WO2021134289A1 - Solution de conservation de tissu adipeux, son procédé de préparation et son application - Google Patents

Solution de conservation de tissu adipeux, son procédé de préparation et son application Download PDF

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Publication number
WO2021134289A1
WO2021134289A1 PCT/CN2019/130092 CN2019130092W WO2021134289A1 WO 2021134289 A1 WO2021134289 A1 WO 2021134289A1 CN 2019130092 W CN2019130092 W CN 2019130092W WO 2021134289 A1 WO2021134289 A1 WO 2021134289A1
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WIPO (PCT)
Prior art keywords
adipose tissue
preservation solution
adipose
human albumin
cell
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PCT/CN2019/130092
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English (en)
Chinese (zh)
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李文东
宋云庆
黎波
卢瑞珊
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广东华夏健康生命科学有限公司
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Priority to PCT/CN2019/130092 priority Critical patent/WO2021134289A1/fr
Publication of WO2021134289A1 publication Critical patent/WO2021134289A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts

Definitions

  • the invention relates to the field of biological technology, in particular to an adipose tissue preservation liquid, a preparation method and an application.
  • Adipose tissue is mainly composed of a large number of clustered fat cells, which are divided into lobules by loose connective tissue. According to the structure and function of fat cells, adipose tissue is divided into two types. Yellow (white) adipose tissue is yellow (white in some mammals), which is commonly referred to as adipose tissue. It is made up of a large number of single-vesicle adipocytes. There is a large lipid droplet in the center of the cell, and the cytoplasm is a thin layer, located at the periphery of the cell, surrounding the lipid droplet. On HE slices, lipid droplets are dissolved into a large vacuole.
  • the nucleus is oblate, pushed to the side of the cell by lipid droplets, and part of the cytoplasm is crescent-shaped.
  • Yellow adipose tissue is mainly distributed in the subcutaneous, omentum and mesangium, accounting for about 10% of adult body weight. It is the largest energy storage in the body and participates in energy metabolism. It also has the ability to generate heat, maintain body temperature, buffer protection and support filling, etc. effect.
  • adipose tissue contains a large number of adipose stem cells, but the viability of adipose tissue will gradually decrease in vitro, which directly affects the quality of the cells after separation. How to maintain the vitality of stem cells in adipose tissue has become the primary problem to be solved. Prior to this, people added penicillin, streptomycin, human albumin and other substances on the basis of basic medium such as DMEM/F12 to preserve isolated adipose tissue.
  • the stem cells in the adipose tissue exhibit low vitality or even death during long-term storage or transportation.
  • composition system of the preservation solution adding several kinds of amino acids and antibiotics to the buffer solution composed of inorganic ions to maintain the activity of the isolated adipose tissue and reduce the infection rate.
  • these measures have maintained the viability of seed cells for a long period of time, the composition of the preservation solution is complicated, and the preparation needs to go through the steps of weighing ingredients and sterilization. The preparation process is cumbersome and cannot be conveniently applied; Adding a variety of antibiotics can reduce the chance of infection, but it is toxic to adipose stem cells; in addition, a certain amount of amino acids and human albumin are often added to the existing preservation solution to provide nutrition for the isolated placenta, but the nutrients are single And the cost is higher.
  • the technical problem to be solved by the present invention is to provide an adipose tissue preservation solution, preparation method and application.
  • the prepared preservation solution can well maintain the viability of adipose stem cells without causing cell functional variation.
  • the present invention provides an adipose tissue preservation solution, including: polyethylene glycol 400, human albumin, norfloxacin, low molecular dextran and Ac-DEVD-CHO.
  • the present invention uses polyethylene glycol 400 as the basic component, adds anti-apoptotic substances, and also uses the broad-spectrum antibacterial substance norfloxacin, which solves all aspects of the natural layering between the preservation solution and the tissue. This phenomenon can also achieve the true preservation effect through the full fusion of the solution and the tissue.
  • the above-mentioned adipose tissue preservation solution uses polyethylene glycol 400 as the basic preservation solution.
