Note: Descriptions are shown in the official language in which they were submitted.
Z ~L~ 7
¦BACKGROUND OF THE INVENTION
¦ Many types of research rely on tissue culture techniques
¦to produce cells for experimental purposes. For example,
¦tissue culture ~echnlques are employed in the production of
¦vaccines, in the assay of viruses, in antibody assay and
production, in interferon assay and production, in virus
¦isolation procedures, in propagation of established virus
strains, and in other procedures. Widespread use of tissue
l cultures has given economic significance to various cell
I lines propagated in vitro which are employed in assay pro-
cedures, genetic studies, vaccine production, and a number
of other areas.
Numerous media for the propagation and maintenance of
l cells in tissue culture are known. One type of culture
¦ medium is chemically defined medium which has known chemical
¦composition, both quantitatively and quali~atively, in
¦contrast to natural or undefined media containing natural
¦products such as animal serum, embryo extracts, yeast hydro-
¦lysates, and the like of unknown or incompletely known
~0 ¦chemical composition. Examples of chemically defined media
are Medium 199 of Morgan, Morton, and Parker, Proc. Soc._ Exptl.
Biol. and Med. 73, 1-8 (1950), Eagles Basal Medium, Science
122, 501-504 (1955), Science 123, 845-847 (1956), J. Biol.
l Chem. 226, 191-206 (1957), and Eagle's Minimum Essential
¦ Medium, Science 130, 432-437 (lg59). Such media may also
include various balanced salt solutions (BSS) such as Hanks
BSS, Earle BSS, Dulbecco Phosphate-buffered saline, and Puck
Saline G. Details for preparation and formulation of such
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¦ media are found in standard texts such as Merchant et al.,
Handbook of Cell and Organ Culture, Burgess Publishing co.,
~Minneapolis (19~4). Such chemically defined media consist
¦of defined amounts of carbohydrates, amino acids, minerals,
¦vitamins, salts and other components. Other examples of
¦ chemically defined media are described in U.S. Patent Nos.
¦ 4,072,565; 4,055,466 and 3,887,430.
¦ Media are also classified with respect to their ability
¦to support cell metabolism and proliferation. Those which
¦support cellular proliferation are generally referred to as
¦ "growth media". Most growth media have as their main component
¦ a BSS or chemically defined medium which must be supplemented
¦with one or more natural products such as fetal bovine
¦ serum (FBS), sometimes referred to as fetal calf serum. At
¦ the present state of the art most cell types cannot be grown
in chemically defined media alone. It is believed that this
may be due to the absence of an unknown natural growth
factor or combination of natural factors. Typical growth
media include either Eagles Basal Medium or Medium 199
supplemented with 10 to 20 percent fetal calf serum.
One example of a growth medium comprising a serum
supplement was described by Earle et al. in U.S. Patent No.
2,653,021. The medium employed was 40% horse serum, 20%
chick embryo extract and 40% saline which was conditioned
by growing a massive culture of the same or similar tissue
ell type in the medium for a time long enough for the cells
to adapt the nutrient medium environmentally to the desired
cell type and enable the growth therein, by cloning, of a
massive culture of the desired cell type from a single
living cell. Such conditioning was accomplished by growing
~.~7'~
about 1% by volume of a similar tissue cell type therein for
about 24 hours. The conditioning cells were then removed and
sterile glucose added to the solution.
Fetal bovine serum is currently widely used to supplement
chemically defined media. For cells known to be difficult to
culture, especially endothelial cells, fetal calf serum has
heretofore been deemed a necessary medium component. The most
widely used medium for endothelial cells consists of 80
chemically defined medium and 20~ fetal bovine serum.
Unfortunately, there are disadvantages to this need for fetal
calf serum. Recently ~here has been a severe fetal calf serum
drought which is adversely affecting tissue culture work and
recombinant DNA research, Nature 285, 63 (1980). The shortage
is the result of changes in world agricultural policies and
practices along with an increase in the research uses for, and
the amount of research done that requires, fetal bovine serum.
Further, large quantities of fetal bovine serum are consumed
by the hybridoma technique whereby specific antibodies are
produced on a large scale. Due to the short supply, serum
prices have escalated to the point where some research must be
cut back or else alternatives to fetal bovine serum must be
found.
