A method for obtaining svf by mechanical treatment of fat

Abstract

The application provides a method for obtaining SVF by mechanically treating fat, and a connecting device for obtaining SVF by mechanically treating fat, which comprises a shell, an interface pipe, a wheel disc, and a roller; the wheel disc is rotatably connected to the inside of the shell through a shaft, the roller protrudes from the shell and is engaged with the wheel disc; a limiting structure is in rolling engagement with a limiting block. In use, two injection devices are connected to the interface pipe of the connecting device at the same time, the wheel disc is rotated to select a corresponding processing hole to be communicated with the interface pipe, and the injection device is pushed to make fat pass through the connecting device for obtaining SVF by mechanically treating fat and enter the other injection device. The connecting device has a convertible hole diameter, can adjust the hole diameter shape and quantity between the injection devices, and achieves the purpose of cutting and emulsifying fat in different degrees.

Description

[0001] This invention is a divisional application, parent application number: 2025104628465, application date: April 14, 2025, invention title: A connecting device and method for mechanically processing fat to obtain SVF. Technical Field

[0002] This invention relates to the field of medical device technology, and more specifically to a method for mechanically processing fat to obtain SVF. Background Technology

[0003] Human fat is rich in vascular matrix components (SVF), a mixture of various cells and cytokines with repair functions. These include adipose-derived stem cells (ADSCs), hematopoietic stem cells, endothelial progenitor cells, endothelial cells, vascular smooth muscle cells, fibroblasts, lymphocytes, macrophages, mast cells, and platelets. ADSCs, in particular, possess excellent immunomodulatory and angiogenesis-promoting capabilities, and have been reported in numerous clinical applications in adjuvant fat grafting and osteoarthritis treatment.

[0004] The fat used to prepare SVF (Small Vein Fat) is mostly obtained from liposuction, including the tumescent fluid added during the procedure. Before SVF preparation, a centrifugation step is usually performed. After centrifugation, the fat obtained from liposuction separates into four layers: oil phase, fat layer, tumescent fluid, and tissue fragments. The fat layer (generally referred to as "Coleman's fat") is then collected for further processing. This processing can generally be divided into enzymatic and mechanical methods. Enzymatic hydrolysis uses collagenase to digest and break down the extracellular matrix components, allowing SVF-related cells to be collected by centrifugation. Mechanical separation uses methods such as ultrasound and vibration to emulsify the fat, destroying the extracellular matrix components and separating the SVF-related cells. However, enzymatic hydrolysis has disadvantages such as being time-consuming and complex; the use of collagenase introduces residual risks; and the need to operate outside the surgical environment increases the risk of contamination. Mechanical separation has advantages such as being time-efficient and simple; avoiding the introduction of exogenous substances that could cause residual risks; and retaining some extracellular matrix components in a gel-like state, making it more suitable for tissue filling. In summary, mechanical separation is characterized by its short operation time and simplicity, making it particularly suitable for use in the operating room. This means that SVF (short-terminated fat grafting) can be prepared shortly after liposuction for fat grafting or other treatments, eliminating the need for a second surgery. This offers advantages in terms of convenience and risk control. However, the yield and viability of SVF obtained through mechanical separation are currently not high.

[0005] In summary, the existing technology has the following problems: how to improve the yield and activity of SVF obtained by mechanical separation. Summary of the Invention

[0006] This invention provides a connection device and method for mechanically processing fat to obtain SVF, and the technical problem it solves is how to improve the yield and activity of SVF obtained by mechanical separation.

[0007] To achieve the above objectives, in one aspect, the present invention proposes a connection device for mechanically processing fat to obtain SVF, comprising:

[0008] The housing, the interface pipes at both ends of the housing, the wheel that is rotatably connected inside the housing, the roller that meshes with the wheel, the window opened on the housing, and the limiting block fixed inside the housing;

[0009] Multiple wheel disks are provided, and the wheel disks are rotatably connected to the inside of the housing via shafts. Each wheel disk is provided with a limit structure and a processing hole. The wheel disks can be pressed together through the processing hole, and the processing hole penetrates the wheel disk and can communicate with the interface pipe.

[0010] The limiting structure is disposed on the side of the wheel, so that the limiting block makes rolling contact with the limiting structure;

[0011] The roller protrudes from the housing, and the disk is rotated by rolling the roller, so that the processing hole is connected to the interface pipe.

[0012] Specifically, there are cavities between the roulette wheels; there are three roulette wheels, including: a first roulette wheel; a second roulette wheel; and a third roulette wheel.

[0013] The rollers are provided in three parts: a first roller, a second roller, and a third roller.

[0014] The first roller engages with the first disc, the second roller engages with the second disc, and the third roller engages with the third disc.

[0015] Specifically, the limiting structure is disposed on both sides of the wheel, and the limiting structure includes: a limiting annular groove and a limiting groove;

[0016] The limiting annular groove is formed on two sides of the wheel with the shaft as the center;

[0017] The limiting groove is hemispherical and is disposed on the limiting ring groove. The diameter of the limiting groove is greater than the width of the limiting ring groove.

