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WO1990003429A1 - Micro-emulsions de lipides pour milieux de culture - Google Patents

Micro-emulsions de lipides pour milieux de culture Download PDF

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Publication number
WO1990003429A1
WO1990003429A1 PCT/US1989/003985 US8903985W WO9003429A1 WO 1990003429 A1 WO1990003429 A1 WO 1990003429A1 US 8903985 W US8903985 W US 8903985W WO 9003429 A1 WO9003429 A1 WO 9003429A1
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Prior art keywords
lipid
cell culture
culture media
cells
component
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PCT/US1989/003985
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English (en)
Inventor
Duane Inlow
Brian Maiorella
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Novartis Vaccines and Diagnostics Inc
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Cetus Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]

Definitions

  • This invention is in the field of fermentation and cell culture. More particularly, the invention concerns cell culture media wherein lipids are supplied in the form of microemulsions and methods to disperse lipids in cell culture media.
  • Lipids are an apparently essential requirement for most animal cell culture. [See Yamane et al., Proc. Japan Acad. 57B(10):385-389 at p. 388 (1981); and Iscove, "Culture of Lymphocytes and Hemopoietic Cells in Serum-Free Medium," In Barnes et al. (eds.), Methods for Serum-Free Culture of Neuronal and Lymphoid Cells, pp.
  • lipids may turn out to be a universal requirement for freshly explanted cells.
  • the direct addition of lipids to media is not practical due to their low solubility.
  • lipids are provided to the medium in the serum wherein the lipids are carried as water soluble lipoproteins; whereas conventionally, in serum-free media, lipids are carried by albumin to which the lipid is coupled through a process employing a solvent, such as chloroform.
  • lipids as liposomes to cells in culture (1,2,3). These liposomes are most typically prepared by sonicating the lipid mix in the presence of a protein (albumin) and result in particles of size typically 25 nm (4).
  • Iscove id. at page 179-181, provides an example of the prior art process used to supply lipids to serum-free culture.
  • lipids arc mixed by being dissolved in chloroform; the chloroform is then evaporated and the lipids are "dried onto the bottom of the beaker.”
  • a suspending medium containing albumin is then added and the mixture is sonicated to disperse the lipids in the form of vesiclesliposomes small enough to pass through the pores of a sterilizing filter (0.45 ⁇ m).
  • the resulting suspensions are stored at 4°C. They may become somewhat more opaque in the first day or two of storage. This change probably reflects some coalescence of the liposomes to larger average size but has no influence on their effectiveness in culture....
  • each individual lipid preparation should be titrated in culture to determine its optimal concentration. Filter-sterilization of medium which has already been supplemented with lipid should be avoided, since there is a risk of retaining an
  • lipids to cell culture media enhances the availability of lipids to the cells and avoids globular lipid aggregates that can be lost in filtration and thus become unavailable to the cells.
  • This invention further provides the option of not having to filter sterilize the lipid component and the rest of the media components separately.
  • This invention concerns stable lipid microemulsions which can be added to cell culture media to provide essential lipids in a bioavailable form.
  • the invention further concerns cell culture media in which lipids are supplied in the form of such lipid microemulsions.
  • This invention further provides for methods to disperse hpids in culture media by the use of one or more emulsifiers.
  • the emulsifiers are preferably phospholipids and/or non-toxic polymeric detergents, and still more preferably lecithin and/or Pluronic polyols and/or polysorbate compounds.
  • This invention further provides for a lipid solution component comprising lipids, an organic solvent and one or more emulsifiers, and an aqueous component comprising one or more emulsifiers and water. It is the addition with agitation as by vortexing of the aqueous component to the lipid solution component which results in the formation of a lipid microemulsion.
  • Figure 1 shows the growth of Sf9 cells in the presence or absence of lipid supplement.
  • Figure 2 shows the particle size diameter profile of the lipid emulsion.
  • a microemulsion is herein defined as a preparation wherein one liquid is distributed in small globules throughout the body of a second liquid wherein said small globules are herein described as micelles.
  • the lipid microemulsions of this invention are prepared by combining with agitation an aqueous component and a lipid solution component, wherein the aqueous component is herein defined as comprising one or more emulsifiers in water, and the lipid solution component is herein defined as comprising one or more lipids and one or more emulsifiers in an organic solvent.
  • the lipid microemulsions of this invention provide a means of eliminating lipid carriers such as serum, serum albumin, or other proteins from culture media.
  • lipid carriers such as serum, serum albumin, or other proteins from culture media.
  • media which correspond to conventional culture media except that the serum moiety is partially replaced with at least one lipid nutrient in the form of a microemulsion of this invention. Therefore, serum or other proteinaceous substances such as albumin of conventional media can be reduced or dispensed with according to this invention.
