CA1177424A - Composition for cell culture - Google Patents

Abstract

ABSTRACT
A cell culture medium not relying on fetal bovine serum as a necessary component is disclosed. The medium optimally comprises from about 80-90% of a chemically defined nutrient medium and from about 10-20% of a natural biological fluid which has been treated by contact with "feeder" culture of smooth muscle cells for about 24 hours. The treatment may involve only natural fluid or such fluid in admixture with nutrient medium. Thymidine is then added in an amount such that its final concentration is between 10-4M and 10-6M. Such medium has been found suitable for the growth of mammalian cells, including mammalina endothelial cells. Additionally, lots of fetal bovine serum found unsuitable to support cell growth may be treated by contact with smooth muscle cells for about 24 hours and thereby converted to acceptable lots.

Description

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 ~ ~'7'7~

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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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 - ~7'7~ ~

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 ~ ~'7~ ~

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 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.

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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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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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Claims (28)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition for use in culturing mammalian cells in vitro, which composition is capable of supporting the growth of mammalian endothelial cells, comprising:
(1) a natural mammalian biological fluid of the serum type, (2) a chemically defined nutrient medium, and (3) thymidine or a thymidine precursor in an amount such that the final concentration of thymidine in said composition is equal to from about 10-4M to about at least said natural mammalian biological fluid but not said thymidine or thymidine precursor having been contacted for a period of from about 18 hours to about 36 hours with a feeder culture of mammalian smooth muscle cells.

2. A composition according to claim 1 in which said natural mammalian biological fluid is present in an amount of from about 10-20% by volume and the balance of the ingredients are from about 90% to about 80% by volume of said chemically defined nutrient medium, said thymidine or thymidine precursor in the amount specified, and trace amounts of antibiotics.

3. A composition according to claim 1 or 2 in which said natural mammalian biological fluid consists of calf serum, mammalian amniotic fluid, human plasma derived serum or a mixture of two or more of such fluids.

4. A composition according to claim 1 or 2 in which at least a part of said natural mammalian biological fluid is fetal bovine serum from a lot found unacceptable, prior to contact with a mammalian smooth muscle cell feeder cul-ture as specified in claim 1, for use in culturing endo-thelial cells.

5. A composition according to claim 1 or 2 in which said natural mammalian biological fluid is present in an amount of from about 8% to about 12% by volume to which, after contacting with a mammalian smooth muscle cell feeder culture for a time in the order of 24 hours, from about 2%
to about 8% by volume of fetal calf serum is added.

6. A composition according to claim 2 in which at least a part of said chemically defined nutrient medium has not been subjected to mammalian smooth muscle cell feeder culture treatment.

7. A composition according to claim 2 in which said mammalian smooth muscle cell feeder culture treatment is conducted for a period of essentially 24 hours.

8. A lyophilized composition according to claim 1, 2 or 6.

9. A composition according to claim 1, 2 or 6 in the frozen state.

10. A process of making a composition for use in culturing mammalian cells in vitro, which composition is capable of supporting the growth of mammalian endothelial cells, comprising:
(I) conditioning a natural mammalian biological fluid of the serum type by contacting said natural mammalian biological fluid with a feeder culture of mammalain smooth muscle cells, under culturing conditions, for a period of from about 18 hours to about 36 hours, (II) admixing said natural mammalian biological fluid, either prior to or subsequent to said conditioning period, with a chemically defined nutrient medium, and (III) admixing said natural mammalian biologi-cal fluid, subsequent to said conditioning period, with thymidine or a thymidine precursor in an amount such that the final concentration of thymidine in said composition is equal to from about 10-4M to about 10-6M.

11. A process according to claim 10 in which the composition produced comprises about 10-20% by volume of said natural mammalian biological fluid, about 90-80%
by volume of said chemically defined nutrient medium, said thymidine or thymidine precursor in the amount specified and trace amounts of additives.

12. A process according to claim 10 in which the nautral mammalian biological fluid being conditioned com-prises essentially only said natural mammalian biological fluid and trace amounts of additives.

13. A process according to claim 11 or 12 in which said additives comprise antibiotics.

14. A process according to claim 10, 11 or 12 in which said natural mammalian biological fluid is calf serum, mammalian amniotic fluid, human plasma derived serum or a mixture of two or more of such fluids.

15. A process according to claim 10, 11 or 12 in which at least a part of said natural mammalian biological fluid is fetal calf serum from a lot found unacceptable, prior to contact with said mammalian smooth muscle cell feeder cul-ture, for use in culturing endothelial cells.

16. A process according to claim 10, 11 or 12 in which said natural mammalian biological fluid is present during said conditioning period in an amount of about 8% to 12% by volume and fetal bovine serum in an amount of from 2% to 8% by volume is added before use of the finished composition as a cell culture medium.

17. A process according to claim 11 in which at least a part of said chemically defined nutrient medium is not subjected to said conditioning period.

18. A process according to claim 10, 11 or 12 wherein said conditioning period is essentially 24 hours.

19. A process according to claim 10, 11 or 12 in which said composition is lyophilized and stored before use in tissue culture.

20. A process according to claim 10, 11 or 12 in which said composition is quick frozen prior to storage or use in tissue culture.

21. A method of culturing mammalian cells in vitro which comprises:
(I) isolating a desired type of mammalian cell from a mammalian specimen, and (II) adding to said cell isolate a composition comprising:
(1) a natural mammalian biological fluid of the serum type, (2) a chemically defined nutrient medium, and (3) thymidine or a thymidine precursor in an amount such that the final concentration of thymidine in said composition is equal to from about 10-4M
to about 10-6M, at least said natural mammalian biological fluid but not said thymidine or thymidine precursor having been contacted for a period of from about 18 hours to about 36 hours with a feeder culture of mammalian smooth muscle cells, prior to being added to said cell isolate.

22. A method according to claim 21 in which said natural mammalian biological fluid of the serum type is calf serum, mammalian amniotic fluid, human plasma-derived serum, or a mixture of two or more of such fluids.

23. A method according to claim 21 in which at least a part of said natural mammalian biological fluid is fetal calf serum from a lot found unacceptable for culturing endo-thelial cells prior to contact with a mammalian smooth muscle cell feeder culture.

24. A method according to claim 21, 22 or 23 in which the mammalian cells isolated and thereafter cultured are mammalian endothelial cells.

25. A method according to claim 21, 22 or 23 in which the mammalian cells isolated and thereafter cultured are mammalian smooth muscle cells.

26. A method according to claim 21 in which said natural mammalian biological fluid is present in about 8% to 12% by volume and, after contacting said natural mammalian biologi-cal fluid with mammalian smooth muscle feeder culture, from about 2% to about 85 by volume of fetal calf serum is added thereto.

27. A method according to claim 21, 22 or 23 in which trace amounts of antibiotics are present in said composition.

28. A method according to claim 21, 22 or 23 in which said natural mammalian biological fluid is contacted with mammalian smooth muscle feeder culture for a period of essentially 24 hours.