[0018] Specifically, the limiting block is internally provided with a limiting spring and a limiting ball; the limiting ball can protrude from the limiting block;

[0019] The limiting spring and the limiting ball are pressed together, and the limiting ball is in rolling contact with the limiting structure on the wheel.

[0020] Specifically, the processing holes of the first wheel include: a first cutting hole, a second cutting hole, and a third cutting hole;

[0021] The processing holes of the second wheel include: a first extrusion hole, a second extrusion hole, and a third extrusion hole.

[0022] 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 multiple wires are arranged radially in the second cutting hole, with both ends of the wires fixed to the inner wall of the second cutting hole.

[0023] Specifically, the diameter of the first extrusion hole is smaller than the diameter of the second extrusion hole, and the diameter of the second extrusion hole is smaller than the diameter of the third extrusion hole.

[0024] Specifically, the limiting block includes: a first limiting block, a second limiting block, a third limiting block, and a fourth limiting block;

[0025] 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.

[0026] The second limiting block is disposed in the cavity between the first wheel and the second wheel, 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, and the second limiting block is in rolling contact with the limiting structure on one side of the second wheel.

[0027] The third limiting block is disposed in the cavity between the second wheel and the third wheel, and the third limiting block is fixed on the shaft. The third limiting block is in rolling contact with the limiting structure on the other side of the second wheel; the third limiting block is in rolling contact with the limiting structure on one side of the third wheel.

[0028] 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.

[0029] Specifically, the interface tube is connected to the injection device.

[0030] On the other hand, the present invention proposes a method for mechanically processing fat to obtain SVF, using the above-mentioned connecting device for mechanically processing fat to obtain SVF, and connecting the interface tube of the connecting device to the injection device.

[0031] The rotating disk connects to the corresponding processing hole and interface pipe;

[0032] The injection device is pushed to allow fat to pass through the connecting device of the mechanical method for processing fat to obtain SVF and enter into the injection device at the other end;

[0033] SVF is obtained from processed fat.

[0034] The connecting device of this invention connects two syringes, ensuring a completely sealed connection. This connecting device features an adjustable orifice size, allowing adjustment of the orifice shape and number between the syringes to achieve different degrees of fat cutting and emulsification. No connector replacement is required during orifice adjustment, ensuring that the fat inside the syringe is not repeatedly exposed, reducing the risk of SVF contamination during operation. Furthermore, the orifice size and shape of the connecting mechanism can be customized according to the SVF separation and preparation process. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the connection device for mechanically processing fat to obtain SVF according to an embodiment of the present invention;

[0036] Figure 2 This is a schematic diagram illustrating the installation and use of a connection device for mechanically processing fat to obtain SVF according to an embodiment of the present invention;

[0037] Figure 3 This is a top view of a connecting device for mechanically processing fat to obtain SVF according to an embodiment of the present invention;

[0038] Figure 4 This is a side view of a connecting device for mechanically processing fat to obtain SVF according to an embodiment of the present invention;

[0039] Figure 5 This is a front cross-sectional view of the internal structure of a connecting device for mechanically processing fat to obtain SVF according to an embodiment of the present invention.

[0040] Figure 6 This is a cross-sectional side view of the internal structure of a connecting device for mechanically processing fat to obtain SVF according to an embodiment of the present invention.

[0041] Figure 7 This is a schematic diagram of the structure of the first wheel in an embodiment of the present invention;

[0042] Figure 8 This is a schematic diagram of the structure of the second wheel in an embodiment of the present invention;

[0043] Figure 9 This is a front view of the second wheel according to an embodiment of the present invention;

[0044] Figure 10 This is a schematic diagram showing the percentage of SVF in Coleman fat in the examples and comparative examples;

[0045] Figure 11 Typical fluorescence images of AO / PI staining for examples and comparative examples;

[0046] Figure 12 This is a schematic diagram illustrating the cell viability of SVF in the examples and comparative examples;

[0047] Figure 13 Cell density diagrams of SVF in each group in the examples and comparative examples;

[0048] Figure 14 This is a diagram of cell morphology from Example 3;

[0049] Figure 15 This is a graph showing the results of a first flow cytometry sample detection according to an embodiment of the present invention;

[0050] Figure 16 This is a graph showing the results of a second type of flow cytometry sample detection according to an embodiment of the present invention;

[0051] Figure 17 This is a graph showing the results of a third type of flow cytometry sample detection according to an embodiment of the present invention;

[0052] Figure 18 This is a graph showing the results of a fourth type of flow cytometry sample detection according to an embodiment of the present invention.

[0053] Figure 19 This is a graph showing the fifth type of flow cytometry sample detection results according to an embodiment of the present invention;

[0054] Figure 20 This is a statistical chart of flow cytometry sample detection according to an embodiment of the present invention;

[0055] Figure 21 This is a flow cytometry result of the first type of isotype control standard according to an embodiment of the present invention.

