System for the cell culture and cryopreservation of marine invertebrates

A system for the culture of eukaryotic cells of marine invertebrates employing a culture medium containing a mixture of sodium, magnesium, chlorine, potassium, calcium, bromine, and sulfate is disclosed, as well as a system for the cryopreservation of such cells in which the medium contains dimethyl sulfoxide.

TECHNICAL FIELD 
The present invention relates to marine cell culture, and, more 
particularly, to a system for the in vitro culture and cryopreservation of 
marine invertebrate cells. 
BACKGROUND OF THE INVENTION 
A number of synthetic basal media have been developed for animal cell 
culture, especially for mammalian and insect cells. Commercially available 
media include: Eagle media, M 199, Dulbecco's modified Eagle's medium 
(DME), Ham's F10 and F12, RPM1 1640, Grace's Insect media and Leibovitz 
L15. These media, in conjunction with bovine serum or specific growth 
factors, are often sufficient to support animal cell growth. The 
formulation of these media, however, are insufficient for sustained growth 
and proliferation of marine invertebrate cells. 
Historically, there has been no flexible basal nutrient media available 
which can support the in vitro proliferation or preservation of cells from 
a diverse number of multicellular, eukaryotic, marine invertebrate 
species. 
Recent advances in cellular biology have demonstrated the importance of the 
synergistic involvement of a variety of media components, including ions, 
nutrients, growth factors and attachment matrices in the development and 
proliferation of higher eukaryotic cells. 
The inorganic salts in culture media have two major functions. First, salt 
concentrations are created to approach the natural salt concentration 
levels in the environment from which the cells are derived. This will 
minimize any deviation in osmotic pressure on the cells that would require 
energy consuming ionic pumps to maintain cellular integrity. 
Secondly, these salts include many ions which are utilized as enzymatic 
cofactors and intracellular messengers. Often, for ions to be effectively 
transported into the cell, additional carrier molecules are needed in the 
medium. In mammalian systems, transferrin is a carrier of iron. In marine 
invertebrate systems, it has been demonstrated that metal chelators like 
diethylenetetramine pentaacetic acid ("DTPA"), citrate and amino acids 
(lysine and taurine) help to transport metal ions into molluscan tissue. 
See Coombs, T. C. (1977) Proc. Anal. Div. Chem. Soc. 14:219. 
Although most mammalian culture media utilize glucose as the main carbon 
source, others have demonstrated the benefits of alternative carbon 
sources for invertebrate culture. See Grace, T. D. C. (1962) Nature 
195:788-789, and Leibovitz, A. (1983) Am J. Hyg. 78:173-180. 
It is known that many animal cells grow best when attached to natural 
substrates like collagen, laminin, and fibronectin. It has been shown that 
the addition of ascorbic acid to culture medium increased the production 
and deposition of collagen by mammalian cells. See Engvall, E., et al 
(1986) J. Cell Biol. 102:703-710. The production of collagen by cells in 
culture helps to create a natural matrix for growth. This type of matrix 
is very important for many marine invertebrate cells. Sponges, for 
example, are mostly cells on a collagen mesh. 
In a general basal media it is very difficult to define all the 
requirements animal cells need for growth. The use of bovine or fetal 
bovine serum as a supplement, as used in mammalian culture systems, does 
not work with marine invertebrate cells. Serum or hemolymph from marine 
gastropods, such as Haliotis sp. and Strombus sp., offer an alternative 
and effective source of additional lipids, trace metals, growth factors 
and nutrients. These improve the cultured growth of marine invertebrates. 
The preservation of cultured cells and a method for preserving and shipping 
field samples of marine invertebrates offer both economical and 
environmental savings by reducing or eliminating large scale 
re-collections of organisms of interest. Serman, J. K. (1964) Proc. Soc. 
Exptl. Biol. Med. 117:251-264 discloses the value of adding dimethyl 
sulfoxide (DMSO) to medium in order to prevent ice crystals from rupturing 
cells during freezing. 
Accordingly, it is an object of the present invention to provide a system 
which employs a basal nutrient medium which is designed to facilitate the 
in vitro growth, proliferation, and cryopreservation of multicellular, 
marine invertebrate cells. 
It is another object of the present invention to provide a system that will 
accommodate a wide variety of species and cell types, rather than optimize 
for any single specific cell line. 
It is a further object of the present invention to provide a system 
employing media that is reliable, convenient to use, and cost effective in 
its manufacture. 
