CA2183980A1 - Method for culturing mammalian cells and insect cells in a medium containing fish serum - Google Patents

Abstract

A method for culturing mammalian cells using fish serum. The method uses serum extracted from the blood of fishes to culture mammalian cells for various purposes. The technique has the key advantages of 1) freedom from mammalian infectious agents that could contaminate cell lines or endanger researchers or recipients of therapeutants derived from mammalian cell culture; 2) consistent and reproducible serum content and quality; 3) low cross-reactivity; and 4) provision of the appropriate serum nutrients to maintain the growth of mammalian cells. Fish serum is used together with designated defined media to allow mammalian cells to grow and populations of these cells to be maintained. Blood serum is derived from captive stocks of fish raised under controlled conditions such that the diet, habitat genetics, life history, and reproductive status of the fish remains substantially consistent and reproducible.

Description

2 21 8 3 9 8 0 1 ` ~ PCT/US94/14221 . . . .
METHOD FOR CULTURING MAMMALIAN CELLS AND INSECT CELLS
IN A MEDIUM CONTAINING FIS~ SERUM
.
Field sf the Invent; on The present invention relates generally to cell culture, and more Spe?~; fi ?~ y to the culture of l j ;~n cells and insect cells using a serum derived from fish. The technique 5 has si?~nif icant advantages over the more commonly used technique of using blood serum derived from fetal calves or other mammals as more fully set forth below.
B l~k?~rOlln?l Of ~he Invent;on Animal cell culture is a basic technique in the fields of 10 biology and ~i ri nf:~. The production of living cells in vitro, in the laboratory, permits numerous applications that would be ?~iff;?-l~lt or impossible in vivo, in the living animal. The culture of animal cells requires a defined medium containing spe?-;fi?- quantities of certain chemicals, and in 15 addition for most cells, up to 1596 of an llnrl.of;n-~?l nutrient medium usually fetal bovine serum (FBS). Serum from newborn calves and other mammals is also used, but FBS is preferred because of its high level of growth factors and low cross-reactivity with other animal cells . FBS or other l; ~n 20 products are also used to coat the surface of culture-ware to promote cell attachment.
The production of FBS in this country is an estimated 700, 000 liters annually worth $300 to $400 million. The industry obtains fetal calves for bleeding from slaughter 25 houses, or in some cases, rears herds of cattle for this purpose. These herds are held in as isolated a situation as possible in order to prevent disease. Whole blood is obtained aseptically (by syringe) from an animal, centrifuged to separate cells from serum, and the serum filtered to 0.22 30 microns to remove most bacteria. Often, serum is heated to 56C to inactivate the complement system, a group of immune proteins .
Insect cell culture has also been conducted for many years to develop control methods for this important animal 35 group, and has been used as a model for biological processes WO95/23212 2183~8i~ - pcrNs94~l422l in humans and higher animals. More recently, insect cell culture and a virus vector have become valuable tools for the eYpression of foreign genes. This technique is superior to the production of foreign proteins by bacteria as higher 5 yields, better ~copies", and more comple~ eukaryotic proteins can be obtained (Smith et al., 1983). Some examples of the recombinant proteins produced by insect cell expression systems are human interf eron and interleukin-2, substances that are injected as therapy in human subjects.
An example of the current method of protein production includes the following steps: 1 ) culture of an insect cell line ( S~odo~tera f ruC~; per~l~ ); 2 ) insertion of the desired f oreign gene in the baculovirus, Autographa Calif ornica Polyhedral Virus (Ac NPV); 3) infection of the insect cell line with this virus; and 4 ) e~traction of the resulting foreign protein from the infected insect cells.
Contamination of cell cultures because of inf ectious organisms in serum can be a serious problem. Bacteria, fungi, viruses, and mycoplasma have been isolated from bovine serum.
In the period from 1960-1980, mycoplasma from bovine serum was the second major group of contaminants found in cell culture (Barile, 1977). Now, FBS is usually screened for mycoplasma and most viruses. However, a more serious cause for concern is an all-protein infectious agent called a prion for which no test is available (Ringman, 1993 ) . This organism causes a fatal brain disease in mammals called Bovine Spongiform Encephalopathy (BSE), or Umad cow disease". BSE occurs in sheep, cows, and other mammals, and is most likely the cause of similar neuro-degenerative diseases in humans. In Britain in 1986, BSE resulted in the destruction of cattle and caused fears for the safety of the meat supply and other bovine products. Since then, the disease has turned up in cattle in many other countries. Consequently, serum from these countries cannot be imported for use in the U.S.
During insect cell culture procedures, most insect cells are maintained in a defined medium plus 10% FBS. Therefore, any inf ectious agent in the FBS could contaminate a Wo 95/23212 218 3 9 8 0 PCTIUSg4/14221 recombinant protein made by the cells, and could be transmitted to humans receiving this protein as the}apy.
Basic texts in cell culture teach a like-for-like approach, or r -lii?n sera, especially FBS, for culture of 5 r ~ qn cells ~Freshney, 1986). So firmly esti?hl;~hPcl is this dogma of FBS, that some cell culture publications refer only to "serum", when FBS is intended (Pollard and Walker, 1984). Likewise, major serum suppliers such as Sigma rhpmici~l Co. and Hy-Clone Laboratories inc1ude only bovine and some 10 equine sera in their cell culture catalogs. In practice during the past thirty years, only r l ii?n sera have been used for the culture of 1 i iqn cells.
U.S. Patent No. 4,449,480 to Isom et al. discloses freshwater mussels in an artificial habitat ut;1;7;n~ growth 15 media. However, the Isom et al. invention is directed to larviculture, that is, the provision of food and habitat for the larval stage of young animals such as aquatic invertebrates. Isom et al. are not concerned with cell culture. Larviculture is an interim technique, having the 20 goal of keeping the larval animal alive until it can progress to the next stage, or in this particular case, until it can Ll~..s~u-.,. from a parasitic stage and feed ;n~PpPnrlPntly on its own (Pennack 1953). Thus, the Isom et al. invention is not applicable to the instant problem, as cell lines do not grow 25 and transform to become independent of their culture medium and learn to feed on their own.
Further, the method taught by Isom et al. can only operate on the parasitic stage of whole, mult;l pll~ qr animals (Barnes, 1963), and is not applicable to cell lines derived 30 from the organs and tissues of a whole animal. A flln~ ql purpose of cell culture is to permit processes and P-TPr; iqt;r~n in cells that would not be possible to perform on a whole animal. Isom et al. borrow certain techniques from cell culture (for example, a modified solution or medium of 35 salts and proteins) and from bacteriology (for example, antibiotics). The use of these tools, however, does not render the Isom et al. method a cell culture or bacteriology process, and the disclosed method cannot function as such.

