Mechanical method for processing fat to obtain svf

Abstract

The application provides a connecting device and method for obtaining SVF by mechanically treating fat, and the connecting device comprises a shell, an interface pipe, a wheel disc, a roller, a window and a limiting block; the window is arranged on the shell; the interface pipe is arranged at two ends of the shell; the wheel disc is rotationally connected to the inside of the shell through a shaft, the roller protrudes from the shell and is engaged with the wheel disc; the limiting block is fixed to the inside of the shell and is in rolling contact with the side surface of the wheel disc; a limiting structure and a treatment hole are arranged on the wheel disc, the wheel discs can be tightly connected through the treatment hole, the treatment hole penetrates the wheel disc and can be communicated with the interface pipe; and the limiting structure is in rolling cooperation with the limiting block. The connecting device has a transformable hole diameter, can adjust the hole diameter shape and quantity between syringes, and achieves the purpose of cutting and emulsifying fat in different degrees. Meanwhile, the hole diameter size and shape of the connecting device can be customized according to the separation and preparation process of SVF.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical devices, in particular to a connecting device and method for obtaining SVF by mechanical treatment of fat. BACKGROUND

[0002] The human body fat is rich in stromal vascular fraction (SVF), which is a mixture of various cells and cytokines with repair functions. It includes adipose-derived stem cells (ADSCs), hematopoietic stem cells, endothelial progenitor cells, endothelial cells, vascular smooth muscle cells, fibroblasts, lymphocytes, macrophages, mast cells, platelets, etc. In particular, the ADSCs therein have good immune regulation ability and promote blood vessel regeneration ability, and have been reported in many clinical applications in the aspects of assisting fat transplantation and treating osteoarthritis.

[0003] The fat used for preparing SVF is mostly obtained by liposuction surgery, which contains the swelling solution added during the liposuction surgery process. Before SVF preparation, a centrifugation step is usually performed. The fat obtained by liposuction surgery is divided into four layers after centrifugation, i.e., oil phase, fat layer, swelling solution and tissue fragments. The fat layer is taken (the fat obtained in this step is generally referred to as "Coleman fat") for subsequent processing. The subsequent processing can be divided into enzymatic method and mechanical method. The enzymatic method uses collagenase to digest the extracellular matrix components, so that the SVF-related cells can be collected by centrifugation. The mechanical separation method uses ultrasonic waves, shock waves, etc. to emulsify fat and destroy the extracellular matrix components, so that the SVF-related cells are separated. However, the enzymatic method has the disadvantages of long time consumption and complex operation. The use of collagenase brings about the risk of residue. The additional operation outside the surgical environment increases the risk of contamination caused by operation. The mechanical separation method has the advantages of short time consumption and simple operation. No foreign substances are introduced to cause residue risk. Part of the extracellular matrix components is preserved in the form of gel, which is more suitable for tissue filling. In summary, the mechanical separation method has the characteristics of short time consumption and simple operation, which is particularly suitable for operation in the operating room, i.e., SVF preparation can be performed in a very short time after the completion of liposuction surgery, for fat backfilling or other treatment surgery, so that the patient does not need to undergo a second surgery, which has advantages in treatment convenience and surgical risk control. However, the SVF yield and SVF viability obtained by the mechanical separation method are not high.

[0004] In summary, the existing technology has the following problems: how to improve the SVF yield and SVF viability obtained by mechanical separation. SUMMARY

[0005] The application provides a connecting device and method for obtaining SVF by mechanically treating fat, and solves the problem of how to improve the yield and activity of SVF obtained by mechanical separation.

[0006] To achieve the above object, in one aspect, the application provides a connecting device for obtaining SVF by mechanically treating fat, comprising:

[0007] a shell, an interface pipe arranged at both ends of the shell, a wheel disc rotatably connected inside the shell, a roller engaging with the wheel disc, a window opened on the shell, and a limiting block fixed inside the shell;

[0008] The wheel disc is provided with a plurality of limiting structures and treatment holes, and the wheel discs are connected by the treatment holes.

[0009] The limiting structure is arranged on the side surface of the wheel disc, so that the limiting block and the limiting structure are in rolling contact.

[0010] The roller protrudes from the shell, and the rotation of the wheel disc is realized by rolling the roller, so that the treatment hole is communicated with the interface pipe.

[0011] Specifically, the wheel discs have cavities therebetween, and the wheel discs are provided with three, including a first wheel disc, a second wheel disc, and a third wheel disc.

[0012] The roller is provided with three, including a first roller, a second roller, and a third roller.

[0013] The first roller engages with the first wheel disc, the second roller engages with the second wheel disc, and the third roller engages with the third wheel disc.

[0014] Specifically, the limiting structure is arranged on both sides of the wheel disc, and the limiting structure comprises a limiting ring groove and a limiting recess.

[0015] The limiting ring groove is arranged on both side surfaces of the wheel disc with the shaft as the center.

[0016] The limiting recess is semispherical, the limiting recess is arranged on the limiting ring groove, and the diameter of the limiting recess is greater than the width of the limiting ring groove.

