U.S. patent number 4,440,860 [Application Number 06/239,523] was granted by the patent office on 1984-04-03 for stimulating cell growth.
This patent grant is currently assigned to The Children's Medical Center Corporation. Invention is credited to Michael Klagsbrun.
United States Patent |
4,440,860 |
Klagsbrun |
April 3, 1984 |
**Please see images for:
( Certificate of Correction ) ** |
Stimulating cell growth
Abstract
Compositions and methods for promoting cell growth featuring, in
one aspect, cell culture media containing milk or colostrum and
fibronectin; fibronectin is preferably pre-coated onto the culture
substrate.
Inventors: |
Klagsbrun; Michael (Newton,
MA) |
Assignee: |
The Children's Medical Center
Corporation (Boston, MA)
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Family
ID: |
26810824 |
Appl.
No.: |
06/239,523 |
Filed: |
March 2, 1981 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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113229 |
Jan 18, 1980 |
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953491 |
Oct 23, 1978 |
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Current U.S.
Class: |
435/384;
424/85.1; 435/289.1; 435/387; 435/389; 435/391; 435/402; 435/406;
435/408 |
Current CPC
Class: |
C12N
5/0068 (20130101); C12N 2533/52 (20130101) |
Current International
Class: |
C12N
5/00 (20060101); C12N 005/00 (); C12N 005/02 ();
C12M 003/00 (); C12M 003/04 () |
Field of
Search: |
;435/240,241,284,285,286 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Journal of Cell Physiology, vol. 109, No. 2, pp. 223-234; 1981.
.
George H. Rothblat et al. editors, Growth, Nutrition and Metabolism
of Cells in Culture, vol. II, p. 379; 1972. .
Jakoby et al., editors, Methods in Enzymology, vol. LVIII Academic
Press, p. 267; 1979. .
Maciaz et al., "Hormonal Requirements of Baby Hamster Kidney Cells
in Culture", Cell Biology International Reports, Jan. 1980. .
Groves (1965), Biochem. Biophys. Acta. 100, 154-162. .
Querinjean et al. (1971), Eur. J. Biochem. 20, 420-425. .
Butler et al., In Lactation, Larson et al. ed., pp. 217-255,
(Academic Press, New York, 1974). .
Hayashi et al. (1976) Nature 259, 132-134. .
Hutchings et al. (1978), Proc. Nat'l. Acad. Sci. U.S.A. 75,
901-904. .
Rizzino et al. (1978), Proc. Nat'l. Acad. Sci. U.S.A. 75,
1844-1848. .
Mather et al., Proceedings of the International Workshop for Cell,
Tissure and Organ Cultures in Neurobiology, Federoff, ed., pp.
619-630, (Academic Press, New York 1978). .
Pena et al. "Fibroblast to Substratum Contacts Mediated by the
Different Forms of Fibronectin", Cell Biology International
Reports, vol. 2, No. 4, 1978. .
Grinnel et al. (May 1979), Initial Adhesion of Human Fibroblasts in
Serum-Free Medium: Possible Role of Secreted Fibronectin, Cell.,
vol. 17, 117-120..
|
Primary Examiner: Warden; Robert J.
Parent Case Text
BACKGROUND OF THE INVENTION
This application is a continuation-in-part of Klagsbrun,
"Stimulating Cell Growth," Ser. No. 113,229, filed Jan. 18, 1980,
which in turn is a continuation-in-part of Klagsbrun, "Stimulating
Cell Growth," Ser. No. 953,491, filed Oct. 23, 1978 both now
abandoned.
Claims
I claim:
1. A cell culture medium comprising mammalian milk and
fibronectin.
2. The medium of claim 1, further comprising at least one of the
additives transferrin, insulin, epidermal growth factor, and
fibroblast growth factor.
3. The medium of claim 1 wherein
said milk is bovine milk, and
said fibronectin is human plasma fibronectin.
4. The cell culture medium of any one of claims 1, 2, or 3, wherein
said mammalian milk comprises, by volume, between about 0.5% and
25% of said medium.
5. The medium of claim 4 wherein said mammalian milk comprises, by
volume, about 10% of said medium.
6. A cell culture medium comprising colostrum and fibronectin.
7. The medium of claim 6, further comprising at least one of the
additives transferrin, insulin, epidermal growth factor, and
fibroblast growth factor.
8. The medium of claim 6 wherein said colostrum is bovine
colostrum, and said fibronectin is human plasma fibronectin.