WO95/23212 4 PCr/US94114221 Thus, the Isom et al. method cannot provide a solution to the instant problem.
The method of the present invention is Pcrer~ ly timely as BSE has recently been found in rAnArliAn cattle (Ci '^11, 5 1993 ) and is strongly implicated as the cause of death in W; ~ronf:; n mink which were fed protein meal made from dairy cow carcasses (Marsh, 1993 ) . In Europe there is evidence of the disease in humans receiving contaminated human growth hormone (Knauer, 1993 ) .
10 S -ry of the Invention It is therefore an object of the present invention to provide a method of using fish serum to culture r ~1 i An cells and insect cells.
It is a further object of the present invention to use a 15 serum for 1 i An cell culture and insect cell culture that is free from various 1 iAn infectious agents, such as BSE, that could c~nrlAn~r researchers or the recipients of cell culture products.
It is another object of the present invention to provide 20 a serum for cell culture that is free from ~ 1 iAn infectious agents that can invalidate the results of scientific testing relating to the culture of 1 i i~n cells and insect cells.
It is a further object of the present invention to create 25 a new serum supply of consistent quality for use in cell culture .
It is still a further object of the present invention to provide a means to enhance the supply of serum that is available for research and commercial production in the 30 biotechnology field ~r~ri f i CAl 1 y for the culture of r 1 i An cells and insect cells.
These and other objects and advantages of the present invention will be apparent to those of ordinary skill in the art upon inspection of the detailed description, drawings, and 35 Arr~nrl~d claims.
The preisent invention is a method of using fish serum instead of bovine or other r ~l i An sera for ~ n cell culture and for insect cell culture. This method would appear _~= . _ . = . .

