JPS61209585A - Medium for cell culture - Google Patents

Abstract

PURPOSE:To provide the titled medium having excellent cell-proliferation property, and suitable for the proliferation of animal cell to produce a valuable material such as interferon, hormone, monoclonal antibody, etc., and composed mainly of a liquid component extracted from the embryo of a fertilized egg. CONSTITUTION:After sterilizing the surface of the shell, a fertilized egg of e.g. chicken is incubated in an incubator at 38-39 deg.C and 85-95% RH for 10-11 days. The embryo is separated from the incubated egg, washed with water, added with clean water or dilute aqueous solution of NaCl, homogenized with a homogenizer in the form of paste, and filtered with a filter such as a filter press or centrifuged to collect a liquid component. The liquid is again filtered with a micro-filter to remove microorganisms, etc., and the filtrate is stored as a medium for cell culture at <=-30 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cell culture medium suitable for growing animal cells that produce useful substances such as interferon and hormones.

[Conventional technology]

As proposed in Japanese Patent Publication No. 58-111682, such a cell culture medium is obtained by adding serum collected from a specific higher animal to a basic medium consisting of vitamins and saccharides.

Serum, which is a component of the above-mentioned cell culture medium, is preferably collected from fetal cows or adult chickens among higher animals, and serum from cows, calves, old chickens, chickens, roosters, and waste chickens. It is considered unsuitable because it contains substances that inhibit the growth of animal cells.

[Problem that the invention seeks to solve]

By the way, the serum obtained by the above-mentioned conventional method is used to supplement the nutrients of the medium by adding 5 to IOX to the basic medium consisting of vitamins, saccharides, etc., and the serum itself does not serve as a medium.

Moreover, the source of this type of serum is dairy cows and beef cows.

In Japan, adult birds are a valuable resource for supplying milk and beef and producing eggs, and we cannot afford to use this resource for serum collection.

Therefore, at present, this type of serum is exclusively imported from resource-rich countries such as the United States.

Therefore, as a result of various researches, the present inventors attempted to obtain a medium equivalent to a culture medium supplemented with fetal bovine serum and adult chicken serum from the abundant resources available in Japan. The extract medium is comparable to commercially available cell culture media.
The present invention was completed after discovering that it has a spasmodic effect.

[Means for solving problems]

The present invention relates to a cell culture medium, and is characterized in that it mainly contains a liquid-like component extracted from fertilized egg embryos.

The cells referred to in the present invention are cells that produce useful substances such as interferon and hormones, such as cells excised from animal internal organs or muscle tissues such as human IJnnoF globoid cells and GH1.

To obtain the cell culture medium of the present invention, first, fertilized eggs are prepared. As fertilized eggs, it is preferable to use poultry eggs such as chicken, duck, or turkey eggs, which are easily available in large quantities.

In Japan, eggs for brooding are commercially available as "vrII eggs" with a guarantee, so it is convenient to use this commercially available product. However, commercially available products have a fertilization vehicle of approximately 80X, so if you find unfertilized egg gold in the incubator that will be used later, it will be necessary to remove it.

Next, the surface of the eggshell of the fertilized egg is wiped with absorbent cotton soaked with iodine solution or alcohol solution to remove any deposits, and the surface of the eggshell is sterilized. put in.

It is preferable to maintain the temperature within the hatching body at 38-95N and the carbon dioxide concentration at approximately 5XK. To maintain the humidity inside the incubator, a container filled with water (a dish-shaped container is preferable) may be placed inside the incubator.

Fertilized eggs are hatched under the above conditions, but in order to prevent fertilized eggs from becoming a death penalty, fertilized eggs are hatched at approximately 180 degrees two to three times a day (for example, in the morning and evening). It is desirable to rotate it one by one.

Next, the fertilized eggs that have been hatched for 10 to 11 days are removed from the hatching weight, and the embryos are separated from the fertilized eggs. As shown in the later test examples, embryo extracts from fertilized eggs whose hatching period is less than 10 days or longer than 11 days,
Since the effect of multiplying animal cells is weak, it is desirable to complete hatching within the above period. When chicken eggs are used as fertilized eggs, the size of the chicken embryo before hatching is so small that it cannot be seen with the naked eye, but since it hatches during the above period, it is about 2I.
/grows to the size of one. The yield of embryos from fertilized eggs hatched during the above period is approximately 3 times the egg weight.

In addition, in order to separate embryos from fertilized eggs that have completed hatching during the above period, the fertilized eggs are flat-cracked or machine-cracked to remove the eggshell, and then the embryos are collected from the resulting egg fluid. However, when processing a small amount, it is better to make a hole in the eggshell at the blunt end of the fertilized egg and remove the embryo from this hole with tweezers.