Another problem associated with the use of fetal bovine
serum is the variation between different lots of serum. In
the past, scientists have been allowed to test a particular
lot of the serum on the particular cells to be cultured before
accepting such lot for the desired use, but with the current
shortage, such testing is no longer permitted by
suppliers who can sell as much of this serum as they can
get. Testing of fetal bovine serum lots has generally been
~:~'7'7~ ~
performed with demanding cells such as endothelium. Endothelium
was first successfully grown in culture as of about 1973
See Jaffe, E. A., Nachman, R. L., Becker, C. G. and Minick,
C. R., "Culture of Human Endothelial Cells Derived from
Umbilical ~eins," J. Clin. Invest., 52, 2745-2756 (1973);
and Gimbrone, M. A., Cotran, R. S. and Folkman, J., "Human
Vascular Endothelial Cells in Culture, "J. Cell Biol., 60,
673-684 (1974). Endothelial cells are especially difficult
to grow in culture relative to most other cell types. Their
cultures are not considered successful unless they meet
certain distinguishing morphological characteristic standards --
e.g. they must exhibit contact inhibition at confluence with
resultant monolayer growth, and must further exhibit a
smooth "cobblestone" appearance in the monolayer. The
cobblestone is due to the small size and cuboidal shape of
endothelial cells. It has accordingly been reasoned that a
fetal bovine serum lot on which an endothelial culture can
successfully be grown is one on which almost any type of
cell will successfully grow. Since such testing is no longer
allowed, a need exists to find substitutes for fetal bovine
serum and to render acceptable those lots of fetal bovine
serum that do not come up to standard. Moreover, while
other types of biological fluids may support the growth of
some cell types, usually they do so only with poorer growth
rates.
In short, not only the increasing ~navailability of
fetal bovine serum but the known wide variations in its
effectiveness as a growth medium from lot to lot, particularly
with demanding cell types such as endothelium, lends im-
portance to the discovery of a uniform, reliable cell growth
medium of natural biological origin.
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The present invention provides an answer to the supply
and variability problems of fetal bovine serum. It has been
found that the need of pulmonary endothelial cell cultures
for fetal bovine serum of acceptable quality is obviated if
biological natural fluids heretofore believed unacceptable
such as calf serum, amniotic fluid or human plasma-derived
serum, in admixture with Medium 199 or another of the chemically
defined media, is trea~ed for about 24 hours by contact with
bovine pulmonary artery smooth muscle cells, followed by a
suitable addition of thymidine. Using a treated medium of
this invention, endothelial cells can surprisingly be cloned
without the presence of acceptable-tested fetal bovine serum.
There have been marketed products said to contain a
growth factor or growth supplement (Collaborative Research,
Waltham, MA) for use in culture media, but the associated
trade literature suggests a culture method in which 20%
fetal bovine serum is added to the commercial product contain-
ing growth supplement.
The use of thymidine alone to enhance the growth of
endothelial cell cultures has previously been reported by
McAuslan, B. R. et al., J~ Cell PhYsiol. 100, 87 (1979).
The article states that Medium 199 with 2.5-20% fetal calf
serum supplemented with 10-5M thymidine ~ se, or with a
chemical agent such as a folinic or folic acid that will
generate thymidine in situ, enhances the cell growth of
bovine corneal and aortic endothelial cells. Confluence
appears to have been achieved at fetal calf serum concen-
trations of the order of 10% in the presence of thymidine.
Other culturing techniques for endothelial cells have
been reported but all substantially differ from that of this
invention and all require the use of large quantities of
fetal bovine serum. Examples of such techniques are found
in the Jaffe et al. and Gimbrone et al. reference supra and
in Ryan, U.S. et al., Tissue and Cell, 12, 4 (1980), Ryan,
U.S. et al., Science, 208, 748 (1980), Ryan, U.S. et al.,
Tissue and CP11, 8, 125 (1976), and Habliston, D.L. et al.,
Amer. Rev. of Res Dis., 119, 853 (1979).