  • Use of the culture media of this invention makes it easier to isolate and purify the product accumulated in the media. Mass production of culture media is also made easier, according to this invention.
  • Another advantage of this invention is that preferred emulsifiers of this invention, Pluronic polyols, are already used as physical protectants in mammalian cultures, and have been shown therein to be non-toxic to cells and non-inhibitory to cell growth.
  • PCT US 89/01029 published February 9, 1989 and PCT US 89/01028, published February 9, 1989 provide examples of successful cultivation of insect cells in media containing Pluronic polyols and the expression of recombinant proteins by such insect cells.
  • PCT US 89/01028, supra describes the use of lipid microemulsions herein disclosed in serum-free, low protein or essentially no protein media for insect cell culture.
  • PCT US 89/01029, supra discloses the successful culturing of insect cells in airlift fermentors.
  • Pluronic polyols preferred emulsifiers of this invention, serve dual purpose in providing not only a physical protectant for agitated and/or sparged cultures but also in providing an emulsifying system for lipids to be supplied to the culture. Therefore, media of this invention provide an enhanced nutritional environment for the growth of cells under any culture conditions but especially for cells growth under agitated and/or sparged conditions, and, in general, for cultures that are well-aerated. As a consequence, the media thus provide an enhanced nutritional environment for the production of recombinant, viral and/or natural products, wherein examples of natural products are native proteins or monoclonal antibodies.
  • protein is herein defined to exclude the term “peptones.”
  • the media of this invention preferably contain very low concentrations of protein, that is, preferably less than about 1000 ⁇ /ml, more preferably less than about 50 ⁇ g/ ml, still more preferably less than about 5 ⁇ /ml. If certain optional components are determined to be desirable as indicated below, for example, growth factors such as insulin, transferrin or catalase, they are present preferably at low concentrations and do not function as lipid carriers.
  • Lipids are defined herein as any of a heterogeneous group of fats and fatlike substances characterized by being water-insoluble and being extractable by nonpolor (or fat) solvents such as alcohol ether, chloroform or benzene, among others.
  • Lipids include unsaturated fatty acids, saturated acids, steroids, lipid soluble vitamins and phospholipids and their esters. Examples of unsaturated fatty acids include linoleic acid, linolenic acid, oleic acid, arachidonic acid, and uric acid and their glycerides and esters.
  • Preferred fatty acids also include fatty acid esters, preferably
  • polyunsaturated fatty acid methyl esters as for example, those found in fish hver oil, preferably cod liver oil.
  • saturated fatty acids include palmitic acid and stearic acid and their glycerides.
  • lipid soluble vitamins include vitamins A, D and E.
  • steroids that is lipids which contain a hydrogenated cyclopentophenanthrene-ring system, include progesterone, adrenocortical hormones, the gonadal hormones, cardiac aglycones, bile acids and sterols (such as cholesterol).
  • Preferred steroids are sterols, preferably cholesterol.
  • lipids to be supplied to cell culture media of this invention depend on the choice of cells to be cultured. Also, lipids are supplied in
  • lipid components appropriate for the particular cell line being cultured wherein the concentration is not toxic to the cells and is non-inhibitory to cell growth.
  • insect cells appear to be able to tolerate a higher concentration of lipids than mammalian cells.
  • Exemplary lipid components for a mammalian cell culture are shown in Examples 1 and 2, below, whereas an exemplary lipid component of Example 1 for the mammalian cells is shown in Example 3.
  • the lipid component of Example 1 for the mammalian cells is the same as that for the insect cells of
  • Example 3 except in the respect that the concentrations of the lipid and the emulsifier Tween 80 are ten times less for the mammalian lipid component that for the insect lipid component.
  • Preferred ranges for lipids for insect cells are as follows: A mixture of polyunsaturated fatty acid methyl esters, such as, fish liver oil, preferably cod liver oil, is preferably present in the media at a concentration of from about 1 mg/l to about 50 mg/l, preferably from about 5 mg/l to about 15 mg/l, and most preferably about 10 mg/l. Said concentrations of cod liver oil further contain the preferred concentrations of the lipid soluble vitamin A.
  • the sterol preferably cholesterol, is at a concentration from about 2 mg/l to about 7 mg/l, more preferably from about 3 mg/l to about 5 mg/l, and most preferably about 4.5 mg/l.
  • the alpha- tocopherol of the lipid component is at a concentration of from about 0.5 mg/l to about 4 mg/l, more preferably about 2 mg/l.
  • insect cell culture media containing the lipid microemulsions of this invention are those disclosed in claimed in U.S. Serial No. 077,303.
  • the compositions of three of such preferred serum-free, low protein or essentially no protein media for insect cell culture used in the examples below are outlined in Table 1.