Wo 9Y23212 ~ ~ ~8 ~ PCrNSs4/l422 contradictory to those skilled in the art of cell culture.
Because of the great phylogenetic dif f erences between f ish and mammals, and between fish and insects, there is a reasonable expectation of failure. Searches of the non-patent literature 5 show no prior use of fish serum for r ~ 1; ~n cell culture or f or insect cell culture .
The l; ~n cell lines used in the method of the present invention are commonly used for a number of experimental and commercial applications. These applications include toxicology, biochemistry, cancer research, and the production of re: ` in~nt proteins for therapy in humans and domestic animals. There is growing concern that these therapeutants could carry the infectious organisms found in bovine serum (Anon. 1994). Therefore, the use of fish serum instead of ~ n serum in the culture of these cells provides important safety advantages for the recipients of these rP: i n~nt products.
Fish serum can be used for cell culture ~rr~ ;~ at;t~nq that now employ bovine or other -1; An sera, and for applications where these sera are ineffective or unsafe.
The ideal serum for cell culture would provide the nutrients and growth f actors that maintain cells and support their growth. In addition, the serum should be (A) consistent in quality, (B) have serum immune proteins (immunoglobulins), either at very low levels (as in FSB) or unlike those of mammals and insects for low cross-reactivity, and (C) be free of l; An infectious agents that would contaminate cell lines or cell culture products, or endanger researchers.
It has been found that serum from fishes is effective for 1; ~n cell culture and for insect cell culture, and meets the characteristics of the ideal serum with respect to, consistency, low cross-reactivity, safety, and control of content. In particular, sera from several species of aquacultured fishes can be used to grow insect cells.
Research was conducted with four species of aquacultured fish, representing three families of teleosts; two salmonids, the rainbow trout and its seawater form the stPPl hP;~l (Oncorhynchus mykiss); the Atlantic Salmon (Salmo salar); one WO95123212 2i~ o Pcrluss~ll422l cyclopterid, the lumpfish (Cyclopterus lumpus); and one ictalurid, the channel catfish (Ictalurus punctatus). Serum from these fishes is effective for insect cell culture and meets the characteristics of the ideal serum including 5 consistency, low cross-reactivity, safety, and control of content .
A. Consistency Until the past few years, serum from fish would have been inconsistent in quality, because wild stocks (even within the lO same species) vary in diet, habitat, genetics, life history, and reproductive status. This incr~nc;~tency would influence the l~L~ ;h;l;ty of cell culture P~)Pr; Ls, and make fish serum unsuitable for cell culture research. Now, by using domesticated stocks reared in aquaculture facilities, fish lS serum can be obtained with product consistency similar to serum from herds of cattle reared for this purpose. The essential requirement is for donor fish to be reared under consistent and therefore rel~roduc;hle conditions, n~
necessarily the nature or 8rPr; f i ~-s of these conditions. This 20 reproducibility of conditions reduces variability in serum content, and yields lot-to-lot consistency of serum -- an important factor in cell culture.
The preferred specific rearing conditions for the donor fish are disclosed below. However, other conditions are 25 desirable to alter a ~-nPnt in fish serum for culture of specific 1 i ~n cells and insect cellg, ag described below.
B. Low Cross-Reactivity The fishes are a distinct evolutionary group from the mammals and from insects. As such, many of their enzyme and 30 immune system proteins, Psreci~lly; t-~lobulin8, differ from those of mammals and insects. For example, most fishes are cold-blooded vertebrates having a body t clLule that approximates that of the waters where they live. Cold water fishes normally live in water with a temperature range of 0C
35 to 18C, and do not survive long above 20C. Therefore, their serum proteins, including antibody proteins, function within a range far below the 37C and higher body temperatures characteristic of the warm-blooded vertebrates such as humans 21 83~9~8 0 Wo 95123212 PcTIUSg4/14221 and other mammals . This f unctional temperature dif f erence strongly implies a difference in protein structure.