SUMMARY OF THE INVENTION
Unlike the culture media in current use, especially for
endothelial cells, which generally comprise 10-20% fetal
bovine serum and 80-90% chemically defined medium, the
present invention provides a uniform and reliable culture
medium. The medium of this invention is not subject to
fluctuation in availability or quality of any particular
natural serum-type biological fluid and hence can be kept in
ready and stable supply.
The new medium comprises, in addition to chemically
defined medium, a natural biolo~ical fluid such as calf
serum, amniotic fluid, human plasma-derived serum, fetal
bovine serum found upon testing to be ~ se inadequate to
support the desired cPll growth or another fluid heretofore
deemed per se unusable, which natural fluid has been subjected
to a special treatment. The treatment involves contacting
the natural fluid alone or in admixture with chemically de-
fined medium, with a culture of bovine pulmonary artery
smooth muscle cells for a period in the order of about 24
hours, followed by the addition of thymidine or a thymidine
precursor in an amount to provide a final concentration of
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thymidine of from about 10 4 to about 10-6M, preferably 10-5M.
It is not known precisely what function the smooth muscle cells
play. It is theorized that they may serve to detoxify the
fluids, to add specific growth factors, to remove antigens
and antibodies that might inhibit cell growth or they may
perform a combination of such functions and others not yet
perceived. Similarly, the precise function of thymidine is
not completely understood, though it is clear that the medium
functions more satisfactorily after this treatment than if it
is omitted.
The growth mediurn of the present invention usually com-
prises no more than about 30%, and preferably about 10-15%
of a mammalian natural serum type fluid and about 70-90%
of chemically defined medium plus antibiotics and other de-
contaminants, etc. in minor amounts. It has been found that a
medium comprised of 10% calf serum and 90% Mediurn 199 treated
for about 24 hours with bovine pulmonary artery smooth muscle
cells and 10-5M thyrnidine functions as well as an otherwise
similar culture medium containing 20% of an acceptable growth
supporting lot of fetal bovine serum. Similarly 15% amniotic
fluid or 15% PDS and 85% of Medium 199 has been similarly
treated with smooth muscle cells and thereafter supplemented
with thymidine of 10-4 to 10-6M concentration to yield a
very acceptable growth supporting mediurn. Culture media
containing less than about 10% of natural fluid, however, do
not usually appear to function well, nor in most instances
do media containing substantially in excess of 20% natural
fluid, though greater and lesser amounts may be used in
special situations. This is consistent with past observations
when the natural fluid was untreated fetal bovine serum from
an acceptable lot.
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~ ~17'7~2~
A special embodiment of the invention enables the con-
version of unacceptable lots of fetal bovine serum into
acceptable lots using the treatment of this invention. In
addition, using the medium of the invention, acceptable lots
of fetal bovine serum can be stretched, e.g. by reducing the
amount used in an endothelial culture to about 2 to 8% of
the total culture medium, employed in conjunction with from
about 18 to about 8% of other natural fluid treated in
accordance with this invention. This is in contrast to normal
use of 10-20% fetal calf serum in culture media for endothelial
cells.
Natural biological fluids heretofore thought to be
unacceptable for endothelial cell growth and unacceptable
lots of fetal bovine serum, when treated according to the
teaching of this invention, are not only rendered capable of
supporting endothelial cell cultures but become generally
useful for culture of less demanding cell lines with less
strict growth condition requirements than those for endothelial
cells.
DETAILED DESCRIPTION OF THE INVENTION
The cells used for the treating process are vascular
smooth muscle cells obtained from bovine pulmonary artery,
and usually from the same batch of vessels from which
bovine endothelial cultures are derived.
Typically, 12 veal plucks (heart, lungs and attached
great vessels of young calf) are obtained from a local
slaughterhouse. The pulmonary artery is removed, washed
with several changes of sterile saline containing 3 x the
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recommended strength of antibiotics and then slit down the
center so that it lies flat with the luminal surface uppermost.
Under a laminar flow hood, the cells of the endothelial layer
are removed by gently scraping with a scalpel and collected
into centrifuge tubes containing Medium 199 and antibiotics.
These isolates are subsequently seeded into flasks and become
endothelial cultures.
