TW201400612A - Service platform for isolation, cultivation, extraction and frozen storage of adipose cells - Google Patents

Abstract

This invention provides a service platform for isolation, cultivation, extraction and frozen storage of adipose cells, which comprises a collection process, a treatment process and an application process. The collection process is used to collect adipose tissues and place them in a sterile container; the treatment process is used to treat the adipose tissues obtained from the collection process and comprises: an adipose tissue isolation procedure, a cultivation procedure, an extraction procedure, and a freezing and storage procedure; the application process is used to apply the adipose tissues, adipose stem cells, nucleated cells, growth factors and cytokines obtained from the treatment process. The service platform can be used to fully exploit adipose tissues and develop various medical uses through the frozen storage of adipose tissues and adipose stem cells.

Description

Separation, culture, extraction and freezing of service platforms for storing fat cells

The invention relates to a service platform for treating adipose tissue and adipose stem cells, in particular to a service platform for fully utilizing adipose tissue and adipose stem cells by separating, culturing, extracting and freezing storage.

In the prior art autologous fat transplantation, fat is transplanted from a part where fat is accumulated, and the fat is transplanted to a part with less fat, but after the fat is transplanted to a new part, part of the fat is excreted and gradually shrinks, resulting in It is necessary to perform multiple operations, and after multiple operations, it is not only easy to cause local tissue fibrosis, but also a very painful process for the patient. Furthermore, after fat transplantation to a new site, it may be due to lack of oxygen and nutrients, resulting in fat calcification and formation of lumps.

Yoshimura et al. found that the stromal vascular fraction (SVF) of adipose tissue contains Platelet-derived Growth Factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (fibroblast). Growth factor, FGF), vascular endothelial growth factor (VEGF), insulin-like growth factors (IGF) and other growth factors, and have the ability to promote angiogenesis, so Yoshimura et al. In fat transplantation, adipose tissue and stromal vascular fraction (SVF) are transplanted together to improve the survival rate after fat transplantation, so it is also called cell assisted lipotransfer (CAL).

However, the volume of fat extracted from existing autologous fat transplantation surgery is much larger. The amount required for autologous fat transplantation, and the remaining fat is often discarded by medical waste. The adipose stem cells rich in vascular matrix are not only capable of promoting the function of angiogenesis, but also anti-apoptosis and regulation. The immune response and the potential to differentiate into tissues, the lack of a method to improve the use of fat cells, especially the lack of a method of "extraction, cryopreservation of adipose stem cells and adipose tissue."

In view of the fact that the prior art lacks a system that is efficient and can reuse fat cells, the object of the present invention is to provide a service platform that can fully utilize adipose tissue and fat stem cells, which can not only avoid multiple liposuction, but also re-use medical waste. Reuse.

To achieve the above object, the present invention provides a service platform for separating, culturing, extracting and freezing stored fat cells, which comprises a collection program, a processing program and an application program.

The collection procedure collects adipose tissue and is housed in a sterile container.

The treatment program processes the adipose tissue obtained by the collection procedure and includes the following processes: an adipose tissue separation process, a culture process, an extraction process, and a freezing and storage process. The adipose tissue separation process is performed after the treatment process, and the adipose stem cells and the nucleated cells are separated from the adipose tissue; wherein the culture process is to culture the adipose stem cells and the nucleated cells obtained by the adipose tissue separation process; wherein the extraction process The cells are extracted by adipose stem cells of a culture process to obtain growth factors and cytokines; wherein the freezing and storing process freezes and stores the adipose tissue, adipose stem cells, nucleated cells, growth factors and cytokines obtained by the above procedures. .

The application is applied to adipose tissue, adipose stem cells, nucleated cells, growth factors and cytokines obtained by the aforementioned treatment procedures in the fields of autologous fat transplantation, wound repair, skin repair, cell therapy, and regenerative medicine.

In accordance with the present invention, the "service platform" as used herein refers to a method of collecting programs, processing programs, and applications.

Preferably, the adipose tissue separation process comprises an adipose tissue separation step and a nucleated cell separation step. More preferably, the adipose tissue separation step comprises: removing tissue fluid of adipose tissue, and separating the adipose tissue by an enzyme capable of separating the adipose stem cells to obtain a fat stem cell. More preferably, the step of separating the nucleated cells comprises: treating the adipose tissue with a proteolytic enzyme to separate the nucleated cells.

According to the present invention, proteolytic enzymes include, but are not limited to, collagenase, trypsin, neutral protease, elastase, papain, and dispase. ).

