Note: Descriptions are shown in the official language in which they were submitted.
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Title of the Invention
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METHOD OF TISSUE CULTURE
Back~round of the Invention
1. Field of the Invention
The present invention relates to a method of tissue
culture of animal tissue and more particularly to a method of
tissue culture which enables an efficient successive
cultivation of somatic cells of animal~to be maintained.
2. Description of the Prior Art
It has heretofore been necessary to employ so-called
successive cultivation of animal tissue for wide categories
of biological studies including fields such as medicine, pharmacology
and so on as an effective and practicable means of research.
For this reason, the successive cultivation of animal tissue has
found its application in various fields of research, such
as in elucidating the mechanisms of cell differentiation,
cancer etc. in recent years.
For successive cultivation of animal tissue, it is
necesqary to use, excluding a few exceptions, a serum of, such
as, adult, neonate or fetus of cattle, horse, chicken, rabbit
and so on, in addition to the chemically defined synthetic
culture medium consisting of amino acids, vitamins, minerals
and so on. The reason for the indispensability of serum is based
on the fact, that a causal matter of cell multiplicative growth
factor ~a substance other than the general nutritive materials)
is contained therein, so that a multiplication of animal tissue
will be impossi~le without serum, owing to the lack of the said
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causal matter. There are yet many unknown aspects besides the
problem of instability of the matter. In addition, serum is expen-
sive due to the insufficient supply system therefore. For these
reasons, a tissue culture has remained as difficult.
S BRIEF SUMMARY OF THE INVENTION
Thus this invention is concerned with a method of tissue
culture which permits the resolution of difficulties described
above.
It is hoped to provide a method of tissue culture in
which a whole or a part of the animal serum to be incorporated in
the culture medium can be dispensed with.
It is also of concern to provide a method of tissue
culture which enables successive cultivation in an efficient manner.
The invention will appear more clearly from the following
description with the accompanying drawings.
The method according to the present invention is charac-
terized in cultivating animal tissue under the employment of a
culture medium containing an extract of micro algae.
More particularly, the invention provides a method
for culturing cells or tissue of lower animals comprising culturing
a sample of cells or tissue of a lower animal in a nutritive
culture medium for cells and tissues of lower animals, said culture
medium containing an effective amount of an extract of algae
capable of causing cell multiplication of said cells or tissue.
In a more preferred aspect, the invention provides a method
for culturing tissues and cells of lower animals, which comprises:
; providing a nutrient culture medium containing, per ml of said
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culture medium, from 0.3 to 400~/g, calculated as the solids, of an
aqueous extract of an algae selected from the group consisting of
Chlorella, Scenedesmus, and Spirulina, said extract having been
prepared by extracting said algae with water at a temperature of
from 50C to 150C, for from 0.5 to 120 minutes, so that said
extract contains sugars, proteins, polysaccharides and nucleic
acids having molecular weights in the range of from 1,000 to
1,000,000 and exhibits an activity for promoting multiplication of
animal cells; then placing and culturing on said culture medium
a sample which is a piece of animal tissue or animal cells obtained
from a lower animal, which sample is free of human cells and human
tissues.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a chart of molecular fractionation of Al-
component of hot water extract of Chlorella cells.
Figs. 2 and 3 show graphically the results obtained in
the experiments given in Examples 1 and 2 respectively.
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_ tailed Description of the Invention
In this specification, the term "micro algae" means
unicellular algae or its near relatives, such as for
example, Chlorella, Scenedesmus, Spirulina or so on inclusive
of natural living and cultivated ones.
The extract of micro algae as indicated in this speci-
fication denotes an extract which is from an extraction
of cells of one or more of said micro algae using an adequate
solvent. As the solvent, an aqueous one is especially preferred.
Among the aqueous solvents, such as for example, water itself
and aqueous solutions containing dissolved therein acids, bases
and organic solvent~, may be exemplified.
In order to effect the extraction, algae cells are
brought into contact with the solvent which is heated or kept
at ordinary temperature. It is preferable to employ a hot water
extraction, in which the algae cells are suspended in water in
an amount of 1 - 1000 grams in dry weight of algae per liter
of water and are ~ept at 50 - lS0 C for 0.5 - 120 minutes,
preferably at 100 C for more than 1 minute, and then are removed
from water by, for example, centrifugation etc., to leave an extract.
The extract may be refined on requirement by means of, for example,
gel filtration, dialysis and so on.
The thus obtained extract of micro algae contains sugars,
proteins, polysaccharides, nucleic acid and 80 on having molecular
weight~ in the range from 1,000 to 1,000,000, and exhibits a
multipllcation promoting activity in cultivating animal tissue.
The extract obtained as above can be used per se, as a
fraction of molecular weight fractionation thereof or in a form
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of concentrate or dry powder obtained by freeze-drying, spray-
drying etc. Especially, a high molecular fraction containing
sugars, proteins and polysaccharides and dry powder thereof
is preferred.