[0017] Specifically, the limiting block is provided with a limiting spring and a limiting ball inside; and the limiting ball can protrude from the limiting block.

[0018] The limiting spring and the limiting ball are in close contact, and the limiting ball is in rolling contact with the limiting structure on the wheel disc.

[0019] Specifically, the processing holes of the first wheel disc include: a first cutting hole, a second cutting hole and a third cutting hole;

[0020] The processing holes of the second wheel disc include: a first extrusion hole, a second extrusion hole and a third extrusion hole.

[0021] Specifically, a well-shaped wire mesh is provided in the first cutting hole; a cross-shaped wire mesh is provided in the third cutting hole; and a plurality of wires are radially provided in the second cutting hole, with both ends of the wires fixed on the inner wall of the second cutting hole.

[0022] Specifically, the aperture of the first extrusion hole is smaller than the aperture of the second extrusion hole, and the aperture of the second extrusion hole is smaller than the aperture of the third extrusion hole.

[0023] Specifically, the limiting block includes: a first limiting block, a second limiting block, a third limiting block and a fourth limiting block;

[0024] The first limiting block is fixed on the inner wall of the housing, and the first limiting block is in rolling contact with the limiting structure on one side of the first wheel disc;

[0025] The second limiting block is arranged in the cavity between the first wheel disc and the second wheel disc, and the second limiting block is fixed on the shaft, the second limiting block is in rolling contact with the limiting structure on the other side of the first wheel disc, and the second limiting block is in rolling contact with the limiting structure on one side of the second wheel disc;

[0026] The third limiting block is arranged in the cavity between the second wheel disc and the third wheel disc, and the third limiting block is fixed on the shaft, and the third limiting block is in rolling contact with the limiting structure on the other side of the second wheel disc; the third limiting block is in rolling contact with the limiting structure on one side of the third wheel disc;

[0027] The fourth limiting block is fixed on the inner wall of the housing, and the fourth limiting block is in rolling contact with the limiting structure on the other side of the third wheel disc.

[0028] Specifically, the mouthpiece is connected to the injection device.

[0029] In another aspect, the present invention provides a method for mechanically processing fat to obtain SVF, using the aforementioned connecting device for mechanically processing fat to obtain SVF, and connecting the interface tube of the connecting device to an injection device;

[0030] Rotate the wheel to select the corresponding processing hole to connect to the interface tube;

[0031] Pushing the injection device makes the fat from one injection device pass through the connecting device for treating fat by mechanical method to obtain SVF into the other end of the injection device;

[0032] Obtaining SVF from the treated fat.

[0033] The connecting device of the application can ensure complete sealing when connecting two syringes. The connecting device of the application has a convertible pore size, which can adjust the pore size shape and number between the syringes to achieve the purpose of cutting and emulsifying the fat to different degrees. When adjusting the pore size, the connector does not need to be replaced, which can ensure that the fat in the syringe will not be exposed multiple times, reducing the risk of SVF contamination during operation. At the same time, the pore size and shape of the connecting mechanism can be customized according to the separation and preparation process of SVF. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a structural schematic diagram of a connecting device for treating fat by mechanical method to obtain SVF according to an embodiment of the application;

[0035] Figure 2 is a schematic diagram of the installation and use of a connecting device for treating fat by mechanical method to obtain SVF according to an embodiment of the application;

[0036] Figure 3 is a top view of a connecting device for treating fat by mechanical method to obtain SVF according to an embodiment of the application;

[0037] Figure 4 is a side view of a connecting device for treating fat by mechanical method to obtain SVF according to an embodiment of the application;

[0038] Figure 5 is a front view of the internal structure of a connecting device for treating fat by mechanical method to obtain SVF according to an embodiment of the application;

[0039] Figure 6 is a side view of the internal structure of a connecting device for treating fat by mechanical method to obtain SVF according to an embodiment of the application;

[0040] Figure 7 is a structural schematic diagram of a first wheel according to an embodiment of the application;

[0041] Figure 8 is a structural schematic diagram of a second wheel according to an embodiment of the application;

[0042] Figure 9 is a front view of a second wheel according to an embodiment of the application;

[0043] Figure 10 is a schematic diagram of the percentage of SVF in Coleman fat in the examples and comparative examples;

[0044] Figure 11 Typical fluorescence images of AO / PI staining of examples and comparative examples;

[0045] Figure 12 Graphs of cell viability of SVF in examples and comparative examples;

[0046] Figure 13 Graphs of cell density of SVF in each group in examples and comparative examples;

[0047] Figure 14 Graphs of cell morphology in Example 3;

[0048] Figure 15 Graphs of detection results of first flow cytometry sample of examples of the present application;

[0049] Figure 16 Graphs of detection results of second flow cytometry sample of examples of the present application;

[0050] Figure 17 Graphs of detection results of third flow cytometry sample of examples of the present application;

[0051] Figure 18 Graphs of detection results of fourth flow cytometry sample of examples of the present application;

[0052] Figure 19 Graphs of detection results of fifth flow cytometry sample of examples of the present application;

[0053] Figure 20 Graphs of detection statistics of flow cytometry sample of examples of the present application;

[0054] Figure 21 Graphs of detection results of first flow cytometry sample of isotype control of examples of the present application;

[0055] Figure 22 Graphs of detection results of second flow cytometry sample of isotype control of examples of the present application;

[0056] Figure 23 Graphs of detection results of third flow cytometry sample of isotype control of examples of the present application;

[0057] Figure 24 Graphs of detection results of fourth flow cytometry sample of isotype control of examples of the present application;

[0058] Figure 25 Graphs of detection results of fifth flow cytometry sample of isotype control of examples of the present application;

[0059] Figure 26 Graphs of detection statistics of flow cytometry sample of isotype control of examples of the present application.

