JPH06181740A - Culture vessel and its production - Google Patents

Abstract

PURPOSE:To obtain the subject vessel capable of easily and surely peeling a small piece after cultivation and fixing the piece on a slide glass, etc., and suitable for the small piece culture without causing the flotation of small piece during cultivation by forming a covering part acting as the small piece for culture on the bottom of a vessel in a specific manner. CONSTITUTION:A liquid silicone rubber (preferably rubber curable at room temperature) is poured into the main body 2 of a vessel consisting of a molding of a transparent plastic (preferably polystyrene, polyester, etc.) and cured to form a covering part 3. The covering part is preferably hydrophilicized by a physicochemical means such as corona discharge treatment to obtain the objective vessel having the transparent rubbery covering part on the bottom. The covering part can easily be peeled off to form a small piece for cultivation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture container used for tissue culture or cell culture on a small piece for culture.

[0002]

2. Description of the Related Art In the fields of tissue culture, cell culture, etc., microscopic specimens of cultured tissues and cells are manufactured,
After culturing, when you want to move the culture part to another system as it is and continue culturing, generally place a glass cover glass used when making a microscope preparation in the well of a Petri dish or plate. Then, the tissues, cells and the like are cultured on the cover glass in the medium. After culturing, take out the cover glass, fix it on a slide glass and use it as a sample for a microscope, or transfer multiple cover glasses in which tissues and cells have been cultured to one culture system and co-cultivate them to See the interaction of transmitters.

At this time, the cover glass used is very easily damaged and often breaks during handling. Therefore, recently, a small piece for culture in which the surface of a transparent plastic film is hydrophilized has been used. However, since the plastic film has a smaller specific gravity than the glass and the medium has a large specific gravity, there is a problem that the plastic film does not float or completely sink in the medium. Therefore, even if the cells are seeded, there are disadvantages that the cells do not adhere to the intended small piece for culture or adhere to both sides.

Further, when used for co-culture, even if a cover glass made of glass having a high specific gravity is used, the cover glass moves in the culture container when carrying it, and the directionality of the messenger between cells is checked. It becomes an obstacle when you want to.

Therefore, there is also a method of applying an adhesive or the like on one surface of a cover glass or a small piece of a plastic film for culture and fixing it in a container. However, when preparing a preparation as a specimen after the culture, the adhesive is removed. I need it. In addition, it has been attempted to incubate on a sticky sheet, but when trying to mass-produce such a sheet, the sheets stick to each other and cause a trouble in handling. Therefore, it is necessary to sandwich the sheet with release paper.

[0006]

DISCLOSURE OF THE INVENTION The present invention is intended to solve such a problem in culturing tissue or cells on a small piece, and its object is to prevent cracking.
It does not float in the medium and can be easily and reliably moved and fixed on another culture bed or slide glass after culturing, and is structurally simple and inexpensive, suitable for culturing on versatile small pieces. To provide a container.

[0007]

Means for Solving the Problems That is, the present invention is characterized in that a transparent rubber-like covering portion, which is a small piece for culture that can be easily peeled off, is provided on the bottom surface of the container body for culture. A container, which is a transparent plastic molded product, is filled with liquid silicone rubber and cured to form a coating, and the surface of the coating is made hydrophilic by physicochemical means. It is a method for manufacturing a culture container, which is characterized by adding the culture container.

The present invention will be described in detail below with reference to the drawings. FIG. 1 is a cross-sectional view of a culture container which is an embodiment of the present invention.

The shape of the target culture vessel is not particularly limited, but generally, as shown in FIG. 1, a petri dish (a) and a plurality of petri dishes (a) consisting of a vessel body (2) and a lid (1). Plates (b) with wells are used. In the present invention, the presence or absence of the lid does not matter. The material for these culture vessels is required to have transparency, no cytotoxicity, water resistance, etc.,
In addition, taking into account the releasability of the coating portion (3) described later, it is preferable that the surface of the formed article has a small polarity. Examples of such resin include polystyrene, polyester, polypropylene, polyethylene, polyvinyl chloride, TPX resin and the like.

As a material for forming the covering portion (3), it is transparent, has no cytotoxicity, has elasticity and flexibility like rubber, and has the same material as the above-mentioned surface of the container body. It is not particularly limited as long as it can be easily peeled off and has water resistance and the like, but since it may be treated with various solvents after culturing, it has solvent resistance. As the silicone rubber, it is preferable. As the silicone rubber used, a two-component liquid silicone rubber composed of a main agent and a curing agent is used. If possible, a type that cures at room temperature is preferable.

Next, the production method of the present invention by coating with silicone rubber will be described. First, a predetermined amount of the main agent and the curing agent are mixed well. Since air bubbles are involved during mixing, degassing is performed under reduced pressure after mixing. The liquid silicone rubber thus prepared is dispensed into the bottom surface of the container body (2) by a predetermined amount. Then, while keeping horizontal, leave at room temperature. The liquid silicone rubber spreads evenly on the bottom of the container and eventually hardens.

The cured coating (3) is of uniform thickness and
It is transparent and has a smooth surface. The thickness of the coating part (3) can be adjusted by the amount of liquid silicone rubber dispensed into the container, and is preferably in the range of 0.05 to 2 mm.
When the thickness is less than 0.05 mm, it is difficult to maintain the shape of the sheet-shaped covering portion (3) when peeled from the bottom surface of the container after culturing, and it becomes difficult to handle at the time of post-treatment. On the other hand, if it is thicker than 2 mm, the distance between the sample to be observed and the objective lens becomes large during microscopic observation, which makes it difficult to observe at high magnification, which is not preferable.

