Method for extracting cells by enzymolysis of vitreous body

Abstract

The invention belongs to the technical field of cell extraction, and particularly relates to a method for extracting cells by enzymolysis of vitreous bodies, which comprises the following steps: centrifuging a vitreous body sample at room temperature, discarding supernate, and adding digestive enzyme into precipitate; after adding digestive enzyme, digesting in a water bath for 30 minutes; and after the enzymolysis is finished, centrifuging the enzymatic hydrolysate at room temperature, discarding the supernate, and collecting the precipitate, namely the enzymolysis vitreous cells. According to the technical scheme, a simple and efficient vitreous body enzymolysis method is provided, and a large number of cells can be provided for follow-up research.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell extraction, and particularly relates to a method for enzymatically digesting vitreous to extract cells. Background Art

[0002] The vitreous body is a loose connective tissue located between the lens and the retina. Its structure is gel-like and mainly composed of water, collagen fibers, hyaluronic acid, and a small amount of cells. As an important refractive medium in the eye, the vitreous tissue plays an important role in regulating eye development, transporting nutrients, maintaining the eye structure, and stabilizing the blood-eye barrier. Abnormalities in the vitreous body can lead to the occurrence of various eye diseases, such as persistent hyperplastic primary vitreous hyperplasia, retinal detachment, etc. Although the cell content in normal vitreous is scarce, in different types of retinal diseases, the vitreous body contains a large number of infiltrating and proliferating cells, resulting in vitreous opacity and severely affecting vision.

[0003] Studies have found that the cell components in the vitreous body are closely related to the occurrence and development of vitreoretinal diseases. For example, when intraocular inflammation such as uveitis occurs, a large number of immune cells infiltrate into the vitreous body. These cells secrete cytokines related to inflammation and are finely regulated by cytokines. In addition, vitreous hyalocytes play an important role in regulating the proliferation and migration of retinal vascular endothelial cells, participate in the complex interaction process between cells under pathological conditions, and are closely related to the pathogenesis of many eye diseases. Therefore, cells derived from human vitreous tissue have become an important tool for exploring human eye diseases. Obtaining the cell population components in the vitreous body for analysis and detection is crucial for scientific research and clinical diagnosis and treatment.

[0004] Few studies on the cell components in human vitreous body can only record the existence of cells in the vitreous body and conduct morphological studies on them. There are still many challenges in obtaining living cells in the vitreous body for cytological analysis. On the one hand, the collagen fibers and hyaluronic acid in the vitreous body are interconnected to form a three-dimensional network structure, which can absorb water to form a gel-like structure, greatly enhancing the viscoelasticity of the vitreous tissue and providing structural support for the eyeball. This dense and intertwined gel-like fiber network causes great interference to the acquisition of cells in the vitreous body. On the other hand, the main component of the vitreous body is water, accounting for about 98% of the total volume of the vitreous body. As an important refractive medium in the eye, in order to maintain its good optical transparency, the number of cells in it is limited, and the number of cells in the vitreous body is extremely small, which also poses new challenges to the acquisition of cell components in the vitreous body. Summary of the Invention

[0005] The object of the present invention is to provide a method for extracting cells by enzymatically digesting vitreous humor, which is a new method capable of effectively collecting viable cell components in human vitreous humor, and is helpful for deepening the understanding of the role of vitreous cell components in the occurrence and development of eye diseases. Further research on vitreous cells may also provide new ideas for the development of vitreous substitutes after vitrectomy.

[0006] To achieve the above object, the present application is realized through the following technical solutions:

[0007] A method for extracting cells by enzymatically digesting vitreous humor, comprising the following steps:

[0008] S1. Centrifuge the vitreous sample at room temperature and discard the supernatant. Add a complex enzyme to the precipitate.

[0009] S2. After adding the complex enzyme, digest in a water bath for 30 minutes.

[0010] S3. After the enzymatic digestion is completed, centrifuge the enzymatic digestion solution at room temperature, discard the supernatant, and collect the precipitate, which is the enzymatically digested vitreous cells.

[0011] Further, the vitreous sample in step S1 is the vitreous humor separated after vitrectomy.

[0012] Further, in step S1, the room temperature centrifugation is carried out at 1000×g for 5 minutes at room temperature.

[0013] Further, in step S1, before adding the complex enzyme, add phosphate buffer solution to the precipitate.

[0014] Further, the complex enzyme is hyaluronidase, heparinase, collagenase and DNase.

[0015] Further, the addition order of the complex enzyme is to sequentially add hyaluronidase, heparinase, collagenase and DNase to the precipitate after adding phosphate buffer solution, or a mixed solution of hyaluronidase, heparinase, collagenase and DNase in a set ratio.

[0016] Further, the added hyaluronidase, heparinase, collagenase and DNase are each diluted at 1:1000.

[0017] Further, in step S2, digest in a 37°C water bath for 30 minutes and mix once every 10 minutes.

[0018] Further, in step S3, the room temperature centrifugation is carried out at 1000×g for 5 minutes at room temperature.

[0019] The beneficial effects of the present invention are:

[0020] This technical solution provides a simple and efficient method for vitreous enzymolysis, which can provide a large number of cells for subsequent research. Description of the Drawings

[0021] Figure 1 It is a sample after vitrectomy.

[0022] Figure 2 It is a schematic diagram of enzymolysis of the vitreous sample of the present invention. Among them, A is the digestive enzyme group, and B is the control group without digestive enzyme.

[0023] Figure 3 It is a diagram of the cell precipitation after enzymolysis. Among them, there is more cell precipitation in tube A and less cell precipitation in tube B.