9. The medium of any one of claims 6, 7, or 8, wherein said
colostrum comprises, by volume, between 0.5% and 25% of said
medium.
10. The medium of claim 9 wherein said colostrum comprises, by
volume, between 2% and 5% of said medium.
11. The medium of claim 10 wherein said colostrum comprises, by
volume, about 5% of said medium.
12. The medium of claim 10 wherein said colostrum comprises, by
volume, about 2% of said medium.
13. The medium of claim 12 wherein said medium contains insulin and
transferrin.
14. The medium of claim 13 wherein said insulin and transferrin are
each present in said medium in the approximate concentration of 10
.mu.g/ml.
15. A cell culture means comprising nutrient medium comprising
mammalian milk, and a cell culture surface which is precoated with
fibronectin.
16. The cell culture means of claim 15 wherein
said milk is bovine milk, and
said fibronectin is human plasma fibronectin.
17. A cell culture means comprising
nutrient medium comprising colostrum, and
a cell culture surface which is pre-coated with fibronectin.
18. The cell culture means of claim 17 wherein
said colostrum is bovine colostrum, and
said fibronectin is human plasma fibronectin.
19. A process of culturing cells comprising
providing a cell culture substrate which is coated with
fibronectin,
introducing said cells onto said substrate, and
providing the proper conditions for growth, including feeding said
cells with nutrient medium comprising mammalian milk.
20. The process of claim 19 wherein said mammalian milk comprises
by volume, between 0.5% and 25% of said medium.
21. The process of claim 20 wherein said mammalian milk comprises,
by volume, about 10% of said medium.
22. The process of any one of claims 19, 20, or 21 wherein said
medium further comprises at least one of the additives transferrin,
insulin, epidermal growth factor, and fibroblast growth factor.
23. A process of culturing cells comprising
providing a cell culture substrate which is coated with
fibronectin,
introducing said cells onto said substrate, and
providing the proper conditions for growth, including feeding said
cells with nutrient medium comprising colostrum.
24. The process of claim 23 wherein said colostrum comprises, by
volume, between 0.5% and 20% of said medium.
25. The process of claim 24 wherein said colostrum comprises, by
volume, about 2% of said medium.
26. The process of claim 24 wherein said colostrum comprises, by
volume, about 5% of said medium.
27. The process of any one of claims 23, 24, 25, or 26 wherein said
medium further comprises at least one of the additives transferrin,
insulin, epidermal growth factor, and fibroblast growth factor.
28. A method of promoting proliferation of transformed or cancerous
cells, said method comprising feeding said cells medium containing
colostrum or mammalian milk.
29. The method of claim 28 wherein said medium comprises colostrum
in an amount between 0.5% and 25% (by volume).
30. The method of claim 28 wherein said medium comprises mammalian
milk in an amount between 1% and 25% (by volume).
31. A transformed or cancerous cell-proliferation medium comprising
colostrum or mammalian milk.
32. The medium of claim 31 wherein said medium comprises between
0.5% and 25% (by volume) colostrum.
33. The medium of claim 31 wherein said medium comprises between
1.0% and 25% (by volume) mammalian milk.
Description
This invention relates to cell growth and proliferation.
It is known that mammalian serum contains an ingredient ("factor")
capable of inducing growth of cells in culture; bovine serum is
widely used for that purpose. The growth factor in human serum has
been isolated and found to be a protein having a molecular weight
of about 13,000 and an isoelectric point of about 9.7. Growth
factors have also been found in the pituitary gland, in the
submaxillary gland, in brain and in cartilage.
Groves (1965) Biochem. Biophys. Acta. 100, 154-162 has reported the
presence of the blood iron-binding protein transferrin in milk.
Querinjean et al. (1971) Eur. J. Biochem. 20: 420-425 have found
that lactoferrin, an iron-binding protein similar to transferrin,
is present in milk.
Butler et al. in In Lactation, Larson et al. ed., pp. 217-255
(Academic Press, New York 1974) have found that the lactoferrin
level of colostrum declines rapidly during the postpartum
period.
Five articles report that several cell types can grow in serum-free
media which contain various mixtures of hormones, mitogens and
other factors including, in all the mixtures, transferrin: Hayashi
et al. (1976) Nature 259, 132-134; Hutchings et al. (1978) Proc.