Preferably, the culture process comprises a fat stem cell and a nucleated cell culture step. More preferably, the step of culturing and nucleating cells of the adipose stem cells comprises: culturing the adipose stem cells and the nucleated cells in a suitable environment.

In accordance with the present invention, the phrase "appropriate environment for the cultivation of adipose stem cells" is any environment in which adipose-derived stem cells can be cultured, including any medium capable of culturing adipose-derived stem cells, including, but not limited to, Dulbecco's modified eagle medium (DMEM), Duchen Modified Ingle-12's modified eagle medium-12 (DMEM-12), Iscove's modified Dulbecco's medium (IMDM) And RPMI 1640 culture solution; and incubating the adipose stem cells at 37 ° C and containing 5% carbon dioxide.

Preferably, the growth factor obtained by the extraction process and the substance secreted by the cells during the cytokine culture process.

More preferably, the growth factors and cytokines include, but are not limited to, EGF, VEGF, and the like.

Preferably, the freezing and storing process comprises: adding adipose tissue, adipose stem cells, and nucleated cells to an antifreeze protection agent, respectively, and storing at a low temperature. The cell culture medium containing the growth factor and the cytokine is treated at a low temperature and then stored at a low temperature.

According to the present invention, the cryoprotectant is used to protect cells in a low temperature environment, and the cells are prevented from being damaged by ice crystals. The types of the cryoprotectant include, but are not limited to, glycerol, dimethyl sulfoxide. , DMSO), ethanediol, propanediol, acetarmide, and methanol.

According to the invention, cryogenic storage refers to storage in a liquid nitrogen tank.

Preferably, the application program uses adipose tissue, adipose stem cells, nucleated cells, growth factors and cytokines obtained by the above-mentioned collection procedure and processing program to match each other according to the purpose of the application, taking autologous fat transplantation as an example. Adipose tissue and nucleated cells are co-implanted into specific parts of the receptor such as the chest or face; for wound repair or skin repair, for example, growth factors and cytokines are placed on the surface of the receptor; cell therapy and regeneration In the medical field, for example, the differentiation ability of adipose stem cells is used to achieve regeneration of cells, tissues or organs.

The service platform of the present invention includes a collection program, a processing program, and An application, wherein the processing program processes the adipose tissue obtained by the collecting program, and comprises three processes, wherein the first process separates the adipose tissue and comprises an adipose tissue separation step and a nucleated cell separation step; The method comprises the steps of: adipose stem cells and a nucleated cell culture step; the third process comprises an extraction step; and the fourth process comprises a freezing and storage step.

Based on the above characteristics, patients can obtain adipose tissue, adipose stem cells, nucleated cells, growth factors and cytokines simultaneously after autologous fat transplantation, and simultaneous transplantation of adipose tissue and nucleated cells can improve the survival rate of fat transplantation. It can also cryopreserve adipose tissue, adipose stem cells, nucleated cells, growth factors and cytokines to facilitate the use of the next autologous fat transplantation, which not only avoids the pain of patients undergoing multiple liposuction, but also the resulting adipose stem cells. , growth factors and cytokines are used in a variety of medical applications to achieve the full use of adipose tissue.

Referring to FIG. 1, the service platform for separating, culturing, extracting and freezing stored fat cells of the present invention comprises a collection program 10, a processing program 20 and an application program 30.

The collection procedure 10 collects adipose tissue and is housed in a sterile container.

The treatment program 20 processes the adipose tissue obtained by the collection procedure and includes an adipose tissue separation process 21, a culture process 22, an extraction process 23, and a freezing and storage process 24.

The adipose tissue separation process 21 is followed by the treatment program 20, and the adipose stem cells and the nucleated cells can be separated from the adipose tissue, as shown in FIG. 2, and includes an adipose tissue separation step 211 and a nucleated cell separation step. Step 212. The adipose tissue separation step 211 comprises: washing the adipose tissue with a phosphate buffer salt solution containing penicillin/streptomycin to remove the tissue fluid of the adipose tissue.

The nuclear cell separation step 212 comprises treating the adipose tissue with a proteolytic enzyme to isolate the nucleated cells.

The culture process 22 is to culture the adipose stem cells and nucleated cells obtained by the adipose tissue separation step 21, as shown in FIG. 3, and includes an adipose stem cell and a nucleated cell culture step 221, which comprises containing penicillin/streptococcus. After washing the adipose tissue with the phosphate solution, collagenase is added to separate the adipose tissue stem cells S1, and the collagen degrading enzyme activity is neutralized by fetal bovine serum (FBS), and then centrifuged to separate into fat. Adipocyte and stromal vascular fraction (SVF), after removing the fat cells, adding a hypotonic solution to dissolve the red blood cells (RBC); After washing, the adipose stem cells S2 are cultured in an appropriate environment.