C . Saf ety As previously stated, a major advantage of fish serum for 5 cell culture is safety. Serum from fishes is unlikely to contain infectious agents harmful to mammals, including humans. Fish are cold-blooded animals with body temperatures that approximate the waters where they live. Therefore their pathogens, ~peciAl ly those of cold-water fishes, prefer temperatures well below the body temperatures of most mammals.
Both evolutionary and temperature differences provide important saf ety qualities to f ish serum . Very f ew bacteria or viruses that infect li~ fishes can infect mammals.
Instead, viruses of cold-water fishes are often controlled by raising water temperature ( and therefore the fishes ' body t~ e~Lurt:) above 18C (Wolf, 1988). Therefore, infection of l; An cell cultures or of researchers by an agent in fish æerum is highly unlikely.
D. Control of Content An additional advantage of using fish serum for cell-culture applications is control o~ serum content. Levels of certain substances in fish serum can be controlled by procedures that would be ; rlr~ ; hle with mammals f or biological or regulatory reasons. For example, ~ l; An genetic triploids are not viable, but in SAl ;~lc, triploids live and grow normally and serum from the female triploid contains no sex steroids (Schreck and Moyle, l990 ) .
Conversely, the sexual maturity of donor fish can be induced by light or hormone injections if high sex steroid serum is desired. Also, fish can be held under conditions unacceptable for mammals, such as total darkness, to increase certain hn :e~ such as melatonin in serum.
DetA; 1 ed Descri~tion of the Present Invention Using the present invention, the culture of two commonly used lines of ~ n cells in fish serum has been Led, as has the culture of two commonly used lines of insect cells. The s~l; An cell lines used were Chinese hamster ovary cells (CHOs), supplied by the ATCC (#CCL-61), Wo 95/23212 2 I S 3 g 8 ~ PCr/US94/14221 and monkey kidney cells (VEROs), also supplied by the ATCC
#CCL-81. S~odoptGra cells and Dro~ h; 1~ sp cells are used as the insect cell lines.
The fish serum used was taken from two species of 5~ nirlc, the rainbow trout and the SteplhpArl (Oncorhynchus mykiss), and the Atlantic salmon (Salmo salar), the lumpfish (Cyclopterus lumpus), and the channel catfish (Ictalurus punctatus ) . These species were used because consistent and reproducible methods for their production are well established, large numbers of these species are reared in commercial aquaculture and therefore large amounts of serum can be obtained, and individual fish are large enough (over two pounds ) so that blood can be drawn easily . other species of aquacultured fish fit these criteria, particularly the sturgeon and the striped bass.
The process begins with the consistent and reproducible conditions in which donor fish are reared. All fish used as serum sources are 1) progeny of domesticated broodstock, 2) inspected for disease according to the American Fisheries Society Blue Book standards, 3 ) sexually immature, 4 ) in the log phase of growth, 5) larger than two pounds (range from 2-12 pounds ), 6 ) reared by standard h1lch~nrlry methods appropriate to the species as described in Piper ( 1988 ), and 7) fed commercially manufactured pelleted feed of a composition consistent with that rec ' 1 by Halver (1972) and commonly used for each species. Rainbow trout and catfish are reared in freshwater; ste~lhPA~ salmon and lumpfish are reared in seawater.
Water ~t aLUL~ at the time of bleeding is normally 8 to 12C, but water t~ LUL~S of 4 to 14C are suitable for rainbow trout, salmon, S~PPlhP~I, and lumpfish, and temperatures of 4 to 30C for catfish; the objective being to avoid h~nrll in~ stress in the donor fish. R~nfll;nrJ stress is further reduced by starving fish for 48 hours before bleeding.
Each fish is stunned by administering a sharp blow to the fish's head, by immersion in ice-water, or by immersion in water containing C02 -or other fish anesthetic, the objective being to stun the fish to a level of loss of reflex activity WO 95/23212 2 1 8 3 9 g O PCT/US94/14221 ( loss of consciousness) as defined by Schreck and Moyle (1990). Whole blood is then drawn by syringe from the dorsal aorta, or the caudal vein. No more than 20% of the fish's total blood volume is drawn at each bleeding. After a 5 recovery time of approximately two weeks, the same f ish can be bled again. Repeated bleeding of the same group of fish (up to the time they become sexually mature) results in low lot-to-lot variability in the serum. Serum parameters for all fish species used and a comparison sample of FBS are given in 10 Table 1. For the method described, fish serum content must fall within the range given in Table 1.