Natl. Acad. Sci. U.S.A. 75, 901-904; Mather et al. in Proceedings
of the International Workshop for Cell, Tissue and Organ Cultures
in Neurobiology, Federoff, ed. pp. 619-630 (Academic Press, New
York 1978); Taub et al. (in press) Proc. Nat'l. Acad. Sci. U.S.A.;
Rizzino et al. (1978) Proc. Nat'l. Acad Sci. U.S.A. 75,
1844-1848.
A problem encountered in the culture of some cell types, e.g.,
uncloned epithelial cells, is fibroblast overgrowth. An approach to
the problem has been the use of selective media which inhibit
fibroblast growth.
SUMMARY OF THE INVENTION
We have discovered a potent, new growth factor, with 50-100 times
greater specific activity than even that in serum. The invention
thus features, in one aspect, a concentrate of this growth factor,
which is a protein derived from mammalian (e.g., human, bovine,
sheep) milk obtained less than about 56 hours post-partum
(hereinafter "colostrum"), and the method of concentrating it based
on its physical properties, which include a molecular weight below
about 20,000 (at least for human and bovine colostrum) and an
isoelectric point of between about 4.4 and 4.8.
In preferred embodiments human or bovine colostrum is collected a
short time after birth, fat and inactive protein are removed, and
gel filtration, isoelectric focussing and preparative
polyacrylamide gel electrophoresis are carried out. The concentrate
is useful in cell growth media for tissue culture, and, e.g., as a
food additive for humans and other animals.
We have also found that defatted colostrum which has not been
further purified is useful in cell growth media for tissue culture
of certain cell types, e.g., kidney, gastrointestinal, and bile
duct epithelial cells, and myoblasts. We have also found that
colostrum added to conventional serum-based media provides a new
medium which is more effective in promoting cell proliferation than
either serum or colostrum alone and has the added advantage of
being cheaper than conventional serum-based media.
We have found that some cell types, e.g., primary and early passage
fibroblasts (fibroblasts which have been transferred to fresh
medium fewer than about 12 times following removal from an animal)
and normal rat embryo cells, unlike epithelial cells, fail to
proliferate in colostrum-containing media. This finding provides
the basis for a further aspect of the invention, the use of
colostrum in tissue culture media to prevent fibroblast overgrowth
while promoting the proliferation of a desired cell type.
We have also discovered that, although milk which is not colostrum
(hereinafter "milk") by itself fails to stimulate significant
proliferation in normal cells, the addition of the iron-binding
protein transferrin (T) to milk results in media which very
effectively stimulate such proliferation. The invention thus
features, in another aspect, inexpensive (compared to serum-based
media) cell culture media containing either milk or colostrum, and
transferrin. One or more of the additives insulin (I), epidermal
growth factor (EGF) and fibroblast growth factor (FGF) can also be
added to the media to further stimulate cell proliferation.
We have also discovered that an additional factor, the serum
protein fibronectin, also enables milk-based media to support the
proliferation of cultured cells, and also enhances the ability of
colostrum to support cultured cell growth. An additional aspect of
the invention therefore provides cell culture media containing
colostrum or milk plus fibronectin, as well as, for some cell
types, transferrin, insulin, epidermal growth factor, and
fibroblast growth factor. In preferred embodiments, the fibronectin
is coated onto the cell culture substrate (e.g., plate or dish)
prior to plating the cells.
The finding that colostrum does not promote the proliferation of
some cell types, and our discoveries that milk, in the absence of
fibronectin or transferrin, does not promote proliferation of any
cell types, and that both colostrum and milk do promote
proliferation in malignant (cancerous and transformed) cells,
provide the foundation for an additional aspect of the invention, a
method of measuring the concentration of malignant cells in a
sample. Colostrum or milk is added to nutrient medium which by
itself does not kill or stimulate proliferation in eukaryotic
cells. Cells from the sample are introduced into or onto the medium
and the rate of cell proliferation is determined. Because
practically the only cells which can grow in the milk-based medium
are malignant cells, the proliferation rate is directly
proportional to the concentration of malignant cells in the
sample.
Other advantages and features of the invention will appear from the
following description of preferred embodiments thereof, including
the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a gel filtration graph in which elution volume is
measured on the horizontal axis; molecular weights for specific
calibration markers are indicated along the top. On one vertical
axis there is measured counts per minute.times.10.sup.-3 for
thymidine incorporation into DNA (a standard measure of DNA
synthesis activity) for Balb/c 3T3 target cells (open connected
dots) and human skin fibroblast target cells (solid connected
dots). On the other vertical axis there is measured (dashed line)
absorbance of UV light of 280 nm wavelength, giving a measure of
the total protein content in each fraction.