[0056] Figure 22 This is a flow cytometry result of the second type of isotype control standard according to an embodiment of the present invention.

[0057] Figure 23 This is a flow cytometry result of the third type of isotype control in an embodiment of the present invention.

[0058] Figure 24 This is a flow cytometry result of the fourth type of isotype control in an embodiment of the present invention.

[0059] Figure 25 This is a flow cytometry result of the fifth type of isotype control in an embodiment of the present invention.

[0060] Figure 26 This is a statistical graph showing the flow cytometry detection of isotype control samples in an embodiment of the present invention.

[0061] Explanation of icon numbers:

[0062] 1. Housing; 2. Injection device; 11. Roller; 12. Interface tube; 13. Window; 14. Wheel; 15. Limiting block; 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 wheel; 142. Second wheel; 143. Third wheel; 1411. First cutting hole; 1412. Second cutting hole; 1413. Third cutting hole; 1414. First indicator area; 1415. Second indicator area; 1416. Third indicator area; 1421, First extrusion hole; 1422, Second extrusion hole; 1423, Third extrusion hole; 1424, Fourth indication area; 1425, Fifth indication area; 1426, Sixth indication 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 Implementation

[0063] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] This invention provides a connecting device for mechanically processing fat to obtain SVF, such as... Figure 1 As shown, it includes:

[0065] The housing 1, the interface pipes 12 disposed at both ends of the housing 1, the wheel 14 rotatably connected inside the housing 1, the roller 11 meshing with the wheel 14, the viewing window 13 opened on the housing 1, and the internal limiting block 15 fixed to the housing 1;

[0066] A viewing window 13 is disposed on the housing 1; the viewing window 13 is used to display the type or size of the aperture on the wheel 14. Interface pipes 12 are disposed at both ends of the housing 1, such as... Figure 2 As shown, the interface tube 12 is connected to the injection device 2, and the preferred connection method is a threaded connection.

[0067] The wheel 14 is rotatably connected inside the housing 1 via the shaft 16, as shown below. Figure 4As shown, roller 11 protrudes from housing 1 and engages with disk 14; the disk 14 rotates by rolling roller 11, connecting the processing hole to interface pipe 12. Limiting block 15 is fixed inside housing 1 and rolls in contact with the side of disk 14; disk 14 has a limiting structure and processing hole, allowing disks 14 to be tightly connected via the processing hole, which penetrates disk 14 and connects to interface pipe 12; the limiting structure and limiting block 15 roll in cooperation. A marking area is provided on disk 14, the position of which corresponds to the position of window 13. When the marking area rotates to the position of window 13, the operator can identify which processing hole is connected to interface pipe 12 by seeing the corresponding marking area.

[0068] There are cavities between the discs 14; there are three discs 14, including: a first disc 141; a second disc 142; and a third disc 143; preferably, the number of discs 14 is 2, 4, or 5. The rollers 11 are used to adjust the internal diameter of the connecting device, wherein, for example... Figure 3 As shown, three rollers 11 are provided: a first roller 111, a second roller 112, and a third roller 113. The first roller 111 engages with a first disc 141, the second roller 112 engages with a second disc 142, and the third roller 113 engages with a third disc 143. Rotating the roller 11 causes the disc 14 to rotate, allowing the processing hole connected to the interface pipe 12 to be changed. In other words, rotating the roller 11 allows selection of the desired processing hole type or size.

[0069] The limiting structure on the wheel 14 is provided on both sides of the wheel 14. The limiting structure includes a limiting ring groove and a limiting groove 1442. The limiting ring groove is opened on the two sides of the wheel 14 with the shaft 16 as the center. The limiting groove is hemispherical and is provided on the limiting ring groove. The diameter of the limiting groove is greater than the width of the limiting ring groove.

[0070] The limiting block 15 is internally equipped with a limiting spring and a limiting ball 155; 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 14. The limiting block 15 is used to limit the rotational displacement of the wheel, so that the hole of the wheel corresponds to the hole of the other two wheels in the connecting device.

[0071] like Figure 5 As shown, 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;

[0072] The first limiting block 151 is fixed on the inner wall of the housing 1, and the first limiting block 151 makes rolling contact with the limiting structure on one side of the first wheel 141.

[0073] The second limiting block 152 is disposed in the cavity between the first wheel 141 and the second wheel 142, and the second limiting block 152 is fixed on the shaft 16 by a fixing rod. The second limiting block 152 is in rolling contact with the limiting structure on the other side of the first wheel 141, and the second limiting block 152 is in rolling contact with the limiting structure on one side of the second wheel 142.

[0074] The third limiting block 153 is disposed in the cavity between the second wheel 142 and the third wheel 143, and the third limiting block 153 is fixed on the shaft 16 by 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 142; the third limiting block 153 is in rolling contact with the limiting structure on one side of the third wheel 143.

[0075] 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 143.