Serum Analysis ~m Bovine ~*
15 Glucose 50-100 mg/dL 92 Blood urea nitrogen 1-3 mg/dL 18 Creatinine 0 . 3-3 mg/dL 2 . 8 Sodium 130-170 mEq/dL 135 Chloride 130-140 mEq/dL 96 20 Calcium 10-18 mg/dL 14 Phosphorus 10-20 mg/dL 11 Uric acid 0 . 2-2 mg/dL 1. 8 Total protein 3-6 g/dL 3 . 5 Albumin 0 . 5-2 . 8 g/dL 2 . 6 25 Globulin 0 . 6-3 . 6 g/dL 0 . 9 Bilirubin 0.1-1 mg/dL 0.1 Iron 20-100 ug/dL 191 Cholesterol 300-500 mg/dL 39 Triglycerides ~ 300-600 mg/dL 74 * FBS lot obtained from Sigma Chemical Co.

21 ~98~
Wo 95/23212 PCT/US94/14221 Blood is allowed to clot for not less than 15 minutes or more than 2 hours, and is then centrifuged at 1100 x g for at least 10 minutes and for no more than 20 minutes.
Serum is removed from the collection tubes and st~r; l; ~r~r~
5 by passing first through a .45 ~ filter, and then through a . 22~ filter.
Serum from 6 or more fish of the same species is ~ ` inPd as a numbered lot, and frozen at -70C. ~o heat treatment such as that commonly used f or bovine serum to denature 10 complement immune system proteins is needed.
CHO or VERO cells shipped frozen from the American Type Culture Collection are thawed, counted, and seeded immediately in 25 cm2 sterile tissue culture flasks containing 5.0 ml of medium. The medium is RPMI-1640, a widely-used defined medium 15 (which may be purchased from Sigma Chemical Co. ), plus 10%
(v/v) FBS. This flask containing cells and medium is placed in an i nmlhator at 37C and 5% CO . The insect cells are grown in f lasks or other vessels in Grace ' s medium supplemented with TC Yeastolate and Lac~lhllmin Hydrolysate and 10% FBS at 27C
20 in a cloged ai ~rhr~re.
M; ~1; An cells are allowed to grow and increase in numbers over a period of one week. During this time, the cells are "split" or subcultured every 48 hours as follows.
After the first 48 hours, cells from the original flask are 25 detached using the standard tryrs; n; 7At; nrl tenhn; q--r~ described by Pollard and Walker (1984) and are transferred to two new 25 cm2 tissue culture flasks. This procedure is repeated every 48 hours until a total of eight f lasks have been seeded.
Cells growing in 10% FBS must be adapted first to lower 30 levels of FBS before they can be cultured in similar low levels of f ish serum .
When the cells in the eight f lasks have reached the log phase of grow~h, the process of weaning to lower FBS levels begins. The RPMI-1640 plus 10% FBS is removed by aspiration 35 or pipette from each flask, and replaced with RPMI-1640 plus 7 . 5% FBS. These flasks are placed in the incubator and the cells are allowed to grow and increase in number for 48 hours.-At this time the RPMI-1640 with 7 . 5% FBS is removed from the 2~.83g8;0 Wo 95/23212 -. - PCT/US94114221 eight flasks and replaced with RPMI-1640 plus 5% FBS. The process may be repeated to lower FBS levels to 2 . 5% or 1% .
The weaning process takes up to 10 days to acclimate cells to the lower concentrations of FBS.
Cells growing in the flasks containing RPMI-1640 plus 5%
or 2 . 5% or 1% FBS are then harvested using standard trypsinization techniques, washed with serum-free RPMI-1640, centrifuged at 300g for 3 . 5 minutes at room temperature, counted in a hemocytometer, and rac~ p~n~ d in new 25 cm2 tissue culture flasks containing serum-free RPMI-1640.
Aliquots from these flasks containing 5 x 109 cells/ml (by nA1clllAtinn) are then seeded in new flasks containing 5 ml of RPMI-1640 plus thawed fish serum as described above, at a concentration of 2.5% or 5% (v/v). Flasks containing the cells and media are then incubated at 37C in 5% C02/95% air.
Insect cell culture follows the same ~Lu.xd.lr e:. When the cells in the flasks have reached the log phase of growth, the process of weaning to lower FBS levels begins. The medium plus 10% FBS is removed by aspiration or pipette from each flask, and replaced with medium plus 7.5% FBS. These flasks are placed in an incubator and the cells are allowed to grow and increase in number for 48 hours. At this time the medium with 7 . 5% FBS is removed from the flasks and replaced with medium plus 5% FBS. The process may be repeated to lower FBS
levels to 2 . 5% or 1% . The weaning process takes up to 10 days to acclimate cells to the lower concentrations of FBS.
Cells growing in the flasks containing medium plus 5% or 2 . 5% or 1% FBS are then harvested using standard tryp~; n; 7at; nn techniques, washed with serum-free medium, centrifuged at 300g for 3.5 minutes at room temperature, counted in a hemocytometer, and resuspended in new 25 cm2 tissue culture flasks containing serum-free medium. Aliquots from these flasks cnntAinin~ 5 x 10~ cells/ml (by calculation) are then seeded in new flasks containing 5 ml of medium plus thawed fish serum as described above, at a concentration of 2.5% or 5% (v/v). Flasks containing the cells and media are then incubated at 37C in 5% CO2/95% air.

