WO2019237812A1 - Adipose tissue digestive juice and method for rapidly obtaining stromal vascular fraction cells - Google Patents

Abstract

A adipose tissue digestive juice and a method for rapidly obtaining stromal vascular fraction (SVF) cells are provided. The adipose tissue digestive juice is composed of a type I collagenase, a HEPES buffer solution and a DMEM/F12 culture medium, and the adipose tissue can be digested to obtain SVF.

Description

Adipose tissue digestive fluid and method for quickly obtaining stromal vascular component cells

This application claims the priority of a Chinese patent application filed on June 15, 2018 with the Chinese Patent Office, application number 201810618076.9, and the invention name "Adipose Tissue Digestive Fluid and Method for Rapidly Obtaining Matrix Vessel Component Cells", the entire content of which is approved Citations are incorporated in this application.

Technical field

The invention relates to the technical field of cell separation, in particular to adipose tissue digestive juice and a method for quickly obtaining stromal vascular component cells.

Background technique

Refractory wounds are common in surgical diseases. They have the characteristics of long course, many complications, and great influence on appearance. In recent years, they have been increasing year by year, and have become a factor that seriously affects the quality of life of patients. How to promote the healing of refractory wounds has been an explored problem, but so far there is no ideal method. Mesenchymal stem cells (MSCs) were found in the bone marrow for the first time. They are multipotent cells with the potential to promote tissue regeneration. MSCs can accelerate wound healing by promoting angiogenesis and tissue regeneration in local tissues. However, the bone marrow-derived MSCs are low in content and difficult to extract, which limits the clinical application of bone marrow-derived MSCs. Recent studies have found that there are also a large number of MSCs in adipose tissue, and adipose tissue has the characteristics of abundant sources and little damage to tissues. And the content of stem cells in fat, cell expansion and proliferation ability are significantly better than bone marrow tissue.

Stromal vascular component cells (SVF) are adipose-derived regenerative cells found in particles aspirated from fat, and more than 30% of them are adipose-derived stem cells. SVF is obtained after adipose tissue is digested, filtered, and centrifuged to remove mature adipocytes. In 2001, Zuk first discovered that SVF contains a variety of cellular components, including MSCs, endothelial precursor cells, macrophages, smooth muscle cells and other components, and can induce differentiation into adipocytes, osteoblasts, chondrocytes, etc.

Recent research suggests that SVF can promote the healing of difficult-to-heal wounds by differentiating into vascular endothelial cells, secreting pro-angiogenesis and anti-apoptotic growth factors. Adding SVF in the process of transplanting fat found that its effect is very considerable: it can improve the survival rate of adipocytes, and it can also significantly improve the texture of the skin.

At present, the conventional method for obtaining SVF is to use a certain concentration of type I collagenase for constant temperature shock digestion. The conventional isolation method uses type I collagenase digestion with a final concentration of 0.25% to obtain SVF in about 40 minutes. Each milliliter of fat can be digested by collagenase to obtain SVF of 1.0-2.5 × 10 ⁵ , and the activity rate is 70% -85%. It can be seen that it takes a long time to obtain cells with stromal vascular components, and the viability is low. Therefore, there is a need to provide a digestion method with shorter digestion time and higher cell viability.

Summary of the Invention

In view of this, the present invention provides adipose tissue digestive fluid and a method for quickly obtaining stromal vascular component cells. This method can quickly obtain stromal vascular component cells, shortening the time to 15-20 minutes, and at the same time, the cell viability has been significantly improved.

In order to achieve the above-mentioned object, the present invention provides the following technical solutions:

The invention provides adipose tissue digestive juice, which is composed of type I collagenase, HEPES buffer solution and DMEM / F12 medium.

The traditional method of digestion did not pay attention to the change of the system pH during the digestion process. This study found that the type I collagenase in the conventional method is not in the optimal pH environment, so the time to obtain SVF is longer. The method of the invention adds a buffer system of HEPES buffer solution to the digestion system, so that the digestion system can be stably maintained between pH 6.8 and 8.2, and the type I collagenase activity is optimized to achieve complete digestion in a short time. The SVF obtained by the traditional digestion scheme takes 40 minutes, and the digestion scheme added with additional buffering components according to the present invention can obtain high quality and quantity of SVF within 15-20 minutes.

Preferably, the amount of each component in the digestive juice is:

Type I collagenase: 0.1 wt% to 0.3 wt%;

HEPES buffer: 1 ～ 30mM;

DMEM / F12 medium: To complement.

Preferably, the amount of each component in the digestive juice is:

Type I collagenase: 0.25% by weight;

HEPES buffer: 5 ～ 25mM;

DMEM / F12 medium: To complement.