[0060] Description of Figure Numbers:

[0061] 1. Housing; 2. Injection device; 11. Roller; 12. Interface tube; 13. Window; 14. Disc; 15. Stopper; 16. Shaft; 17. Fixing rod; 21. Push rod; 111. First roller; 112. Second roller; 113. Third roller; 131. First window; 132. Second window; 133. Third window; 141. First disc; 142. Second disc; 143. Third disc; 1411. First cutting hole; 1412. Second cutting hole; 1413. Third cutting hole; 1414. First prompt area; 1415, second prompt area; 1416, third prompt area; 1421, first extrusion hole; 1422, second extrusion hole; 1423, third extrusion hole; 1424, fourth prompt area; 1425, fifth prompt area; 1426, sixth prompt area; 1417, first limiting ring groove; 1441, second limiting ring groove; 1442, limiting groove; 151, first limiting block; 152, second limiting block; 153, third limiting block; 154, fourth limiting block; 155, limiting ball; 145, shaft hole. DETAILED DESCRIPTION

[0062] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0063] The embodiment of the present invention provides a connection device for obtaining SVF by mechanically processing fat, such as Figure 1 Shown, including:

[0064] A housing 1, mouthpieces 12 provided at both ends of the housing 1, a wheel 14 rotatably connected to the interior of the housing 1, a roller 11 engaged with the wheel 14, a viewing window 13 provided on the housing 1, and an internal stopper 15 fixed to the housing 1;

[0065] The window 13 is provided on the housing 1; the window 13 is used to display the aperture type or size on the wheel 14. The mouthpiece 12 is provided at both ends of the housing 1, such as Figure 2 As shown, the mouthpiece 12 is connected to the injection device 2, and the connection method is preferably a threaded connection.

[0066] The wheel 14 is rotatably connected to the interior of the housing 1 via the shaft 16. Figure 4As shown, the roller 11 protrudes from the shell 1 and engages with the wheel disc 14; the wheel disc 14 rotates by rolling the roller 11, and the processing hole is communicated with the interface pipe 12. The limiting block 15 is fixed inside the shell 1 and in rolling contact with the side of the wheel disc 14; the wheel disc 14 is provided with a limiting structure and a processing hole, and the wheel discs 14 can be connected by pressing through the processing hole, and the processing hole penetrates the wheel disc 14 and can be communicated with the interface pipe 12; the limiting structure is in rolling contact with the limiting block 15. The wheel disc 14 is provided with a mark area, and the position of the mark area can correspond to the position of the window 13. When the mark area rotates to the position of the window 13, the operator can distinguish which processing hole is communicated with the interface pipe 12 by seeing the corresponding mark area.

[0067] There is a cavity between the wheel discs 14; the wheel discs 14 are provided with three, including: the first wheel disc 141; the second wheel disc 142; the third wheel disc 143; preferably, the number of wheel discs 14 is 2, 4, 5. The roller 11 is used to adjust the inner hole diameter of the connecting device, wherein, as shown in the figure, Figure 3 As shown, the roller 11 is provided with three, including: the first roller 111, the second roller 112, and the third roller 113; wherein, the first roller 111 engages with the first wheel disc 141, the second roller 112 engages with the second wheel disc 142, and the third roller 113 engages with the third wheel disc 143. Rotate the roller 11, the roller 11 rotates with the wheel disc 14, and the processing hole communicated with the interface pipe 12 can be replaced. That is, rotating the roller 11 can select the required processing hole type or size.

[0068] The limiting structure on the wheel disc 14 is provided on both sides of the wheel disc 14, and the limiting structure includes: a limiting ring groove and a limiting groove 1442; the limiting ring groove is provided on both sides of the wheel disc 14 with the shaft 16 as the center; the limiting groove is semispherical, and the limiting groove is provided on the limiting ring groove, and the diameter of the limiting groove is greater than the width of the limiting ring groove.

[0069] The limiting block 15 is provided with a limiting spring and a limiting ball 155 inside; the limiting ball can protrude from the limiting block 15; the limiting spring and the limiting ball are in close contact, and the limiting ball is in rolling contact with the limiting structure on the wheel disc 14. The limiting block 15 is used to limit the rotational displacement of the wheel disc, so that the hole of the wheel disc corresponds to the hole of the other two wheel discs in the connecting device.

