KR20040053651A - Cell culture dish formed with a lattice - Google Patents

Abstract

PURPOSE: Cell culture dish formed with lattice is provided, thereby directly numbering cultured cells without separating the cells from the culture dish through a microscope. CONSTITUTION: The cell culture dish formed with lattice has a cell growth layer on the lattice. The method for preparing the cell culture dish formed with lattice comprises the steps of: patterning the lattice having a constant area on the metal membrane of a substrate using photolithography; and pressurizing the lattice patterned metal membrane on a substrate of the cell culture dish to copy the metal membrane lattice pattern to the substrate of the cell culture dish, wherein a compound may be coated on the lattice formed substrate of the cell culture dish to form a cell growth layer; the compound is polylysine, polyethylene, polypropylene, copolymer thereof or glass; and plasma surface modification under vacuum for proliferation of cells may be further induced.

Description

Cell culture dish formed with a lattice}

The present invention relates to a cell culture vessel having a lattice formed therein and a method for producing the same.

Conventional cell culture vessels consisted only of physicochemical treatments in which cells could grow. Therefore, in order to count the number of cells grown in a conventional cell culture vessel, the cells had to be separated from the culture vessel and transferred to a tool for cell counting. Cell counting was performed directly under a microscope using an automated device such as a Coulter counter or FACS or using a tool such as a hematocytometer. However, these cell counting methods are inconvenient because the cells must be separated from the cell culture vessel, and there is a problem that a separate cell counter must be prepared. In addition, due to the process of separating the cells from the culture vessel, an error could occur in the contamination by the external microorganisms and the number of cells measured.

It is an object of the present invention to provide a cell culture vessel capable of counting cells directly from the culture vessel without separating the cells from the cell culture vessel.

It is also an object of the present invention to provide a method of making said cell culture vessel capable of counting cells directly from the vessel.

1 is a diagram schematically showing an example of a process of directly forming a lattice in a cell culture vessel using photolithography.

Fig. 2 is a diagram schematically showing an example of a process of forming a lattice in a cell culture container using a metal lattice as a template.

Figure 3 is a photograph showing the cells cultured in the cell culture vessel of the present invention prepared according to the process shown in Figure 1, Figure 3a is SupT1 cells, Figure 3b is Hela cells.

Figure 4 is a photograph showing the cells cultured in the cell culture vessel of the present invention prepared according to the process shown in Figure 2, Figures 4a to 4c are photographs observed at 70, 160 and 320 magnification, respectively.

The present invention provides a cell culture container including a grid pattern having a predetermined area on a substrate.

The substrate is a base material of the lattice pattern and forms the basis of the lattice pattern. The substrate may be, for example, polyethylene, polypropylene, copolymers or glass thereof, but is not limited thereto. Preferably, the substrate is transparent. The lattice has a uniform area and has various shapes, for example radial or circular or checkered shapes. The lattice may be manufactured in the form of a pattern formed by a pattern forming process used in a conventional semiconductor manufacturing process, for example, photolithography and thermal relief. The lattice is used to count cells present in the lattice under a microscope, and multiply the obtained cell number by the ratio of the area of the unit lattice to the culture vessel to finally count the total number of cells in the culture vessel. The area of the unit grid is not particularly limited as long as it has a constant area, but is preferably 1 mm ² which is commonly used. In addition, the substrate may be surface-treated to help the adhesion or growth of cells. For example, it may be surface treated by gas plasma treatment in a vacuum phase.

The cell culture vessel of the present invention may further include a growth layer formed on the grid pattern. The growth layer is a layer that supports growth by allowing cells to grow by sticking or floating. The growth layer is in the form of a very thin film made of a material that is not toxic to cells and can support cell growth. The growth layer is, for example, polylysine. In addition, the growth layer includes a material capable of supporting the growth of cells growing in suspension, as well as a material that cells can adhere to and grow. Preferably, the growth layer is transparent.

The present invention provides a method for producing the cell culture container comprising the step of patterning a grid having a predetermined area using photolithography on the culture vessel substrate.

The method of the present invention may further include forming a growth layer by applying a compound capable of growing cells on the culture vessel substrate on which the lattice is patterned.

The culture vessel substrate may support a lattice pattern or growth layer formed thereon, and the patterning may have an embossed or engraved form. In addition, the grating includes one formed by a photoresist material or a material constituting the substrate. The compound in which the cells can grow refers to a compound which is not toxic and can grow by attaching or floating cells. In addition, the substrate may be surface-treated to help the adhesion or growth of cells. Moreover, the substrate after the said patterning can also be surface-treated. The surface treatment may be, for example, surface treatment by gas plasma treatment in a vacuum.