WO 9s/232l2 218 3 ~ ~b PCT/US94/14221 At these concentrations of fish serum, 1; An cells will grow normally with an approximate doubling time of 24 hours, the same as would be expected if they were cultured in F8S. When the cells are stained with a hematoxylin stain for 5 observation, those cultured with 1% or 2.5% fish serum are normal and qualitatively similar to those cultured in media t~nnt;l;n;ng FBS. Cells cultured at 5% fish serum appear normal except for small lipid-filled vacuoles in the cytoplasm.
Results show that insect cells survive and grow in sera lO from all species of fishes tested. Insect cells in lumpfish serum survived and did not attach for 48 hours but attached normally when FBS was then added. Compared to the FBS
control, insect cells grown in fish serum showed no obvious difference in appearance, and for lumpfish and steelhead 15 serum, cell growth was similar to growth in FBS. Compared to cells in FBS, final cell numbers were lower and att~` L
less for cells grown in sera from the other fish species tested .
At this stage, 1 i ~n cells and insect cells can be 20 subcultured for experimental or commercial purposes using fish serum as a r~rl~ L for FBS, in either flasks or suspension cul ture .
The effectiveness of fish serum for growth of insect cells is almost certainly inf luenced by lipid content, sexual 25 maturity of the donor fish (as reflected in serum steroids), 'and the growth rate of donor f ish .
Serum lipid in the fish species tested was high. Typical triglycerides were greater than 400 mg/dL and cholesterol was greater than 450 mg/dL, ten times or more higher than those of 30 FBS. High serum lipoproteins are potentially growth inhibitory (Ito et al., 1982), therefore it is inferred that f or some insect species lower lipid content in f ish serum would improve cell growth.
The best performing sera were from fishes in their 35 maximum growth phase, and b~inn;n~ (3-4 months before) sexual maturity .
Although only two insect cell line were tested with sera from several species of aquacultured fish, similar results can wo 95/23212 2 ~ 8 ~ ~ 8 0 PC~/US94/14221 be expected with other closely related lepidopteran cell lines such as r ~tra brA~; cae or 3 ~ sI~ . (Davis et al ., 1993 ) .
In addition to Droso~h; 1 a and Spodoptera, other important insect cell lines and sera f rom other species of f ish may be -5 substituted for those disclosed here.
Preferred and alternate ~ '; ~ Ls have now been described in detail. It is to be noted, however, that this description is merely illustrative of the principles underlying the inventive concept. It is therefore contemplated that various modifications of the disclosed Ls will, without departing f rom the spirit and scope of the present invention, be apparent to those of ordinary skill in the art. For e~ample, other species of fish and other l; An cells may be substituted for those disclosed herein.
The species of f ish used as a source of serum may vary depending on what -1; An cells are to be cultured.
However, the reprodur;h; ~ ;ty of fish species and strain used and of fish culture conditions required for consistent serum quallty limits donor species to those grown in aquaculture.
Content of serum is influenced by such factors as hllchAnrlry, feed, L~:~Ludu~Live status, genetics, age, and hAnr11;ng. Also, some f ishes containing trimethyl amine oxide or high levels of other non-protein nitrogen ( ammonia or amines ) in their serum may be unsuitable as donors.
~he method of obtaining the serum is not critical as long as the fish are treated in a humane manner and serum is obtained and processed (centrifuged, filtered, rA~kA~d, etc. ) aseptically .
Although the method is described using CHOs and VEROs, later e~perimentation has shown that normal diploid fibroblasts can be cultured using the fish serum method.
For C310s, VEROs, or other cell lines, the defined medium (RPMI-1640) may be replaced with other media selected from the group consisting of Eagles MEM, BME, Medium l99, or McCoys, all widely-used defined media.
The apparent limitation of no more than 5% fish serum for culturing - -1; An cells is most likely the result of the WO 95/23212 2 i~ O PCT~US94114221 very high levels of lipid in the sera of most bony fishes (Teleosts). Such a large excess of any essential -nPrt in the cell culture media is likely to be inhibitory. Cholesterol levels of 300-500 mg/dL and triglycerides of 300-600 mg/dL
5 were routinely measured in the sera of the bled fishes. This compares with cholesterol of 30-60 mg/dL and triglycerides of 50-80 mg/dL in FBS. Lowering lipid levels in fish serum through physical or chemical removal or changes in f ish diet would most likely remove the 5% limitation.
10 ReferPnrPq Anonymous, 1994. "Manufacturers warned not to use bovine-origin materials from BSE countries". Fn~ Veterin~3riAn, March/April 1994, page 8.
Barile, M.F. 1977. "Mycoplasma Contamination of Cell 15 Cultures - a Status Report". Cell Clllture ~nrl its ~?~71iCati''nq (R-T- Acton and J.D. Lynn, Eds. ) pages 291-334.
Barnes, R.D. 1963. InvertPhrate Zoologv. W.B. Saunders Co. Philadelphia, Pa. pages 297-298.
Freshney, R.I. 1987. ~nir~l Cell Cult1lre - A ~nll~l of 20 Blq;c Tprhniclues. J. Wiley & Sons, N.Y.
Halver, J.E . 1972 Fi r~h Nutrition. ~r~riPm; r Press, ~ew york .
Kingman, S. 1993. "London Meeting Explores the Ins and Outs of Prions". Science 262: 180-181.
Pennak, R.W. 1953. Freshwater Invertebrates of the U~; ted States.
Piper, R.G. 1983. Fich Hatcher~ n;~ . U.S.
Department of the Interior, Fish and Wildlife Service.
Wa8hington, D . C.
Pollard, J.W. and Walker, J.M. l990. "Basic Cell Culture". Meth~iq in M~leclll~r Biolo~7y, Volume 5; Animal Cell Culture. Humana Press, Clifton, ~.J. pages 1-12.
Schreck, C.B. and Moyle, P.B., 1990. Methr-~lq in Fiqh BiQlor,JSr, pp. 223-232. ~r-r;r~n Fisheries Society, Bethesda, 35 Md.
Wolf, K. 1988. Figh Viruqes ~nri Fish Vir~l DiqP~qP, p.
108. Cornell University Press, Ithaca, ~.Y.
._,. _. ,=~= . = -- . .