[0070] As shown in the figure, Figure 5 The limiting block 15 includes: a first limiting block 151, a second limiting block 152, a third limiting block 153, and a fourth limiting block 154;

[0071] The first limiting block 151 is fixed on the inner wall of the shell 1, and the first limiting block 151 is in rolling contact with the limiting structure on one side of the first wheel disc 141;

[0072] The second limiting block 152 is disposed in the cavity between the first wheel disc 141 and the second wheel disc 142 and is fixed to the shaft 16 via a fixing rod. The second limiting block 152 is in rolling contact with the limiting structure on the other side of the first wheel disc 141 and in rolling contact with the limiting structure on one side of the second wheel disc 142.

[0073] The third limiting block 153 is disposed in the cavity between the second wheel disc 142 and the third wheel disc 143 and is fixed to the shaft 16 via the fixing rod 17. The third limiting block 153 is in rolling contact with the limiting structure on the other side of the second wheel disc 142; the third limiting block 153 is in rolling contact with the limiting structure on one side of the third wheel disc 143;

[0074] The fourth limiting block 154 is fixed on the inner wall of the housing 1 , and the fourth limiting block 154 is in rolling contact with the limiting structure on the other side of the third wheel disc 143 .

[0075] like Figure 5 , the sliding limit blocks at the far left and far right are fixed to the housing 1 of the connecting device, and the two middle limit blocks are fixed to the shaft 16. The spring inside the limit block, which is always in a compressed state, provides a force for the limit ball (pinball). At the same time, the outer shell of the limit block limits the displacement of the pinball so that only the tip leaks out of the outer shell and contacts the limit ring groove. When the wheel rotates and the optional aperture corresponds to the connection port, the pinball will enter the deeper limit groove to achieve displacement limitation of the wheel component. When the optional aperture needs to be changed, use greater force to rotate the roller, and the pinball will slide out of the deeper limit groove into the limit ring groove. When the next optional aperture corresponds to the interface, the pinball will enter the next deeper limit groove. When the pinball enters the limit groove, the mark of the aperture currently connected to the interface 12 will be displayed on the window 13. As shown Figure 3 , the windows 13 are window 131, window 132, and window 133 from left to right, for example, Figure 9 When the limiting ball 155 in the second limiting block 152 enters the limiting groove 1442 of the second wheel 142, the window 132 will display the mark of the aperture currently connected to the interface tube 12 (interface).

[0076] like Figure 7 As shown, the processing holes of the first wheel 141 include: a first cutting hole 1411, a second cutting hole 1412 and a third cutting hole 1413; a well-shaped wire mesh is arranged in the first cutting hole 1411; a cross-shaped wire mesh is arranged in the third cutting hole 1413; a plurality of wires are arranged radially in the second cutting hole 1412, and both ends of the wires are fixed on the inner wall of the second cutting hole 1412.

[0077] like Figure 8As shown, the second wheel disc 142 is provided with three through-hole structures, the through-holes have different diameters, and the through-holes are used as processing holes to process fat. The fat has been mechanically divided by the left and right two wheel discs with the hole diameter of the screen. The fat is extruded and emulsified. Specifically, the processing holes of the second wheel disc 142 include: the first extrusion hole 1421, the second extrusion hole 1422, and the third extrusion hole 1423. The hole diameter of the first extrusion hole 1421 is smaller than the hole diameter of the second extrusion hole 1422, and the hole diameter of the second extrusion hole 1422 is smaller than the hole diameter of the third extrusion hole 1423. Preferably, the first extrusion hole 1421 is a 1.4mm diameter hole, the second extrusion hole 1422 is a 2mm diameter hole, and the third extrusion hole 1423 is a 2.4mm diameter hole. Near the wheel disc outer edge of the through-hole, there is a character sticker near the through-hole mark for display on the connection mechanism window, prompting that the through-hole corresponding to the interface pipe 12 of the connection device at present is which number, that is, the fourth prompt area 1424 marks the corresponding first extrusion hole 1421, the fifth prompt area 1425 marks the corresponding second extrusion hole 1422, and the sixth prompt area 1426 marks the corresponding third extrusion hole 1423. When the limiting ball 155 in the limiting block rolls into the limiting groove in the second limiting ring groove 1441, the mark of the hole diameter corresponding to the interface pipe 12 will be displayed on the window 132.

[0078] The wheel disc has a hole in the center, which is the shaft hole 145. The shaft 16 passes through the shaft hole 145 to connect the three wheel discs. Figure 6 As shown, the wheel disc can rotate on the shaft 16 under the condition of the dial roller 11. Figure 5 As shown, Figure 5 It is a cross-sectional front view of the internal structure of the connection device. The rollers correspond to the left wheel disc, the middle wheel disc, and the right wheel disc in the adjusting connection mechanism, respectively. The central part of the three wheel discs is penetrated by the shaft. The sliding limiting mechanism at the leftmost and rightmost ends is fixed on the connection mechanism shell, and the two limiting blocks in the middle are fixed on the shaft. There are annular grooves on both sides of the wheel disc. There are corresponding deeper circular grooves on the annular grooves on the same radius connection line of the selectable hole diameter. The limiting block 15 is a sliding limiting mechanism. The limiting ball 155 in the limiting block 15 can roll in the annular groove. When the selectable hole diameter corresponds to the interface pipe 12, the limiting ball 155 slides into the circular groove and is blocked from rotating, providing mechanical feedback and achieving positioning. When it is necessary to continue to select the hole diameter, a slightly larger force is used to dial the roller (limiting ball 155) corresponding to the wheel disc, which can continue to rotate to the target hole diameter and achieve positioning. The limiting structure on the first wheel disc 141 and the third wheel disc 143 is the same as that on the second wheel disc 142.