[0076] like Figure 5 The leftmost and rightmost sliding limit blocks are fixed to the housing 1 of the connecting device, while the two middle limit blocks are fixed to the shaft 16. A spring inside the limit block, always under compression, provides a force to the limit ball (spark). Simultaneously, the housing of the limit block restricts the displacement of the ball, ensuring only its tip protrudes from the housing and contacts the limit ring groove. When the wheel rotates and the selectable aperture corresponds to the connection port, the ball will enter a deeper limit groove, thus limiting the displacement of the wheel component. When the selectable aperture needs to be changed, a greater force is used to rotate the roller, causing the ball to slide out from the deeper limit groove into the limit ring groove. When the next selectable aperture corresponds to the interface, the ball will enter the next deeper limit groove. When the ball enters the limit groove, the window 13 displays a mark indicating the aperture currently connected to the interface pipe 12. Figure 3 Window 13, from left to right, is window 131, window 132, and window 133, for example, as shown in the image. 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 hole currently connected to the interface tube 12 (interface).

[0077] 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 provided in the first cutting hole 1411; a cross-shaped wire mesh is provided in the third cutting hole 1413; multiple wires are arranged radially in the second cutting hole 1412, and the two ends of the wires are fixed on the inner wall of the second cutting hole 1412.

[0078] like Figure 8As shown, the second disc 142 has three through-holes with different diameters. These through-holes serve as processing holes to process fat, extruding and emulsifying the fat that has already been mechanically segmented by the mesh openings on the left and right discs. Specifically, the processing holes of the second disc 142 include: a first extrusion hole 1421, a second extrusion hole 1422, and a third extrusion hole 1423. The diameter of the first extrusion hole 1421 is smaller than that of the second extrusion hole 1422, and the diameter of the second extrusion hole 1422 is smaller than that of the third extrusion hole 1423. Preferably, the first extrusion hole 1421 has a diameter of 1.4 mm, the second extrusion hole 1422 has a diameter of 2 mm, and the third extrusion hole 1423 has a diameter of 2.4 mm. On the outer edge of the wheel near the through hole, there are character stickers marking the nearby through holes for display on the connecting mechanism window, indicating the number of the through hole currently corresponding to the interface tube 12 of the connecting device. Specifically, the fourth indicator area 1424 corresponds to the first extrusion hole 1421, the fifth indicator area 1425 corresponds to the second extrusion hole 1422, and the sixth indicator area 1426 corresponds to the 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 window 132 will display the marking of the diameter of the hole currently connected to the interface tube 12.

[0079] There is a hole in the center of the wheel, which is the axle hole 145. The axle 16 passes through the axle hole 145 to connect the three wheels. Figure 6 As shown, the wheel can rotate on the shaft 16 by turning the roller 11. Figure 5 As shown, Figure 5 This is a front cross-sectional view of the internal structure of the connecting device, where the rollers correspond to the left, middle, and right discs of the adjusting connecting mechanism, respectively. A shaft passes through the center of each of the three discs. The leftmost and rightmost sliding limiting mechanisms are fixed to the connecting mechanism housing, while the two middle limiting blocks are fixed to the shaft. There are annular grooves on both sides of the discs. On the annular grooves, there are corresponding deeper circular grooves on the same radius connecting line as the selectable aperture. The limiting block 15 is a sliding limiting mechanism; the limiting ball 155 inside the limiting block 15 can roll within the annular groove. When the selectable aperture corresponds to the interface pipe 12, the limiting ball 155 slides into the circular groove, its rotation is hindered, and mechanical feedback is provided, achieving the positioning purpose. When it is necessary to continue changing the selectable aperture, a slightly larger force is used to move the corresponding roller (limiting ball 155) on the disc, allowing it to continue rotating to the target aperture and achieving positioning. The limiting structures on the first disc 141 and the third disc 143 are the same as those on the second disc 142.

[0080] The processing holes on the third disc 143 are of the same type as those on the first disc 141, both containing wire mesh blades within their aperture structures to physically cut the passing fat. The intermediate disc, the second disc 142, has a different aperture. The first roller 111, the second roller 112, and the third roller 113 are used to adjust the first disc 141, the second disc 142, and the third disc 143 within the connecting device, respectively. In use, both injection devices 2 (syringes) are simultaneously connected to the interface tube 12 of the connecting device. The syringe plungers 21 are pushed alternately, causing the fat to be first cut by the wire mesh blades as it passes through the connecting device, and then mechanically emulsified by fluid compression through the aperture structure.

[0081] The connecting device of this invention connects two syringes, ensuring a completely sealed connection. This connecting device features an adjustable orifice size, allowing adjustment of the orifice shape and number between the syringes to achieve different degrees of fat cutting and emulsification. No connector replacement is required during orifice adjustment, ensuring that the fat inside the syringe is not repeatedly exposed, reducing the risk of SVF contamination during operation. Furthermore, the orifice size and shape of the connecting mechanism can be customized according to the SVF separation and preparation process.

[0082] By designing and changing the aperture of the connector device, Coleman fat is first divided into smaller fat blocks by the wire mesh in the mesh aperture, and then emulsified by the large aperture and small aperture of the connector. This reduces the resistance generated during fat emulsification, making the operation easier; at the same time, it reduces damage to SVF, increases SVF yield, and increases cell concentration in SVF.