  • the polyethylene glycol 400 can be purchased from a reagent company to ensure the stability of the quality of the preservation solution and avoid quality differences caused by artificial configuration.
  • the adipose tissue preservation solution includes:
  • the adipose tissue preservation solution includes:
  • the content of the human albumin is based on 100% polyethylene glycol 400.
  • the content of human albumin mentioned above is the mass content.
  • the concentration of the aforementioned norfloxacin, low-molecular-weight dextran and Ac-DEVD-CHO is based on 100% polyethylene glycol 400 containing 5%-10% human albumin.
  • the human albumin added in the adipose tissue preservation solution can be directly utilized by the cells, which improves the resistance ability of the cells and maintains the activity of the cells.
  • the content of the human albumin is preferably 5%-10%, more preferably 5% or 10%.
  • the concentration of norfloxacin is 1 ⁇ -2 ⁇ norfloxacin, and more preferably 1 ⁇ norfloxacin.
  • the aforementioned norfloxacin is a generally commercially available medical grade injection product.
  • the concentration of norfloxacin is 100 ⁇ , and the final concentration is controlled to be 1 ⁇ -2 ⁇ during the preparation process.
  • the content of the low molecular dextran is preferably 1 wt%.
  • 1wt% of low-molecular-weight dextran is added to the preservation solution, which can maintain the osmotic pressure of the preservation solution, so that the fat is stored in an isotonic and isotonic environment, which is beneficial to maintain the activity of adipose mesenchymal stem cells.
  • the low-molecular-weight dextran is a generally commercially available finished product.
  • the concentration of the low-molecular-weight dextran is 6.0%, and during the preparation process, the final concentration is controlled to 1%.
  • the above content is the mass content.
  • the Ac-DEVD-CHO is also known as Caspase 3 inhibitor, which acts as the initiator of the apoptosis signal pathway. It inhibits the initiation molecule of apoptosis, enhances the anti-apoptotic effect of cells, directly inhibits cell apoptosis, and preserves the vitality of cells. Biological characteristics.
  • the concentration of the Ac-DEVD-CHO is preferably 5-20 ng/mL, and more preferably 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, or the interval of any two of the above values.
  • polyethylene glycol 400 human albumin, norfloxacin, low molecular dextran and Ac-DEVD-CHO can all be generally commercially available.
  • the present invention provides a method for preparing the above-mentioned adipose tissue preservation solution, which includes the following steps:
  • step S2) The human albumin-containing polyethylene glycol 400 obtained in step S1) is mixed with norfloxacin, low molecular dextran and Ac-DEVD-CHO to obtain adipose tissue preservation solution.
  • the content of human albumin is controlled to be 5%-10%.
  • the concentration of norfloxacin is 100 ⁇ , and during the mixing process, the concentration of norfloxacin in the adipose tissue preservation solution is controlled to be 1 ⁇ -2 ⁇ .
  • the obtained adipose tissue preservation solution is filtered through a 0.22 ⁇ m membrane for use.
  • the present invention provides the application of the adipose tissue preservation solution in the preservation of adipose tissue.
  • the present invention provides a method for preserving adipose tissue.
  • the adipose tissue is stored in the adipose tissue preservation solution or the adipose tissue preservation solution prepared by the preparation method.
  • the volume ratio of the adipose tissue to the adipose tissue preservation solution is 1:1 ⁇ 3. More preferably, it is 1:1.
  • the storage temperature is 4°C.
  • the specific method is:
  • the adipose tissue that has been extracted from the body is allowed to stand and the swelling fluid is discarded.
  • the transportation condition of the adipose tissue is 4°C for transportation.
  • Preserving fat at low temperature can prevent the denaturation of nutrients on the one hand, and on the other hand maintain the biological activity of seed cells.
  • the adipose tissue preservation solution provided by the present invention can maintain the original biological characteristics of adipose mesenchymal stem cells within 72 hours.
  • the present invention provides an adipose tissue preservation solution, including: polyethylene glycol 400, human albumin, norfloxacin, low molecular dextran and Ac-DEVD-CHO.
  • the present invention uses polyethylene glycol 400 as the basic component for the first time. This substance has good compatibility with oils and fats, and can also separate the lipids after being compatible with water substances; human albumin is used as the fat inter-fat substance.