The embryos separated by the above method have egg weight etc. attached, so
These deposits are washed away by placing the embryo in clean water or a dilute aqueous sodium chloride solution.Finally, the cell culture medium of the present invention can be obtained by extracting the liquid component from the embryo. For extraction of liquid components.

First, fresh water or a dilute aqueous sodium chloride solution is added to the embryo, and this is turned into a paste using a homogenizer or a crusher K.

Next, the paste obtained is filtered through a filter such as a filter press.
If you do this, you will get a liquid that has been separated from the paste.

In addition, a centrifugal separator can also be used to separate the liquid from the paste, and in this case, centrifugation t-2 to t-3
A transparent liquid can be obtained by repeating the process several times.

The obtained liquid is used as a cell culture medium by filtration with a microfilter to remove microorganisms, and is stored at -30°C or lower, preferably -60°C or lower.

〔Example〕

20 fertilized chicken eggs (breed: Dekalb) at a temperature of 38-3
It was placed in an inflator maintained at 9°C, humidity 85-90X, and carbon dioxide concentration 5X.

Then, while maintaining the above conditions, add eggs once in the morning and once in the evening.
The eggs were rotated 80 degrees and incubated for 10 days.

Next, when the eggs were taken out of the incubator and inspected with a transparent seal, 4 eggs were found to be guilty of death, and 16 eggs were found to be normal.

The eggshell surface of each of these 16 normal eggs was wiped clean with alcohol, a crack was made at the blunt end of the eggshell, the eggshell at the blunt end was removed with tweezers, and a hole was made in the eggshell.

From this hole, remove the chicken embryo inside the eggshell with tweezers.
The chicken embryos were washed in a dilute aqueous sodium chloride solution to remove deposits, and a total of 30.9 chicken embryos were obtained.

An equal amount of O19% sodium chloride aqueous solution was added to this chicken embryo 3011, and the mixture was run in a homogenizer for 5 minutes to make the chicken embryo tissue fine and paste-like.

The obtained paste was first diluted with 15.00 Or, pem.
After centrifugation for an hour, remove the supernatant, then this supernatant t'
After centrifugation at 30,000 r, p, m for 4 hours, the supernatant was drained to obtain a liquid component of 47II.

This liquid component was passed through a microfilter to remove microorganisms and the like to obtain cell culture medium 33#t.

[Effect]

Although it has not been thoroughly investigated what kind of structure the cell culture medium of the present invention consists of nutrients such as proteins, lipids, carbohydrates, etc., as shown in the later test examples, the cell culture medium of the present invention has the following structure:
Since it shows the same cell growth power as a commercially available cell culture medium without adding all nutrients, it is presumed that it contains most of the nutrients necessary for cell growth and does not contain any substances that inhibit cell growth. .

Although the cell culture medium of the present invention shows the same effect as commercially available products, when special nutrients are required, the basic medium (
By adding the cell culture medium of the present invention (which consists of vitamins, salts, sugars, etc.), it is possible to obtain a medium with even greater growth effects.

Test example 1 The following four types of samples were prepared.

Control group 2: Adult chicken serum medium (same as above) In control groups 1 and 2, unknown nutrients were added to the serum.

Then, for each sample, the above test samples 0, 5
rttl Ic L-929 cells (cell concentration 2.OX
10' Ce1ls/R/ ) t- After adding and suspending, transfer the suspension to a Petri dish, place the Petri dish in a thermostat maintained at a carbon dioxide concentration of 5X and a temperature of 37°C, and leave as shown in Table 1. When the proliferation state of L-929 cells was observed every day, the results shown in Table 1 were obtained.

Table 1 In the table, the number of days indicates the number of days the sample was left in the thermostat. In addition, the numbers in the table are determined by measuring the number of cells in the sample using a microscope.
s/1111).

Test example 2 The following four types of samples were prepared.

Test group 1: Example medium (10 days 8 medium) and 9.5 m of Y-1 cells (cell concentration 1.2
/aj) was added and suspended, 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% and a temperature of 37°C. -1
When the proliferation state of the cells was observed, the results shown in Table 2 were obtained.

Table 2 In the table, Δ indicates that the cells died, and the meanings of other symbols and numbers are the same as in Table 1.

〔Effect of the invention〕

As described above, the cell culture medium of the present invention has the ability to proliferate vitamin spores. Moreover, since the cell culture medium of the present invention uses eggs, which can be obtained at low cost, as food, it can be produced on an industrial scale.

Therefore, the cell culture medium of the present invention is suitable as a cell culture medium for producing useful substances such as interferon, hormones, monoclonal antibodies, and human transferrin.

Claims (3)

[Claims] (1) A cell culture medium characterized by mainly containing a liquid component extracted from a fertilized egg embryo. (2) The cell culture medium according to claim 1, which uses fertilized eggs that have been hatched for 10 to 11 days. (3) Claim 1 that uses chicken eggs as fertilized eggs
The cell culture medium according to item 1 or 2.