  • the Pluoronic Polyol-Lipid Microemulsions in Table 1 are prepared according to Example 3, infra.
  • lipids being supplied in the form of a microemulsion in addition to enhancing the availability of the lipids to the insect cells, is in providing the option of not having to filter sterilize the lipid fraction and the rest of the media components separately.
  • the lipid microemulsions of this invention can be added to the media without being filter sterilized, and the entire media can then be filter sterilized without the concern of lipid loss during the filter sterilization process.
  • such an advantage is significant
  • the lipid solution component of the media of this invention is prepared by combining the appropriate amount of a mixture of lipids, for example, a mixture of polyunsaturated fatty acids, alpha-tocopherol acetate and cholesterol, and the emulsifier(s), in an appropriate amount of an organic solvent, preferably a C 1 -C 3 alcohol, more preferably ethanol, to form a solution. It is important that the final concentration of the organic solvent is non-toxic and noninhibitory to cell growth. For example, a preferred concentration of ethanol as the organic solvent in preferred media of this invention would be approximately 1 ml/l.
  • the lipid solution is prepared by combining the appropriate amount of a mixture of lipids, for example, a mixture of polyunsaturated fatty acids, alpha-tocopherol acetate and cholesterol, and the emulsifier(s), in an appropriate amount of an organic solvent, preferably a C 1 -C 3 alcohol, more preferably ethanol, to form a solution. It is important that the
  • the lipid solution component is then optionally filter sterilized.
  • the lipid solution component can be stored under nitrogen in a temperature range of from about -20°C to about -80°C.
  • the aqueous component containing one or more emulsifiers, preferably protective agent/emulsifiers, is similarly optionally filter sterilized.
  • the aqueous component which is preferably of greater volume than the lipid solution than the lipid component is slowly and, optionally aseptically, added to the lipid solution component with agitation as by vortexing.
  • the lipid microemulsion is formed upon the addition with agitation of the aqueous component to the lipid solution component. (In general, it is preferred to add the aqueous component to the lipid solution component rather than vice versa.)
  • the optionally filter sterilized lipid microemulsion can then be added to the media. If the option of aseptically forming the lipid microemulsion from filter sterilized components is not taken, the option remains of filter sterilizing the entire medium after all additions are completed.
  • Emulsifiers preferred for preparing the lipid microemulsions of this invention are non-toxic polymeric detergents, bile salts, such as, deoxycholate, and/or phospholipids.
  • the emulsifiers are non-toxic polymeric detergents and/or phospholipids. It is further preferred that the non-toxic polymeric detergents are non- ionic and that the phospholipid emulsifier is lecithin. It is further preferred that there be one or more emulsifiers present in both the aqueous and lipid solution
  • McCutcheon Division of MC Publishing Co., 175 Rock Road, Glenn Rock, NJ, U.S.A.) are examples of a source of finding non-toxic, non-ionic polymeric detergent candidates for use as emulsifiers of this invention. Infra, a simple test is outlined for selecting appropriate emulsifiers within the scope of this invention.
  • the emulsifiers are present at final concentrations in the media which are non- toxic to cells and noninhibitory to cell growth, reproduction and expression of products thereby.
  • the emulsifiers are considerably more concentrated in the aqueous and lipid solution components than in the media in which they are diluted, preferably by a factor of 100 or 1000, respectively.
  • the solubility limits of the volumes of the aqueous or lipid solution components could be reached before the maximum, non-toxic and beneficial concentration of a particular emulsifier has been reached for the medium as a whole. If such an event should occur, the emulsifier could be added directly to the medium, that is, outside of the hpid microemulsion, if it is determined that such an addition would be beneficial to the cells, as, for example, by enhancing the protective and/or emulsifying effects of a protective agent/emulsifier. Therefore, a preferred
  • concentration (weight/volume) range for Pluronic polyols in the media of this invention would be from about 0.01% to about 1%; however, the solubility limit of Pluronic F68 is about 10% in water, and therefore, a 10 ml aqueous component would not be sufficient to accommodate the high end of the emulsifier concentration range in the final media. Additional Pluronic F68 could thus be added directly to the media if such additions would be considered necessary to optimize the
  • the concentration of Pluronic F68 in a 10 ml aqueous component would be preferably from about 1% to about 10%, more preferably from about 5% to about 10%, and still more preferably about 10%.
  • the emulsifiers of the aqueous component be protective agents.
  • Protective agents are herein defined as non-toxic, water soluble compounds that functionally act to protect cells from damage and death in agitated and/or sparged cultures.