DISCLOSURE OF THE INVENTION 
The present invention provides a system for the proliferation of marine 
invertebrate cells in in vitro culture, and the production of cell 
metabolites therefrom. 
In one aspect, the present system comprises a culture of viable eukaryotic 
cells or tissue derived from at least one marine invertebrate organism in 
association with a culture medium which comprises components in the 
following approximate ranges: From 7.875 to 13.125 g/L of sodium, from 1.0 
to 1.69 g/L of magnesium, from 14.25 to 23.8 g/L of chlorine, from 0.29 to 
0.48 g/L of potassium, from 0.3 to 0.5 g/L of calcium, from 0.0225 to 
0.0375 g/L of bromine and from 2.0 to 3.38 g/L of sulfate. 
Another embodiment of the culture medium of the present system includes 
carbon sources, in which the carbon sources are selected from the group 
consisting of galactose, mannose and fructose. Further embodiments of the 
media include from approximately 30 to 300 mg/L of taurine, and from 
approximately 10 to 100 mg/L of ascorbic acid. The culture media may also 
comprise from approximately 3 to 20% by volume gastropod plasma or 
components fractionated therefrom. 
A further aspect of the invention provides a method for employing the 
present system to produce desirable metabolites from the cells established 
in culture. 
A still further aspect of the invention includes the present system in 
which propagating cells are attached to microcarriers, such as of 
polystyrene. 
In yet another aspect of the invention, a system is provided for the 
cryopreservation of cultured cells and tissue from marine invertebrates. 
In this aspect, the system comprises a culture of viable eukaryotic cells 
or tissue derived from at least one marine invertebrate organism in 
association with the present culture medium to which has been added 
components in the following approximate ranges: From 5 to 20% by volume 
gastropod plasma and about 7.5% by volume dimethyl sulfoxide. 
The novel features of this invention, as well as the invention itself, both 
as to its structure and operation, will be best understood from the 
accompanying drawings taken in conjunction with the accompanying 
description.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a system for the proliferation of marine 
invertebrate cells in in vitro culture, and the production of cell 
metabolites therefrom. 
In one aspect, the present system comprises a culture of viable eukaryotic 
cells or tissue derived from at least one marine invertebrate organism in 
association with a culture medium which comprises components in the 
following approximate ranges: From 7.875 to 13.125 g/L of sodium, from 1.0 
to 1.69 g/L of magnesium, from 14.25 to 23.8 g/L of chlorine, from 0.29 to 
0.48 g/L of potassium, from 0.3 to 0.5 g/L of calcium, from 0.0225 to 
0.0375 g/L of bromine and from 2.0 to 3.38 g/L of sulfate. 
A further embodiment of the culture medium of the present system includes 
carbon sources, in which the carbon sources are selected from the group 
consisting of galactose, mannose and fructose. Further embodiments of the 
media include from approximately 30 to 300 mg/L of taurine, and from 
approximately 10 to 100 mg/L of ascorbic acid. The culture media may also 
comprise from approximately 3 to 20% by volume gastropod plasma or 
components fractionated therefrom. 
A further aspect of the invention includes the present system in which 
propagating cells are attached to microcarriers, such as beads made of 
polystyrene. This aspect of the invention facilitates the transfer of 
marine invertebrate cell cultures from low volume plasticware to larger 
suspension systems to permit larger volume production of the cell 
cultures, or the metabolites derived therefrom. The utilization of plastic 
microcarriers also allows the direct transfer of attached cells to a flask 
or bioreactor. 
In another aspect of the invention, a system is provided for the 
cryopreservation of cultured cells and whole pieces of tissue obtained 
from marine invertebrates. In this aspect, the system comprises a culture 
of viable eukaryotic cells or tissue derived from at least one marine 
invertebrate organism in association with the present culture medium to 
which has been added components in the following approximate ranges: From 
5 to 20% by volume gastropod plasma and about 7.5% by volume dimethyl 
sulfoxide (DMSO). 
The basal medium employed in the present system is a new formulation of 
nutrients, salts and other components which make possible the growth, 
proliferation and cryopreservation of multicellular marine invertebrate 
cells. The medium contains balance amounts of amino acids, carbohydrates, 
vitamins, ions and other components, which allow its flexible use in 
growing a large variety of marine invertebrates. This is a basal medium 
designed to effectively culture a wide variety of proliferating cell and 
tissue cultures. This invention is particularly, though not exclusively, 
useful for generating a medium to store frozen field or culture samples in 
a viable state, eliminating the need for expensive and ecologically 
destructive re-collection of rare sample organisms. 