The silicone rubber coating portion (3) thus formed has a hydrophobic surface, and if it remains as it is, it is difficult for tissues and cells to adhere to the coating portion (3), so that the surface is made hydrophilic. As a method of hydrophilic treatment, corona discharge treatment,
It is preferable to use physicochemical means such as low temperature plasma treatment and ultraviolet irradiation treatment. Finally, do the packaging,
Sterilize. As a sterilization method, a method using ethylene oxide gas, gamma ray, electron beam or the like can be applied.

Further, in the primary culture, as in the case of a general culture vessel, fibronectin, on the coating portion (3), that is, the culture surface, depending on the type and purpose of the cells to be cultured,
By coating a cell adhesion factor such as vitronectin or laminin or an extracellular matrix derived from a living body such as collagen or gelatin, cell spreading and adhesion are further improved. In addition, in culturing cells of the nervous system, coating with synthetic substances with positive charges such as polylysine, polyornithine, polyethyleneimine, and aminated silane promotes cell extension and adhesion as well as neurite outgrowth. In addition, the survival rate of cells during long-term culture is also increased.

The extracellular matrix or the compound having a positive charge is coated with the extracellular matrix or a compound having a positive charge so as to cover the entire surface of the coating portion (3) hydrophilically treated as described above. , Water, methanol,
A solution dissolved in ethanol, a mixed solvent of water and methanol, or a mixed solvent of water and ethanol is filled. Then, while the extracellular matrix or the synthetic substance having a positive charge is adsorbed on the rubber-like coating portion, the solution is allowed to stand at room temperature for, for example, about 10 minutes, then the solution is discharged, washed with pure water, and air-dried or It is carried out by drying at a temperature of 40 ° C. or lower.

Next, a method of using the culture container according to the present invention will be described. The culture method may be carried out according to the culture in a general tissue culture petri dish or plate. At this time, the covering part (3) plays the same role as the small piece for culturing, and since the covering part (3) is in close contact with the bottom surface of the container body (2), the seeded cells and tissues are not covered by the surface of the covering part (3). It adheres only to the top and grows. Observation with an inverted microscope during culturing can be performed in the same manner as in a general tissue culture container, and there is no problem during observation such as floating or moving of a small piece when using a conventional small piece for culture.

After culturing, if the end of the covering portion (3) is pinched with tweezers and lifted, the covering portion (3) is easily peeled off. The peeled coating portion is immersed in a solution for dehydration, fixation, and staining, dried, fixed on a slide glass, and stored as a sample. Since the covering portion is a rubber-like flexible sheet, it can be easily adhered to the slide gas surface.

In addition, when examining the influence of secretions from cells on other cells, a plurality of target cells are separately cultured in the culture container of the present invention, and each coating portion (3) is peeled off. hand,
The cells are co-cultured by arranging them on the bottom surface of one culture container and closely contacting them. Also in this case, the covering part (3) firmly adheres to the bottom surface of the culture container, and there is no movement of the small pieces during handling as in the conventional culture on small pieces, and the small pieces do not come into contact with each other. In addition, the direction of secretion can be surely seen. It is also possible to simultaneously observe a plurality of types of cells.

[0019]

EFFECTS OF THE INVENTION According to the present invention, the covering portion provided on the bottom surface of the container body and serving as a small piece for culturing has no fear of floating or shifting during culturing, damage during handling, and the like.
After culturing, it can be easily removed with tweezers,
When preparing a slide by fixing it on a slide glass, or when transferring it to another culture container for culturing, it can be easily and reliably fixed without applying an adhesive or the like. It does not hinder, and is suitable as a culture container used for multipurpose small piece culture.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of a culture container that is an embodiment of the present invention.

[Explanation of symbols]

 1 lid 2 container body 3 cover

Claims (6)

[Claims] 1. A culture container, which is provided with a transparent rubber-like coating on the bottom surface of the container body, which is a small piece for culture that can be easily peeled off. 2. The culture container according to claim 1, wherein the coating portion is a silicone rubber coating having a thickness of 0.05 to 2 mm, and the surface thereof is subjected to hydrophilic treatment. 3. Collagen, gelatin, and
The culture container according to claim 1 or 2, which is coated with an extracellular matrix such as laminin, fibronectin, vitronectin, and the like. 4. The surface of the coated portion is coated with a compound having a positive charge, such as polylysine, polyortinine, polyethyleneimine, aminated silane, and the like.
The culture container according to claim 1 or 2. 5. A container body made of a transparent plastic molded product is filled with liquid silicone rubber and cured to form a coating portion, and the surface of the coating portion is rendered hydrophilic by physicochemical means. A method for producing a culture container, comprising: 6. A solution obtained by dissolving an extracellular matrix or a compound having a positive charge in water, methanol, ethanol, a mixed solvent of water and methanol, or a mixed solvent of water and ethanol is described in claim 5. A method for producing a culture container, which comprises filling the surface of the covering portion of the culture container and leaving it at room temperature, discharging the solution, washing with pure water, and air-drying or drying at a temperature of 40 ° C. or lower. .