[0079] The processing hole type provided on the third disc 143 is the same as the processing hole of the first disc 141, that is, the hole diameter structure has a wire mesh blade, which can physically cut the passing fat. The second disc 142, that is, the middle disc has a different hole diameter. The first roller 111, the second roller 112 and the third roller 113 correspond to the first disc 141, the second disc 142 and the third disc 143 in the adjusting connecting device respectively. In use, two injection devices 2 (syringes) are connected to the interface pipe 12 of the connecting device respectively, and the plunger 21 of the syringe is pushed alternately, so that the fat is cut by the wire mesh blade first and then subjected to fluid extrusion to realize mechanical emulsification when passing through the connecting device.

[0080] The connecting device of the application can ensure complete sealing when connecting two syringes. The connecting device of the application has a convertible hole diameter, which can adjust the hole diameter shape and number between the syringes to achieve the purpose of cutting and emulsifying the fat to different degrees. When adjusting the hole diameter, the syringe fat does not need to be exposed for multiple times, which reduces the pollution risk of SVF in the operation process. At the same time, the hole diameter size and shape of the connecting mechanism can be customized according to the separation and preparation process of SVF.

[0081] That is, by designing and converting the hole diameter of the connecting device, the Coleman fat is first divided into smaller fat pieces by the wire mesh in the wire mesh hole diameter, and then emulsified by the large hole diameter and the small hole diameter of the connecting device, so as to reduce the resistance generated in the fat emulsification process, facilitate operation, reduce the damage of SVF, improve the SVF yield, and increase the cell concentration in SVF.

[0082] In addition, the embodiment of the application also provides a method for obtaining SVF by mechanically processing fat, which adopts the connecting device for obtaining SVF by mechanically processing fat, and connects the interface pipe of the connecting device with an injection device.

[0083] The rotating disc selects a corresponding processing hole to be connected with the interface pipe;

[0084] The injection device is pushed to make the fat pass through the connecting device for obtaining SVF by mechanically processing fat from one injection device to another injection device; and SVF is obtained from the processed fat.

[0085] For example: Transfer fat to a syringe, connect the syringe to one end of the connector via a Luer port, and then connect another empty syringe to the other end of the connector. Then, adjust the dial so that the holes corresponding to the connector tube, from left to right, are the crosshair hole, the 2.4mm hole, and the crosshair hole. Rapidly push the syringe through the connector and into the syringe at the other end. Continue pushing the syringe through the connector and back into the syringe at the original end, marking this as one cycle. Repeat this cycle multiple times. The fat is then cut and emulsified, ultimately yielding SVF.

[0086] like Figure 2 As shown, the connecting device has multiple rollers 11 for adjusting the internal aperture of the connecting device. Preferably, three rollers are provided: a first roller 111, a second roller 112, and a third roller 113, corresponding to the left, center, and right wheels within the connecting device. The two interface tubes 12 of the connecting device are connected to the syringe by a threaded connection, but other connection methods, such as a sliding connection, are also possible. The connecting device has a window 13 that displays the aperture number of the currently used wheel. The first window 131, the second window 132, and the third window 133 correspond to the left, center, and right wheels within the connecting device, respectively. The aperture structure of the left wheel (first wheel 141) and the right wheel (third wheel 143) contains wire mesh blades, which can physically cut fat passing through. The middle wheel (second wheel 142) has a different aperture structure. The rollers 11 are used to adjust the left, center, and right wheels within the connecting device. When in use, two syringes 2 are connected to the interface tubes 12 of the connecting device at the same time, and the push rods 21 of the syringes are pushed alternately, so that the fat is first cut by the wire mesh blade when passing through the connecting device, and then squeezed by the fluid through the aperture structure to achieve mechanical emulsification. Figure 7 It is the structure of the left wheel (first wheel 141) inside the connecting mechanism. The limiting groove 1417 on the wheel is used to cooperate with the limiting mechanism (limiting block 15) to limit the rotational displacement of the wheel so that the hole of the wheel corresponds to the openings at both ends of the connecting mechanism. There are three through-hole structures on the first wheel 141. The through-holes have metal wire meshes of different shapes for mechanically dividing the fat passing through. The diameters of the three apertures are all 2 to 4 mm, and the preferred diameter is 2.4 mm. The first cutting hole 1411 is a well-shaped wire mesh, the second cutting hole 1412 is a star-shaped wire mesh, and the third cutting hole 1413 is a cross-shaped wire mesh. There is a prompt area on the outer edge of the wheel near the processing hole, where character stickers marking the nearby processing holes can be affixed. They can be displayed on the window of the connecting mechanism to prompt the number of the through hole currently corresponding to the connecting mechanism interface tube 12.