This invention defines such media which can be used to produce biomass for 
the study and production of metabolites and other natural products from a 
genetically diverse array of marine species, such as but not limited to 
porifera, gastropods, and tunicates. 
It is the balance of these parameters, with specific focus on the 
requirements of multicellular marine invertebrates, that is the essence of 
this invention. Key elements provided in this medium for the culture of 
marine invertebrate cells are derived from the state-of-the-art 
correlation that animal cells have certain ionic, nutrient, matrix protein 
attachment and growth factors. These elements are related to the 
requirements for growth and development. 
The basal nutrient medium in this invention is designed to facilitate the 
in vitro growth, proliferation and cryopreservation of multicellular, 
marine invertebrate cells. This is intended to be a general medium that 
will accommodate a wide variety of species and cell types. The invention 
recognizes major inorganic ions be adjusted to concentrations approaching 
that of their natural marine environment. See Hughs, K. D. (1993) 
Analytical Chem. 65(20):888-889. In addition, the invention includes metal 
binding agents, such as taurine, to aid in the transport of important ions 
into the cells. 
Moreover, the invention recognizes that carbon sources in addition to 
glucose, such as mannose, fructose and galactose, can be utilized as 
alternative energy sources in certain invertebrates. Still another feature 
of the invention is the addition of ascorbic acid as an anti-oxidant and 
rate limiting cofactor of collagen production. The invention also 
recognizes the desirability of supplements, and in particular the use of 
gastropod hemolymph, plasma or serum, most usually plasma (the cell free 
hemolymph or blood), from marine gastropods, including but not limited to 
Haliotis sp. and Strombus sp. 
An additional advantage provided by the present invention is a system for 
freezing and storing viable field and culture samples of marine 
invertebrates. This is accomplished by the use of the media containing 
gastropod plasma and DMSO, as further described below. 
A number of commercially available synthetic media can serve as the base 
starting point for the final media formulations of this invention. Each of 
the following commercially-available media are known to function in the 
present system, after being adjusted to the disclosed formulation 
requirements of the invention, although a range of performance is to be 
expected: Eagle, DME, M 199, Ham's F10, F12, RPMI 1640 and Leibovitz L15. 
The formulation which performed best is given in Table 1 and is based on 
RPMI 1640. Cell growth was not obtained in any of these media when used 
without the presently-described modifications. 
Utilizing any of the above mentioned commercial media, amino acid 
concentrations can also be supplemented with commercially available 
non-essential amino acids, in particular glutamine and taurine. 
Inorganic salts are added to adjust the final approximate amounts in a 
liter of media to be: Sodium (Na) 10.5 g, magnesium (Mg) 1.35 g, chlorine 
(Cl) 19.0 g, potassium (K) 0.38 g, calcium (Ca) 0.4 g, bromine (Br) 0.03 g 
and sulfate (SO.sub.4) 2.7 g. Proper ionic balance is desirable to realize 
the full benefits of the invention. Because of the large number of 
components and flexible nature of the media, it is to be noted that these 
ion concentrations should lie within an approximate range of plus or minus 
25% of the stated values. 
In addition to the glucose (1-4.5 g/L) in the starting media, alternative 
sugars can be added to serve as carbon sources, desirably for example 
mannose at 1.44 g, fructose at 1.44 g and galactose at 4.32 g per liter of 
media. Again the final amount of these components should be substantially 
within .+-.25% of the above values. 
Ascorbic acid is added to the media desirably at 0.05 g/L. A main goal is 
to improve the rate of post-translational collagen modification, and in so 
doing, to increase the transport of collagen out of the cell and into a 
matrix formation suitable to enhance cell growth. 
Hemolymph is obtained from commercially-harvested 0.5-3 kg gastropods (e.g. 
Haliotis sp. or Strombus sp.). Known techniques for collecting hemolymph 
are employed to obtain serum or plasma, e.g. by arterial or venous 
puncture. All media containing gastropod plasma are filtered, e.g. through 
a standard 0.2 m filter, to remove cell contaminants. Subfractions of 
gastropod hemolymph, and the constituent components thereof, are obtained 
by known methods, such as salt precipitation, column chromatography, size 
exclusion (filtration, dialysis, or chromatography) or solvent extraction. 