  • the nutrient content of mesenchymal stem cell preservation solution is used as the adipose tissue preservation solution.
  • the added apoptosis inhibitor can greatly improve the resistance of cells and maintain cell viability, so that the adipose tissue can still maintain the original cell morphology and biological characteristics of the adipose tissue under the condition of in vitro and long-term transportation.
  • the final prepared adipose tissue preservation solution can well maintain the viability of adipose-derived mesenchymal stem cells without causing cell functional variation. It can be used for long-term preservation of isolated adipose tissue and can still maintain adipose mesenchyme within 72 hours. The vitality of cytoplasmic stem cells.
  • the preparation method of the cell tissue preservation solution provided by the present invention is simple and can be prepared in a large amount in a short time.
  • the preservation solution has low cost, stable composition, safety, and no toxic side effects.
  • Figure 1 is a cytoscopy image of adipose tissue in Comparative Example 1 after being stored in the preservation solution for 48 hours;
  • Figure 2 is a diagram of the particle size distribution of adipose tissue after being stored in the preservation solution for 48 hours in Comparative Example 1;
  • Figure 3 is a cytoscopy image of adipose tissue in Comparative Example 2 after being stored in the preservation solution for 48 hours;
  • Figure 4 is a diagram of the particle size distribution of adipose tissue after being stored in the preservation solution for 48 hours in Comparative Example 2;
  • Figure 5 is a cytomicroscopic view of adipose tissue after being stored in the preservation solution for 48 hours in Example 1;
  • Figure 6 is a diagram of the particle size distribution of adipose tissue after being stored in the preservation solution for 48 hours in Example 1;
  • Figure 7 is a cytoscopy image of adipose tissue after being stored in the preservation solution for 48 hours in Example 2;
  • Figure 8 is a diagram of the particle size distribution of adipose tissue after being stored in the preservation solution for 48 hours in Example 2;
  • Figure 9 is a cytomicroscopic view of adipose tissue after being stored in the preservation solution for 48 hours in Example 3;
  • Fig. 10 is a diagram showing the particle size distribution of adipose tissue in Example 3 after being stored in the preservation solution for 48 hours.
  • the adipose tissue that has been extracted from the body is allowed to stand and the swelling fluid is discarded.
  • preservation solution (V:V) 1:1
  • the adipose tissue is transferred to the prepared adipose tissue preservation solution for storage and transported to In the laboratory.
  • the isolated fat enters the experimental program within 24h, 48h, 72h (maintain 4°C during transportation).
  • adipose-derived mesenchymal stem cells After separation and culture of adipose-derived mesenchymal stem cells, adjust the density to 5 ⁇ 10 5 cell/mL, inoculate 8 mL into a 9cm-diameter plate, wait for it to grow naturally adherent for 48 hours, remove the non-adherent cells 0.25% trypsin is used for enzymolysis, the number of adherent cells is calculated, and the adherence rate is obtained.
  • Morphology detection Take adipose stem cells in logarithmic growth phase and observe the morphology of the cells under an inverted microscope.
  • the medium is exchanged; a dish of cells is taken every 48 hours for enzymatic hydrolysis until the confluence of the batch of cells reaches 100%, and the number of cells is counted to obtain the cell proliferation rate.
  • adipose stem cells in the logarithmic growth phase adjust the cell density to 1 ⁇ 10 6 cells, and take 2.5 ⁇ L of monoclonal antibodies against human CD105, CD90, CD73, CD45, CD34, and HLA-DR, respectively, and add 500 ⁇ L of cell suspension, incubated for 20min at room temperature in the dark, and set up a blank isotype control; centrifuged at 1500r/min for 5min, discarded the supernatant, washed twice with PBS containing 10% FBS, resuspended with 500 ⁇ L of 1640, and tested on the machine.
  • the extracted P1 adipose stem cells is greater than 22 ⁇ m, accounting for 2.3%.
  • the extracted P1 adipose stem cells is greater than 22 ⁇ m, accounting for 0.7%.
  • the extracted P1 adipose stem cells is greater than 22 ⁇ m accounting for 2.6%.
  • the extracted P1 adipose stem cells is greater than 22 ⁇ m accounting for 5.2%.
  • the extracted P1 adipose stem cells is greater than 22 ⁇ m accounting for 7.3%.
  • ⁇ means P ⁇ 0.05 compared with the 24h in Example 1, which is statistically significant.
  • ⁇ It means that compared with the 24h of Example 2, P ⁇ 0.05, which is statistically significant.
  • the adipose tissue preservation solution provided by the present invention can better maintain cell survival rate and maintain its biological characteristics.

