Low-temperature preservation method for autologous fat particles
阅读说明:本技术 一种自体脂肪颗粒低温保存方法 (Low-temperature preservation method for autologous fat particles ) 是由 张红芳 刘中国 张丽 于 2019-10-09 设计创作,主要内容包括:本发明涉及一种自体脂肪颗粒低温保存方法,所述方法包括如下步骤:第一步,将经过提纯清洗过滤后的脂肪颗粒,装载到密封容器(2)内部;第三步,将装有脂肪颗粒的密封容器(2)放入本申请提供的低温保存设备中,开始冷冻降温;第四步,在冷冻降温的初始阶段,对密封容器(2)内部空间进行气体清洗;第五步,在气体清洗步骤之后,脂肪完全进入冷冻状态之前,对密封容器(2)进行气体充盈;第六步,在脂肪完全进入冷冻状态后,通过控制器(13)关闭输入气体的气泵和抽出气体的气泵,并且关闭位于气体输送支管(33)和气体排放支管(34)的电磁阀,使得密封容器(2)内部的气氛稳定,进入稳定气氛冷冻状态。(The invention relates to a low-temperature preservation method of autologous fat particles, which comprises the following steps: firstly, loading the fat particles after purification, cleaning and filtration into a sealed container (2); thirdly, the sealed container (2) filled with the fat particles is placed into the low-temperature preservation equipment provided by the application, and the freezing and cooling are started; fourthly, in the initial stage of freezing and cooling, the internal space of the sealed container (2) is cleaned by gas; fifthly, filling the gas into the sealed container (2) after the gas cleaning step and before the fat completely enters the frozen state; and sixthly, after the fat is completely in the frozen state, closing the gas pump for inputting gas and the gas pump for extracting gas by the controller (13), and closing the electromagnetic valves positioned on the gas conveying branch pipe (33) and the gas discharge branch pipe (34) to stabilize the atmosphere inside the sealed container (2) and enter a stable atmosphere frozen state.)
1. An autologous fat granule cryopreservation device, which is characterized in that: the equipment comprises a refrigerated cabinet (1), a bearing plate (21) positioned in the refrigerated cabinet (1), an air path system, a sealed container (2) and a controller (13); the refrigerator (1) body comprises an accommodating space and a gas supply cavity, the accommodating space forms a plurality of independent spaces for accommodating the sealed containers (2) through a plurality of partition plates (11), and the gas path system comprises a gas conveying main pipe (31), a gas discharge main pipe (32), a gas conveying branch pipe (33) and a gas discharge branch pipe (34), wherein one end of the gas conveying branch pipe is embedded into the partition plates, and the other end of the gas conveying branch pipe is connected with the main pipe; the sealing container (2) comprises a bearing plate (21), a sealing plate (22), an internal bearing bowl (23) and an upper cover (24), wherein two air ducts (25) are arranged on the bearing plate (21), and the two air ducts (25) are respectively communicated with a gas conveying branch pipe (33) and a gas discharging branch pipe (34).
2. The autologous fat particle cryopreservation apparatus of claim 1, wherein: a gas delivery branch pipe (33) and a gas discharge branch pipe (34) are embedded inside the carrying plate (21), one end portion of each of the gas delivery branch pipe (33) and the gas discharge branch pipe (34) is embedded inside the partition plate (11), and the gas delivery nozzle (36) and the gas discharge nozzle (37) penetrate the surface of the partition plate (11) from inside the partition plate (11) and extend upward from the upper surface of the partition plate (11).
3. The autologous fat particle cryopreservation apparatus of claim 1, wherein: and a gas flow meter and an electromagnetic valve are also arranged at the connection part of the gas delivery manifold (31)/the gas discharge manifold (32) and the gas delivery branch pipe (33)/the gas discharge branch pipe (34).
4. The autologous fat particle cryopreservation apparatus of claim 3, wherein: the gas flowmeter and the electromagnetic valve are connected with a controller (13), thereby realizing the opening and closing of the gas path and the control of the gas flow.
5. The autologous fat particle cryopreservation apparatus of claim 3, wherein: the gas inlet end of the gas delivery main pipe (31) is connected with a gas pump for inputting gas, and the gas outlet end of the gas discharge main pipe (32) is connected with a gas pump for pumping gas; the gas pump for inputting gas is connected with a nitrogen gas bottle (35).