Such subfractions and components are routinely screened for beneficial 
effects in the present system and media. 
Samples of gastropod hemolymph are collected from live animals (such as 
Haliotis sp.) These samples are centrifuged at 20,000 times gravity for 30 
minutes to remove the cells, and are stored at -10.degree. to -20.degree. 
C. This plasma is added to the media at levels between 3% and 20% by 
volume. This plasma is a desirable component of the invention in the sense 
that all cell types tested grew, multiplied and withstood the rigors of 
cryopreservation better in the presence of this plasma. The exact 
components in the gastropod plasma responsible for the above-mentioned 
advantages have not been identified. This invention recognizes, however, 
that selected subfractions of gastropod hemolymph will also be expected to 
prove beneficial and hence are considered to be included in the invention. 
TABLE 1 
______________________________________ 
Amount Amount 
Compound (mg/L) Compound (mg/L) 
______________________________________ 
Salts and Buffers 
NaCl 25,430 KCl 740 
MgSO.sub.4.7 H.sub.2 O 7,030 MgCl.sub.2.6 H.sub.2 O 5,070 
CaCl.sub.2 1,100 Ca(NO.sub.3).sub.2 100 
NaHCO3 2,000 NaH.sub.2 PO.sub.4.7 H.sub.2 O 1,512 
NaBr 42 HEPES 5,957.5 
Carbon Sources 
glucose 2,000 galactose 4,320 
fructose 1,440 mannose 1,440 
Vitamins, Amino Acids 
ascorbic acid 
50 glutathione 1 
L-arginine 200 L-asparagine 1,372 
L-aspartic acid 1,350 L-cystine 20 
L-glutamic acid 1,490 glycine 760 
L-histidine 15 hydroxy L-proline 20 
L-isoleucine 50 L-leucine 50 
L-lysine 40 L-methionine 15 
L-phenylalanine 15 L-proline 1,170 
L-serine 1,080 L-threonine 20 
L-tryptophan 5 taurine 122 
L-tyrosine 20 L-valine 20 
p-aminobenzoic 1 d-biotin 0.2 
D-pantothenate 0.25 choline chloride 3 
folic acid 1 i-inositol 35 
nicotinamide 1 pyridoxine 1 
riboflavin 0.2 thiamine 1 
vitamine B12 0.005 
Haliotis sp. plasma 10% 
______________________________________ 
The ability to store and ship field samples and cultures is another 
important attribute of this invention, since large scale re-collection of 
marine invertebrates is a difficult and ecologically destructive process. 
It is known that many of these organisms have either open or no 
circulation systems. This fact dictates that their tissues be relatively 
permeable to allow nutrient and excrement transport. It is this tissue 
construction that can be exploited to deliver cryopreservation solutions 
to the cells while they are still in "blocks" of tissue. Consequently, 
small pieces of tissue can be cut from these animals, placed in a medium 
of the invention that contains approximately 20% by volume of gastropod 
plasma and approximately 7.5% by volume of DMSO, and frozen slowly at 
-10.degree. C. to -20.degree. C. The DMSO protects the cells from the 
formation of ice crystals during freezing, and the media with the plasma 
protect the cell membranes during thawing. Frozen samples can be stored at 
-10.degree. C. for several weeks, but long term storage will desirably be 
at -70.degree. C. or below. One embodiment of the media according to the 
present invention is described in Table 1, containing approximately 20% 
gastropod plasma and approximately 7.5% DMSO. 
A further embodiment of the invention is the adaptation of these marine 
invertebrate cell cultures from low volume plasticware to larger 
suspension systems. This is accomplished by allowing the propagating cells 
to attach to polystyrene microcarriers (Solohill Labs Inc.) which are 
small round spheres or beads, similar to grains of sand in appearance. Use 
of such microcarriers allows the direct transfer of attached cells to a 
stirred flask of bioreactor vessel. 
The following examples serve to illustrate certain preferred embodiments 
and aspects of the present invention and are not to be construed as 
limiting the scope thereof. 
EXPERIMENTAL 
In the experimental disclosure which follows, all weights are given in 
grams (g), milligrams (mg), micrograms (.mu.g), nanograms (ng), or 
picograms (pg), all amounts are given in moles (mol), millimoles (mmol), 
micromoles (.mu.mol), nanomoles (nmol), picomoles (pmol), or femtomoles 
(fmol), all concentrations are given as percent by volume (%), proportion 
by volume (v:v), grams per liter (g/L), molar (M), millimolar (mM), 
micromolar (.mu.M), or normal (N), all volumes are given in liters (L), 
milliliters (mL), or microliters (EL), and linear measurements are given 
in millimeters (mm), or nanometers (nm) unless otherwise indicated. 