[0083] In addition, this embodiment of the invention also provides a method for obtaining SVF by mechanically processing fat, using the above-mentioned connecting device for obtaining SVF by mechanically processing fat, and connecting the interface tube of the connecting device to the injection device.

[0084] The rotating disk connects to the corresponding processing hole and interface pipe;

[0085] The injection device is pushed to allow fat to pass through a connecting device that describes the mechanical method of fat processing to obtain SVF and enter into another injection device; SVF is obtained from the processed fat.

[0086] For example: Transfer fat into a syringe, connect one end of the syringe to one end of the aforementioned connecting device via a Luer interface, and then connect another empty syringe to the other end of the connecting device. Adjust the dial so that the holes corresponding to the interface tubes, from left to right, are a crosshair hole, a 2.4mm hole, and another crosshair hole. Quickly push the syringe to inject the fat through the connecting device into the syringe at the other end. Then continue operating the syringe to push the fat back through the connecting device to the initial syringe, marking one cycle. Repeat this process multiple times. This cuts and emulsifies the fat, ultimately yielding SVF.

[0087] 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, middle, and right discs within the connecting device. The two interface tubes 12 of the connecting device are connected to the syringe via threaded connections, but other connection methods, such as sliding connections, are also possible. The connecting device has windows 13 that display the aperture number of the currently used disc, where the first window 131, the second window 132, and the third window 133 correspond to the left, middle, and right discs within the connecting device, respectively. The left disc (first disc 141) and the right disc (third disc 143) have wire mesh blades within their aperture structures, capable of physically cutting through passing fat. The middle disc (second disc 142) has a different aperture structure. The rollers 11 are used to adjust the left, middle, and right discs within the connecting device, respectively. In use, the two injection devices 2 are connected to the interface tube 12 of the connecting device at the same time. The plunger 21 of the syringe is pushed alternately so that the fat is first cut by the wire mesh blade when it passes through the connecting device, and then mechanically emulsified by the fluid compression through the aperture structure. Figure 7 The structure of the left wheel (first wheel 141) inside the connecting mechanism is described. The wheel has a first limiting annular groove 1417 for engaging with a limiting mechanism (limiting block 15) to restrict the rotational displacement of the wheel, ensuring that the holes on the wheel align with the openings at both ends of the connecting mechanism. The first wheel 141 has three through-hole structures, each containing metal wire mesh of different shapes for mechanically cutting the passing grease. The diameter of each of the three holes is 2–4 mm, preferably 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. A marking area is located on the outer edge of the wheel near the processing holes, where character stickers marking the nearby processing holes can be affixed and displayed on the connecting mechanism's window to indicate the number of the through hole currently corresponding to the interface tube 12 of the connecting mechanism.

[0088] The wheel 14 has a sliding limiting mechanism, namely, a limiting block 15. It consists of a spring, a ball, and a housing. The outer housings of the leftmost and rightmost sliding limiting mechanisms are fixed to the inner wall of the housing 1 of the connecting device, while the two middle sliding limiting mechanisms are fixed to the shaft 16. The spring, always under compression, provides a force to the ball, while the housing restricts the ball's displacement, ensuring only the tip protrudes from the housing and contacts the annular groove (limiting annular groove). When the wheel rotates and the selectable aperture corresponds to the connection port of the interface tube, the ball will enter a deeper circular groove (limiting groove 1442), thus limiting the displacement of the wheel component. When the selectable aperture needs to be changed, a larger force is used to rotate the roller, and the ball will slide from the deeper circular groove into the annular groove. When the next selectable aperture corresponds to the interface tube, the ball will enter the next deeper circular groove. When the ball enters the circular groove, the window 13 displays a mark indicating the aperture currently corresponding to the interface tube 12. For example, when the limiting ball (ball) rolls from the first limiting ring groove 1417 into the circular groove of the first wheel 141, the window 131 will display the markings of the current hole diameter corresponding to the interface tube 12. That is, 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.

[0089] The connecting device between the two syringes can be made of metal, such as pure titanium, TC4 titanium alloy, 316L stainless steel, etc., or of polymer materials, such as polyethylene, polypropylene, polyacetate, polymethyl methacrylate, etc. Each component can be made of different materials and then assembled together.

[0090] The syringe material should be able to withstand one or more of the following methods: high temperature and high pressure, irradiation sterilization, and ethylene oxide sterilization lamp sterilization. Figure 2 The interface tube 12 shown is the connection port between the connecting device and the syringe. The connection method shown here is a threaded interface, but it can also be connected by other connection methods, such as a sliding interface.

[0091] The accompanying drawings only show one form of a connecting device for preparing SVF that can connect two syringes, where each disc structure has only three selectable apertures and mesh shapes. However, the invention is not limited to the shape and number of apertures; for example, the discs may also have two, four, or other numbers of apertures. The shape and size of the apertures are also not limited to those shown in the figures.