After removal of the endothelial layer (intima), the
underlying layer of the vessel (media) consists mainly of
smooth muscle cells. In order to obtain smooth muscle
cultures the outer layer of the vessel (adventitia) is dis-
sected away and explants of the media layer are made. These
are small blocks of tissue approximately 1-2 mm square ob-
tained by mincing. Six to eight explants are placed into a
25 cm3 flask, well separated. The flask is upended and 2
mls of culture medium are added. The flask is left standing
for at least two hours at room temperature to facilitate
attachment of explants to the base of the flask. The flask
is then gently laid flat on the incubator shelf, such that
culture medium slowly covers the bottom and does not dislodge
the explants. After one week the explants are observed and
fed with 5 mls of culture medium. Smooth muscle cells grow
out from the explants and colonize the flask. The flasks
are usually ready to be split 1:2 in 14 days. When confluence
is reached in 25 cm2 (T25) flasks, the smooth muscle cells
are transferred to 75 cm2 ~T75) flasks. Explants float out
during culture medium changes. Use of the culture to treat
medium according to this invention, by contacting therewith
for about 24 hours, can begin when the flask is mostly
covered by cells (approximately 1 million). Normally, T75
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post-confluent cultures containing three or four layers of
smooth muscle (34 million cells) are used to treat 20 mls
culture medium over a 24 hour period.
A. Treating Procedure
The growth medium used for the propagation of bovine
vascular smooth muscle cells may, in accordance with this
invention, be the same as that placed on smooth muscle cultures
for treating. It consists of:
Medium 199 Made up from powder using sterile water.
Passed through a 0.2 ~ filter. Stored
in refrigerator for 2 weeks maximum.
NaHC03 Necessary to stabilize pH in 'open'
systemJ4.35 gms/liter
Serum Heretofore 10% of an acceptable lot
of fetal bovine serum (FBS) has been
used. It has been found according to
this invention that various combina-
tions and percentages of unacceptable
FBS, calf serum (CS), human amniotic
fluid (HAF), and human plasma derived
serum (HPDS~, treated according to
this invention may be substituted.
Antibiotic of choice e.g. Gentamycin (50 ,ug/ml)
Smooth muscle cultures are normally fed twice weekly.
Thus, a typical schedule would be as follows:
Day l~a.m. Feed smooth muscle cultures (SMC).
20 mls - 30 mls per 75 cm3 flask.
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Day 2, a.m. Harvest 2~ hour treated medium from
SMC, pass through 0.22u filter to
eliminate any cells or cellular
debris or contaminants. Add
thymidine to 10 5M (1 ml of 10 3
stock per 100 mls Ml99).
Feed treated medium + thymidine to
endothelial cultures (10 mls per
T25), 20 mls per T75, 8 mls per 60 mm
dish, 3 ml per 35 mm dish or
multiplate.
Any treated medium not immediately fed to endothelial cells
can be stored in the refrigerator for one week. To store for
longer periods, deep freezing or lyophilization is
recommended.
If larger amounts of treated medium are required, medium
can be harvested daily from the same culture of smooth muscle
cells (SMC) for at least a week with no apparent diminution of
treating effect. The SMC cultures require a minimum of care.
Flasks of SMC providing the medium of this invention may be
passaged as seldom as once per month.
In an alternative embodiment of this invention, only the
serum or other natural fluid is treated with the SMC culture
and the treated product is kept chilled, then frozen or
lyophilized, packaged and shipped. In this instance, the user
adds nutrient medium to bring the total medium to the
appropriate ingredient concentration levels.
B. Isolation and Culture of Endothelial Cells
Cal~ pulmonary artery endothelial cells are obtained by
the method of Ryan, U.S., et al, Science 208, 7~8 (1980).
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Reagents for the growth medium of the invention for use
with endothelial cells are: Medi~ 199 made up as 10 X in
deionized sterile water from powder filtered through a 0.22u
filter and stored in a refrigerator, serum (which has either
been stored over the long term in deep freeze or simply re-
frigerated over a short term such as a week or less), thymidine
made up as 10-3M in sterile deionized water, filtered through
a 0.22,u filter, (which may be stored in small amounts frozen),
and antibiotics (kept frozen except for gentamycin, which is
refrigerated).