Abstract

L'invention concerne une solution de conservation de tissu adipeux, comprenant : du polyéthylène glycol 400, de l'albumine humaine, de la norfloxacine, du dextrane de faible poids moléculaire et de l'Ac-DEVD-CHO. La présente invention utilise le polyéthylène glycol 400 en tant que composant de base pour la première fois ; ladite substance présente une bonne compatibilité avec une substance de type huile et peut également séparer une substance de type lipide après avoir été compatible avec une substance de type eau ; et de l'albumine humaine est utilisée en tant que composant nutritionnel de solution de conservation de cellules souches mésenchymateuses adipeuses. En outre, un inhibiteur d'apoptose ajouté peut considérablement améliorer la résistance au stress d'une cellule et assurer la viabilité cellulaire, de telle sorte que le tissu adipeux à l'état de transport in vitro et à long terme peut toujours garantir qu'une cellule souche adipeuse conserve la morphologie cellulaire et les caractéristiques biologiques d'origine. Dans le même temps, le procédé de préparation d'une solution de conservation de tissu cellulaire fourni est simple, et la viabilité d'une cellule souche mésenchymateuse adipeuse peut toujours être parfaitement assurée dans les 72 heures.
PCT/CN2019/130092 2019-12-30 2019-12-30 Solution de conservation de tissu adipeux, son procédé de préparation et son application WO2021134289A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014051173A1 (fr) * 2012-09-27 2014-04-03 (주)세포바이오 Composition comprenant de la sérum-albumine humaine recombinante dérivée d'une plante, des lipides et des hydrolysats de protéines végétales comme ingrédients actifs pour la cryoconservation de cellules souches ou de cellules primaires
CN105325402A (zh) * 2015-11-13 2016-02-17 黄林海 一种用于玻璃化冻存骨髓间充质干细胞的冻存保护剂
CN106719600A (zh) * 2016-11-30 2017-05-31 广州赛莱拉干细胞科技股份有限公司 一种脂肪组织冻存液
CN109619088A (zh) * 2018-12-27 2019-04-16 广州赛莱拉干细胞科技股份有限公司 一种脂肪间充质干细胞的保存液
WO2019103529A2 (fr) * 2017-11-24 2019-05-31 주식회사 차바이오랩 Composition de congélation et de conservation de cellules, et procédé de congélation et de conservation de cellules l'utilisant
CN110612978A (zh) * 2019-09-26 2019-12-27 广东华夏健康生命科学有限公司 一种用于人月经血的保存液及其制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014051173A1 (fr) * 2012-09-27 2014-04-03 (주)세포바이오 Composition comprenant de la sérum-albumine humaine recombinante dérivée d'une plante, des lipides et des hydrolysats de protéines végétales comme ingrédients actifs pour la cryoconservation de cellules souches ou de cellules primaires
CN105325402A (zh) * 2015-11-13 2016-02-17 黄林海 一种用于玻璃化冻存骨髓间充质干细胞的冻存保护剂
CN106719600A (zh) * 2016-11-30 2017-05-31 广州赛莱拉干细胞科技股份有限公司 一种脂肪组织冻存液
WO2019103529A2 (fr) * 2017-11-24 2019-05-31 주식회사 차바이오랩 Composition de congélation et de conservation de cellules, et procédé de congélation et de conservation de cellules l'utilisant
CN109619088A (zh) * 2018-12-27 2019-04-16 广州赛莱拉干细胞科技股份有限公司 一种脂肪间充质干细胞的保存液
CN110612978A (zh) * 2019-09-26 2019-12-27 广东华夏健康生命科学有限公司 一种用于人月经血的保存液及其制备方法

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