Claims (30)

We claim:

1. A method of culturing living cells, comprising culturing the cells in a culture medium comprising a defined culture medium supplemented with prepared fish serum, wherein said fish serum is prepared by:
a. raising fish under controlled conditions such that the diet, habitat, genetics, life history, and reproductive status of the fish remains substantially constant and reproducible;
b. starving the fish for up to about forty-eight hours;
c. stunning the fish by non-toxic methods until the fish is unconscious;
d. withdrawing whole blood from the fish;
e. allowing the blood to clot;
f. centrifuging the blood until the blood serum is separated from the blood cells;
g. removing the serum; and h. sterilizing the serum.

2. The method of claim 1, wherein said fish is stunned by administering a sharp blow to the head of the fish.

3. The method of claim 1, wherein said fish is stunned by immersing the fish in water containing a fish anesthetic.

4. The method of claim 3, wherein the fish anesthetic is carbon dioxide.

5. The method of claim 1, wherein said fish is stunned by immersing the fish in ice water.

6. The method of claim 1, wherein the blood is allowed to clot for up to about two hours.

7. The method of claim 1, wherein the blood is centrifuged at 1100 x g for at least about ten minutes.

8. The method of claim 1, wherein the blood is centrifuged for about ten minutes to about twenty minutes.

9. The method of claim 1, wherein:
a. said serum is frozen after sterilization to a temperature of about -70 degrees C; and b. wherein said frozen serum is thawed prior to culturing the cells.

10. The method of claim 1, wherein:
a. said cells are mammalian cells;
b. said defined culture medium is RPMI-1640; and c. said fish serum is present at a concentration of about 1%.

11. The method of claim 1, wherein:
a. said cells ar mammalian cells; and b. said defined culture medium is selected from the group consisting of RPMI-1640, Eagles MEM, BME, Medium 199, and McCoys Medium.