Typically the reagents for the growth medium for endo-
thelial cells consist of 90 ml sterile water, 10 ml of lOX
Medium 199, 5.8 ml of NaHC03 (4.35 g/l) which is necessary to
stabilize the pH in calf pulmonary artery endothelial cultures,
10-5M thymidine (1% total or less by volume, usually 1 ml of
10-3M stock), an antibiotic of choice (50 ,ug/ml gentamycin),
10% serum in various combinations of FBS (unacceptable lots),
CS, HAF, HPDS and the like. The medium may be stored in the
refrigerator up to one week.
According to this invention up to 20% of the total
medium, (defined for this application as natural fluid plus
chemically defined nutrient medium, with or without other
additives in minor amounts) can be any natural biological
serum-type fluid of mammamian origin such as unacceptable
fetal bovine serum, calf serum, human, or other mammalian
amniotic fluid, human plasma derived serum, whole animal
serum, or another similar natural fluid or mixture of such
fluids. The preferred amount of the natural fluid is 10%,
but up to 20% or even 30% or more may be used in some cir-
cumstances. Usually at least 80% of the total medium is a
7'7~
chemically defined nutrient medium with the preferred amount
in the range of 80-90~. The invention contemplates the use of
95~ or even 98~ chemically defined nutrient and a
correspondingly lesser amount of natural fluid in special
circumstances, or with some cell lines. The chemically
defined medium may be a chemically defined medium such as
Medium 199, or another standard medium such as those discussed
above and others of the type generally referred to by workers
in the cell culture art as basal media.
It is preferred that an an-tibiotic be added to the
culture medium before the smooth muscle treatment process so
that bacterial contamination is avoided. Examples of suitable
antibiotics are gentamycin, penicillin, streptomycin,
fungizone, and others.
Either the natural fluid alone or a mixture of natural
fluid and nutrient medium is placed on "feeder" culture of
smooth muscle cells for about 24 hours, removed and filtered
through a 0.22~ filter. The "feeder" treated mixture may then
be fed to the endothelial cells. Where only natural fluid has
been "feeder" treated, nutrient medium is first added in
requisite amounts and the resulting mixture is used to feed
endothelial cells. It should be noted that thymidine is not
added to the smooth muscle cell culture, but should be added
immediately before feeding endothelial cells for maximum
effect, in cases where the medium is not to be stored at all,
or to be stored in unfrozen condition for no more than a week.
Alternatively, thymidine may be added just before the medium
is quick-frozen and the medium may then be packaged and stored
or shipped.
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The feeding schedule for endothelium is semi-weekly,
every 3-4 days in amounts of 10 ml per T25, 25-30 per T75,
8.0 ml per 60 mm dish. The cells are cultured a~ 37C in a
5% C02, 95% air incubator at approximately pH 7.4.
The endothelial cells are subcultured by first washing
with calcium and magnesium free Puck's saline and then by
washing with two small volumes of 0.05% trypsin and 0.02%
ethylenediaminetetraaceticacid (EDTA) which in less than 1
minute detaches the endothelium. GIBC0 1/ TRYP/EDTA is diluted
10-fold in calcium magnesium free Puck's and frozen and
filtered through a 0.22 ,u filter. The cells are then taken
up in fresh growth medium, triturated gently to break up clumps,
and divided among new flakes. For a confluent layer the
split is 1:2 and for a heavy culture, the split is 1:3.
All procedures described above for culture of endothelium
and treating of medium on smooth muscle cells can be conducted
using standard equipment common to all tissue culture facil-
ities, namely C02 incubator, laminar flow hood, autoclave or
sterilizing apparatus, plastic disposable cultureware, dis-
secting instruments, and filtering apparatus.
C. General
It is to be understood that the invention contemplates
culturing mammalian cells of any desired type with the medium
of the invention.
~ 1/ Trad ame - Grand Island Biolo~ical Company
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In addition to plating cells in flasks as described in
detail above, any effective cell culturing technique may be
utilized including culturing on glass, plastic and other types
of beads such as polylysine-treated glass beads, Sephadex*;
polystyrene or polyacrylamide beads sold by Bio-Rad
Laboratories under the trade name Bio Carrier beads and by
Pharmacia under the trade name Cytodex*.