EXAMPLE 1 
A specimen of the marine sponge Haliclona sp. is cut into strips measuring 
5.times.5.times.40 mm, placed in cold freezing media containing 20% 
gastropod plasma and 7.5% DMSO, and then frozen at -10.degree. C. 
One month later the sample is thawed, placed in 1% sodium hyperchlorite 
using synthetic sea water (26.22 g/L NaClO and 1 g/L KCI)(SSW) for 60 
seconds, rinsed with synthetic sea water, and passed through a metal mesh. 
Cells are collected by pipette and placed in medium containing 5% 
gastropod plasma, 0.5 mg/mL gentamicin, 1.25 .mu.g/mL fungizone, and 0.5 
mg/mL penicillin/streptomycin, for one hour at 20.degree. C. The cells are 
then washed with media and placed into a plastic T75 culture flask at 
1.times.10.sup.6 cells/mL, in 15 mL of media, with 4% gastropod plasma, at 
20.degree. C. Primary cultures contained the same antifungal and 
antibiotic agents at 10% of the above stated levels. These agents are then 
diluted and washed out as the cultures are expanded. 
One month later, after several generations of cell reproduction, or 
passages, one T75 flask is treated with 0.5 mg/mL trypsin and 0.2 mg/mL 
EDTA in synthetic sea water to detach the cells from the flask. These 
cells (1.times.10.sup.8) are washed in media and then split into two T75 
flasks. Two days later both flasks are again harvested yielding 
3.2.times.10.sup.8 cells. These cells are placed into a 1 liter stir flask 
containing 100 mL of media, with 6% gastropod plasma and 5 g of 
polystyrene microcarriers (Solohill #104-1521). The system is allowed to 
incubate for two hours, stirred and then allowed to incubate for another 
hour. The system is then continuously stirred, with 200 mL of media 
containing 5% gastropod plasma being added every other day. After ten days 
in the stirred flask, 4.46 g of cells are recovered from the system. 
The cells are extracted with methanol and then hexane. Both these extracts 
demonstrate antibiotic activity, similar to the natural sponge, against 
Staphylococcus aureus, in a qualitative bacterial growth bioassay. 
EXAMPLE 2 
Mantle epithelium tissue samples are cut from the green abalone, Haliotis 
fulgens, in strips measuring 2.times.5.times.20 mm and frozen in freezing 
medium at -10.degree. C. One week later, the samples are thawed and placed 
in 1% sodium hyperchlorite, in synthetic sea water, for 60 seconds. 
The samples are then rinsed with synthetic sea water, cut into 
2.times.2.times.2 mm pieces, and placed in trypsin/EDTA/synthetic sea 
water for one hour, at 20.degree. C. The cells are then passed through a 
metal screen and rinsed with media. The primary cultures are started in 24 
well TC plastic plates, in medium plus 10% gastropod plasma, 50 .mu.g/mL 
gentamicin, 0.125, .mu.g/mL fungizone and 50 .mu.g/mL 
penicillin/streptomycin, at 20.degree. C. The samples are also split into 
two groups, one with 5% CO.sub.2 and one in room air. 
Both groups are expanded to T75 flasks and then split again after one week. 
At this point the cultures are allowed to become confluent. On the third 
passage, one month after the start of the culture, the confluent cells 
will began grouping into dense clusters. These epithelial cells will began 
to form a nucleus and thereafter deposit a hard outer wall in the shape of 
a circle onto the culture plastic which resembles a shell-like structure. 
EXAMPLE 3 
Cells (9.3.times.107) from the sixth passage of the marine sponge, Aplysina 
fistularis are added to 12 g of polystyrene microcarriers and 50 mL of 
media without gastropod plasma. The system is allowed to incubate for one 
and one half hours at 20.degree. C, and then 150 mL of media with 4.3% 
gastropod plasma is added with constant stirring. Every 2 to 3 days an 
aliquot of the culture is taken, the cell number is determined by counting 
on a hemocytometer, and new media is added to the system. As shown in FIG. 
1, the curve reflects a plot of the total number of cells times ten 
million versus day in suspension culture. 