[0024] Figure 4 It is a comparison photo of cell viability. Among them, the left photo is the photo of cell viability after enzymolysis, and the right photo is the photo of cell viability without enzymolysis.

[0025] Figure 5 It is a flow cytometry detection diagram of vitreous cells after enzymolysis. A large number of immune cells, such as T cells and macrophages, can be detected after enzymolysis, and immune cells do not exist in normal vitreous. Detailed Embodiments

[0026] The technical solution of the present invention will be described in detail below with reference to the drawings. The following embodiments are only exemplary and can only be used to explain and illustrate the technical solution of the present invention, rather than being construed as a limitation to the technical solution of the present invention.

[0027] Materials involved in this application:

[0028] Vitreous separated after vitrectomy, hyaluronidase (1 mg / mL), heparinase (1 mg / mL), type I collagenase (10 mg / mL), type I DNase (1 mg / mL).

[0029] This application provides a method for extracting cells by enzymolyzing vitreous, which adopts the following steps:

[0030] S1. Transfer the sample after vitrectomy soaked in the lavage fluid to a 50 mL centrifuge tube, centrifuge at 1000×g for 5 minutes at room temperature, discard the supernatant, add 1 mL of phosphate buffer solution (PBS), then sequentially add hyaluronidase (diluted 1:1000), heparinase (diluted 1:1000), collagenase (diluted 1:1000) and DNase (diluted 1:1000). The addition amount of each enzyme is determined according to the sample. In this embodiment, the addition amount of each enzyme is 1 mL. Specifically, whether the addition amount of each enzyme is more or less can achieve the technical solution of this application. The difference is only in the number of cells, and excessive use of the above various enzymes for enzymatic digestion does not result in a reduction in the number of cells. Therefore, in the technical solution of this application, the addition amount of various enzymes is not restricted.

[0031] S2. Digest in a 37-degree water bath for 30 minutes, and mix well every ten minutes.

[0032] S3. After 30 minutes of enzymatic digestion, centrifuge at 1000×g for 5 minutes at room temperature, discard the supernatant, and collect the precipitate, which is the enzymatically digested vitreous cells.

[0033] Perform cell resuspension, staining and microscopic imaging detection on the above enzymatically digested vitreous cells:

[0034] 1. Resuspend with an appropriate amount of PBS according to the size of the cell mass, and take 10 μL for staining.

[0035] 2. Prepare a live cell staining solution:

[0036] 1 mL of PI solution (10 μM), add 1 μL of Calcein AM (1:1000 stock solution).

[0037] 3. Add 100 μL of live cell staining to the sample, incubate at 37 °C for 20 - 30 minutes, centrifuge at 1000×g for 5 minutes at room temperature, discard the supernatant, and wash once with PBS.

[0038] 4. Take pictures using a live cell workstation to obtain the ratio of live cells (green) to dead cells (red).

[0039] Flow cytometry detection:

[0040] 1. Take an appropriate amount of resuspended cells, add anti-CD16 / CD32 antibody, and block at room temperature for 20 minutes.

[0041] 2. Then add various fluorescently labeled flow antibodies, and incubate at room temperature in the dark for 20 minutes.

[0042] 3. Wash the cells with PBS to remove the excess antibody.

[0043] 4. Perform flow cytometry detection.

[0044] A large number of free cells are obtained after enzymatic digestion. Vitreous enzymatic digestion is as follows Figure 1 and Figure 2 shown. After enzymatic digestion, a large number of cells are obtained.

[0045] The cell viability is not lost after enzymatic digestion. As shown in Figure 4 , in the live / dead cell staining, the vitreous enzymatic digestion operation does not significantly change the cell viability. On the contrary, the cell number increases greatly.

[0046] Normal operation of flow cytometry after enzymatic digestion:

[0047] Flow cytological examination is performed on the cells after enzymatic digestion. The enzymatic digestion does not cause damage to the cell viability and surface markers. As shown in Figure 5 .

[0048] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting cells by enzymatic hydrolysis of vitreous body, characterized in that: Use the following steps: S1. Centrifuge the vitreous sample at room temperature, discard the supernatant, and add digestive enzymes to the precipitate; S2, after adding digestive enzymes, digest in a water bath for 30 minutes; S3. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis solution is centrifuged at room temperature, the supernatant is discarded, and the precipitate is collected as the enzymatic hydrolyzed vitreous cells.

2. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 1, characterized in that: The vitreous body sample in step S1 is the vitreous body separated after vitrectomy.

3. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 1, characterized in that: In step S1, room temperature centrifugation is performed at 1000×g for 5 minutes.

4. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 1, characterized in that: In step S1, phosphate buffer is added to the precipitate before adding digestive enzymes.

5. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 1, characterized in that: The digestive enzymes are hyaluronidase, heparinase, collagenase and DNA enzyme.

6. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 5, characterized in that: The order of adding the digestive enzymes is to add hyaluronidase, heparinase, collagenase and DNA enzyme to the precipitate after adding phosphate buffer, or to add a mixture of hyaluronidase, heparinase, collagenase and DNA enzyme in a set ratio.

7. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 6, characterized in that: The added hyaluronidase, heparinase, collagenase and DNase were diluted 1:1000 each.

8. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 1, characterized in that: In step S2, digest in a 37°C water bath for 30 min, mixing every 10 min.

9. The method for extracting cells by enzymatic hydrolysis of vitreous body according to claim 1, characterized in that: In step S3, room temperature centrifugation is performed at 1000×g for 5 minutes.

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