The amount of thymidine added must be controlled narrowly
for best results. In general, thymidine addition should be so
controlled that the final concentration is between 10 and
10 6 molar. When Medium 199 is the chemically defined medium,
all thymidine is added unless the cells to be cultured contain
thymidine or a related compound such as folic or folinic acid.
With other chemically defined media, it is appropriate to
determine the content of thymidine or thymidime precursors and
then calculate the amount of thymidine, if any, to be added to
keep the final concentration within the limits specified.
Careful control is necessary because too much thymidine
adversely affects the cells whereas too little fails to
provide the desired degree of growth.
The time of smooth muscle cell contact treatment with
either the natural fluid or total medium of this invention
must be kept to approximately 24 hours. Variance of two to
three hours in either direction appears to have no significant
effect, but when treatment'time is 12 hours or less, the
medium is relatively ineffective for growth and when treatment
time is 48 hours, deleterious effects upon cells are ohserved.
As used herein "approximately 24 hours" encompasses from about
18 to about 36 hours.
* Trademark
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The tempera~ure of medium treatment and cell growth may
be somewhat variable. Ambient conditions may be utilized~
but the preferred temperature is 37+2C, as maintained in a
standard C02 incubator. The medium treatment should further
be conducted at essentially neutral p~I, with pH 7.4 as
obtained by phenolphthalein endpoint adjus~ment, being
preferred. The pH for cell culture will depend upon preferred
pH for the cell type to be grown.
Moreover, it is contemplated that smooth muscle cells
of pulmonary arteries of mammalian species other than the
bovine may be utilized for the treating process. Also, smooth
muscle from other mammalian organs is deemed usable.
Where the medium is to be packaged and shipped or stored,
it is contemplated that it be concentrated by removal of at
least 90% of its water content after smooth muscle treatment,
preferably by lyophilization. The lyophilized material may
then be quic~ frozen or may be packaged and shipped in lyo-
philized form. Alternatively, to treating either natural
fluid alone or total medium with SMC, a mix containing less
than optimum nutrient may be so treated. Whenever the SMC
treatment involves anything other than total medium, careful
attention must be given to the smooth muscle cell culture to
insure that its ability for treating new material is main-
tained -- or if not so maintained that fresh smooth muscle
culture is promptly substituted for spent culture. In this
regard, ability to support the culture of endothelium may
be utilized to test treated medium to determine whether its
smooth muscle treating culture remains effective or is spent.
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It is within the scope of the invention to lyophilize
and/or quick-freeze, and then package treated medium, whether
or not concentrated to low water content, either before or
after thymidine addition. Quick frozen concentrated medium
without thymidine is made ready for use by thawing, diluting
to the appropriate water content for culturing and adding
thymidine. Otherwise similar lyophilized medium needs only
to be diluted to appropriate water content and thymidine
treated. It is further contemplated, according to this in-
vention, that when the natural fluid to be used in the culture
medium, at suitable dilution, either without nutrient medium
or with a reduced amount is contacted with smooth muscle cells,
the treatment may optionally be in the presence of antibiotics
or other desired decontaminating agents and that in such cases
the material recovered from the SMC culture is concentrated
if desired and then lyophilized and/or quick frozen before or
after thymidine addition. In such cases, chemically defined
nutrient medium is added to the appropriate strength after
thawing the treated component.
One notable aspect of the invention is that it enables
the ~rowth of cell cultures from one species on medium
containing treated natural serum-type fluid from another speci.es,
e.g. growth of human endothelium on treated calf serum, and
the like.
In the following examples, all starting media and enzymes
are commercially available. FBS and CS can be purchased,
HAF was obtained from a private source, HPDS was prepared
in the laboratory using outdated human plasma from the blood
bank.
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Example 1
Bovine smooth muscle cells were cultured as described
previously. The growth medium consisted of 90% Medium 199,
10% calf serum and 50 ~ug/ml gentamycin. After contact with
the smooth muscle culture for approximately 24 hours, the
treated medium was removed, filtered through a 0.22~u filter,
and thymidine was added to a final concentration of 10 5M.