Preferably, the amount of each component in the digestive juice is:

Type I collagenase: 0.25% by weight;

HEPES buffer: 10 ～ 25mM;

DMEM / F12 medium: To complement.

More preferably, the amount of each component in the digestive juice is:

Type I collagenase: 0.25% by weight;

HEPES buffer: 20mM;

DMEM / F12 medium: To complement.

More preferably, the amount of each component in the digestive juice is:

Type I collagenase: 0.25% by weight;

HEPES buffer: 25mM;

DMEM / F12 medium: To complement.

The invention also provides a method for quickly obtaining stromal vascular component cells, including the following steps:

The digestive fluid provided by the present invention is used to digest adipose tissue to obtain stromal vascular component cells; the digestive fluid is composed of type I collagenase, HEPES buffer solution and DMEM / F12 medium.

Preferably, the digestion temperature is 36 to 38 ° C.

In the embodiment provided by the present invention, the temperature of digestion is 37 ° C.

Preferably, the rotation speed of the digestion is 100 to 200 rpm.

In the embodiment provided by the present invention, the rotation speed of the digestion is 150 rpm.

Preferably, the digestion time is 15 to 25 minutes.

In the embodiment provided by the present invention, the digestion time is 20 minutes.

In the embodiment provided by the present invention, the steps of centrifugation to terminate the digestion and washing are further included after the digestion.

The invention provides adipose tissue digestive fluid and a method for quickly obtaining stromal vascular component cells. The adipose tissue digestive fluid is composed of type I collagenase, HEPES buffer and DMEM / F12 medium. The invention has one of the following advantages:

1. The adipose tissue digestive fluid provided by the present invention can stably maintain the digestive system between pH 6.8 and 8.2, optimize the type I collagenase activity, and achieve complete digestion in a short time. The SVF obtained by the traditional digestion scheme takes 40 minutes, and the digestive juice of the present invention can be used to digest adipose tissue, and a high quality and quantity of SVF can be obtained in 15-20 minutes. The test results show that the SVF activity rate obtained by digesting adipose tissue with the digestive juice of the present invention is above 85%.

2. The digestive juice formula of the invention is simple and suitable for industrial production.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the expression of surface markers on each cell;

Figure 2 shows the effect of adipogenic staining of cells.

detailed description

The invention discloses an adipose tissue digestive fluid and a method for quickly obtaining stromal vascular component cells. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can modify or appropriately modify and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention. Apply the technology of the present invention.

Explanation of terms:

Adipose tissue (adipose tissue issue): mainly composed of a large number of clusters of fat cells, agglomerated fat cells are separated by a thin layer of loose connective tissue into small leaflets. Reticulated fibers in adipose tissue are well developed. Yellow (white) adipose tissue is yellow (white in some mammals), which is commonly referred to as adipose tissue. It consists of a large number of single-vesicle adipocytes. The adipocytes are round or polygonal. There is a large lipid droplet in the center of the cell, and the cytoplasm is a thin layer. It is located around the periphery of the cell and surrounds the lipid droplet. On HE slices, the lipid droplets were dissolved into a large vacuole. The nucleus is oblate and is pushed to one side of the cell by lipid droplets. It is crescent-shaped with part of the cytoplasm. The yellow adipose tissue is mainly distributed in the subcutaneous tissue, omentum, and mesentery. In adult men, it usually accounts for about 10% to 20% of body weight, and women tend to have more. The largest "energy bank" in the body. It has the functions of storing fat, maintaining body temperature and participating in fat metabolism. It is involved in energy metabolism and has the functions of generating heat, maintaining body temperature, buffering protection and supporting filling. Brown adipose tissue is brown, which is characterized by rich capillaries in the tissue. Fat cells are scattered with many small lipid droplets. The mitochondria are large and rich. The nucleus is round and located in the center of the cell. Such adipocytes are called polyfoam adipocytes.

Stromal vascular component cells (SVF): Adipose tissue is mainly composed of adipocytes, ASCs, vascular endothelial cells, pericytes, fibroblasts, macrophages and extracellular matrix. Each cubic centimeter volume of adipose tissue includes 1 × 10 ⁶ adipocytes, 1 × 10 ⁶ ASCs, 1 × 10 ⁶ vascular endothelial cells, and (1 ～ 2) × 10 ⁶ other cells (such as blood source Cells) provide a rich source of stromal stem cells for research and applications in tissue engineering and regenerative medicine. Researchers have now used enzyme digestion to separate and extract stromatal vascular component cells (SVF) from adipose tissue discarded by abdominal wall plastic surgery or liposuction. SVF is a population of adipose-derived stem cells mixed with other cell components. After adherent culture in vitro, pure adipose-derived stromal / stem cells (ASCs) can be obtained.