[0092] Example 1: Preparation of SVF using the connection device of the present invention.

[0093] Step 1: Centrifuge the fat obtained from liposuction at 1200×g (1200 times the acceleration due to gravity) for 3 minutes. Take the fat layer (Coleman's fat) for further processing.

[0094] Step 2: Transfer the Coleman fat into a syringe, connect the syringe to one end of the device via a Luer interface, and then connect another empty syringe to the other end of the device.

[0095] Step 3: Adjust the connector wheel of this device so that the corresponding holes on the interface, from left to right, are "crosshair hole - 2.4mm hole - crosshair hole". Quickly push the syringe to allow the Coleman fat to pass through this connecting mechanism into the syringe at the other end. Then continue operating the syringe to push the Coleman fat back into the initial syringe through this connecting mechanism, which is counted as one cycle. Repeat this process 10 to 30 times.

[0096] Step 4: Adjust the connector wheel of this device so that the corresponding holes on the interface are, from left to right, "star-shaped wire hole - 2mm hole - star-shaped wire hole". Following step 3, repeatedly inject Coleman grease through the connector of this device for 10-30 cycles.

[0097] Step 5: Adjust the connector wheel of this device so that the corresponding holes on the interface are arranged from left to right as "well-shaped wire hole - 1.4mm hole - well-shaped wire hole". Following Step 3, reciprocate the injection of Coleman grease through the connector of this device for 10-30 cycles.

[0098] Step 6: Centrifuge the emulsified fat to discard the oil phase and the liquid layer containing broken tissue components, retaining the SVF-rich components.

[0099] Example 2: A comparative study was conducted to compare the differences in SVF volume, cell density, and cell viability between SVF obtained using the above preparation method and SVF obtained by treating only one pore size.

[0100] First, the fat obtained from liposuction is centrifuged at 1200×g for 3 minutes. The fat layer (Coleman's fat) is then taken for further processing.

[0101] Method A, prepared using the method described in Example 1, wherein in steps 3, 4 and 5, the Coleman fat is circulated 10 times through the connecting mechanism.

[0102] Method B involves passing Coleman fat through the existing syringe connector (hole diameter 2 mm) 30 times.

[0103] Method C involves passing Coleman fat through the existing syringe connector (hole diameter 1.4 mm) 30 times.

[0104] The processed fats were then centrifuged to remove the oil phase and the liquid layer containing broken tissue components, while retaining the SVF-rich components, which were designated as SVF A, SVF B, and SVF C, respectively.

[0105] In the three processing methods A, B, and C, the Coleman fat was passed through the device described in this patent a total of 30 times during the processing. Each processing method was performed 3 times, obtaining 3 sample data. The obtained SVF components of categories A, B, and C were analyzed as follows.

[0106] Volume ratio: Calculate the percentage of SVF obtained in Coleman fat and plot a graph for comparison, such as... Figure 10 As shown.

[0107] Cell density: 1 ml samples from each of SVF A, SVF B, and SVF C were mixed thoroughly with 1 ml of phosphate buffer solution and then filtered through a 100-micron cell sieve. 20 μL of the filtrate was taken and mixed with 20 μL of acridine orange / propidium iodide (AO / PI) staining (the staining principle is that AO can pass through intact cell membranes and embed into the nuclei of both living and dead cells, exhibiting green fluorescence; PI can only pass through incomplete cell membranes, i.e., the cell membranes of dead cells, embedding into the nuclei of all dead cells, exhibiting red fluorescence). The stained samples were counted using an automated fluorescence cell counter to obtain cell viability and cell density. Based on the cell density detection results, the original cell density in SVF A, SVF B, and SVF C was calculated. Typical fluorescence images of AO / PI staining are shown below. Figure 11 As shown, the cell viability of each SVF group is as follows: Figure 12 As shown, the cell density of each SVF group is as follows: Figure 13As shown in the results, the percentage of SVF volume relative to Coleman fat prepared using the apparatus of the present invention (method A) falls between that of methods B and C. This is because method B, using only a larger fixing aperture, results in limited emulsification of Coleman fat, leaving some adipocytes undamaged; while method C, using only a smaller fixing aperture, experiences consistently higher shear forces on the Coleman fat, leading to greater adipocyte damage. This result is confirmed by the cell viability test results, where method C yields the lowest SVF cell viability among the three methods, while the SVF prepared using the apparatus of the present invention (method A) shows a significantly improved cell viability. Furthermore, because the apparatus of the present invention (method A) causes less cell damage (higher cell viability) and achieves a higher degree of fat emulsification (greater volume compression), the resulting SVF cell density is the highest. In other words, for the same volume of SVF, the SVF prepared using the apparatus of the present invention (method A) contains more effective cells.

[0108] Example 3: The SVF prepared in Example 1 was cultured in an adherent culture, and the mesenchymal stem cells therein were directionally expanded.

[0109] The specific method is as follows: the SVF prepared by the method described in this patent (Example 1) is washed with phosphate buffered saline (PBS), that is, 1 ml of the SVF is mixed with 9 ml of PBS and shaken evenly, centrifuged at 300×g for 5 minutes, and the supernatant in the centrifuge tube is discarded to obtain cell pellet.