FIG. 2 is an isolectric focussing graph in which fraction numbers
are measured on the horizontal axis. On one vertical axis there is
measured (solid line connecting open dots) counts per
minute.times.10.sup.-4 for thymidine incorporation into DNA for
Balb/c 3T3 target cells. On the other vertical axis is measured
(dashed line) the corresponding pH for each fraction.
FIG. 3 is a graph of counts per minute.times.10.sup.-4 for
thymidine incorporation into DNA, against concentration (in
volumetric percent) of bovine colostrum and calf serum, showing the
greater specific activity of the colostrum.
FIG. 4 is a graph of numbers of canine kidney epithelial (MDCK)
cells counted after 12 days against concentration of: colostrum
obtained 8 hours after birth of a calf (day 1 colostrum) (o); milk
obtained 176 hours after the birth of the same calf (day 8 milk)
(.DELTA.); and calf serum (.).
FIG. 5 is a graph of numbers of MDCK cells counted against time in
days for: 2.5% (by volume) day 1 colostrum (o); 2.5% (by volume)
day 8 milk (.); and unsupplemented medium (.DELTA.).
FIG. 6 is a graph of numbers of early passage fibroblasts counted
against time in days for 2.5% (by volume) day 1 bovine colostrum
(o); 2.5% (by volume) serum (.); and 2.5% (by volume)
colostrum+2.5% (by volume) serum (.DELTA.).
FIG. 7 is a graph of numbers of early passage fibroblasts counted
against time in days for 2.5% (by volume) day 1 bovine colostrum
(o) and 2.5% (by volume) serum (.).
FIG. 8 is a bar graph showing relative numbers of six cell lines
grown in serum, colostrum and colostrum plus fibronectin.
FIG. 9 is a bar graph showing relative numbers of eleven cell lines
grown in serum, milk plus fibronectin, and milk plus fibronectin
plus additives.
DESCRIPTION OF PREFERRED EMBODIMENTS
Growth Factor Purification
Growth factor concentrate is obtained from colostrum by the
following preferred process.
Fat is first removed, e.g., by centrifugation, by delipidization
with alcohol-ether or alcohol-acetone, or by chloroform
extraction.
Inactive proteins are then grossly removed, e.g., by contacting the
colostrum with acetic acid, pH 4.3, for 2 hours and the removing
the precipitate by centrifugation.
Isoelectric focusing and gel filtration are then carried out, in
either order, to concentrate growth promoting protein.
FIG. 1 shows that in human colostrum the growth factor has a
molecular weight between 14,300 and 17,800. However, it is possible
that in the work leading to FIG. 1 a multimer form of the protein
was measured, so that the basic protein unit could have a molecular
weight 1/2 or even 1/4 as large. Since the inactive protein in milk
is mostly of molecular weight well above that of the growth factor
(as shown by the dashed line in FIG. 1, measuring the total protein
content in each fraction), a safe dividing line for molecular
weight is about 20,000 for both human and bovine colostrum.
Purification based on molecular weight is carried out with a
1.5.times.94 cm gel filtration column equilibrated with 0.055 M
NaCl and 0.001 M KH.sub.2 PO.sub.4, pH 7.0, and calibrated with
proteins of known molecular weight. The sample is applied to the
column and eluted with buffer at a flow rate of 11 ml/hr.
FIG. 2 shows that the growth factor in human colostrum has an
isoelectric point between 4.42 and 4.68; this range is identical to
the range we obtained for bovine colostrum. Purification based on
isoelectric point is carried out with a vertical electrofocussing
column with a capacity of 110 ml. The partially purified colostrum
is dialyzed against distilled water, lyophilized, and introduced
into 50% sucrose. A linear gradient of the sucrose is then formed
(5-50% wt./vol.) and carrier ampholytes, at pH 4-6, are distributed
in the gradient. The anode is placed in the lower electrode
solution, which contains 0.16 M H.sub.3 PO.sub.4 (pH 1.2)) and the
cathode is placed in the upper electrode solution, which contains
0.25 M NaOH (pH 11.6). Isoelectric focusing is carried out at a
constant voltage of 1650 V for 23 hr. at 4.degree. C. In the case
of human colostrum, the fraction having an isoelectric point
between 4.4 and 4.8 (a range slightly wider, to provide a margin
for error, than the 4.42 and 4.68 range) is collected.