The present invention also comprises the steps of applying a photoresist on the culture vessel substrate;

Applying a mask on which a grid having a predetermined area is drawn on the culture vessel substrate to which the photoresist is applied; And

Exposing and developing to form a photoresist pattern of the grating.

The method may also be a method comprising forming a growth layer by applying a compound capable of growing cells on the photoresist pattern of the lattice.

Here, the culture vessel substrate is polyethylene, polypropylene, copolymers or glass thereof, preferably in a transparent form. In addition, the compound in which the cells can grow is preferably a polylysine-based material having a positive charge, but is not limited thereto, and materials commonly used in the art may be used. In addition, the patterned substrate may be surface treated to aid in cell adhesion or growth. The surface treatment may be, for example, surface treatment by gas plasma treatment in a vacuum phase.

In the present invention, the photolithography means a method of a pattern forming process used in a conventional semiconductor manufacturing process. Photolithography typically involves cleaning the substrate, applying photoresist, attaching a mask, developing and / or etching. In addition, the photoresist refers to a photosensitive material.

An example of a process of forming a lattice on a culture vessel substrate using the above photolithography will be described with reference to FIG. 1.

First, the photoresist 3 is coated on the culture vessel substrate 2. The photoresist-coated culture vessel substrate is covered with a mask with a lattice, and exposed. When the exposed portion is developed after exposure, the lattice pattern 4 is formed in the photoresist. Here, by forming a growth layer 6 by coating a compound in which cells can grow, a culture vessel in which the lattice of the present invention is drawn is prepared.

The present invention also includes the steps of: patterning a grating having a constant area using photolithography on a metal film on a substrate; And transferring the metal membrane lattice pattern by pressing the lattice patterned metal membrane onto the culture vessel substrate to transfer the metal membrane lattice pattern.

The method may further include forming a growth layer by applying a compound capable of growing cells on the culture vessel substrate on which the lattice pattern is transferred.

The method includes depositing a metal film on a substrate;

Applying a photoresist on the substrate on which the metal film is deposited;

Covering, exposing and developing a mask in which a grid having a predetermined area is drawn on the photoresist-coated substrate;

Etching the metal film according to the lattice pattern;

Transferring the lattice pattern to the culture vessel substrate using the template for the mold having the obtained metal film lattice pattern as a template; And

It may be a method comprising the step of applying a compound that can grow cells on the culture vessel substrate having the grid pattern. In addition, the surface treatment may be such that the cells can be attached to the culture vessel substrate having the grid pattern. The surface treatment may be, for example, treating a gaseous plasma in a vacuum.

Here, the mold substrate may be glass, silicon, or metal, but is not limited thereto. In addition, the culture vessel substrate is preferably polyethylene, polypropylene, copolymers or glass thereof. The metal is not particularly limited but is preferably copper, platinum, steel, gold or aluminum. In addition, the compound capable of growing the cell refers to a compound that is not toxic and can grow by attaching or floating the cell. Such compounds include, for example, polylysine. It is preferable that the culture vessel substrate is transparent so that the cells can be directly counted under a microscope.

An example of a process of forming the lattice on the cell culture vessel substrate using the metal lattice as a template will be described below with reference to FIG. 2.

First, the metal 10 is deposited on the metal lattice substrate 8. A mask on which a lattice is drawn is placed on the substrate 8 on which metal is deposited, and exposed after development. After the development is completed, etching is performed to form the metal lattice 12. The obtained metal lattice 12 and the culture vessel substrate 2 are heated to a constant temperature, and the metal grid 12 is pressed onto the culture vessel substrate 2 to transfer the pattern of the grid to the culture vessel substrate 2. The metal lattice 12 is removed, and a growth vessel 6 is formed by coating a compound capable of growing cells to prepare a culture vessel in which the lattice of the present invention is drawn.

On the other hand, the process of forming the growth layer described in the present invention or the surface treatment of the substrate with a plasma may be performed even before the pattern of the metal grid is formed on the substrate.

Hereinafter, the manufacturing process of the cell culture container of the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

Example 1: Preparation of the culture vessel by the process of forming the grating directly on the culture vessel substrate through the photolithography method

First, the surface of the cell culture vessel (polystyrene, Falcon Co., Ltd.) in which cells are grown was washed with deionized water and dried with nitrogen. The photosensitive material was spin-coated with a promoter (HMDS; hexamethyldisilazane) at 500 rpm, and then the photosensitive material (AZ5314) (manufactured by Clariant Industries Ltd. Korea) was spin-coated at 1.3 rpm with a thickness of 5,000 rpm.