[0110] Mix the cell pellet with 5 ml of complete culture medium (a-MEM basal medium + 10% fetal bovine serum) and place it in a T75 culture flask, ensuring the liquid completely covers the flask. Place the culture flask in an incubator for incubation (incubator conditions: 5% CO2, 95% humidity, 37°C).

[0111] On the third day of culture, add 5 ml of complete culture medium to the culture flask.

[0112] On day 5 of culture, adherent cells were observed in the culture flask, with a confluence of over 70%. The complete culture medium in the culture flask was aspirated and discarded. 3 ml of trypsin digestion solution was added, and the flask was treated at 37°C for 2 minutes. Microscopic observation showed that the adherent cells had completely detached from the bottom surface. Then, 7 ml of complete culture medium was added to stop the digestion.

[0113] Collect the mixture of trypsin digestion solution, complete culture medium and adherent cells into a centrifuge tube, centrifuge at 300×g for 5 minutes, discard the supernatant in the centrifuge tube and obtain the cell pellet.

[0114] The cell pellet was resuspended in complete culture medium, and the cell suspension was counted using AO / PI dye. 3.75 × 10⁻⁶ cells were added to a new T75 culture flask. 5 10 cells, and the total culture medium volume was increased to 10 ml.

[0115] On day 4 of culture, adherent cells were observed in the culture flask, and the confluence of adherent cells reached over 70%. Photos were taken. Cell morphology is as follows. Figure 14 As shown, the cells adhere to the bottom of the culture flask and grow, exhibiting a spindle shape.

[0116] Remove and discard the complete culture medium from the culture flask, add 3 ml of trypsin digestion solution, treat at 37°C for 2 minutes, observe under a microscope that the adherent cells have completely detached from the bottom surface, and then add 7 ml of complete culture medium to stop the digestion.

[0117] Collect the mixture of trypsin digestion solution, complete culture medium and adherent cells into a centrifuge tube, centrifuge at 300×g for 5 minutes, discard the supernatant in the centrifuge tube and obtain the cell pellet.

[0118] Cells were stained with surface markers using the MSC Marker Flow Cytometry Kit (BD 562245) according to the manufacturer's instructions, and then analyzed using a flow cytometer (BD Canto II). Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 , Figure 20 , Figure 21 , Figure 22 , Figure 23 , Figure 24 , Figure 25 , Figure 26 As shown, the analysis results of cell surface markers are as follows:

[0119] Table 1. Flow Cytometry Detection Data

[0120]

[0121] The test results show that the SVF prepared using the method described in this application (Example 1) contains cells capable of adherent culture. Furthermore, the proportion of positive staining for CD73, CD90, and CD105 on the surface markers of these adherent cells is greater than 95%, while the positive staining for CD11b, CD19, CD34, CD45, and HLA-DR surface markers is less than 2%. This indicates that these cells are adipose-derived mesenchymal stem cells.

[0122] The present invention has the following beneficial effects:

[0123] The connecting device of this invention connects two syringes, ensuring a completely sealed connection. This connecting device features an adjustable orifice size, allowing adjustment of the orifice shape and number between the syringes to achieve different degrees of fat cutting and emulsification. No connector replacement is required during orifice adjustment, ensuring that the fat inside the syringe is not repeatedly exposed, reducing the risk of SVF contamination during operation. Furthermore, the orifice size and shape of the connecting device can be customized according to the SVF separation and preparation process.

[0124] The above description is merely an illustrative embodiment of the present invention and is not intended to limit the scope of the invention. The various components of the present invention can be combined with each other without conflict. Any equivalent changes and modifications made by those skilled in the art without departing from the concept and principles of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for obtaining SVF by mechanical treatment of fat, characterized in that, The application relates to a connecting device for mechanically treating fat to obtain SVF, which comprises the following parts: A shell (1), an interface pipe (12) arranged at both ends of the shell (1), a wheel disc (14) rotationally connected in the shell (1), and a roller (11) engaged with the wheel disc (14); The wheel disc (14) is rotationally connected in the shell (1) through a shaft (16), the wheel disc (14) is provided with a limiting structure and a treatment hole, the wheel discs (14) can be tightly connected through the treatment hole, the treatment hole penetrates the wheel disc (14) and can be communicated with the interface pipe (12); The wheel disc (14) is provided with three wheel discs (141), (142) and (143); The roller (11) is provided with three rollers (111), (112) and (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 treatment hole of the first wheel disc (141) comprises a first cutting hole (1411), a second cutting hole (1412) and a third cutting hole (1413); The treatment hole of the second wheel disc (142) comprises a first extrusion hole (1421), a second extrusion hole (1422) and a third extrusion hole (1423); When used, two injection devices (2) are connected with the interface pipes (12) of the connecting device at the same time, the wheel disc is rotated to select the corresponding treatment hole to be communicated with the interface pipe; The injection device is pushed to make the fat pass through the connecting device for mechanically treating fat to obtain SVF and enter the other injection device; The SVF is obtained from the treated fat; The first cutting hole (1411) is provided with a well-shaped wire mesh, the third cutting hole (1413) is provided with a cross-shaped wire mesh, and a plurality of wire lines are arranged in the second cutting hole (1412) in the radial direction, and the two ends of the wire lines are fixed on the inner wall of the second cutting hole (1412); The hole diameter of the first extrusion hole (1421) is smaller than that of the second extrusion hole (1422), and the hole diameter of the second extrusion hole (1422) is smaller than that of the third extrusion hole (1423); The treatment hole arranged on the third wheel disc (143) is of the same type as the treatment hole of the first wheel disc (141), that is, the hole diameter structure has a wire mesh blade, and the fat passing through can be physically cut.