The growth promoting concentrate can be further purified by
preparative polyacrylamide gel electrophoresis, e.g., using the
standard gel system of Ornstein and Davis.
The final concentrate, preferably in dry powder form, is
particularly useful for tissue culture work, as its chemical makeup
(unlike that of serum, e.g.) is known.
The amount of concentrate used will vary, depending on such factors
as purity and cell type. Useful concentrations ordinary range
between 1 ng and 5 g of concentrate per ml of medium.
Another possible use for the concentrate is as a food additive,
e.g., in baby feeding formulas for humans and livestock.
Use of Colostrum in Growth Media
Bovine colostrum obtained eight hours after the birth of a calf is
centrifuged at 12,000 g for 30 minutes to remove fat and cellular
debris, diluted in Dulbecco's modified Eagles Medium (DMEM) to a
concentration of 10 percent (v/v) or less, and sterilized by
filtration. Diluted colostrum is used to supplement DMEM which
contains 4.5 g of glucose per liter, 50 U of penicillin per ml and
50 mg of streptomycin per ml.
The graph of FIG. 3 shows that bovine colostrum stimulates far
greater mitogenic activity in BALB/c 3T3 cells than equal
concentrations of bovine serum.
There is shown in FIG. 4 a dose response curve of MDCK cell
proliferation in colostrum, milk and serum. Canine kidney
epithelial cells (MDCK) in DMEM are seeded sparsely (10.sup.4
cells/well, 5.times.10.sup.3 cells/cm.sup.2) into 24-well
microliter plates. Six hours after plating the DMEM is removed and
the attached cells are fed with MEM supplemented with varying
concentrations of day one colostrum, day eight milk and calf serum.
Each medium is changed every third day, and after 12 days the cells
are trypsinized and counted in a Coulter counter. Two and a half
percent colostrum (by volume), the concentration producing the
greatest effect, produces approximately the same cell count after
12 days as 5 percent serum.
There is shown in FIG. 5 a time course curve of MDCK cell
proliferation in day one colostrum, day eight milk and
unsupplemented medium. MDCK cells in DMEM are seeded sparsely
(10.sup.4 cells/well, 5.times.10.sup.3 cells/cm.sup.2) into 24-well
microliter plates. Six hours after plating the DMEM is removed and
cells are fed with DMEM supplemented with 2.5 percent (v/v) day one
colostrum, DMEM supplemented with 2.5 percent (v/v) day eight milk,
and unsupplemented DMEM. Every third day cells in duplicate are
counted and the remaining cells refed with fresh medium. After
thirteen days the cell count for cells grown in 2.5 percent
colostrum increases by a factor of between 10.sup.2 and 10.sup.3,
while practically no proliferation is observed for cells grown in
the other two media.
Table I below shows the number of MDCK cells counted after 9 days
of feeding cells with colostrum in varying concentrations; cell
numbers obtained with milk are also shown for purposes of
comparison. MDCK cells are plated sparsely (10.sup.4 cells per
well, 5.times.10.sup.3 cell/cm.sup.2) into 24-well microliter
plates. Three hours after plating, the DMEM is removed and attached
cells are then fed every third day. It can be seen that colostrum
very effectively promotes cell proliferation, and that 2.5%
colostrum is more effective than 1.25% colostrum. Useful colostrum
concentrations will vary, depending on the cell type cultured, but
will ordinarily range between 0.5% and 20% of the medium, v/v.
TABLE I ______________________________________ Proportion of Milk
or Colostrum (% (v/v) in DMEM) Final Cell Number
______________________________________ Day 1 colostrum (1.25%)
360,000 Day 8 milk (1.25%) 3,800 Day 1 colostrum (2.5%) 440,000 Day
8 milk (2.5%) 4,200 ______________________________________
There is shown in FIG. 6 a time course curve of early passage
fibroblast proliferation in 2.5% day 1 bovine colostrum, 2.5%
serum, and 2.5% bovine colostrum plus 2.5% serum all in DMEM.