[0087] The wheel disc 14 has sliding limit mechanism, i.e. limit block 15. It is composed of spring, ball and shell. The shell of the sliding limit mechanism at the leftmost and rightmost end is fixed on the inner wall of the shell 1 of the connecting device, and the shell of the two middle sliding limit mechanisms is fixed on the shaft 16. The spring in a constant compression state provides a force for the ball, and the shell limits the displacement of the ball so that only the tip of the ball leaks out of the shell and contacts the annular groove (limit ring groove). When the wheel disc rotates and the optional aperture corresponds to the connecting port of the interface pipe, the ball will enter the deeper circular groove (limit groove 1442) to realize the displacement limitation of the wheel disc component. When it is necessary to replace the optional aperture, the roller is rotated with a larger force, and the ball will slide out of the deeper circular groove 1442 to the annular groove. When the ball enters the circular groove, the mark of the aperture corresponding to the interface pipe 12 will be displayed on the window 13. For example, when the limit ball (ball) rolls into the circular groove of the first wheel disc 141 from the first limit ring groove 1417, the mark of the aperture corresponding to the interface pipe 12 will be displayed on the window 131, i.e. the first prompt area 1414 marks the first cutting hole 1411, the second prompt area 1415 marks the second cutting hole 1412, and the third prompt area 1416 marks the third cutting hole 1413.

[0088] The material of the connecting device of the two syringes can be metal, such as pure titanium, TC4 titanium alloy, 316L stainless steel, etc., or high polymer material, such as polyethylene, polypropylene, polyacetate, polymethyl methacrylate, etc. Each component can be composed of different materials and then assembled together.

[0089] The material of the syringe should be able to withstand high temperature and high pressure, radiation sterilization, ethylene oxide sterilization and other sterilization methods. Figure 2 The interface pipe 12 shown in the figure is the connecting port of the connecting device and the syringe. The connection mode shown here is a threaded interface, and other connection modes such as a sliding interface can also be used.

[0090] Only one form of the connecting device for preparing SVF that can connect two syringes is shown in the figure, in which each wheel disc structure has only three optional apertures and a wire mesh shape. However, the present application does not limit the shape and number of apertures, such as two, four or other number of apertures on the wheel disc. The shape and size of the aperture can also not be limited to those shown in the figure.

[0091] Example 1: Preparation of SVF using the connecting device of the present application.

[0092] Step 1, centrifuge the lipoaspirate at 1200xg for 3 minutes. Take the fat layer (Coleman's fat) for further processing.

[0093] Step 2, transfer the Coleman's fat into a syringe, connect the syringe to one end of the device via a luer fitting, and then connect another empty syringe to the other end of the device.

[0094] Step 3, adjust the device connector wheel to have the following sequence of holes from left to right: crosshatch hole - 2.4mm hole - crosshatch hole. Rapidly inject the syringe to pass the Coleman's fat through the connection mechanism into the other syringe. Then continue to inject the syringe to pass the Coleman's fat back through the connection mechanism into the original syringe, which is considered as one cycle. Repeat the process for 10-30 cycles.

[0095] Step 4, adjust the device connector wheel to have the following sequence of holes from left to right: star hole - 2mm hole - star hole. Repeat the process of step 3 for 10-30 cycles.

[0096] Step 5, adjust the device connector wheel to have the following sequence of holes from left to right: well hole - 1.4mm hole - well hole. Repeat the process of step 3 for 10-30 cycles.

[0097] Step 6, centrifuge the emulsified fat, discard the oil phase and the liquid layer containing the broken tissue components, and keep the SVF-rich component.

[0098] Example 2: Comparative study to compare the SVF obtained using the above preparation method with the SVF obtained using only one size of hole in terms of SVF volume, cell density, and cell viability.

[0099] First, centrifuge the lipoaspirate at 1200xg for 3 minutes. Take the fat layer (Coleman's fat) for further processing.

[0100] Method A, use the preparation method described in Example 1, and when performing steps 3, 4, and 5, the number of cycles of the Coleman's fat passing through the connection mechanism is 10.

[0101] Method B, pass the Coleman's fat through the current existing syringe-to-syringe connector (hole diameter of 2mm) only, and the number of cycles of passing through the connector is 30.

[0102] Method C, Coleman fat was only passed through the current existing syringe interconnector (hole diameter of 1.4mm) for 30 times.

[0103] Then the treated fat was centrifuged respectively, and the oil phase and the liquid layer containing broken tissue components were discarded, and the SVF-rich components were retained, and were recorded as SVF A, SVF B, and SVF C respectively.

[0104] In the three treatment methods, the total number of times that the Coleman fat passed through the device during the treatment process was consistent, and was 30 times. Each treatment method was performed 3 times to obtain 3 sample data. The obtained SVF components of A, B, and C were analyzed as follows

[0105] Volume ratio: calculate the percentage of obtained SVF in Coleman fat, and draw a chart for comparison as shown in Figure 10 .

