plurality of samples to be extracted can be processed at the same time, and the processing efficiency is high.
Referring to fig. 4, according to an embodiment of the present invention, the bottom end of the mounting seat 701 has an external thread, and the centrifugal groove 301 and the spiral groove 302 have screw holes respectively matched with the external thread.
In the above embodiment, the mounting seat 701 is detachably connected to the centrifugal groove 301 and the spiral groove 302 through the external threads and the internal threads on the screw hole, which are matched with each other, so that the position of the third motor 7 can be adjusted and fixed conveniently.
Referring to fig. 4, according to an embodiment of the present invention, a spring 8 is further included, and both ends of the spring 8 are fixedly connected to the base plate 1 and the mounting plate 2, respectively.
In the above embodiment, the elastic action of the spring 8 is used to support the mounting plate 2, reducing the pressure of the cam 501 and the roller 502.
According to one embodiment of the invention, the device further comprises a protective cover for covering the whole fat-derived stromal vascular fraction extraction means.
In the above embodiments, the protective cover is used to provide a sterile and temperature-controlled environment, so that the extract to be extracted can be extracted or stored at 4-37 ℃, and the pollution of the environment to the extract is reduced.
Example 1
A method for extracting blood vessel components of fat-derived stroma comprises the following steps:
And 2, adding normal saline (the normal saline adopts a sodium chloride solution with the concentration of 0.9 percent, and the normal saline is used for immersing the particles), and performing vortex oscillation for 15min under the condition that the frequency is 30Hz to obtain suspension.
And 3, centrifuging the suspension for 2min at the rotating speed of 1000rpm to obtain a centrifugal substance.
And 4, filtering the centrifugate by adopting a 100-mesh filter screen to obtain the matrix blood vessel component at the bottom of the tube, thus obtaining the composition.
Example 2
A method for extracting blood vessel components of fat-derived stroma comprises the following steps:
And 2, adding normal saline (the normal saline adopts a sodium chloride solution with the concentration of 0.9 percent, and the normal saline is used for immersing the particles), and performing vortex oscillation for 2min under the condition that the frequency is 50Hz to obtain suspension.
And 3, centrifuging the suspension for 5min at the rotating speed of 500rpm to obtain a centrifugal substance.
And 4, filtering the centrifugate by adopting a 100-mesh filter screen to obtain the matrix blood vessel component at the bottom of the tube, thus obtaining the composition.
Example 3
A method for extracting blood vessel components of fat-derived stroma comprises the following steps:
And 2, adding normal saline (the normal saline adopts a sodium chloride solution with the concentration of 0.9 percent, and the normal saline is used for immersing the particles), and performing vortex oscillation for 25min under the condition that the frequency is 5Hz to obtain suspension.
And 3, centrifuging the suspension for 1min at the rotating speed of 1500rpm to obtain a centrifugal substance.
And 4, filtering the centrifugate by adopting a 100-mesh filter screen to obtain the matrix blood vessel component at the bottom of the tube, thus obtaining the composition.
Example 4
A method for extracting blood vessel components of fat-derived stroma comprises the following steps:
And 2, adding normal saline (the normal saline adopts a sodium chloride solution with the concentration of 0.9 percent, and the normal saline is used for immersing the particles), and performing vortex oscillation for 20min under the condition that the frequency is 20Hz to obtain suspension.
And 3, centrifuging the suspension for 1.5min at the rotating speed of 1200rpm to obtain a centrifugal substance.
And 4, filtering the centrifugate by adopting a 100-mesh filter screen to obtain the matrix blood vessel component at the bottom of the tube, thus obtaining the composition.
Preparing the obtained Stromal Vascular Fraction (SVF) into suspension, and mixing with the fat mass to obtain fat; fat is injected into the implanted area. The prepared matrix blood vessel component has the characteristics of easy vascularization and good cell activity after injection. The volume retention rate after the autologous fat transplantation can be improved by matching with the autologous fat transplantation technology. In addition, SVF can be used in combination with various drugs and materials for treating osteoarthritis, cartilage repair, and other diseases. Meanwhile, the research of the application shows that the vortex oscillation has the best effect on extracting the matrix vascular components under the conditions that the frequency is 30Hz and the time is 15 min.
1) Test method
Extracting fat tissues on the back of the rabbit under the aseptic condition, removing fascia, dividing the fat tissues into four equal parts, respectively processing the fat tissues by adopting methods of low-frequency vortex oscillation (sonication), high-speed centrifugation (centrifugation), enzyme digestion (enzyme digestion) and nano emulsification (nano emulsification), extracting SVF, preparing into 1mL SVF suspension, observing the number of prepared immediate living cells by Trypan blue staining (Trypan blue staining), and obtaining the test result shown in FIG. 9; the proliferation capacity of the cells was observed by CCK-8 assay, and the results are shown in FIG. 10.
2) Test results
As can be seen from FIG. 9, the number of immediate viable cells of SVF extracted by low frequency vortex shaking is significantly higher than the number of viable cells extracted by high speed centrifugation, enzymatic digestion and nanoemulsification. As can be seen from FIG. 10, the CCK8 test result shows that the SVF proliferation activity extracted by low-frequency vortex shaking is significantly higher than that of the SVF proliferation activity extracted by high-speed centrifugation and nano-emulsification, and is close to that of the SVF proliferation activity extracted by enzyme digestion.
