JPS6153037B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for cell fusion. More specifically, the present invention relates to a method for fusion of cells with monomers in the presence of water-soluble polymers.

In recent years, with the development of cell engineering and genetic engineering, cell fusion techniques have come into widespread use. In immunology, specific cells that produce antibodies that attack antigens are fused with actively proliferating cancer cells to produce large amounts of antibodies, which are then used for treatment. We are achieving results. In addition, cell fusion technology has been applied to the production of useful products in the fusion of animal cells and the improvement of plant cell varieties.

By the way, in order to fuse cells, a substance is needed that acts as a bridge between the cells to be fused.
This is called a "fusing agent." There are Sedai virus and chemical substances that correspond to fusion agents. The most important requirement for a fusion agent is the ability to efficiently fuse all cells in the system. Various fusion agents have been developed to date, but the ideal one has not yet been perfected. However, many fusion agents have been tested for their effectiveness. Among these, polyethylene glycol (DEG) is currently the most widely used fusion agent and is considered to be the most likely to induce fusion. A suitable cell fusion agent is one that first aggregates cells and stimulates (activates) the cell membrane appropriately without destroying the cell membrane. Even with PEG, the fusion rate varies somewhat depending on the cell type, but is around 10-30%. Further, even if fusion of different types of cells A and B is performed, fusion of cells of the same type and fusion of two or more cells will occur, and the probability that the two target cells of A and B will fuse is low. Therefore, it is of great significance to discover a fusion agent that causes fusion at a high rate and creates a fusion body consisting of two cells.

While the present inventors were proceeding with research on culturing various cells on various substrates, it became necessary to have a fusion method that can fuse cells together at a high rate. Therefore, we searched for fusion agents that can fuse cells at a high rate, mainly using vitrifying monomers, and as a result, we discovered a monomer that causes fusion at a considerably higher rate than PEG. However, when attempting to fuse cells using only monomers, there is a drawback that most cells become inactivated before they fuse. Therefore, when carrying out cell fusion using a monomer alone, it is necessary to complete the fusion within an extremely short time of one minute.
However, it is quite difficult to perform the fusion operation uniformly in such a short time. Therefore, in order to perform fusion sufficiently reliably without producing inactivated cells, a protective agent is required to extend the fusion time. The present invention is characterized in that a water-soluble polymer is used as the protective agent. That is,
It is an object of the present invention to provide a method for fusing cells using a polymer that prevents cell deactivation and a monomer as a fusing agent. According to the present invention, if the fusion is performed within 10 minutes, the fusion can be efficiently completed without inactivating the cells.
Furthermore, according to the present invention, the fusion time can be extended, so that the fusion agent sufficiently diffuses to the cell membrane surface, sufficient contact between cells occurs, and the fusion efficiency increases.

Hereinafter, the configuration of the present invention will be explained in detail.

First, the serum-containing medium was removed by centrifugation.
100-300× ¹⁰⁵ cells/ml (cell pack) [A]
A solution containing a mono- or di-acrylate or mono- or dimethacrylate as a coalescing agent represented by the general formula of the group and a water-soluble polymer as a protective agent exemplified in group [B] was added while gently rotating the test tube. Afterwards, warm to 37°C to fully fuse the cells. Then, 10 ml of medium is added and the water-soluble polymer and fusion agent are removed by centrifugation. The present invention can be further developed by growing the fused cells in a serum-containing medium from which water-soluble polymers and fusion agents have been removed.

The monomer concentration of the coalescing agent used in carrying out the present invention is preferably 40 to 50% by weight. However, the concentration of monomer is determined in large part by the molecular weight of the monomer. That is, if the molecular weight of the monomer is large, the viscosity in the system becomes too high when it is dissolved, and conversely, the fusion efficiency decreases. Therefore, when the molecular weight of the monomer is large, a concentration of about 40% is desirable. The concentration of the water-soluble polymer serving as the protective agent is desirably 5 to 10% by weight, and when the molecular weight of the monomer is high, the polymer concentration of the protective agent is preferably as low as about 5%. The fusion time is preferably about 5 to 10 minutes; if it is too short, the fusion will not be completed. According to the present invention, homologous and heterologous animal and plant cells are fused together at a high rate. Therefore, the present invention is not restricted to cell types or the like.

The monomer used as a protective agent in the present invention is an acrylate or diacrylate represented by the general formula of group [A] below.

[A] group, n of the oxyethylene group of the acrylate and diacrylate exemplified herein is 10 or more. n
If it is 9 or less, there is no fusion ability at all even in the presence of water-soluble polymers, and the cells are also inactivated. In particular, if the number of n is specified, n=23 and n=50. When n=23, fusion only occurs in the presence of a water-soluble polymer. When n=50, fusion can be achieved with almost no cell deactivation even in the absence of a water-soluble polymer.

The water-soluble polymers used as the protective agent in the present invention are exemplified by the following group [B]; sodium polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, sodium cellulose glycolate, poly-
DL-alanine, poly-L-arginine hydrochloride,
Poly-L-glutamic acid, poly-L-histidine, poly-D-lysine hydrobromide, poly-3-hydroxypropyl-L-glutamine, poly-DL-
Lysine hydrobromide, poly-L-lysine hydrobromide, poly-L-lysine hydrochloride, poly-L-orinithine hydrobromide, poly-L-proline, poly-L
-Tyrosine, poly N-methylglycine, etc. In addition to those listed above, the protective agent may be any synthetic polymer, semi-synthetic polymer, or natural polymer as long as it is a water-soluble polymer that dissolves in water.

The structure and effects of the present invention will be specifically explained below using Examples.

[Example 1] 0.1 g of polyvinylpyrrolidone and methoxypolyethylene glycol methacrylate Dissolve 0.4 g in 0.5 g of MEM medium, centrifuge it to remove the serum-containing medium, add it to Molt T-cell packed with cells while rotating the test tube gradually, and incubate at 37°C for 5 minutes. Basion.
Thereafter, 10 ml of the medium was changed and centrifuged to remove the polymer and fusion agent. The fusion rate reached 43.7%, of which more than 70% produced fusions consisting of two cells.

[Example 2] Methoxypolyethylene glycol methacrylate was added to 0.6 g of MEM medium solution containing 50 mg of polyvinylpyrrolidone. 0.4g of M-50G) was dissolved. The solution was centrifuged in advance to remove the serum-containing medium.
The test tube was added to the cell pack of T-cell while rotating, and incubated at 37°C for 10 minutes. Thereafter, 10 ml of medium was added and centrifuged to remove the polymer and fusion agent (monomer). The fusion rate reached 45.3%, of which 77% consisted of two cells.

The results of Examples 1 and 2 are similar to those of the conventional
Fusion occurs with about 15% higher efficiency than fusion with PEG, and the proportion of fusions consisting of two cells, which is advantageous for producing useful substances, is significantly higher.

Claims (1)

[Claims] 1. In a method for fusing cells of the same or different species; mono(meth)acrylate or di(meth)acrylate as a cell fusion agent represented by the following general formula and highly water-soluble as a protective agent. Cell fusion methods consisting of mixing cells of the same or different types in a system consisting essentially of molecules; However; n is an integer of 10 or more. 2 Methacrylate is methoxypolyethylene glycol methacrylate The method according to claim 1. 3 Methacrylate is methoxypolyethylene glycol methacrylate The method according to claim 1. 4. The method according to claim 1, wherein the concentration of the cell fusion agent is 40 to 50% by weight. 5. The method according to claim 1, wherein the concentration of the protective agent is 5 to 10% by weight.