Phosphate buffer: PBS, full name Phosphate Buffered Saline. In medical terms, it means phosphate buffer solution, used for molecular cloning and cell culture, pH = 7.4, isotonic with human blood, the main components are potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride.

Collagenase: specifically hydrolyzes the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. The chemical nature of collagenase is a protein, so it is very sensitive to temperature, pH, and various factors that cause protein denaturation, and it is very susceptible to external conditions to change its conformation and properties. Collagenase I is used for the isolation of epithelial, lung, fat and adrenal tissue cells. It is used to digest the connecting part of the tissue to make it a single cell. It is used for the isolation of mammalian cells. Its optimum pH is 6.3 ~ 8.8.

DMEM / F12 medium: F12 medium is rich in ingredients and contains various trace elements. It is combined with DMEM at a ratio of 1: 1 and is called DMEM / F12 medium (DME / F12medium). It is used as a basis for developing serum-free formula to utilize F12. Contains richer ingredients and DMEM contains higher concentrations of nutrients as advantages. The medium is suitable for mammalian cell culture under conditions of low serum content.

HEPES buffer: non-toxic to cells. It is a hydrogen ion buffer that can control a constant pH range for a longer period of time.

In the following examples, "%" represents mass percentage content.

The adipose tissue digestive fluid provided by the present invention and the reagents or instruments used in the method for quickly obtaining stromal vascular component cells can be purchased from the market.

The following further describes the present invention in combination with the embodiments:

Example 1

1. The digestion formula of this embodiment is:

Collagen type I: 0.25%;

HEPES buffer: 5mM;

DMEM / F12 medium: make up.

The above raw materials are all sterile.

2. Digestion method:

Wash the adipose tissue until no red blood cells are visible to the naked eye, cut it to about 30 cubic millimeters, and digest with the digestive juice, digestion for 20 min, 37 ° C, and shaking at 150 rpm.

Example 2

1. The digestion formula of this embodiment is:

Collagen type I: 0.25%;

HEPES buffer: 10mM;

DMEM / F12 medium: make up.

The above raw materials are all sterile.

2. Digestion method:

Wash the adipose tissue until no red blood cells are visible to the naked eye, cut it to about 30 cubic millimeters, and digest with the digestive juice, digestion for 20 min, 37 ° C, and shaking at 150 rpm.

Example 3

1. The digestion formula of this embodiment is:

Collagen type I: 0.25%;

HEPES buffer: 15mM;

DMEM / F12 medium: make up.

The above raw materials are all sterile.

2. Digestion method:

Wash the adipose tissue until no visible red blood cells are shredded to about 30 cubic millimeters. Digestion is performed using the above digestion solution. Digestion is performed for 20 minutes at 37 ° C with shaking at 150 rpm.

Example 4

1. The digestion formula of this embodiment is:

Collagen type I: 0.25%;

HEPES buffer: 20mM;

DMEM / F12 medium: make up.

The above raw materials are all sterile.

2. Digestion method:

Wash the adipose tissue until no red blood cells are visible to the naked eye, cut it to about 30 cubic millimeters, and digest with the above digestion solution, digest for 20 min, shake at 37 ° C, 150 rpm.

Example 5

1. The digestion formula of this embodiment is:

Collagen type I: 0.25%;

HEPES buffer: 25mM;

DMEM / F12 medium: make up.

The above raw materials are all sterile.

2. Digestion method:

Wash the adipose tissue until no red blood cells are visible to the naked eye, cut it to about 30 cubic millimeters, and digest with the above digestion solution, digest for 20 min, shake at 37 ° C, 150 rpm.

Comparative Example 1

Traditional digestion method: final concentration of 0.25% type I collagenase (PBS solution containing 0.25% type I collagenase) is digested for 40 min, shaking at 37 ° C, 150 rpm.

Comparative Example 2

Traditional digestion method: Final concentration of 0.25% type I collagenase (PBS solution containing 0.25% type I collagenase) is digested for 20 min, shaking at 37 ° C, 150 rpm.

Comparative Example 3

The digestive formula of this comparative example is:

Collagen type I: 0.25%;

DMEM / F12 medium: make up.

The above raw materials are all sterile.

2. Digestion method:

The adipose tissue was washed until no red blood cells were visible to the naked eye, and digestion was performed using the above digestion solution, digestion was performed at 37 ° C, and shaking at 150 rpm for 20 min.