1) Test method
Further, it was verified by animal experiments whether this advantage of vortex oscillation method in vivo environment is still significant. 24 male rabbits were divided into 4 groups of 6 rabbits each, and each rabbit was subjected to the following experiment: and (3) taking the back fat of the rabbit, respectively processing the back fat by adopting the same amount of sterile cellulite by the four methods to obtain SVF suspension, respectively mixing the SVF suspension with 1g of cellulite blocks, implanting the SVF suspension into subcutaneous tissues on the back of the rabbit ear, and constructing a rabbit ear autologous fat transplantation model. Observing the change trend of the volume of the fat mass by adopting B-ultrasonic detection (B-ultrasonic amplification), wherein the test result is shown in figure 11; and according to the groups, the materials are sacrificed and taken at 2 weeks, 1 month, 3 months and 6 months after the operation, and oil red O staining and HE staining are carried out to determine the fat forming effect, wherein the oil red O staining test result is shown in figure 12, and the HE staining test result is shown in figure 13.
2) Test results
As can be seen from FIG. 11, the B-super-junction fruit shows that the time-varying trends of various methods can be seen that the volume maintenance rate of the vortex oscillation group is obviously higher than that of other groups at the long term (6 months) after the fat transplantation, and the vortex oscillation group, the centrifugation group, the enzyme digestion group and the nano-emulsification group are followed.
As can be seen from FIGS. 12 and 13, the lipid formation effect by vortex oscillation and centrifugation is more uniform, while the lipid droplets are smaller and are distributed unevenly in the enzymolytic group and the nanoemulsifying group due to more connective tissue growth.
In conclusion, SVF extracted by vortex oscillation method has higher cell activity and good clinical transformation potential.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such changes and modifications of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such changes and modifications.
Claims (5)
1. An apparatus for extracting a vascular component from a matrix derived from fat, comprising: the test tube rack comprises a bottom plate (1), a mounting plate (2), a tray (3), a test tube rack (4), a first driving device, a second driving device and a third driving device; wherein the content of the first and second substances,
a first limiting pipe (101) is fixed at the upper end of the bottom plate (1), a second limiting pipe (201) matched with the first limiting pipe (101) is fixed at the bottom end of the mounting plate (2), and the first limiting pipe (101) is sleeved into the second limiting pipe (201) in a sliding mode;
the mounting plate (2) is driven to move up and down on the bottom plate (1) through a first driving device; the upper end of the middle part of the mounting plate (2) drives the tray (3) to horizontally rotate through a second driving device; the upper end of the tray (3) drives the test tube rack (4) to horizontally rotate through a third driving device;
the third driving device comprises a third motor (7), the third motor (7) is provided with a mounting seat (701), a centrifugal groove (301) is formed in the center of the tray (3), and a plurality of vortex grooves (302) are uniformly formed in the tray (3) along the radial direction or/and the circumferential direction; the centrifugal groove (301) and the vortex groove (302) are respectively detachably connected with the mounting seat (701); an output shaft of the third motor (7) is connected with the connecting rod (410) through a coupler;
the bottom end of the mounting seat (701) is provided with an external thread, and the centrifugal groove (301) and the vortex groove (302) are respectively provided with a screw hole matched with the external thread;
the test tube rack (4) comprises a connecting rod (410) and a supporting frame (420), and the lower end of the connecting rod (410) is fixedly connected with an output shaft of the third driving device; a support frame (420) is detachably fixed at the upper end of the connecting rod (410), a plurality of clamping rings (421) are symmetrically arranged on the support frame (420) in the circumferential direction, and the clamping rings (421) are used for clamping the test tubes (430);
first supports (202) are fixed at the upper end of the mounting plate (2) in a bilateral symmetry mode, riding wheels (203) are fixed on the first supports (202), and sliding rails matched with the riding wheels (203) are arranged at the bottom end of the tray (3);
the support frame (420) is hinged or welded with the clamping ring (421).
2. The device for extracting adipose-derived stromal vascular fraction as claimed in claim 1, wherein the top end of the connecting rod (410) is provided with a screw hole, the support frame (420) is provided with a through hole (422) at the center, a hand screw (440) is assembled in the screw hole and the through hole (422), and the hand screw (440) passes through the through hole (422) and is screwed into the screw hole to fixedly connect the support frame (420) and the connecting rod (410).
3. The device for extracting adipose-derived stromal vascular fraction as claimed in claim 1, wherein the first driving device comprises a first motor (5), a cam (501) and a roller (502); the middle of the bottom plate (1) is fixedly provided with a first motor (5) through a second support (102), an output shaft of the first motor (5) is fixedly provided with a cam (501), and a roller (502) is arranged on the mounting plate (2) at a position opposite to the cam (501).
4. The device for extracting adipose-derived stromal vascular fraction as claimed in claim 1, wherein the second driving device comprises a second motor (6), the second motor (6) is fixed in the middle of the mounting plate (2), and the output shaft of the second motor (6) is fixedly connected with the tray (3).
5. A method for extracting adipose-derived stromal vascular fraction based on the extraction device of any one of claims 1 to 4, comprising the steps of:
step 1, removing tissue fascia of a body fat mass, and shearing to obtain particles;
step 2, adding physiological saline into the particles, and performing vortex oscillation to obtain a suspension;
in the step 2, the frequency of vortex oscillation is 5-50 Hz, and the time of vortex oscillation is 2-25 min;
step 3, centrifuging the suspension to obtain a centrifugal substance;
in the step 3, the rotating speed of the centrifugation is 500-1500 rpm, and the time of the centrifugation is 1-5 min;
and 4, filtering the centrifugate to obtain the stromal vascular fraction.
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