According to the present invention, animal tissue is
cultivated using a culture medium containing an extract of
micro algae, wherein the procedures of culture, basic culture
medium and so on may be identical with those in the conventional
tissue culture. Thus, a known synthetic culture medium contain-
ing amino acids, vitamins and so on can be used as the basic
culture medium, to which an extract mentioned above is added
to perform the cultivation of animal tissue under aseptic con-
ditions.
The animal tissue to be cultured includes somatic cells
taken out of individual animal body, such as normal tissue
and cancer cells.
The amount of addition of the micro algae extract to
the basic culture medium may be in the range from 0.3 to 400 ~g
and preferably in the range from 1 to 100 ~g in dry weight of
the extracted matter per ml of the medium. The culture medium
to which the micro algae extract has been added can be used
per se for the cultivation, while it is also possible to incor-
porate a serum addition as in the culture medium of prior art
téchniques. Here, it is possible to reduce the amount of addi-
tion of serum, which has heretofore been assumed to amount to
about 10 %, up to a value of 1 %.
When animaltissues are cultivated by a culture medium
containing the micro algae extract, the successive cultivation
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can be maintained by the multiplication promotion effect.
Comparable effects can be achieved, if the amount of serum which
heretofore is considered in general as necessary to add in an
amount of lO % is reduced to l/lO, so that a great reduction
of serum amount can be reached.
Yet the cause for rendering the successive cultivation
of animal tissue possible is not clear. It is to be assumed
that the metabolism of the tissue is stimulated and becomes
prosperous by the action of an unknown biologically active
substance contained in the high molecular fraction of the micro
algae extract, which contains especially glycoprotein, poly-
saccharide and so on.
As described above in detail, the present invention
brings forth substantial advantages, that the culture of animal
tissue can be performed without employment or, even in case of
employment, with only a little amount of addition of animal
~erum which i8 uhstable not only in the composition but also
in the supply thereof and is disadvantageous from an economical
point of view and that the tissue culture can be carried out
ln An efficient and stable manner.
Description of the Preferred Embodiment
The present invention is further explained by the
following Examples.
Exam ~ 1
30 g of Chlorella powder were suspended in l Q of water
and were ~ubjected to hot water extraction at lO0 C for 30
minute8. The suspen~lon was then centrifuged and the
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supernatant (corresponding to a dry extracted matter of 4.5 g)
was caused to undergo a molecular fractionation on Sephadex
G-25 column (trade mark of the firm Phamacia). The fractions
with molecular weight~ above 3,000 were treated by adsorbing
on DEAE-cellulose column (a trade mar~ of the firm Brown).
The so adsorbed column was then eluted by a M/100 carbonate
buffer. A fraction having a component with molecular weight
over 70,000, and in the range from 30,000 to 10,000 which
contains rich neutral sugar and a component with molecular
weight in the range from 10,000 to 3,000 which contains neutral
sugar and protein in nearly equivalent proportion was obtained
~which i5 denoted hereinafter as Fraction Al) and this Fraction
Al was dried by freezing to prepare 0.63 g of dry matter. The
molecular fractionation chart of Fraction Al is given in Fig. 1.
S X 104 of cell strain RLC-10 (rat liver cells) were
cultivated in a culture medium prepared by adding a fetus cattle
~erum (FCS) in an amount of 10 ~ to the basic culture medium
DM-160 indicated in Table 1 ~Sector C). In a similar manner,
the said cell strain RLC-10 were cultivated in a culture medium
prepared by adding said FCS in an amount of 1 ~ and the above
mentioned Chlorella Fraction A~ in an amount of 5 ~g/ml to the
basic culture medium DM-160 (Sector T). The multiplication of
the cell~ were examined by comparing the above two cultivation
sectors. Both sector~ showed a similar trend of multiplication
of cells. The results obtained are shown graphically in Fig. 2.
In a ~imilar manner a~ in Example 1, cell strain JTC-15
(rat ascite~ hepatoma), JTC-2 (rat ascites hepatoma), RLG-l
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(rat lung cells) and RLC-18 ~rat liver cells) were examined
for their multiplication rates in the medium as employed in
Example 1 with variable contents, i.e. 0.5 ~g/ml, 5 ~g/ml and
50 ~g/ml, of Chlorella Fraction Al. The tendencies of the
multiplication of the cells were the same. The results obtained
are shown graphically in Fig. 3.
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Table 1
Composition of Basic Culture Medium DM-160
Amino Acidsmg/l Vi tamins mg/l
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Ala 400 B~ 1.0
Arg 100 B2 1. 0
A5p 25 B 6 1 . O
Asn 25 B 12 0 . 005
Cys-hCl 80 Pantothenic 1.0
acid
Glu 150 Nicotinamide 1.0
Gln 300 Biotin 0.1
Gly 15 Choline HCl 5 . 0
His 30 C 1.0
Ile 150 Folic acid 1.0
Leu 400 Inositol 5.0
Lys 100
Met 80
Phe 80
Pro 12
Ser 8 0
Thr 100
~rp 40
Tyr 5 0
Val 85
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