2. The method of claim 1, wherein the mechanical processing of fat to obtain SVF is characterized by, The following steps are specifically adopted: Step S1, centrifugal treatment is conducted on fat obtained through liposuction, the centrifugal condition is 1200xg, g is the gravitational acceleration, the treatment time is 3 minutes, the fat layer, that is, the Coleman fat, is taken for further treatment; Step S2, transfer the Coleman fat into a syringe, connect the syringe with one end of the device through a luer interface, and then connect another empty syringe with the other end of the device; Step S3, adjust the holes of the connector disc of the device corresponding to the interfaces from left to right as "cross line hole-2.4mm hole-cross line hole", push the syringe to make the Coleman fat pass through the connection mechanism into the syringe at the other end, and then continue to operate the syringe to push the Coleman fat to pass through the connection mechanism back to the syringe at the initial end, which is recorded as one cycle, and the reciprocating operation is carried out for 10-30 cycles; Step S4, adjust the holes of the connector disc of the device corresponding to the interfaces from left to right as "star line hole-2mm hole-star line hole", and carry out the reciprocating push of the Coleman fat through the connector of the device according to the steps of step S3 for 10-30 cycles; Step S5, adjust the holes of the connector disc of the device corresponding to the interfaces from left to right as "well-shaped line hole-1.4mm hole-well-shaped line hole", and carry out the reciprocating push of the Coleman fat through the connector of the device according to the steps of step S3 for 10-30 cycles; Step S6, centrifugal treatment is carried out on the emulsified fat, and the oil phase and the liquid layer containing the broken tissue components are discarded, and the SVF-rich component is retained.

3. The method of claim 1, wherein the mechanical processing of the fat to obtain the SVF is characterized by, The connecting device for treating fat to obtain SVF by mechanical method further comprises a limiting block (15) fixed in the inside of the shell (1), and a limiting spring and a limiting ball are arranged in the inside of the limiting block (15); 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 disc (14).

4. The method of claim 1, wherein the mechanical processing of the fat to obtain the SVF is characterized by, The limiting structure is arranged on both sides of the disc (14), and the limiting structure comprises a limiting ring groove and a limiting groove; The limiting ring groove is arranged on both sides of the disc (14) with the shaft (16) as the center; The limiting groove is semispherical, 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.

5. The method of claim 3, wherein the mechanical processing of the fat to obtain the SVF is performed by a method comprising: 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 shell (1), and the first limiting block (151) is in rolling contact with the limiting structure on one side of the first disc (141); The second limiting block (152) is arranged in the cavity between the first disc (141) and the second 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 disc (141) and the limiting structure on one side of the second disc (142); The third limiting block (153) is arranged in the cavity between the second disc (142) and the third disc (143), and is fixed on the shaft (16), and the third limiting block (153) is in rolling contact with the limiting structure on the other side of the second disc (142); the third limiting block (153) is in rolling contact with the limiting structure on one side of the third disc (143); The fourth limiting block (154) is fixed on the inner wall of the shell (1), and the fourth limiting block (154) is in rolling contact with the limiting structure on the other side of the third disc (143).

6. The method of claim 5, wherein the mechanical processing of the fat to obtain the SVF is performed by a method comprising: The first disc (141) has three through hole structures, and the through holes have metal wire meshes with different shapes for mechanically dividing the passing fat, and the diameters of the three through holes are all 2-4 mm.

7. The method for obtaining SVF by mechanically treating fat according to claim 1, characterized in that, The intermediate disc, i.e., the second disc (142) has different hole diameters, and the first disc (141), the second disc (142) and the third disc (143) in the connecting device are respectively adjusted by the first roller (111), the second roller (112) and the third roller (113); in use, two syringes are respectively connected with the interface pipe (12) of the connecting device, the plunger (21) of the syringe is alternately pushed, so that the fat is first cut by the wire mesh blade and then subjected to fluid extrusion to realize mechanical emulsification when passing through the connecting device.

8. The method of claim 1, wherein the mechanical processing of the fat to obtain the SVF is characterized by, The connecting device for obtaining SVF by mechanically treating fat further comprises a window (13) arranged on the shell (1) and used for displaying the hole diameter type or size on the disc (14).

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