Plating and feeding procedure are as described for FIG. 5. FIG. 6
shows that, for unknown reasons, the fibroblasts proliferate at a
higher rate in a colostrum-serum mixture than in either colostrum
or serum alone. Thus colostrum and serum can be used together to
provide a superior growth medium which is often cheaper than media
containing serum alone. When serum and colostrum are used together,
their concentrations will vary, depending on factors such as cell
type and the desired growth rate. Useful serum concentrations will
ordinarily range between 1 and 20% (v/v), and useful colostrum
concentrations will ordinarily range between 0.2 and 5% (v/v).
Prevention of Fibroblast Overgrowth
There is shown in FIG. 7 a time course curve of early passage
fibroblast proliferation in 2.5% (v/v) bovine colostrum (o) and
2.5% (v/v) serum (.), both in DMEM. Plating and feeding procedure
are as described for FIG. 5. FIG. 7 shows that in colostrum, unlike
in serum, early passage fibroblasts do not proliferate. This means
that colostrum can be used in media to prevent fibroblast
overgrowth, while still promoting the proliferation of a desired
cell type, such as epithelial cells.
Media Containing Milk and Transferrin
Table II below shows the number of MDCK cells counted after 8 days
of feeding cells with the media shown. All media are DMEM based.
Human milk is obtained 4 months and bovine milk 6 days postpartum,
except the last 2 data, which are for 3 month bovine milk. The
ingredients other than milk, DMEM and serum are used in the
following concentrations: Insulin, 5 .mu.g/ml; EGF, 2.5 ng/ml; FGF,
50 ng/ml; Transferrin, 5 .mu.g/ml. The culture method is the same
as that described in relation to Table I above. Human and bovine
milk in DMEM, in the absence of transferrin, fail to stimulate cell
proliferation, as does transferrin, in the absence of milk. The
proliferation-promoting activity of media containing both milk and
transferrin approaches that of serum-containing media. Ten times as
much 3 month milk must be used to obtain the proliferation rate of
6 day milk.
The amount of milk, transferrin, insulin, EGF, and FGF used will
vary, depending on such factors as the age of the milk and the
desired growth rate. Useful milk concentrations will ordinarily
range between 1% and 25% (v/v). Transferrin concentration will
ordinarily range between 1 and 25 .mu.g/ml medium; insulin will
range from 0 to 10 .mu.g/ml medium; EGF will range from 0 to 10
ng/ml medium; and FGF will range from 0 to 100 ng/ml medium.
TABLE II ______________________________________ Medium Cell Number
______________________________________ 5% Human Milk, Insulin 6,660
5% Human Milk, EGF 7,500 5% Human Milk, FGF 7,000 5% Human Milk,
Insulin, EGF 3,600 5% Human Milk, Insulin, FGF 4,560 5% Human Milk,
EGF, FGF 2,700 5% Human Milk, Transferrin 407,000 5% Human Milk,
Transferrin, Insulin 511,180 5% Human Milk, Transferrin, EGF
513,360 5% Human Milk, Transferrin, FGF 443,840 5% Human Milk,
Transferrin, Insulin, EGF 616,000 5% Human Milk, Transferrin,
Insulin, FGF 550,820 DMEM, Transferrin, Insulin, EGF 8,660 DMEM
6,000 1% Bovine Milk, 3,260 1% Bovine Milk, Transferrin 157,260
2.5% Bovine Milk, 4,000 2.5% Bovine Milk, Transferrin 95,000 5%
Bovine Milk, 7,220 5% Bovine Milk, Transferrin 105,000 2.5% Bovine
Colostrum, 377,280 5% Bovine Serum, 556,000 2% Bovine milk 3,600 2%
Bovine milk., insulin 2,320 2% Bovine milk, EGF 1,600 2% Bovine
milk, FGF 2,300 2% Bovine milk, insulin, EGF, FGF 3,460 2% Bovine
milk, insulin, EGF 2,900 2% Bovine milk, insulin FGF 4,400 2%
Bovine milk, EGF, FGF 3,960 2% Bovine milk, transferrin 441,220 2%
Bovine milk, transferrin, insulin 563,420 2% Bovine milk,
transferrin, EGF 470,600 2% Bovine milk, transferrin, FGF 500,720
2% Bovine.milk, transferrin, insulin EGF 641,800 2% Bovine milk,
transferrin, insulin FGF 607,030 20% Bovine milk (3 month) 6,760
20% Bovine milk (3 month), transferrin 417,550
______________________________________
Colostrum-Based Media in Fibronectin-Coated Plates
There is shown in FIG. 8 relative numbers of six of the cell lines,
listed in Table IV below, grown in three media: 10% calf serum in
DMEM (solid bars); colostrum (between 0.5 and 20%, depending on
cell type) in DMEM in untreated dishes (open bars); and colostrum
(0.5-20%) in DMEM in fibronectin-treated dishes (hatched bars).
