JPS62166883A - Culture medium for animal cell - Google Patents

Abstract

PURPOSE:An inexpensive culture medium for animal cells, obtained by adding a liquid material after removing embryos from fertilized eggs and blood serum to a basal culture medium, capable of mass-production and having improved multiplication ability. CONSTITUTION:A culture medium for animal cells, obtained by blending 100pts. wt. basal culture medium containing nutriments, e.g. vitamin, saccharide, etc., with 0.1-5.0pts.wt. liquid material, prepared by removing shells of fertilized hen's eggs, etc., incubated for 4-15 days, removing embryos therefrom and consisting essentially of egg white and egg yolk and 0.5-2.0pts.w.t. blood serum, e.g. calves, etc., filtering the mixed culture medium through a microfilter to remove microorganisms and filling and hermetically sealing the resultant filtrate in a container, etc.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a novel culture medium for animal cells.

[Conventional technology]

Conventionally, for animal cell culture media, it has been essential to add 5 to 10 parts of beef tallow serum to a basic medium consisting of vitamins and saccharides for nutritional reinforcement.

By the way, tallow baby serum is expensive because it is obtained by removing the fetus from the membranes of a mother cow and collecting serum from this fetus. Therefore, when it is desired to proliferate a large amount of animal cells, a large amount of beef tallow serum is required, which requires considerable expense.

[Problem that the invention seeks to solve]

Therefore, recently, several serum-free media (serum-free media) have been developed, but like these bloodless media, they have the problem of being expensive. After conducting various research to provide a culture medium that can be produced in large quantities, we found that when a liquid component obtained by removing embryos from fertilized eggs is used in combination with serum collected from various animals, excellent cell proliferation ability can be achieved even with a small amount of serum used. The present invention was completed by paying attention to the fact that

[Means for solving problems]

It is characterized by:

To obtain the medium of the present invention, first, a basic medium, a liquid obtained by removing embryos from fertilized eggs, and serum, which constitute the medium, are prepared.

The basic medium may be a commercially available medium to which nutrients such as vitamins and sugars are added, such as MEM medium-19.
9 medium-DMEM medium-F-10 medium, F-12 medium, RPM 1-1640 medium, etc. can be used.

After the eggs have been transformed, the eggs are removed from the shell and the contents are removed, and the main ingredients are egg whites and egg yolks from which the embryos have been removed. Note that good results can be obtained by using fertilized eggs that have been incubated for 4 to 15 days.

Next, the raw materials prepared in this way are mixed at a predetermined ratio.

The mixing ratio of each raw material is preferably 0.1 to 5.0 parts of liquid obtained by removing embryos from fertilized eggs and 0.5 to 2.0 parts of serum to 100 parts of basic medium. .

If the mixing ratio of the above liquid and serum is too low or too high, it will be difficult to obtain a medium with excellent growth ability for animal cells, so it is desirable to keep the mixing ratio within the above range. In the medium of the invention, the amount of serum used can be less than 20% of that of conventional media.

Finally, the resulting culture medium is filtered through a microfilter to remove microorganisms, and the product is completed by filling and sealing the container.

〔Example〕

20 fertilized chicken eggs at a temperature of 38-39℃ and a humidity of 85-9.
0%, and placed in an incubator maintained at a carbon dioxide concentration of 5%.
They hatched for 0 days.

Next, when these eggs were taken out of the incubator and fluoroscopically inspected, it was found that 3 were found to be guilty of death and 17 were normal eggs.G These 17 normal eggs were broken by hand, the eggshell was removed, and the contents were poured into the pad. When 17 embryos were removed from the contents, 726 g of liquid was obtained.

After adding an equivalent amount of physiological saline to the obtained liquid and pomorizing it, the mixture was centrifuged to remove insoluble materials, yielding 653 g of a fertilized egg-derived medium.

Example 1 and GH3 cell medium 100. ! I got i'.

Example 2 Mix 98 g of a commercially available basic medium (DMEM medium), 1 g of the above fertilized egg-derived medium, and 1 g of commercially available adult bovine serum,
79 cell culture medium 100.9 was obtained.

[Effect]

Hereinafter, the effects of the animal cell culture medium of the present invention will be explained while describing tests and experimental examples.

Test example The following three substances were prepared.

B. Commercially available basic medium (MEM DM-160) and fertilized eggs (chicken eggs: Tekalb) incubated for 11 days in an incubator.
The eggs are broken and shelled, and the pressure part is removed to obtain a liquid product.
Add an equal volume of 0.9% saline to this liquid and homogenize for 5 minutes to obtain a suspension...Commercially available fetal bovine serum (FBS) and kotara substances were combined. , the following sample (medium) was made.

Test group 1: To the basic medium of A, 0.2 parts of the oral suspension and 1.0% of the serum of C were added.Test group 2: To the basic medium of A, 1.0 part of the oral suspension was added. Test group 3: Added 1.0 parts of the serum of A and Ha. Test group 3: Added 2.0 parts of the suspension of A and 1.0 part of the serum of Ha to the basal medium of A. Control group 1 (conventional medium) ): Add serum 5 of Ha to the basic medium of A.
Control group 2: Only the basal medium of A. Control group 3 (serum-free medium) A: Basal medium of A. C: Added 2.0 parts of the suspension to the basic medium of A. As the test 1° culture medium, the above sample was added to each sample of Q, 5
Collect ml of v-79 cells (cell concentration 1.8
After stirring, add
The suspension was transferred to a Petri dish, and the Petri dish was placed in a thermostat maintained at a carbon dioxide concentration of 5 parts and a temperature of 37°C, and the proliferation status of V-79 cells was observed for each number of days shown in Table 1. A result of 1 was obtained.

Note that the medium becomes nutrient deficient when cells are grown for a long period of time, so the medium was replaced with a new medium after 5 days of standing.

Table 1 The numbers in the table are the number of cells in the sample converted to the number of combs per millimeter of the sample measured using a microscope, and the unit is 104.

As test 2゜ culture medium, each of the above samples was
, 5 ml each was collected in a petri dish, HeLa cells (cell concentration 6.3 x 10 cells/ml) were added thereto, and the petri dish was placed in a thermostat kept at a carbon dioxide concentration of 5.5 ml and a temperature of 37°C, and the cells were collected as shown in Table 2. HeLa for each number of days left
When the proliferation state of the cells was observed, the results shown in Table 2 were obtained.

Table-2 Note that the numerical values in the table are the same as in Table-1.

Test 3゜ Instead of HeLa cells in Test 2, GH cells (cell concentration 1
．． When the proliferation state of GH3 cells was observed using the same method as in Test Example 2 using the same conditions as in Test Example 2, the results shown in Table 3 were obtained.

Table 3 Mira 1 The medium of the present invention has a greater effect on animal cell growth than conventional media, even if the amount of frame used is less than 20% of that of conventional blood cells-added media.

〔effect〕

Therefore, according to the present invention, a medium with excellent growth ability for animal cells can be produced at low cost and in large quantities.

Claims (2)

[Claims] (1) A culture medium for animal cells, characterized in that it is made by adding a liquid obtained by removing embryos from fertilized eggs and serum to a basic medium. (2) The culture medium for animal cells according to claim 1, which uses fertilized eggs that have been hatched for 4 to 15 days.