After 17 days the 1 liter flask is confluent and the cell count is 
8.18.times.10.sup.9, yielding an 88-fold increase in cell number. Some of 
these cells are preserved in freezing medium and are later thawed to 
initiate new cultures. 
Another aliquot of this culture, which is not treated with trypsin/EDTA, is 
dried onto microscope slides and run through an automated, clinical, 
histology system, with Masson's trichrome stain, for connective tissue. 
The cell clump stained with Masson's trichrome, a clinical connective 
tissue stain which identifies the protein collagen by a deep blue color. 
Thus, the slides stained positive for a large amounts of collagen, an 
important part of the natural sponge. 
EXAMPLE 4 
A marine tunicate, species unknown, is collected from Scripps Canyon in La 
Jolla, Calif. The specimen is cut in 2.20 mm circular slices and stored in 
freezing medium, at -10.degree. C. 
Three days later, one of the samples is thawed, disinfected with NaClO, 
antibiotics and fungicides, disrupted through a metal mesh, and then 
rinsed with synthetic sea water. The resulting cells are placed into 12 
well tissue culture plastic plates containing medium with either 4% 
gastropod plasma or 10% fetal bovine plasma. The gastropod plasma wells 
will begin to proliferate and double their numbers after 4 days. At the 
same time, the cells in fetal bovine plasma will not grow and instead 
begin to die off. By the third passage the gastropod plasma cells are 
doubling every two days, so some wells are expanded to a T75 flask. The 
T75 cells will continue to proliferate and after another month, they are 
harvested and stored by cryopreservation for future use. 
The cells that are left in the 12 well plates for over a month, with 
regular media changes, first grow to confluence and then differentiate 
into honeycomb structures, resembling the cross sectional slices of the 
original animal. 
EXAMPLE 5 
The tropical marine sponge, Acanthella cavernosa is cut into strips 
measuring 4.times.4.times.5 mm and stored in freezing medium at 
-10.degree. C. Weeks later, samples are thawed and disinfected with NaClO, 
antibiotics and fungicides. The cells are then obtained by mechanical 
disruption through a metal screen, and put into culture at 28.degree. C., 
in medium with 5% gastropod plasma. 
Cells from the fourth passage are allowed to attach to plastic 
microcarriers for two hours in media with no gastropod plasma. The system 
is then stirred continuously, with fresh media containing 4% gastropod 
plasma being added in an attempt to keep the culture at a maximum growth 
rate, as illustrated in FIG. 2. The curve reflects a plot of the total 
number of cells times ten million versus days in suspension culture. 
After 10 days the culture is harvested for qualitative analysis of 
antibiotic metabolites. This culture system is repeated several times, 
with the best results yielding 1.0 g of cells per 100 mL of media. 
A sample of these cells is extracted with methanol and hexane. These 
extracts both demonstrate antibiotic properties, similar to extracts from 
the native sponge, against Staphylococcus aureus and Bacillus subtilis, in 
a standard natural product, antibiotic screening bioassay. 
EXAMPLE 6 
The marine nudibranch, Aplysia californica, is anesthetized with 8% 
MgCl.sub.2 and then the albumin gland is surgically removed. The purple 
colored gland is minced with a razor, digested with trypsin/EDTA for one 
hour and then passed through a steel mesh. The resulting cells are placed 
in T75 flasks coated with gastropod plasma, fetal bovine plasma or bovine 
collagen type 1 (Col 1). The cells attached and grew best in the gastropod 
plasma flask. These cultures are expanded four times over 30 days, after 
which the confluent cultures are harvested and cryopreserved in freezing 
medium. 
Thus it is shown that the present system provides for the long term culture 
of eukaryotic cells from marine invertebrates, and for the production of 
important metabolites from the cultures. Significantly, the system will 
function with cells from many different species of organisms, and will 
sustain commercially practical growth rates with concomitant metabolite 
production. 
Furthermore, with appropriate supplementation, the present system provides 
for the long-term cryopreservation of such eukaryotic cells, while 
maintaining the viability and vigor of the cells when re-established in 
culture. 
All patents and patent applications cited in this specification are hereby 
incorporated by reference as if they had been specifically and 
individually indicated to be incorporated by reference. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity and understanding, it 
will be apparent to those of ordinary skill in the art in light of the 
disclosure that certain changes and modifications may be made thereto 
without departing from the spirit or scope of the appended claims.