A mask engraved with an array of grids (50 μm × 50 μm) was coated on the culture vessel coated with the photosensitive material, and exposed to UV (310-405 nm), followed by developer (AZ300MIF) (product of Clariant Industries Ltd. Korea). ) Was immersed for 1 minute to etch portions of the photosensitive material exposed to UV.

In order to allow cells to grow on the culture vessel in which the lattice pattern is formed as described above, a poly-L-lysine (Sigma) solution is treated at room temperature for 10 minutes, and PBS (phosphate- buffered saline).

Using the cell culture vessel formed with the lattice formed as described above, SupT1, which is a kind of human T cells, or Hela cells, which are human cervical cancer cells, were cultured and subjected to cell counting. As a result, it was found that the cells could be counted directly under a microscope on the cultured cell.

3A and 3B show the growth of cells on the lattice of the obtained culture vessel. FIG. 3A shows the growth of SupT1 cells (250 magnification), and FIG. 3B shows the growth of Hela cells (270 magnification).

Example 2: Fabrication of a culture vessel having a lattice by making a metal membrane lattice using photolithography and forming a lattice on a cell culture vessel substrate using the template as a template

A metal of aluminum or gold was deposited to a thickness of 1 μm on a glass or silicon substrate. A promoter (HMDS; hexamethyldisilazane) was spin coated at 5,000 rpm to bond the photosensitive material, and then the photosensitive material (AZ5314) (manufactured by Clariant Industries Ltd. Korea) was spin-coated at 1.3 μm at 5,000 rpm.

A mask engraved with a lattice (50 μm × 50 μm) was coated on the culture vessel coated with the photosensitive material, exposed to UV (310-405 nm), and then placed in a developer (AZ300MIF) (manufactured by Clariant Industries Ltd. Korea). A portion of the photosensitive material exposed to UV was etched by immersion for 1 minute.

Next, the substrate on which the lattice pattern was formed was immersed in a metal visual solution to form a metal lattice, and then the remaining photosensitive material was immersed in acetone and methanol for 1 minute and removed.

The substrate for the mold having the metal lattice prepared as described above was used as a mold for forming the lattice in the culture vessel. The substrate having the metal lattice was heated to 120 ° C., and then pressed on the surface of the culture vessel in which the cells grew to form a lattice.

Using the cell culture vessel formed with the lattice formed as described above, SupT1, which is a kind of human T cells, or Hela cells, which are human cervical cancer cells, were cultured and subjected to cell counting. As a result, it was found that the cells could be counted directly under a microscope on the cultured cell.

4A to 4C show the growth of cells on the lattice of the obtained culture vessel. Figures 4a to 4c are Hela cells grow, it is a photograph observed at 70, 160 and 320 magnification, respectively.

According to the cell culture vessel having a lattice of the present invention, cells can be directly counted through a microscope from the vessel in which the cells are cultured.

According to the manufacturing method of the cell culture container of this invention, the cell culture container which has a lattice can be manufactured.

Claims (11)

Cell culture container comprising a grid pattern having a predetermined area on the substrate. The cell culture container of claim 1, further comprising a growth layer formed on the grid pattern. A method of manufacturing the cell culture container of claim 1, comprising patterning a lattice having a predetermined area by photolithography on a culture vessel substrate. The method of claim 3, further comprising applying a compound capable of growing cells to form a growth layer on the lattice-patterned culture vessel substrate. The method of claim 4 wherein the compound is polylysine, polyethylene, polypropylene, copolymers or glass thereof. 4. The method of claim 3, further comprising inducing surface variation using a gaseous plasma in a vacuum so that the cells can grow on the culture vessel substrate on which the lattice is patterned. Patterning a grating having a constant area using photolithography on a metal film on the substrate; And The method of manufacturing a cell culture vessel of claim 1, comprising the step of transferring the metal membrane lattice pattern by pressing the lattice patterned metal membrane to the culture vessel substrate. The method of claim 7, further comprising applying a compound capable of growing cells to form a growth layer on the culture vessel substrate on which the lattice pattern is transferred. The method of claim 8, wherein the compound is polylysine, polyethylene, polypropylene, copolymers or glass thereof. The method of claim 7, further comprising the step of causing surface variation by using a gaseous plasma in a vacuum so that cells can grow on the culture vessel substrate before transferring the metal membrane lattice pattern or the culture vessel substrate on which the lattice pattern is transferred. How to include more. 8. The method of claim 7, wherein the metal is copper, platinum, gold, steel or aluminum.