  • a protective agent candidate can be selected by first confirming that it is non- toxic to the cells to be cultured by methods known to those skilled in the art of cell culture, for example, by adding it to a suspension or monolayer of the cells of choice for cultivation and comparing the growth of the culture to a control. Then, the non- toxic protective agent candidates can be tested for protective ability by adding the candidate agent to an agitated and/or sparged culture of the cells of choice at small scale and observing viability and growth rate after an appropriate period and comparing the viability and growth rate of the cells of said culture to the viability and growth rate of the cells in a control culture.
  • a source of finding non-toxic, non- ionic polymeric detergent protective agent/emulsifier candidates for the media of this invention can be found in Editions of McCutcheon's Emulsifiers & Detergents, supra.
  • emulsifier or emulsifiers of the aqueous component block copolymers of propylene oxide and ethylene oxide are further preferred.
  • Such protective agent/emulsifiers are Pluronic polyol, such as, Pluoronic F68, F77, F88 and F108, preferably F68 and F88, more preferably F68.
  • the Pluronic polyols are commercially available from BASF Wyandotte Corp. (101 Cherry Hill Road, P.O. Box 181, Parsippany, NJ 07054, U.SA.).
  • the concentration of the protective agent/emulsifier(s) is preferably that which is most effective in protecting the cells from damage, but which concentration is non- inhibitory to cell growth, reproduction and expression of products thereby.
  • the Pluronic polyol protective agent/emulsifiers are present in the media of this invention preferably at a concentration (weight/volume) of from about .01% to about 1%, more preferably from about .05% to about 0.5%, and most preferably about 0.1%.
  • the emulsifier or emulsifiers of the aqueous component it is not necessary for the emulsifier or emulsifiers of the aqueous component to be protective agents to practice this invention. Any non-toxic emulsifiers, that are non-inhibitory to cell growth, reproduction and expression of viral, recombinant or natural products, such as, antibodies or native proteins, can be used.
  • the emulsifier or emulsifiers of the aqueous component are not protective agents, other protective agents would be included in the media if the culture is to be well-aerated.
  • Such other protective agents would be preferably cell surface stabilizing agents and/or viscosifying agents and/or bubble surface tension reducing agents.
  • Examples of preferred protective agents other than non-toxic polymeric detergents are hydroxyethyl starch, methyl cellulose, carboxymethyl cellulose (as, sodium carboxymethyl cellulose), dextran sulfate, polyvinylpynolidone, ficoll, alginic acid and polypropyleneglycol.
  • Preferred emulsifiers in the lipid solution component are phospholipids and non-toxic, non-ionic polymeric detergents.
  • a preferred phospholipid emulsifier is lecithin and preferred non-toxic, non-ionic polymeric detergent emulsifiers are polysorbate compounds having the formula:
  • R is a saturated or unsaturated fatty acid having from 16 to 20 carbons, inclusively;
  • t is an integer between 10 and 30, inclusively;
  • u is an integer between 10 and 20, inclusively.
  • polysorbate emulsifier is polyoxyethylene (20) sorbitan monooleate, otherwise known as polysorbate 80.
  • polysorbate 80 is polyoxyethylene (20) sorbitan monooleate, otherwise known as polysorbate 80.
  • a non-toxic, non-ionic polymeric detergent is commercially available as Tween 80 from ICI Americas Inc. (New Murphy Road & Concord Pike, Wilmington, DE 19897, USA).
  • Another polysorbate 80 is commercially available as Durfax 80 from Durkee Industrial Foods Group/SCM Corp. (900 Union Commerce Bldg., Cleveland, Ohio 44115, USA).
  • Such a non-ionic, non-toxic, polymeric detergent/emulsifier such as, polysorbate 80, is preferably present in the lipid solution components of this invention at a concentration from about 0.5 mg/ml to about 75 mg/ml, more preferably from about 1 mg/ml to about 30 mg/ml, and more preferably from about 2.5 mg/ml to about 25 mg/ml.
  • Optional ingredients that can be added to the lipid solution component include reducing agent/anti-oxidants, such as, dithiothreitol, ⁇ -mercaptoethanol and
  • monothiolglycerol wherein monothiolglycerol is a preferred reducing agent/anti- oxidant
  • Such ingredients are considered amphipathic if they are soluble in both the aqueous and the lipid solution components, and are preferably added to the lipid solution component in appropriate concentrations for the chosen cell line.
  • Testing whether an emulsifier, a protective agent/emulsifier, combination of emulsifiers, or a combination of a protective agent/emulsifier and an emulsifier or emulsifiers is effective in emulsifying the lipids to be supplied to the media of this invention can be simply performed as follows. First, the candidate emulsifier or combination of emulsifiers must be confirmed as non-toxic to the cells of choice in a manner as described above for candidate protective agents. Secondly, the emulsifying ability of the candidate emulsifier(s) is tested at the determined non-toxic
  • the appropriate lipids are dissolved in an appropriate organic solvent, preferably an alcohol (C 1 -C 3 ), more preferably ethanol.