[0106] Cell density: 1ml sample was taken from each of SVF A, SVF B, and SVF C, and mixed uniformly with 1ml phosphate buffer solution, and then filtered using a cell screen with a pore size of 100 microns. 20 microliters of filtrate was taken and mixed with 20 microliters of acridine orange / propyl iodide dye (AO / PI) for staining (the staining principle is that AO can be embedded in the nuclei of live and dead cells through the intact cell membrane, showing green fluorescence; PI can only be embedded in the nuclei of all dead cells through the incomplete cell membrane, i.e. the cell membrane of dead cells, showing red fluorescence), and the stained sample was counted using a fluorescence automatic cell counter to obtain the cell viability and cell density. According to the detection results of cell density, the original cell density in SVF A, SVF B, and SVF C was calculated. The typical fluorescence pictures of AO / PI staining are shown in Figure 11 , the cell viability of each group of SVF is shown in Figure 12 , and the cell density of each group of SVF is shown in Figure 13The results show that the percentage of SVF volume in Coleman fat prepared by the device of the present application (Method A) is between Method B and Method C. The reason is that Method B only uses a large fixed aperture, and the emulsification of Coleman fat is limited, and part of the fat cells are not completely destroyed. Method C only uses a small fixed aperture, and the shear force received by the Coleman fat is always large, and the fat cells are destroyed to a higher degree. This result is confirmed in the detection result of cell viability. The cell viability of SVF obtained by Method C is the smallest among the three methods, and the cell viability of SVF prepared by the device of the present application (Method A) is significantly improved. In addition, since the preparation of SVF by the device of the present application (Method A) causes less damage to cells (high cell viability) and higher emulsification of fat (greater volume compression), the obtained SVF has the highest cell density, that is, under the same volume of SVF, the SVF prepared by the device of the present application (Method A) has more effective cells.

[0107] Example 3: After adherent culture, mesenchymal stem cells in the SVF prepared in Example 1 are directionally expanded.

[0108] Specifically, the SVF prepared by the method of the present application (Example 1) is washed with phosphate buffered solution (PBS), that is, 1 ml of the SVF is mixed with 9 ml of PBS and shaken uniformly, centrifuged at 300 x g for 5 minutes, and the supernatant in the centrifuge tube is discarded to obtain a cell precipitate.

[0109] The cell precipitate is mixed with 5 ml of complete medium (a-MEM base medium + 10% fetal bovine serum) and placed in a T75 culture bottle so that the liquid covers the entire culture bottle. The culture bottle is placed in a culture box for culture (culture box conditions: 5% CO2, humidity 95%, 37°C constant temperature).

[0110] On the third day of culture, 5 ml of complete medium is added to the culture bottle.

[0111] On the fifth day of culture, obvious adherent cells are observed in the culture bottle, and the fusion degree of the adherent cells reaches more than 70%. The complete medium in the culture bottle is aspirated and discarded, 3 ml of trypsin digestion solution is added, treated at 37°C for 2 minutes, and the adherent cells are observed under a microscope to have completely detached from the bottom. Then 7 ml of complete medium is added to terminate the digestion.

[0112] The mixed liquid containing trypsin digestion solution, complete medium and adherent cells is collected into a centrifuge tube, centrifuged at 300 x g for 5 minutes, and the supernatant in the centrifuge tube is discarded to obtain a cell precipitate.

[0113] Resuspend the cell pellet with complete medium, and count the cell suspension using AO / PI dye. Add 3.75 x 10 5 cells to a new T75 culture flask, and supplement the volume of complete medium to 10 ml.

[0114] On the 4th day of culture, observe the culture flask to find that there are obvious adherent cells, and the degree of fusion of the adherent cells is more than 70%, and take a photo. The cell morphology is shown in Figure 14 , which shows that the cells grow by adhering to the bottom of the culture flask, and the cells are spindle-shaped.

[0115] Discard the complete medium in the culture flask, and add 3 ml of trypsin digestion solution to it, and treat it at 37°C for 2 minutes. Under a microscope, observe that the adherent cells have completely detached from the bottom, and then add 7 ml of complete medium to terminate the digestion.

[0116] Collect the mixed liquid containing the trypsin digestion solution, complete medium and adherent cells into a centrifuge tube, and centrifuge it at 300 x g for 5 minutes. Discard the supernatant in the centrifuge tube to obtain a cell pellet.

[0117] Use the MSC Marker flow kit (BD 562245) to stain the surface markers of the cells according to the instructions, and then use the flow cytometer (BD Canto II) to analyze the cells, as shown in Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 、 Figure 20 、 Figure 21 、 Figure 22 、 Figure 23 、 Figure 24 、 Figure 25 、 Figure 26 , the analysis results of the surface markers of the cells are as follows:

[0118] Table 1: Flow cytometry detection related detection data table

[0119]

[0120] According to the detection results, it can be seen that the SVF prepared by the method described in the application (Example 1) contains cells that can be adherently cultured, and the proportion of positive staining of CD73, CD90 and CD105 in the surface markers of these adherently cultured cells is more than 95%, and the positive staining results of CD11b, CD19, CD34, CD45 and HLA-DR surface markers are all less than 2%. This indicates that these cells are adipose-derived mesenchymal stem cells.

