JPH04142441A - Cell separating method - Google Patents

Abstract

PURPOSE:To separate the cell population isolated by one action by overlapping sieves in many stages in the order from a smaller hole diameter to a larger hole diameter, and pouring a cell suspension from above for filtering. CONSTITUTION:Multiple heat-resistant containers 1 opened at the bottom are prepared, and nylon meshes 2 with different hole diameters are stuck and fixed on the open sections respectively with an adhesive to obtain sterilizable sieves 3. They are stacked while the sieve 3 with a smaller mesh is located below and the sieve 3 with a larger mesh is located above. A suspension containing scattered various cells 4 is poured from above, and the cells are separated in the sieves 3 corresponding to the size of the cells. After separation, individual sieves 3 are turned over, a culture solution 5 is poured, and the cells 4 are separated in containers 6. In this method, cell lumps not sufficiently separated are stuck to the upper sieve 3, these cell lumps are isolated via the enzyme processing or vibration processing again, and the solution 5 may be again poured into the containers 1 for separation.

Description

[Detailed Description of the Invention] [Summary] Regarding a method for sorting cultured biological cells, for the purpose of sorting in a simple method, a heat-resistant container with an open bottom is placed with a hole of a diameter of Multiple types of sieves that can be sterilized are made by gluing different nylon meshes together, and the sieves are stacked in order of pore size from small to large, and the cell suspension is poured from the top and filtered. This constitutes a cell sorting method characterized by capturing and separating cells of different sizes using meshes with corresponding pore diameters.

[Industrial application field]

The present invention relates to a method for sorting biological cells.

With the development of biotechnology, it is common practice in the fields of medicine and biology to remove a part of a living body, place it in a sterile container, culture it for a long period of time, and conduct research on the cells.

There are two types of cell culture methods: primary culture, in which cells are taken directly from a living body, and subculture, in which cell lines are transplanted. Cells are separated by enzymatic treatment or mechanical treatment.

However, the isolate thus obtained consists of miscellaneous cells.

For example, when the cerebellum of a chicken is taken out and treated with enzymes, in addition to various nerve cells, dahlia cells and fibroblasts are isolated.

Therefore, it is necessary to sort out the necessary cells from the separated cell population.

[Conventional technology]

As a device for separating arbitrary cells from a group of cells that have been separated separately, the device marks the cells with a dye or fluorescent antibody, then irradiates them with laser light, and separates the cells using scattered light and fluorescence as a guide. Flow cytometry is known.

However, this method requires large-scale and expensive equipment, and is not easy to operate.

On the other hand, the inventors have proposed a method in which nerve cells and fibroblasts are separated and co-cultured by utilizing the difference in adhesion of cells to the surface of a petri dish.

However, since this method cannot be extended to the separation of all cells, it was necessary to develop a method that could easily separate cells.

[Problem to be solved by the invention] Living cells are isolated by enzymatic treatment or mechanical treatment such as shaking to create a cell population, and necessary cells are separated from this miscellaneous population. It is necessary in medical and biological research.

For this reason, there are various sorting methods, but they are expensive and have specific characteristics in cell sorting, so they cannot be universally applied.

Therefore, the challenge is to develop a simple method.

[Means to solve the problem]

The above problem was solved by creating multiple types of sieves that can be sterilized by gluing nylon meshes with different pore sizes to the open part of a heat-resistant container with an open bottom. This problem was solved by configuring a cell sorting method in which cells of different sizes are captured and separated by meshes with corresponding pore diameters by pouring the cell suspension from the top of the stack and filtering it. can do.

[Effect]

The various cells that make up a living body have various shapes in the living body, but cells separated by enzymatic treatment or mechanical treatment such as shaking have a spherical shape with a diameter of several 10 μm.

The inventor focused on this point, and by assembling nylon meshes with different hole sizes in multiple stages, cells can be sorted from each mesh in a single operation from a miscellaneous group of cells with different sizes. be.

FIG. 1 is a sectional view showing a cell sorter according to the present invention and its usage.

That is, a plurality of heat-resistant containers 1 with open bottoms are prepared, and nylon meshes 2 having different hole diameters are adhesively fixed to the open portions using an adhesive to form sterilizable sieves 3.