  • Hie candidate emulsifier or each of the combination of emulsifiers is then combined with the lipid solution or in the separate aqueous solution, depending upon in which solution, that is, the organic or aqueous, the candidate emulsifier is more readily combinable.
  • the aqueous component is added to the lipid solution component and vigorously agitating as by vortexing. A clear to slightly translucent microemulsion should form if the lipids are successfully emulsified.
  • At least one emulsifier should be present in the lipid solution component and one in the aqueous component. If only one candidate emulsifier is being tested, it can be tested with a known emulsifier, such as, for example, if the candidate emulsifier is readily combinable in water, it could be tested in a candidate aqueous component with Tween 80 as the emulsifier in the lipid solution component; whereas, if the candidate emulsifier is readily combinable in a solution of appropriate lipids, it could be tested with Pluronic F68 in an aqueous component Another option would be to test a sole candidate emulsifier in both the aqueous and lipid solution components, that is, wherein the same emulsifier would be present at appropriate concentrations in both a candidate aqueous component and a candidate lipid solution component.
  • One option for emulsifying lipids to be supplied to cell culture media according to this invention is a dual emulsifier system wherein, as noted above, the protective agent is an emulsifier as well as a protective agent and can act in conjunction with an emulsifier or combination of emulsifiers present in the lipid solution component
  • a preferred example of a dual emulsifier system of the media of this invention is the combination of a protective agent/emulsifier, preferably a Pluronic polyol, more preferably Pluoronic F68 or Pluronic F88, and still more preferably Pluronic F68, and a non-toxic non-ionic polymeric detergent, preferably a polysorbate compound, and more preferably polysorbate 80.
  • Another option for emulsifying lipids according to this invention is a system wherein the protective agent is not significantly emulsifying but wherein one or more additional emulsifiers are present in the aqueous component which is added to the lipid solution component and act in conjunction with the emulsifiers present therein to form a microemulsion.
  • lipid microemulsions of this invention are added depends on the particular cells that are being cultured. There are commercially available media for all cell types.
  • IPL-41 is a preferred basal medium.
  • IPL-41 basal medium is commercial available from a number of vendors and is described in Weiss et al., In Vitro. 17(6):495-502 (June 1981) and in Weiss et al., CRC Press, supra, pp. 70-72 (1986).
  • Table 1 of Weiss et al. (in Vitro) at page 496, and Table 3 of Weiss et al., CRC Press, at pages 71-72 outline all the components of IPL-41 and provide their proportions in mg/l; said tables are herein incorporated by reference.
  • IPL-41 the preparation of the complete medium IPL-41 is described wherein tryptose phosphate broth (TPB) and fetal bovine serum (FBS) are added.
  • the IPL-41 basal medium employed in preparing the media of this invention preferably does not contain tryptose phosphate broth (TPB) or fetal bovine serum (FBS).
  • PCT US 89/01028, supra describes preferred replacements for serum and tryptose phosphate broth wherein lipid microemulsions of this invention supply the lipid fraction in insect culture media.
  • basal medium is herein defined as a nutrient mixture of inorganic salts, sugars, amino acids, optionally also containing vitamins, organic acids and/or buffers. Basal media together with supplements provide the nutrients necessary to support cell life, growth and reproduction.
  • the choice of basal media used as the starting point for preparing the media of this invention is not critical, but should be appropriate for the cell line selected and would most preferably contain little or not sown, serum albumin or other lipid carriers.
  • the basal medium can also be considered optional in the sense that appropriate peptone and lipid components can be selected which provide such necessary nutrients as amino acids and vitamins required to support cell life, growth, reproduction, and expression of recombinant or viral products and production of antibodies.
  • DME Dulbecco's Modified Eagle Medium
  • RPMI 1640 Medium also available from Irvine Scientific
  • HL-I Ventrex Labs, Portland, Maine USA
  • HB104 Hana Biologicals, Berkeley, California USA
  • Eagle Minimum Essential Medium and Ham F-12 Medium; RITC 80-7
  • Examples of media for plant cell culture include those described in Murashige et al., Physiol. Plant. 15:473 (1962); Gamborg et al., Exp. Cell Res., 50:151 (1968); and Gamborg et al., In Vitro, 12:473 (1976).
  • culture media for animal cells, algae, bacteria, fungi and protozoa can be found in Cote et al. (eds.), ATCC Media Handbook, first edition 1984 (ATCC 1984).