Prior to exposure to test media, all cell types were detached from
culture dishes by incubation with 1 ml of 0.25% trypsin and 0.2%
EDTA in distilled water. An equal volume of soybean trypsin
exhibitor (Type 1-S, Sigma, 0.25% in phosphate-buffered saline) was
then added to stop the reaction. Cells were diluted in
unsupplemented DMEM and then plated onto untreated or
fibronectin-treated 24-well microliter plates (16 mm diameter,
Costar, Cambridge, MA). Approximately three hours after plating,
the supernatants were removed and the cultures then fed every 3-4
days by removing the culture supernatants and adding 0.5 ml of the
appropriate medium.
To coat plates with fibronectin, human plasma fibronectin
(Collaborative Research, Inc., Waltham, MA) was suspended in DMEM
(25 .mu.g/ml) and 0.4 ml were added to each well of the plate to
attain a final fibronectin concentration of 5 .mu.g/cm.sup.2. The
plates were incubated at room temperature for at least 30 minutes,
the supernatants aspirated, and the cell suspension added
immediately.
FIG. 8 shows that four of the six cell lines grew well in colostrum
in untreated plates, while the two fibroblast lines grew in
colostrum only when the plates had been pretreated with plasma
fibronectin. The last two graphs of FIG. 8 show that fibronectin
allowed the fibroblast cells to achieve a cell density which was
44% of that attained in serum. The colostrum concentration yielding
maximal growth for each cell line (in treated and untreated plates)
was: Vero, CV-1, and F2408, about 2% colostrum; MDCK, 2.5%
colostrum, NRK, about 5% colostrum; NIL8, 2% colostrum with insulin
(10 .mu.g/ml) and transferrin (10 .mu.g/ml). Useful colostrum
concentrations for use with fibronectin will ordinarily range from
0.5% to 25%, v/v. Besides insulin and transferrin, EGF or FGF can
also be added. Useful concentration ranges are: transferrin, 1 to
25 .mu.g/ml medium preferably approximately 10 .mu.g/ml; insulin, 0
to 10 .mu.g/ml preferably approximately 10 .mu.g/ml; EFG, 0 to 10
ng/ml; FGF, 0 to 100 ng/ml.
TABLE III
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Cell Lines and Strains Cell Type Description Source Reference
__________________________________________________________________________
MDCK Madin-Darby canine Flow Laboratories Leighton et al. kidney
epithelial Rockville, MD (1969) Science 163, 472 CV-1 African green
monkey Dr. C. Scher, Sidney Jensen et al. kidney epithelial Farber
Cancer (1964) P.N.A.S. Center, Boston, MA U.S.A. 52, 53 Vero
African green monkey Dr. C. Scher. Simizu et al. kidney cells
(1967) Proc. Soc. Exp. Med. 125, 119 NRK Normal rat kidney Dr. C.
Scher Duc-Nguyen et al. Cells (1966) J. Bacteriol. 92, 1133 F2408
Fischer rat embryo Dr. C. Basilico Frieman et al. cell line New
York University (1973) P.N.A.S. School of Medicine U.S.A. 70, 2415
New York, NY NIL8 Cloned hamster Dr. R. Hynes Diamond et al. embryo
cell line Mass. Institute (1967) Int. J. of Technology, Cancer 2,
143 Cambridge, MA 3T3 Balb/c 3T3, Dr. C. Scher Todaro et al. clone
A31 (1963) J. Cell Biol. 17, 299 LRl Lewis rat embryo Dr. M.
Klagsbrun Steimer et al. cells, Passage 2 (1977) J. Virol. 23, 133
FS4 Human foreskin Dr. D. Giard None fibroblasts, Mass. Institute
Passage 20 of Technology, Cambridge, MA Smooth Bovine aortic Dr. B.