6. The autologous fat granule cryopreservation apparatus of claim 5, wherein: the air pumps for inputting air, the air delivery main pipe (31), the air discharge main pipe (32), the air delivery branch pipe (33), the air discharge branch pipe (34) and the air pump system for pumping air are arranged into two symmetrical groups.
7. The autologous fat particle cryopreservation apparatus of claim 1, wherein: the bearing plate (21) is a circular plate with a concave longitudinal section, internal threads are arranged on the inner side walls of the protrusions on the two sides of the concave shape, and the upper cover (24) is a bell-shaped part with an opening at the lower part. The lower part of the side wall of the box body is provided with an external thread.
8. The autologous fat particle cryopreservation apparatus of claim 1, wherein: the upper part of the air duct (25) is cylindrical at the upper depth, the lower part is a through hole which is matched with the upper part in shape and penetrates through the bearing plate (21), and the upper part and the lower part form a complete and through air duct (25).
9. A cryopreservation method of autologous fat granules based on the cryopreservation apparatus of autologous fat granules claimed in any one of claims 1 to 8, characterized in that: the method comprises the following steps:
firstly, loading the fat particles after purification, cleaning and filtration into a sealed container (2);
thirdly, the sealed container (2) filled with the fat particles is placed into the low-temperature preservation equipment provided by the application, and the freezing and cooling are started;
fourthly, in the initial stage of freezing and cooling, the internal space of the sealed container (2) is cleaned by gas;
fifthly, filling the gas into the sealed container (2) after the gas cleaning step and before the fat completely enters the frozen state;
and sixthly, after the fat is completely in the frozen state, closing the gas pump for inputting gas and the gas pump for extracting gas by the controller (13), and closing the electromagnetic valves positioned on the gas conveying branch pipe (33) and the gas discharge branch pipe (34) to stabilize the atmosphere inside the sealed container (2) and enter a stable atmosphere frozen state.
10. The method for cryopreserving autologous fat particles according to claim 9, wherein the cryopreservation method comprises:
and step six, after the fat completely enters a frozen state, firstly closing the input gas pump and the electromagnetic valve on the gas conveying branch pipe (33), metering by a gas flow meter on the gas discharge branch pipe (34), pumping out gas with the volume of 30% of the internal volume of the sealed container (2) by the gas pump for pumping out gas and the gas discharge branch pipe (34), and closing the gas pump for pumping out gas and the electromagnetic valve on the gas discharge branch pipe (34) to ensure that the atmosphere inside the sealed container (2) is stable and enters a stable atmosphere frozen state.
Technical Field
The invention relates to the technical field of biology, in particular to a low-temperature preservation method for autologous fat particles.
Background
Autologous fat transplantation techniques have been widely used, for example: enlarging breast, enlarging nose or repairing scar. Compared with the traditional artificial tissue substitute, the autologous fat has the advantages of good compatibility, simple and convenient operation, good filling appearance, small surgical wound, rich source and the like. However, the prior art still has the problems of low survival rate of transplanted fat cells, necrosis, absorption, infection and the like, and the transplantation often needs a small amount of repeated injections. Therefore, how to improve the survival rate of the low-temperature preservation of the autologous fat particles is an urgent problem to be solved.
Currently, cryopreservation of fat particles tends to store the fat particles in a cryoprotectant and provide lower temperatures, such as: -80 degrees celsius for storage. However, the atmosphere environment of the storage space is often neglected in the existing storage method, and the performance of fat storage is cracked due to poor atmosphere environment of the storage space.
Therefore, it is desirable to provide a method for cryopreserving autologous fat particles, which can prevent fat contamination and improve the survival rate of later fat transplantation.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a method for preserving autologous fat particles at a low temperature, which can avoid fat pollution and improve the survival rate of later fat transplantation.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a low-temperature preservation device for autologous fat particles comprises a refrigerated cabinet 1, a
Further, a gas delivery branch pipe 33 and a gas discharge branch pipe 34 are embedded inside the
Further, a gas flow meter and a solenoid valve are provided at the junction of the gas delivery manifold 31/gas discharge manifold 32 and the gas delivery branch pipe 33/gas discharge branch pipe 34.
Further, the gas flow meter and the electromagnetic valve are connected with the controller 13, so that the opening and closing of the gas path and the control of the gas flow are realized.