  • the media of this invention can also include other preferably water-soluble ingredients, for example, insulin to enhance glucose uptake, transferrin for iron transport, trace elements as selenium, catalase as a peroxidation protectant, ethanolamine as a lipid precursor, steroid hormones as testosterone, thyroid hormone as triiodothyronine, nucleic acid precursors such as hypoxanthine, thymidine, deoxyadenosine, and deoxycytidine, as well as vitamins, minerals, amino acids and other nutrients such as L-glutamine, pyruvate, alpha-glycerol phosphate, glycerol, folk acid, and inositol which are contained in conventional serum-supplemented or serum free media for cell culture.
  • ingredients are proteins, they are not being supplied for the purpose of carrying lipids and are preferably at low concentrations.
  • the method of preparing a culture medium to which the lipid microemulsions of this invention are added is not critical.
  • the media may be prepared, for example, by dissolving all the ingredients and additives in water in their respective appropriate concentrations first and then filtering the solution on a membrane filter under pressure to get a sterilized culture medium.
  • the method of culturing cells with the media of this invention is also not critical.
  • Cells are cultured in the media of this invention under approximately the same conditions as those for conventional culture media, except that whenever possible, cells should be growth under well-aerated conditions, that is, in agitated and/or sparged cultures.
  • For optimal culture conditions for the growth of insect cells see PCT US 89/01029 and PCT US 89/01028, supra, which are herein incorporated by reference.
  • insect cells grown in the media of this invention are cultured in a temperature range and under conditions appropriate for the particular cell line selected.
  • Spodoptera frugiperda cells preferably Sf9 cells
  • the pH of the culture medium is preferably maintained in a range from about 6 to about 7, more preferably about 6.2 to about 6.4.
  • CSF-1 is produced by host insect cells cultured in media containing lipid microemulsions of this invention.
  • Coldy stimulating factor (CSF-1) refers to a protein which exhibits the spectrum of activity understood in the art for CSF-1, that is, when applied to the standard in vitro colony stimulating assay of Metcalf, J. Cell Physiol., 2.2:89 (1970), it results in the formation of primarily macrophage colonies.
  • Native CSF-1 is a glycosylated dimer, dimerization may be necessary for activity.
  • CSF-1 herein refers to both dimeric and monomeric forms.
  • heterologous proteins that have been expressed in insect cells via a baculovirus expression vector system (BEVS) are outlined in Summers et al.,
  • Exemplary recombinant proteins include, without limitation colony stimulating factors [for example, long and short form CSF-1 or M-CSF, G-CSF, GM-CSF and inte ⁇ ieukin-3 among others], modified pro-urokinase or urokinase, tissue plasminogen activator (TPA), TPA-urokinase hybrids, toxic proteins such as whole ricin toxin, ricin A chain, products containing ricin A, as well as, interferons ⁇ , ⁇ and ⁇ and hybrids thereof), interleukins, tumor necrosis factor, erythropoietin and other hematopoietic growth factors, human growth hormone, as well as porcine, bovine and other growth hormones, epidermal growth factor, insulin, hepatitis B vaccine, superoxide dismutase, Factor VllI, Factor VllI C, atrial natriuretic factor, feline leukemia virus vaccines, as, for example,
  • monoclonal antibodies can be produced by hybridomas cultured in media of this invention and various viral products can be produced from insect cells so cultured.
  • the media of this invention would be used for suspension cultures including microcarrier cultures.
  • the lipid microemulsions of this invention can however be added to any media to which lipids are considered a beneficial addition.
  • cells can be grown successfully in media wherein serum, albumin, other proteinaceous lipid carrier or other non-protein lipid carriers, such as a cyclodextrin or mixture of cyclodextrins (see Yamane et al., supra: U.S. Patent No. 4,533,637), are employed, then the cells can be grown in the media of this invention wherein the lipids are supplied as a lipid microemulsion.
  • lipids are supplied to the cells is not critical. They can be supplied as a microemulsion according to this invention whether they are considered as essential or growth promoting nutrients, physical or chemical protectants or as having other functions, as for example, as a solvolytic agent, as a membrane modifying agent, as a surface tension reducing agent and/or as a cell surface stabilizing agent.
  • the cells can be of animal, microbial or plant origin. If animal cells, they can be from vertebrates or invertebrates. Preferably, the cells are those which can produce recombinant viral and/or natural products. Exemplary vertebrate cells are mammalian cells, for example, lymphocytes, fibroblasts, epithelial cells, ovarian cells, and their transformed cells, various neoplastic cells, and hybridomas derived therefrom.