The extraction process 23 is performed by the adipose stem cells obtained in the culture process 22, as shown in FIG. 4, and is included in a centrifugation step 231 to collect and obtain growth factors and cytokines.

The freezing and storing process 24 freezes and stores the adipose tissue obtained in the collection program 10 and the adipose stem cells, nucleated cells, growth factors and cytokines obtained in the treatment program 20, as shown in FIG. 5, and includes: Adipose tissue adipose stem cells, nucleated cells, growth factors and cytokines are respectively added to the antifreeze protectant S1, and S2 is stored at a low temperature in a liquid nitrogen tank.

10‧‧‧ Collection procedure

20‧‧‧Processing procedures

21‧‧‧Adipose tissue separation process

211‧‧‧Adipose tissue separation step

212‧‧‧With nuclear cell separation step

22‧‧‧Cultivation process

221‧‧‧Adipose stem cells and nucleated cell culture steps

S1‧‧‧Separation of adipose tissue stem cells by proteolytic enzymes

S2‧‧‧Cultivate adipose stem cells in an appropriate environment

23‧‧‧Extraction process

231‧‧‧ Centrifugation step

24‧‧‧Freezing and storage process

S1‧‧‧Adding antifreeze protectant

S2‧‧‧ low temperature storage

30‧‧‧Applications

1 is a schematic diagram of a service platform of the present invention.

Figure 2 is a schematic illustration of the adipose tissue separation process of the service platform of the present invention.

3 is a schematic diagram showing the cultivation process of the service platform of the present invention.

Figure 4 is a schematic illustration of the extraction process of the service platform of the present invention.

Figure 5 is a schematic illustration of the refrigeration and storage process system of the service platform of the present invention.

10‧‧‧ Collection procedure

20‧‧‧Processing procedures

21‧‧‧Adipose tissue separation and freezing process

22‧‧‧Cultivation process

23‧‧‧Extraction process

24‧‧‧Freezing and storage process

30‧‧‧Applications

Claims (7)

A service platform for separating, culturing, extracting and freezing fat cells comprises: a collecting procedure for collecting adipose tissue and accommodating it in a sterile container; and a processing procedure for processing the adipose tissue obtained by the collecting procedure, and comprising the following processes: An adipose tissue separation process, which is a process for separating adipose stem cells and nucleated cells from adipose tissue; a culture process for culturing adipose stem cells and nucleated cells obtained by adipose tissue separation process; a process for extracting adipose stem cells of a culture process to obtain growth factors and cytokines; and, a freezing and storing process, which is carried out by adipose stem cells and nucleated cells obtained by the culture process and adipose tissue obtained by the treatment procedure Freezing and storage; an application is the application of adipose tissue, adipose stem cells, nucleated cells, growth factors and cytokines obtained by the treatment process to the fields of autologous fat transplantation, wound repair, skin repair, cell therapy and regenerative medicine. The service platform of claim 1, wherein the adipose tissue separation step comprises an adipose tissue separation step and a nucleated cell separation step; wherein the adipose tissue separation step comprises: removing adipose tissue tissue fluid, and separating the fat by The stem cell enzyme separates the adipose tissue to obtain a fat stem cell; wherein the nucleated cell separation step comprises: treating the adipose tissue with a proteolytic enzyme to separate the nucleated cells. The service platform as described in claim 2, wherein the proteolytic enzyme is a gelatin Original proteinasease, trypsin, neutral protease, elastase, papain, and dispase. The service platform according to claim 1, wherein the culture process comprises a fat stem cell and a nucleated cell culture step, comprising: adipose stem cells obtained by the adipose tissue separation step and nucleated cells obtained by the nucleation cell separation step are appropriate Environmental training. The service platform according to claim 1, wherein the growth factor and the cytokine are substances secreted by the cells during the culture process. The service platform of claim 1, wherein the freezing and storing process comprises: adding adipose tissue, adipose stem cells, nucleated cells, growth factors, and cytokines to an antifreeze protectant, respectively, and storing at a low temperature. The service platform according to claim 6, wherein the antifreeze protecting agent is glycerol, dimethyl sulfoxide (DMSO), ethanediol, propanediol, acetarmide ) and methanol (methanol).