[0121] The present application has the following beneficial effects:

[0122] The connecting device of the present application can ensure complete sealing when connecting two syringes. The connecting device of the present application has a convertible aperture, which can adjust the shape and number of apertures between the syringes to achieve the purpose of cutting and emulsifying fat in different degrees. When adjusting the aperture, the connector does not need to be replaced, which can ensure that the fat in the syringe is not exposed multiple times and reduce the risk of contamination of SVF during operation. At the same time, the size and shape of the aperture of the connecting device can be customized according to the separation and preparation process of SVF.

[0123] The above description is only a specific embodiment of the present application, and is not intended to limit the scope of the present application. The components of the present application can be combined with each other without conflict, and any equivalent changes and modifications made by those skilled in the art without departing from the concept and principles of the present application shall fall within the scope of the present application.

Claims

1. A connecting device for obtaining SVF by mechanically processing fat, characterized in that: include: A housing (1), mouthpieces (12) provided at both ends of the housing (1), a wheel (14) rotatably connected to the interior of the housing (1), a roller (11) engaged with the wheel (14), a viewing window (13) provided on the housing (1), and a stopper (15) fixed to the interior of the housing (1); A plurality of wheel discs (14) are provided, and the wheel discs (14) are rotatably connected to the interior of the housing (1) via a shaft (16). A limiting structure and a processing hole are provided on the wheel discs (14). The wheel discs (14) can be tightly connected via the processing hole. The processing hole passes through the wheel discs (14) and can communicate with the interface pipe (12). The limiting structure is arranged on the side of the wheel disc (14), so that the limiting block (15) is in rolling contact with the limiting structure; The roller (11) protrudes from the housing (1), and the wheel disc (14) rotates by rolling the roller (11), so that the processing hole is connected to the mouthpiece (12); There is a cavity between the wheel discs (14); the wheel discs (14) are provided with three, including: a first wheel disc (141); a second wheel disc (142); and a third wheel disc (143); The rollers (11) are provided in three numbers, including: a first roller (111), a second roller (112), and a third roller (113); The first roller (111) is engaged with the first wheel disc (141), the second roller (112) is engaged with the second wheel disc (142), and the third roller (113) is engaged with the third wheel disc (143); The limiting structure is arranged on both sides of the wheel disc (14), and the limiting structure comprises: a limiting ring groove and a limiting groove; The limiting ring groove is opened on two side surfaces of the wheel disc (14) with the shaft (16) as the center; The limiting groove is hemispherical, the limiting groove is arranged on the limiting ring groove, and the diameter of the limiting groove is greater than the width of the limiting ring groove; A limiting spring and a limiting ball are provided inside the limiting block (15); the limiting ball can protrude from the limiting block (15); The limiting spring and the limiting ball are in pressing contact, and the limiting ball is in rolling contact with the limiting structure on the wheel disc (14); the processing holes of the first wheel disc (141) include: a first cutting hole (1411), a second cutting hole (1412) and a third cutting hole (1413); The processing holes of the second wheel disc (142) include: a first extrusion hole (1421), a second extrusion hole (1422) and a third extrusion hole (1423).

2. The connecting device for obtaining SVF by mechanically processing fat according to claim 1, characterized in that: A well-shaped wire mesh is provided in the first cutting hole (1411); a cross-shaped wire mesh is provided in the third cutting hole (1413); and a plurality of wires are radially provided in the second cutting hole (1412), with both ends of the wires being fixed on the inner wall of the second cutting hole (1412).

3. The connecting device for obtaining SVF by mechanically processing fat according to claim 1, characterized in that: The aperture of the first extrusion hole (1421) is smaller than the aperture of the second extrusion hole (1422), and the aperture of the second extrusion hole (1422) is smaller than the aperture of the third extrusion hole (1423).

4. The connecting device for obtaining SVF by mechanically processing fat according to claim 1, characterized in that: The limiting block (15) comprises: a first limiting block (151), a second limiting block (152), a third limiting block (153) and a fourth limiting block (154); The first limiting block (151) is fixed on the inner wall of the housing (1), and the first limiting block (151) is in rolling contact with the limiting structure on one side of the first wheel disc (141); The second limiting block (152) is arranged in a cavity between the first wheel disc (141) and the second wheel disc (142), and the second limiting block (152) is fixed on the shaft (16). The second limiting block (152) is in rolling contact with the limiting structure on the other side of the first wheel disc (141), and the second limiting block (152) is in rolling contact with the limiting structure on one side of the second wheel disc (142). The third limiting block (153) is arranged in a cavity between the second wheel disc (142) and the third wheel disc (143), and the third limiting block (153) is fixed on the shaft (16). The third limiting block (153) is in rolling contact with the limiting structure on the other side of the second wheel disc (142); the third limiting block (153) is in rolling contact with the limiting structure on one side of the third wheel disc (143); The fourth limiting block (154) is fixed on the inner wall of the housing (1), and the fourth limiting block (154) is in rolling contact with the limiting structure on the other side of the third wheel disc (143).

5. The connecting device for obtaining SVF by mechanically processing fat according to claim 1, characterized in that: The mouthpiece (12) can be connected to the injection device (2).

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