  • mammalian cells include Chinese hamster ovarian cells, Epstein-Barr Virus (EBV)-transformed human lymphoblastoid cell lines such as UMCL and C51804, human Burkitt's lymphoma-derived Namalwa cells, murine lymphoid cell-derived myeloma SPI cells, human fibroblast cells such as HEL and IMR-90, human tumor-derived epithelial cells such as HeLa-S 3 , Hep-2 and KB, human primary cultured cells, rat Yoshida sarcoma cells, hamster fibroblast cells BHK-21, murine fibroblast cells 3T3, murine lymphoma cells YAC-1, human/mouse hybridomas such as stable cell line D-234-4-27-8 which produces anti-LPS IgM (deposited at the American Type Culture Collection, Rockville, Maryland USA on August 10, 1984 under ATCC Accession No. HB 8598) and hybridomas which produce monoclonal antibodies to human fibroblast
  • EBV
  • Exemplary invertebrate cells are insect cells, preferably cells which can produce viral or recombinant products upon infection, respectively, with either wild- type viruses or recombinant baculoviruses and which have been shown to grow, reproduce and express recombinant and/or viral products in a medium containing serum, albumin, another protein and/or other lipid carriers.
  • insect cells that can be grown in IPL-41 basal medium containing approximately 10% serum, can be grown in media wherein lipids are supplied as microemulsions according to this invention.
  • Such insect cell lines include Bombyx mori. Lymantria dispar.
  • Trichoplusia ni and Spodoptera frugiperda See generally, Granados et al. (eds.), The Biology of Baculoviruses (CRC Press 1986); Vaughn, Adv. Cell. Cult.,
  • insect cells that can be grown in the media of this invention are preferably from any order of the Class Insecta which can be hosts to a baculovirus expression vector system, or other wild-type viruses, but are preferably from the Diptera or Lepidoptera orders.
  • About 300 insect species have been reported to have nuclear polyhidrosis virus (NPV) diseases, the majority (243) of which were isolated from Lepidoptera.
  • NPV nuclear polyhidrosis virus
  • Insect cell lines derived from the following insects are exemplary: Carpocapsa pomonella preferably cell line CP-128); Trichoplusia ni (preferably cell line TN-368); Autographa califomica; Spodoptera frugiperda (preferably cell line Sf9); Lymantria dispar.
  • Mamestra brassicae Aedes albopictus; Orgvia pseudotsugata; Neodiprion sertifer.
  • Preferred insect cells are from Spodoptera frugiperda, and especially preferred is cell line Sf9.
  • the Sf9 cell line used in the examples herein was obtained from Max D. Summers (Texas A & M University, College Station, TX 77843 USA).
  • Other S. frugiperda cell lines, such as IPL-Sf21AE lll, are described in Vaughn et al., In Vitro, 13:213-217 (1977).
  • the preferred insect cell lines cultured in the media of this invention are suitable for the reproduction of numerous insect-pathogenic viruses such as parvoviruses, pox viruses, baculoviruses and rhabdoviruses, of which
  • nucleopolyhidrosis viruses and granulosis viruses (GV) from the group of baculoviruses are preferred.
  • NPV viruses such as those from Autographa spp., Spodoptera spp., Trichoplusia spp., Rachiplusia spp., Galleria spp. and Lymantria spp.
  • European Patent Application 127,839 (published December 12, 1984) to Smith et al. describes a method for producing a recombinant baculovirus expression vector, capable of expressing a selected gene in a host insect cell. Said European application is herein incorporated by reference.
  • the recombinant baculovirus expression vector is cotransfected with wild-type baculovirus DNA into a host insect cell, wherein recombination occurs.
  • Recombinant baculoviruses are then detected and isolated according to methods described in EP 127,839 and Summers et al., "A Manual and Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas
  • PCT US 89/01038, supra specifically describes the construction of baculovirus CSF-1 expression and transfer vectors, including pAcM4 and pAcM6, used to prepare the recombinant baculoviruses AcM4 and pAcM6 by cotransfection with baculovirus DNA in Sf9 cells.
  • Said recombinant baculovirus transfer vectors pAcM4 and pAcM6 referred to in the examples below has been deposited at the American Type Culture Collection (ATCC) [12301 Parklawn Drive, Rockville, MD 20852 (USA))] on June 12, 1987 in E. coli/MM294 under the respective designations ATCC Nos. 67429 and 67438.
  • Said vectors have also been deposited and are maintained in the Cetus Master Culture Collection (CMCC) under the respective designations CMCC No.
  • Recombinant baculovirus AcM4 carries a nucleotide sequence which encodes for a 150 amino acid form of rCSF-1 whereas the
  • recombinant baculovirus AcM6 carries a nucleotide sequence which encodes for a 522 amino acid form of rCSF-1.
  • the effect of timing of the infection of the insect cells with a recombinant baculovirus has been shown to be critical for enhanced specific productivity.
  • the specific production of the recombinant protein was found to be constant during the exponential phase of cell growth under non-oxygen limited conditions. Late infection, under non-exponential growth conditions, resulted in lower specific productivity and lower final titer.