Then, the cell sorter according to the present invention is constructed by placing the small mesh sieve 3 on the bottom and stacking the large mesh sieve 3 one after another.

Then, by pouring the suspension containing the various types of cells 4 separated from the top, it is sorted into a sieve 3 appropriate for the size of the cells.

After the sorting is completed, as shown in Figure 2, each sieve 3 is turned upside down and the culture solution 5 is poured into the cells.
is dispensed into a container 6.

When this method is used, cell clusters that have not been sufficiently isolated will also adhere to the upper sieve, but such cell clusters will be isolated again by enzymatic treatment or shaking treatment, and then separated again. Just pour it into a container and separate it.

Note that it has been conventional practice to filter a homogeneous population of cells by layering several sheets of lens paper or by using a nylon mesh with a fixed pore size.

However, these methods mainly remove undigested tissue that has not been separated by enzymatic treatment or mechanical treatment such as shaking, and instead actively classify cells by size. No fractionation was performed.

〔Example〕

The hole diameter is 20.40.62.82.94.1 at the bottom of the heat-resistant glass with an open bottom and a diameter of 3 On + m.
Six types of sieves were made by adhering cell separation filters made of 48 μm nylon mesh (manufactured by Kyoshin Riko Co., Ltd.) using an epoxy adhesive.

Then, it was placed in an autoclave and sterilized at 121°C for 20 minutes.

Next, the dorsal root ganglion of a chicken embryo 14 days after fertilization was used as a living organism.

Specifically, dorsal root ganglia were aseptically removed from chicken embryos, collagenase enzyme (1 mg/ml) was added, and the mixture was incubated at 37°C for 30
After treatment for minutes, a cell population containing neurons was released.

After removing the collagenase solution from this cell population using a centrifuge, it was resuspended in Eagle's minimal medium, which was then poured into a cell separator as shown in Figure 1 and filtered.

Next, each stage of the sieve was turned upside down and culture solution 5 (Eagle's minimal medium) was poured into it in the manner shown in FIG. 2, and the cells captured on the filter were separated.

Then, the morphology of the fractionated cell fluid was observed using an optical microscope.

Note that cell clusters and extracellular tissue debris that were not sufficiently isolated could be separated from meshes with pore sizes of 62, 82, 94, and 148 μm.

This cell mass was further subjected to enzyme treatment to yield isolated cells.

Note that the second and subsequent enzyme treatments can be performed on the nylon mesh in which the cell mass has been captured, and by immersing only the nylon mesh portion in the enzyme solution, the expensive enzyme solution can be saved.

Further, even when the second and subsequent enzyme treatments were performed using other enzymes such as trypsin and Disper 7, it was possible to easily perform the enzyme treatment by simply immersing the nylon mesh in the enzyme solution.

In this case, the diameter of the isolated cells is 40 μm and 20 μm.
Captured on the Metush.

Next, cells obtained from 40 μm and 20 μm meshes were
0% bovine serum, lμg/100 rn! The cells were resuspended in Eagle's minimal medium containing nerve growth factor and cultured for 24 hours. Taking advantage of the unique morphology of nerve cells and fibroblasts, we counted the number of cells. It was found that nerve cells were captured.

〔Effect of the invention〕

By carrying out the present invention, a cell population that has been isolated can be sorted by size in a single operation.

In addition, cells that are insufficiently isolated can be fractionated again by using the method of the present invention after enzymatic treatment or mechanical treatment.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view showing a cell sorter and its usage, and FIG. 2 is a conceptual diagram of a cell collection method. In the figure, l is a heat-resistant container, 2 is a nylon mesh, and 3 is a heat-resistant container.
is a sieve, 4 is a cell, and 6 is a container.

Claims (1)

[Claims] Multiple types of sieves that can be sterilized were made by gluing nylon meshes with different pore sizes to the open part of a heat-resistant container with an open bottom, and the sieves were stacked in multiple stages in order of pore size from small to large. A method for sorting cells, characterized in that cells of different sizes are captured and separated by a mesh of corresponding pore size by pouring a cell suspension from the top and filtering the cell suspension.