Test example 1 digestion test

Collect adipose tissue (both authorized and consented by volunteers, aged 20-30 years old, no infectious diseases), after washing with normal saline, use a final concentration of 0.25% of the above Examples 1-5 and Comparative Examples 1-3 at 37 ° C, Digestion was performed with shaking at 150rpm for 20min or 40min, and the digestion was terminated by centrifugation, leaving only the bottom cells, and SVF was obtained after washing with physiological saline, and the SVF viability and quantity were detected. The test results are shown in Table 1:

Table 1 SVF activity rate and quantity

It can be seen that the formula of Examples 1-5 of the present invention is significantly better than the formula of Comparative Examples 1-3 in the amount of SVF obtained; the formula of Examples 1-5 is also significantly better than the formula of Comparative Examples 1-3 in terms of SVF activity.

Test example 2 SVF culture test

SVF obtained by digestion of each group in Test Example 1, each 5.0 × 10 ⁵ SVF was inoculated in a 10cm dish, 10mL DMEM / F12 + 10% FBS medium was added, and cultured in a 5% CO ₂ , 37 ° C incubator, every 2 Change the liquid every day (change the liquid on the 3rd and 5th days), observe the confluence of the cells every day, and record the change trend of the confluence. The test results are shown in Table 2:

Table 2 SVF convergence degree change trend

It can be seen that the proliferation ability of the SVF obtained from the formulations of Examples 1-5 of the present invention is significantly better than the SVF obtained from the formulations of Comparative Examples 1-3.

Test Example 3 Immune phenotype analysis of adipose stem cells after SVF culture

SVF third-generation cells digested from the digestive juices of Examples 1-5 and Comparative Examples 1-3 were taken, washed three times after digestion, and made into a cell suspension. Antibodies were added, and CD73, CD90, CD105, and CD11b, CD19, CD34, CD45, HLA-DR expression. The test results are shown in Table 3 and Figure 1:

Table 3 Expression of cell surface markers

The above test results indicate that the cells are adipose-derived mesenchymal stem cells.

Test Example 4 In vitro and In vivo Differential Detection of Adipocytes

SVF third-generation cells obtained by digestion with the digestion liquid of Examples 1-5 and Comparative Examples 1-3 were taken and seeded at a concentration of 2.5 × 10 ⁴ cells in a 24-well plate containing DMEM / F12 + 20% FBS medium to the cells. At 80% confluence, change to a culture solution containing 1 nmol / L dexamethasone, 10 mg / L insulin, 0.5 mmol / L 3-isobutyl-1-methylxanthine, 100 μmol / L indomethacin Change the solution twice, fix with paraformaldehyde 3 weeks later, and stain with oil red O. The adipogenic effect is shown in Figure 2. Visible stained lipid droplets prove that the cells have adipogenic differentiation ability.

The above is only a preferred embodiment of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the principle of the present invention, a number of improvements and retouches can be made. These improvements and retouches also It should be regarded as the protection scope of the present invention.

Claims (10)

1. An adipose tissue digestive fluid, characterized in that it consists of type I collagenase, HEPES buffer and DMEM / F12 medium.
2. The digestive juice according to claim 1, wherein the amount of each component in the digestive juice is:

   Type I collagenase: 0.1wt% ～ 0.3wt%;

   HEPES buffer solution: 1 ～ 30mM;

   DMEM / F12 medium: To complement.
3. The digestive juice according to claim 1, wherein the amount of each component in the digestive juice is:

   Type I collagenase: 0.25% by weight;

   HEPES buffer: 5 ～ 25mM;

   DMEM / F12 medium: To complement.
4. The digestive juice according to claim 1, wherein the amount of each component in the digestive juice is:

   Type I collagenase: 0.25% by weight;

   HEPES buffer: 10 ～ 25mM;

   DMEM / F12 medium: To complement.
5. The digestive juice according to any one of claims 1 to 4, wherein the amount of each component in the digestive juice is:

   Type I collagenase: 0.25% by weight;

   HEPES buffer: 20mM;

   DMEM / F12 medium: To complement.
6. A method for quickly obtaining stromal vascular component cells, comprising the following steps:

   Adipose tissue is digested with the digestive juice according to any one of claims 1 to 5 to obtain stromal vascular component cells.
7. The method according to claim 6, wherein the temperature of the digestion is 36-38 ° C.
8. The method according to claim 6, wherein the rotation speed of the digestion is 100-200 rpm.
9. The method according to claim 6, wherein the digestion time is 15-25 minutes.
10. The method according to any one of claims 6 to 9, characterized in that the digestion further comprises the steps of centrifugation to terminate the digestion and washing.

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