12. The method of claim 1, wherein:
a. said cells are mammalian cells;
b. said defined culture medium is RPMI-1640; and c. said fish serum is present at a concentration of about 2.5%.

13. The method of claim 1, wherein:
a. said cells are insect cells; and b. said defined culture medium is selected from the group consisting of Grace's medium, BML TC-10 medium, and Schneider's insect medium.

14. The method of claim 1, wherein said fish are selected from the group consisting of Oncorhynchus mykiss, Salmo salar, Cyclopterus lumpus, and Ictalurus punctatus.

15. The method of claim 1, wherein:
a. said cells are mammalian cells;
b. said defined culture medium is RPMI-1640; and c. said fish serum is present at a concentration of from about 1% to about 5%.

16. The method of claim 1, wherein:
a. said cells are insect cells; and b. said cells are grown in from about 2 percent to about 10 percent fish serum.

17. The method of claim 1, wherein said serum is sterilized by:
a. filtering the serum through a 0.45 micron filter;
and b. filtering the serum through a 0.22 micron filter.

18. The method of claim 1, wherein said fish serum is sterilized by filtering the serum through a 0.22 micron filter.

19. The method of claim 1, wherein the cells are Spodoptera cells.

20. The method of claim 1, wherein the cells are Drosophila cells.

21. A method of culturing live cells comprising:
a. seeding the cells in a vessel containing a defined culture medium plus 10% fetal bovine serum;
b. incubating the cells and medium;
c. allowing the cells to grow and increase in numbers;
d. subculturing the cells;
e. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with a lower concentration of fetal bovine serum;
f. harvesting the cells;
g. washing the cells with the defined culture medium;
h. centrifuging the cells at 300g for about 3.5 minutes at room temperature;
i. resuspending the cells in a second vessel containing defined culture medium;
j. seeding at least a portion of the cells in a third vessel containing defined culture medium and fish serum in a concentration of about 2.5% to about 5%; and k. incubating the cells and medium.

22. The method of claim 21, wherein the fish serum is prepared by:
a. raising fish under controlled conditions such that the diet, habitat, genetics, life history, and reproductive status of the fish remains substantially constant and reproducible;
b. starving the fish for up to about forty-eight hours;

c. stunning the fish by non-toxic methods until the fish is unconscious;
d. withdrawing whole blood from the fish;
e. allowing the blood to clot;
f. centrifuging the blood until the blood serum is separated from the blood cells;
g. removing the serum; and h. sterilizing the serum.

23. The method of claim 21, wherein removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with a lower concentration of fetal bovine serum includes:
a. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 7.5% fetal bovine serum;
b. incubating the cells and medium;
c. allowing the cells to grow and increase in numbers;
d. subculturing the cells; and e. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 5% fetal bovine serum.

24. The method of claim 21, wherein removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with a lower concentration of fetal bovine serum includes:
a. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 7.5% fetal bovine serum;
b. incubating the cells and medium;
c. allowing the cells to grow and increase in numbers;
d. subculturing the cells;
e. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 5% fetal bovine serum;
f. incubating the cells and medium;

g. allowing the cells to grow and increase in numbers;
h. subculturing the cells; and i. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 2.5% fetal bovine serum.

25. The method of claim 21, wherein removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with a lower concentration of fetal bovine serum includes:
a. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 7.5% fetal bovine serum;
b. incubating the cells and medium;
c. allowing the cells to grow and increase in numbers;
d. subculturing the cells;
e. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 5% fetal bovine serum;
f. incubating the cells and medium;
g. allowing the cells to grow and increase in numbers;
h. subculturing the cells;
i. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 2.5% fetal bovine serum;
j. incubating the cells and medium;
k. allowing the cells to grow and increase in numbers;
l. subculturing the cells; and m. removing the defined culture medium and fetal bovine serum from the vessel and adding defined culture medium with 1% fetal bovine serum.

26. The method of claim 21, further comprising lowering the lipid levels in the fish serum, wherein said cells are mammalian cells.

27. The method of claim 26, further comprising raising the concentration of fish serum to above 5%.

28. The method of claim 21, wherein the cells are Spodoptera cells.

29. The method of claim 21, wherein the cells are Drosophila cells.

30. The method of claim 22, wherein:
a. the sterilized serum is frozen; and b. the serum is thawed prior to adding the serum to the defined culture medium.