The treated medium was then added to subconfluent cultures of
bovine pulmonary endothelial cells in T25 flasks
(approximately 2 x 106 cells per flask) which had previously
been cultured in a growth medium consisting of treated 90%
Medium 199 and 10% FBS and gentamycin (50 ~g/ml). The
endothelial cell cultures were examined daily for a period of
nine days. Flasks which had received treated medium and
thymidine were confluent within 48 hours, were contact
inhibited at confluence, exhibited a uniform "cobblestone"
monolayer morphology, with no discernable vacuolation of the
cells. In addition, (using methods described in Ryan et al,
1978) flasks which had received treated medium and thymidine
were reactive with antibodies to Factor VIII and showed
abundant angiotensin converting enzyme. By contrast, flasks
which had received 90% Medium 199 and 10% calf serum not
treated by contact with smooth muscle cells for 24 hours
and/or which did not receive thymidine, did not reach
confluence, never assumed a cobblestone morphology and showed
cells of irregular size and shape, many containing vacuoles.
Cells of all flasks had a normal karyology.
Example 2
The procedure of Example 1 was repeated employing a
mixture of 85% Medium 199 and 15% human amniotic fluid treated
with~
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~'7'~
smooth muscle cells for about 24 hours. The treated medium
was harvested, whereupon thymidine was added to a concentra-
tion of 10-5M, and transferred to endothelial cells. The
cell cultures were followed as in Example 1. After a nine
day period the endothelial morphology showed confluent
cobblestone monolayers with few vacuolated cells.
Example 3
Human plasma derived serum was prepared by treating 25 ml
of outdated human blood with 0.9 ml of 5M CaC12 and letting
the mixture stand at room temperature overnight. The coagulum
was loosened and the mixture was centrifuged for 10 minutes.
The serum was passed through a 0.45 ,u filter to remove parti-
culate matter. Using the procedure of Example 1, mixtures
of (a) 5% fetal bovine serum plus 5% plasma derived serum
(PDS), (b) 10% PDS, (c) 15% PDS and (d? 20% PDS with genta-
mycin plus (a) 90%, (b) 90%, (c) 85% and (d) 80% Medium 199,
respectively, were each treated with a smooth muscle cell
culture for about 24 hours. Thymidine (10-3M) was then added
to each medium to give a concentration of 10-5M. Each medium,
when added to endothelial cells, produced essentially confluent
cultures within seven days. Cells grown in medium (a) or (b)
were confluent in 72 hours. Cells grown in media (c) and
(d) reached confluence more slowly and tended to be more
vacuolated than those of (a) or (b).
Example 4
The procedure of Example 1 was repeated to convert a
lot of fetal bovine serum found to be unacceptable for
endothelial cell culture into an acceptable lot. A mixture
of ;0% of an unacceptable lot of fetal bovine serum, 90%
Medium 199, and gentamycin (59 ug/ml) was treated with
7'7~
smooth muscle cells for about 2~ hours, whereupon 10 3M
thymidine was added to a concentration of 10 5M. The
resulting medium produced a confluent endothelial cell culture
within 48 hours, haviny the characteristics of the cultures
described in Example 1.
Example 5
Bovine smooth muscle cells were cultured as described
previously. Post-confluent smooth muscle cultures in T75
flasks were treated as follows: normal growth medium was
removed and the cells were refed with 30 mls of calf serum
(CS) (100%, undiluted) containing gentamycin (50 ~g/ml) and
fungizone (0.25 ,ug/ml) and returned to the incubator (37C, 5
CO23. After approximately 24 hours, the treated serum (CS)
was removed and placed in a refrigerator (4C). The treated
CS was then added to Medium 199 to a final concentration of
10% and thymidine was added to give a final concentration of
10 5M. The medium, thymidine and treated serum, were then
filtered through a 0.22,u filter and added to cultures of
pulmonary endothelial cells in T25 flasks, which had
previously been cultured in a growth medium consisting of
Medium 199 (90%), FBS (10% untreated) and thymidine (10 6M).
The endothelial cell cultures were examined and photographed
daily for seven (7) days, at which time cultures were split
2:1 and carried for a further 7 days. A uniform cobblestone
monolayer morphology was preserved with no discernable
vacuolation of the cells.
It is to be understood that the foregoing examples are
merely illustrative rather than limiting. As those of skill
in the tissue culture art will understand, numerous specific
embodiments are possible within the scope of the invention.
It is intended that the invention is not to be limited except
to the extent required by the appended claims.
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