Further, the gas inlet end of the gas delivery main pipe 31 is connected with a gas pump for inputting gas, and the gas outlet end of the gas discharge main pipe 32 is connected with a gas pump for pumping gas; the gas pump for feeding gas is connected to a nitrogen gas cylinder 35.
Further, the gas pumps for inputting gas, the gas delivery manifold 31, the gas discharge manifold 32, the gas delivery branch pipes 33, the gas discharge branch pipes 34, and the gas pump systems for pumping gas are arranged in two symmetrical groups.
Further, the
Further, the upper part of the
The autologous fat particle cryopreservation method of the autologous fat particle cryopreservation equipment comprises the following steps:
firstly, loading the fat particles after purification, cleaning and filtration into a sealed
thirdly, the sealed
fourthly, in the initial stage of freezing and cooling, the internal space of the sealed
a fifth step of filling the sealed
sixth, after the fat is completely frozen, the gas pump for gas input and the gas pump for gas extraction are turned off by the controller 13, and the electromagnetic valves located in the gas delivery manifold 33 and the gas discharge manifold 34 are turned off, so that the atmosphere inside the
Further, step six is specifically that after the fat is completely frozen, the input gas pump and the electromagnetic valve located on the gas delivery branch pipe 33 are firstly closed, the metering is performed through the gas flow meter located on the gas discharge branch pipe 34, 30% of the volume of the gas in the internal volume of the sealed
The low-temperature preservation method of autologous fat particles provided by the invention can avoid fat pollution and improve the survival rate of later-stage fat transplantation.
Drawings
Fig. 1 is a schematic structural diagram of the cryopreservation of autologous fat granules provided by the invention.
Fig. 2 is a schematic structural diagram of the autologous fat granule cryopreservation sealed
Detailed Description
The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown, it being understood that one skilled in the art may modify the invention herein described while still achieving the beneficial results of the present invention. Accordingly, the following description should be construed as broadly as possible to those skilled in the art and not as limiting the invention.
In the interest of clarity, not all features of an actual implementation are described. In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific details must be set forth in order to achieve the developer's specific goals.
In order to make the objects and features of the present invention more comprehensible, embodiments of the present invention are described in detail below with reference to the accompanying drawings. It is to be noted that the drawings are in a very simplified form and are intended to use non-precision ratios for the purpose of facilitating and clearly facilitating the description of the embodiments of the invention.
The low-temperature preservation method of the autologous fat particles is based on the following low-temperature preservation equipment of the autologous fat particles. As shown in fig. 1, the apparatus has a refrigerated cabinet 1 body, a partition 11 located in the refrigerated cabinet 1 body, an air passage system, a sealed
The body of the refrigerated cabinet 1 comprises two parts, an accommodating space at the upper part and an air supply cavity at the lower part. The refrigerator 1 body is provided with necessary refrigeration equipment, a sealing cabinet door and heat insulation parts to ensure the refrigeration effect. The accommodating space is used for accommodating the sealed
The gas path system comprises a gas delivery main pipe 31, a gas discharge main pipe 32, a gas delivery branch pipe 33, a gas discharge branch pipe 34 and a gas pump, wherein one end of the gas delivery branch pipe is embedded in the partition plate, and the other end of the gas delivery branch pipe is connected with the main pipe. Wherein the gas delivery branch pipe 33 and the gas discharge branch pipe 34 are embedded inside the partition plate, one end portion of each of the gas delivery branch pipe 33 and the gas discharge branch pipe 34 is embedded inside the partition plate 11, and the gas delivery nozzle 36 and the gas discharge nozzle 37 penetrate the surface of the partition plate 11 from inside the partition plate 11 to extend upward from the upper surface of the partition plate 11, thereby achieving delivery and discharge of the gas into the sealed
And a gas flow meter and an electromagnetic valve are arranged at the joint of the gas conveying main pipe 31/the gas discharge main pipe 32 and the gas conveying branch pipe 33/the gas discharge branch pipe 34, and the gas flow meter and the electromagnetic valve are connected with the controller 13, so that the opening and closing of the gas path and the control of the gas flow are realized. The gas inlet end of the gas delivery manifold 31 is connected with a gas pump for inputting gas, and the gas outlet end of the gas discharge manifold 32 is connected with a gas pump for pumping gas. The gas pump for feeding gas is connected to a nitrogen gas cylinder 35. The gas pump for supplying gas is capable of introducing clean nitrogen gas into the interior of the apparatus, for example, the interior of the
Preferably, for better control of the gas flow, the gas pump for gas input, the gas delivery manifold 31, the gas discharge manifold 32, the gas delivery branch 33, the gas discharge branch 34, and the gas pump system for gas extraction may be arranged in two symmetrical groups (as shown in fig. 1). Of course, if the volume of the cabinet 1 itself is small, the above-described system may be provided as a single unit for the sake of simplifying the equipment or reducing the cost.