Zetter, Prepared by a muscle Smooth muscle Children's Hospital
modification of the cells cells, Passage 7 Medical Center, method
of Ross (SMC) Boston, MA (1971) J. Cell Biol. 50, 172 Chondo-
Bovine articular Dr. M. Klagsbrun Klagsbrun (1979) cytes (BC)
cartilage chondro- in Methods in cytes, Passage 2 Enzymology LVIII,
Jakoby et al. eds., pp. 560-564
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Milk-Based Media in Fibronectin-Coated Plates
There is shown in FIG. 9 relative numbers of the cell lines listed
in Table IV, above, grown in three media: 10% calf serum (solid
bars); 10% day-80 milk plus fibronectin (open bars); 10% day-80
milk plus fibronectin plus one or more additives (hatched bars;
T=transferrin, I=insulin, E=epidermal growth factor). Cell cultures
and plates were prepared as described above for FIG. 8.
FIG. 9 shows that all 11 cell lines tested grew well in milk-based
media in fibronectin-coated dishes, although some of the lines
required additional factors to attain maximal growth, and one line,
N1L8, did not grow at all in the absence of transferrin and
insulin.
Milk concentrations will vary, depending on cell type cultured, and
on the presence of additives. Useful concentrations will ordinarily
range from 0.5% to 25% of the medium v/v preferably about 10%. FGF
can also be added to the medium. Useful concentration ranges for
additives are: transferrin, 1 to 25 .mu.g/ml medium; insulin, 0 to
10 .mu.g/l; EGF, 0 to 10 ng/ml; FGF, 0 to 100 ng/ml.
Measuring Malignant Cells
Table IV below shows cell counts after 10 days of feeding normal
and 3 types of abnormal rat cell types with media containing 3
month bovine milk, bovine colostrum and bovine serum. Cells are
plated in DMEM (30.times.10.sup.3 cells/well), 35.degree. C., and
after 2 hours DMEM is replaced with the desired medium. Cells are
grown for 12 days. The two transformed cell types are grossly
abnormal malignant cells. The established cell line F2408 is a rat
fibroblast line which has been in culture for several years. These
cells are abnormal, but not to the point of being malignant; they
are analogous to premalignant cells.
Table IV shows that the two malignant cell types proliferate in all
three media, including milk, while normal fibroblast cells fail to
proliferate in milk or colostrum. Thus, for a cell sample
containing both an unknown concentration of malignant cells and
cells which do not proliferate in colostrum, e.g., normal rat
fibroblast cells, the rate of cell proliferation in milk or
colostrum-containing media is directly proportional to malignant
cell concentration. If the sample is suspected of containing normal
cells, e.g., epithelial cells, which proliferate in colostrum, the
method is carried out with milk, not colostrum.
Because the cell line cells proliferate in colostrum but not in
milk, the method can be used, for cell types such as fibroblasts
which do not proliferate in colostrum, to measure premalignant
cells; the rate of cell proliferation in colostrum is directly
proportional to the concentration of premalignant cells. After this
step, the method can be repeated with milk to determine the ratio
of premalignant cells to malignant cells; only the malignant cells
grow in milk.
Concentrations of milk and colostrum used in the method will vary,
depending on such factors as cell type and age of milk. Useful milk
concentrations will ordinarily range between 1% and 25% (v/v), and
useful colostrum concentrations will ordinarily range between 0.5%
and 25% (v/v).
TABLE IV
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TEMPERATURE- SENSITIVE RSV NORMAL RAT ESTABLISHED RAT RSV
TRANSFORMED TRANSFORMED RAT MEDIUM FIBROBLASTS CELLS (F2408) RAT
CELLS CELLS (35.degree.)
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DMEM 3,600 8,000 6,000 3,200 DMEM + 10% Calf 100,000 830,000
1,400,000 1,500,000 SERUM DMEM + 10% 3,200 50,000 500,000 520,000
Bovine Colostrum DMEM + 10% 1,600 7,000 140,000 160,000 Bovine Milk
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Other Embodiments
Other embodiments are within the following claims. For example, the
growth-promoting concentrate purified from colostrum can be used in
any of the aspects of the invention employing unpurified colostrum.
Similarly, milk and colostrum which have been concentrated,
powdered, previously frozen, or otherwise treated in a way which
does not extinguish their potency can be used whenever milk and
colostrum and employed. Fibronectin can be provided in the
milk-based media, as well as on the culture plates. The milk and
colostrum-based media can contain fibroblast growth factor as well
as the other optional additives. Also, colostrum, in addition to
being useful in tissue culture, may be useful in humans, e.g., in
treating ulcers, or in counteracting the side effects of
chemotherapy. Although human plasma fibronectin is preferred,
fibronectin from any source can be used. Similarly, milk and
colostrum from any mammal can be used.
* * * * *