SU821484A1 - Method of making chamber for cell culturing - Google Patents

Description

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The invention relates to microbiological and hematological types of research related to the cultivation of cells in vitro and in. vivo and can be studied in microbiological, hematological, synologic and other studies in creating chambers for the patterns of growth and development of cells (micro, organisms, tissue cells) and determining the influence of various factors on them.

The closest to the invention according to the technical essence is the method of making a cell culture chamber, which consists in forming walls and a bottom with formation of a cavity for cell suspension, is covered with a lid and sealed.

The walls of the chamber according to a known method are made of plastic rings, and the cover is made of nillipore filters by placing a notch on the filter, sterilizing and then cutting circles. The resulting circles were carefully glued to the rings of glueMg by the path of dissolving the filters in acetone so that their matte side was facing the cells (it is considered less toxic). Then the camera in disassembled form is placed in Petri dishes and sterilized under the rays of a bactericidal lamp. Subsequently, filter rings are placed on sterile gauze cloths cleaned with medium 199, B Rings Wear medium 199 to check the quality of the filter and the tightness of the chamber. Under sterile conditions

0 filters in the rings are placed on the test cell suspension, waiting for the culture to pass through the filters and only the cells will remain on them. Then impose rings

5 at each other (smaller to larger), press down from above with a spatula for correlating-en and seal, glue the gap between them with glue. The chambers thus obtained are implanted into animal body I of animal 1.

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The disadvantage of this method is the complexity and laboriousness of manufacturing a large number of chambers, as well as the fact that the obtained measures, although they allow to grow iin vivo and in. vitro cells, however, they are made from: optically opaque materials, which does not allow Observe one and the same individual cells in

the dynamics of their growth and development directly in the chamber; in order to study the cells under study, the chamber in which they were grown must be dismantled, the cells removed and transferred onto a glass slide, which violates the initial interposition of the cells, their shape and size. Some cells may be destroyed. The camera does not allow direct observation of the growth, reproduction and differentiation of the studied cells under the control of a microscope in the state of their development. The chamber has a rigid structure, which adversely affects the body of the animal during implantation. The technique does not allow for the simultaneous production of the required kayer, since each of them is prepared separately.

The purpose of the invention is to simplify, reduce the cost of the method and the possibility of studying the cells in the chamber at different stages of their development.

To achieve this goal, the bottom walls and the cover are made of polyacrylamide gel, while the walls and the bottom form, pouring the working solution into the space between two parallel plates, the top of which has protrusions to form cavities in the bottom plate, the mixture is polymerized to form a gel and remove the top plate, and the chamber is sealed by overlaying the top sheet material with a gap between it and the lid, pour the working solution and polymerize to form a gel, remove the sheet material and punched chamber. Figure 1 shows a cell culture chamber, a general view; in fig. strip formation to place C1 cells; in fig. 3 - preparation of a flat gel plate; figure 4 - the introduction into the cavity of the cell suspension and the imposition of caps; Fig. 5 illustrates the sealing of chambers by fixing caps in a gel mass; in fig. 6 - camera cut-out.

The chamber is prepared as follows.

Take four plexiglass plates 1-4. On the surface of the plate 1 in a staggered order at a distance of about 15-2 mm, glue flat circles 5 with a diameter of 6 mm and a thickness of 1–2 mm (Fig. 2a) that are pre-cut out of plastic or medical rubber and glued with waterproof glue. Plates are sterilized by boiling.

Plate 1 is placed on rectangular Plexiglas coasters b, 2–3 mm thick, located on the sides of plate 2 in such a way that the plane with glued circles 5 of plate 1 faces plate 2 (Fig. 2a, b). Plate 3

impose on the rectangular plexiglass supports 7 with a thickness of 1 2 mm located on the sides of the plate 4 (fig.Za, b). The working solutions filtered through bacterial filters to obtain a polyacrylamide gel are mixed (sterile) and poured into the space between plates 1 and 2 (Fig. 26) and 3 and 4 (Fig. 36). In this case, capillary forces hold the liquid between the plates, the mixture of solutions is polymerized after 1520 minutes (depending on the polymerization mode). After this plate

1 and 2 are removed from the stands. On the plate 2 there is a gel plate with cavities 8 (fig. 2b) -, and on the plate 4 a flat gel plate (Fig. 3b).

Circles are cut out of the flat gel plate with drill bits for the plugs, which function as caps 9 that are 2–3 mm in diameter larger than the cavities 8. The cells in the cavity 8 are inserted (Fig. 4a). On top of the cavity 8 is covered with lids 9.

In the study in the chamber of non-attached kidich cells (for example, motile microorganisms), the latter are pressed to the bottom of the well with a gel circle, the diameter of which corresponds to the diameter of the well. In this case, the camera will be without an internal cavity.

The sealing of the chamber is carried out as follows.

On the sides of the plexiglass plate

2 install supports 10 of such dimensions that, after positioning the sheet material or plate 3 on them, a gap of 1-2 mm is formed between

with this material (plate) and lid 9. A mixture of working solutions is poured into the resulting space, which, polymerizing, connects the plates together (Fig. 56). The sheet material (Plexiglas plate 3) is removed from the supports 10. The chambers are cut out for the plugs with a diameter of 1215 mm.

The manufactured chambers are washed in Hank's NaCl saline solution, medium 199, depending on which cells are to be studied. Subsequently, the chambers are implanted into the animal's body or placed in a nutrient medium — when cultured in vitro.

The technique involves the manufacture of simultaneously cameras of various sizes and in the required quantity, depending on the purpose of the study.

Cameras manufactured by the proposed method from a polyacrylamide gel provide the opportunity to study the same individual cells in the dynamics of their growth and development. For this, cells are microscopically copied directly in the chamber. To facilitate the location of cells in the chamber together with the cells during their

Sowing, it is possible to introduce special markers (particles of activated carbon, killed yeast cells and yes.) After the required time, the chambers are removed from the body or nutrient medium. Under the microscope, the same group of cells is found and examined. If necessary, the Pasteur pipette cells can be extracted. The camera can be re-implanted into the body or labeled in a nutrient medium.

The manufacture of chambers from polyacrylamide gel provides optical transparency, which allows observation of the cells under investigation by light microscopy in the dynamics of their growth and reproduction. The chambers are elastic, xopotdo are transported by animals and have the ability to configure in the cavities when implanted into the body even when simultaneously implanting one animal 2-3 chambers.

The main advantages of the manufactured cameras by this method are the optical transparency, elasticity, biological inertness of the material used to manufacture the cameras. The method of their manufacture is simpler, accessible and allows you to simultaneously receive the required number of cameras.

Claims (1)

1. Evgenieva G.P. Intercellular interactions and their role in evolution of M., Science 1976, pp. 31-37. Phie.1 Cell Suspension fus. if: -t f:  VX