In the individual storage spaces, which are divided by the partitions 11, in the interior of the refrigerated cabinet 1, a sealed
A
During the use process, the bearing
The air path system with the partition plate 11 and the part embedded into the partition plate 11 can effectively separate the space, realize the independent control of the atmosphere inside each sealed
The method for cryopreserving autologous fat particles provided by the present invention based on the above-mentioned cryopreservation apparatus is described in detail below. The method for preserving the autologous fat particles at the low temperature comprises the following steps:
in the first step, the fat particles after purification, washing and filtration are loaded into the sealed
Specifically, to purify, wash the fat particle after filtering, put into inside and bear the
Preferably, the fat particles may be provided with a cryoprotectant, which may be dimethyl sulfoxide and trehalose, or other cryoprotectants commonly used in the art.
In a second step, the sealed
Specifically, the sealed
Preferably, the pre-cooling time is determined according to the volume of the fat particles, and the pre-cooling time is 15 minutes per 5ml of fat.
And thirdly, placing the sealed
Specifically, the gas delivery nozzle 36 and the gas discharge nozzle 37 are inserted into the two
And fourthly, in the initial stage of freezing and cooling, carrying out gas cleaning on the inner space of the sealed
The atmosphere in the internal space of the sealed
Preferably, during this gas purging step, the controller 13 controls the flow rate of the gas flow by controlling gas flow meters located on the gas delivery manifold 33 and the gas discharge manifold 34. During the cleaning step, the gas flow rate in the gas delivery manifold 33 does not exceed the gas flow rate in the gas discharge manifold 34, thereby further facilitating the sufficient discharge of the gas and particles inside the
Fifth, after the gas purging step, the sealed
After the gas purging step, the gas flow rate in the gas delivery manifold 33 is made greater than the gas flow rate in the gas discharge manifold 34 by controlling the gas flow meter by the controller 13 before the fat is completely frozen (i.e., the fat itself reaches a predetermined freezing temperature of-60 to-80 degrees celsius), the gas flow rate in the gas discharge manifold 34 not exceeding 40 sccm. Preferably, the gas flow on the gas delivery manifold 33 is 10-20sccm greater than the gas flow on the gas discharge manifold 34. In addition, in the step of gas filling, under the conditions of lower gas flow rate and higher nitrogen gas pressure, the gas cleaning can be realized, the quick heat transfer can be realized, the fat cooling efficiency is improved, the quick cooling of fat is realized, and the fat quality is ensured.
Furthermore, the judgment of the period of time before the fat has completely entered the frozen state after the gas washing step (i.e. the fat itself has reached a predetermined freezing temperature of-60 to-80) can be calculated from the volume of fat, which has been completely entered the frozen state after a freezing time of 20-30 minutes for every 5ml of fat according to experimental data analysis.
Sixth, after the fat is completely frozen, the gas pump for gas input and the gas pump for gas extraction are turned off by the controller 13, and the electromagnetic valves located in the gas delivery manifold 33 and the gas discharge manifold 34 are turned off, so that the atmosphere inside the
Alternatively, in the sixth step, after the fat is completely frozen, the input gas pump and the electromagnetic valve located on the gas delivery branch pipe 33 are first closed, the gas is measured by the gas flow meter located on the gas discharge branch pipe 34, and the gas inside the sealed
The freshly removed fat was washed and filtered and then split into two portions, one portion was frozen for two weeks by the preservation method provided herein (sample 1), the other portion was frozen by the conventional cryopreservation method (sample 2) after adding the same cryoprotectant and then placing in a-80 ℃ freezer for two weeks), and after two weeks, the
By the method for preserving the autologous fat particles at the low temperature, fat pollution can be avoided, and the survival rate of later fat transplantation is improved.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
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