Fat particle tissue cryopreservation liquid, preparation method and freeze-saving method thereof

A fat particle and cryopreservation technology, applied in the field of cell biology, can solve problems such as toxicity and potential risks, and achieve the effects of safety in use, protection from freezing damage, and reduction of the formation of ice crystals

Active Publication Date: 2019-04-23
米楠 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The object of the present invention is to provide a fat particle tissue cryopreservation solution, which solves the problem that the existing fat particle tissue cryopreservation solution is toxic and has po

Method used

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  • Fat particle tissue cryopreservation liquid, preparation method and freeze-saving method thereof
  • Fat particle tissue cryopreservation liquid, preparation method and freeze-saving method thereof
  • Fat particle tissue cryopreservation liquid, preparation method and freeze-saving method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of fat particle tissue cryopreservation solution

[0035] Specific steps include:

[0036]Add trehalose, hydroxyethyl starch, L-ascorbic acid, human serum albumin, human insulin, basic fibroblast growth factor to DMEM / F12 to obtain a mixed solution, wherein the concentration of trehalose in the mixed solution is 75.6g / L, The concentration of hydroxyethyl starch is 60g / L, the concentration of human serum albumin is 50g / L, the concentration of L-ascorbic acid is 58mg / L, the concentration of human insulin is 12mg / L, and the concentration of basic fibroblast growth factor is 25ug / L;

[0037] (2) Adjust the pH value of the mixed solution to 7.2-7.4, and then use a 0.22um filter to filter and sterilize to obtain a fat particle tissue cryopreservation solution, and store it in the dark at -20°C;

[0038] (3) The mixed solution obtained in step (2) is further tested. When the osmotic pressure obtained from the test is 260-320 mosm, bacteria and fungi a...

Embodiment 2

[0039] Embodiment 2: cryopreservation of human fat granule tissue

[0040] (1) Collection of autologous fat

[0041] Routine disinfection and sterile towels are spread, and local anesthesia with 2% lidocaine is applied to the edge of the liposuction area; an incision of about 0.3 cm is made along the skin, and the hemostatic forceps are slightly bluntly separated under the skin, and swelling anesthesia is evenly injected in the liposuction area; After the anesthesia takes effect, insert the aspiration needle into the subcutaneous fat layer, connect the 20mL syringe, draw the syringe to negative pressure, and then use the syringe needle cap to hold the pulled-out syringe core, keep the negative pressure state, and hold the aspiration needle handle to aspirate. Suction, suction fat particles 100ml.

[0042] (2) Purification of fat particles

[0043] Transfer the pumped fat particles (100ml) to a 250ml sterile bottle, add an equal volume of normal saline, let stand for 30s-1min...

experiment example 1

[0048] Experimental example 1: Creatine kinase detection of cell viability after cryopreservation of fat particles

[0049] Among them, the creatine kinase detection kit was purchased from Zhongsheng Beikong Biological Co., Ltd.

[0050] The specific experimental steps include:

[0051] (1) Take the fat granule tissue frozen for 6 months in Example 2, place it in a 37°C water bath to thaw quickly, absorb the protective agent in time after the frozen storage solution is completely dissolved, and wash it 3 times with normal saline;

[0052] (2) After washing away the residual cryopreservation solution, put it into a fabric grinder, and grind the fat particle tissue at 0°C;

[0053] (3) Centrifuge the fat particle tissue homogenate at 5000r / min for 2min, absorb 0.25ml of the middle layer solution, add an equal volume of normal saline, add 2.5ml of creatine kinase (CK) test solution at 37°C, and add 2min and 2min respectively. At 3 minutes, draw 1ml of the mixed solution to meas...

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Abstract

The invention discloses a fat particle tissue cryopreservation liquid, a preparation method and a freeze-saving method thereof. The fat particle tissue freeze-saving liquid comprises trehalose, hydroxyethyl starch, L-ascorbic acid, albumin, human insulin, a basic fibroblast growth factor and a basic culture medium. The fat particle tissue freeze-saving liquid does not contain dimethyl sulfoxide and fetal bovine serum, has no toxicity to cells, does not denature proteins, is safe to use, can enable frozen fat particle tissue to have higher vitality, improves the survival rate of fat tissue after recovery, can enable fat grafts to maintain higher biological activity and tissue structure, and can be widely applied in clinic.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a fat granule tissue cryopreservation liquid and its preparation method and cryopreservation method. Background technique [0002] It has been more than one hundred years since Billings and Neube first used autologous fat to fill soft tissue defects in the 19th century. In recent years, with the advancement of liposuction technology and the vigorous development of plastic surgery, autologous fat transplantation has become one of the most important treatment methods in plastic surgery. Autologous fat grafting can be used to fill soft tissue defects in almost all parts of the body, such as wrinkles, depressed scars, breast augmentation and facial soft tissue filling. [0003] Autologous fat transplantation is more and more widely used in plastic surgery, but its survival rate after transplantation is not high, and it often requires a small amount of repeated transplantation t...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/021A01N1/0221A01N1/0226
Inventor 米楠
Owner 米楠
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