  • the exponential growth phase be extended to the highest possible cell densities to achieve the highest total productivity of the recombinant protein product
  • Specific productivity (U or mg/cell) of the recombinant protein product is relative independent of cell density at the time of infection as long as the culture is in exponential growth.
  • Spodoptera frugiperda cells are the host insect cells and when preferred media of this invention as described for insect cells in PCT US 89/01028, supra, are used, cell densities of from about 1.0 x 10 6 to about 4.0 x 10 6 cells/ml are preferred for infection with the recombinant baculovirus, more preferably from about 2.5 to about 3.5 x 10 6 cells/ml.
  • the timing of the harvest of the recombinant protein product is critical to avoid contamination of the recombinant protein by viral and cell lysis proteins and to simplify thereby the downstream purification of the recombinant protein. With considerations for the stability of the product, it would be preferred to harvest the recombinant product before significant cell lysis has occurred.
  • each recombinant, viral or native product expressed in the media of this invention should be checked for stability and degradation over the course of the fermentation run. Such considerations should enter into a determination of the optimal harvest time.
  • Example 1 The following examples further illustrate the aqueous and lipid solution components, the lipid microemulsions, media and methods of this invention. These examples are not intended to limit the invention in any manner.
  • Example 1 The following examples further illustrate the aqueous and lipid solution components, the lipid microemulsions, media and methods of this invention. These examples are not intended to limit the invention in any manner.
  • Example 1 The following examples further illustrate the aqueous and lipid solution components, the lipid microemulsions, media and methods of this invention. These examples are not intended to limit the invention in any manner.
  • Example 1 The following examples further illustrate the aqueous and lipid solution components, the lipid microemulsions, media and methods of this invention. These examples are not intended to limit the invention in any manner.
  • Example 1 The following examples further illustrate the aqueous and lipid solution components, the lipid microemulsions, media and methods of this invention. These examples are not intended to limit the invention in any manner.
  • Example 1 The following examples further illustrate the
  • Lipid Microemulsion Preparation for Mammalian Cells 10 mg of cod liver oil, 4.5 mg of cholesterol, 2.0 mg of alpha-tocopherol acetate and 25 mg of Tween 80 were added to 10 ml of ethanol, dissolved therein, and the solution was filter sterilized. To prepare a quantity of lipid microemulsion sufficient for approximately one liter of mammalian culture medium, 10 ml of 10% Pluronic F68 in water was slowly and aseptically added with agitation by vortexing to 1 ml of the lipid solution component A microemulsion was tiiereby formed which can then be added to a culture medium that has already been filter sterilized.
  • Lipid Microemulsion Preparation for Mammalian Cells 10 mg of monothiolglycerol, 10 mg of linoleic acid, 10 mg of lecithin, 5 mg of cholesterol, 5 mg of alpha-tocopherol acetate, and 20 mg of Tween 80 were added to 10 ml of ethanol and dissolved therein. To prepare a quantity of lipid
  • a lipid microemulsion sufficient for approximately 1 liter of mammalian culture medium, 10 ml of 10% Pluronic F68 in water was slowly added with agitation by vortexing to 1 ml of the lipid solution component. A lipid microemulsion was thereby formed which can be then added to a culture medium which medium can then be filter sterilized without a concern of loss of lipids during the process. This emulsion is referred to as the A7 emulsion.
  • cod liver oil 100 mg of cod liver oil, 250 mg of Tween 80, 45 mg of cholesterol and 20 mg of alpha-tocopherol acetate were added to 10 ml of ethanol and dissolved therein. The lipid solution component was then filter sterilized.
  • microemulsion was thereby formed which can then be added to a insect cell culture medium that has already been filter sterilized.
  • ISFM-4 media for a number of passages.
  • Said media contain the lipid microemulsion described above in Example 3.
  • die ISFM-4 culture had a longer lag phase than die ISFM-3 culture, but by the third passage, both cultures grew equally well with short lag phases.
  • Both ISFM-3 and ISFM-4 cultures have longer lag phases than conventional serum containing insect culture media.
  • Lag phase was calculated by extrapolating the exponential phase plot (semi-log) back to the initial cell density (1 x 10 5 cells/ml. The x-intercept hours of culture incubation) is equal to the lag phase.
  • Example 5 A second experiment was conducted to show the effect of the liquid supplement on the growth of Sf9 cells in ISFM-4 serum free media. After two passages in serum free media, the cells were seeded in serum free media with or without the lipid microemulsion of Example 3, and cell growth monitored. It is apparent from figure 1 that the microemulsion is required for Sf9 cell growth.
  • Example 5 A second experiment was conducted to show the effect of the liquid supplement on the growth of Sf9 cells in ISFM-4 serum free media. After two passages in serum free media, the cells were seeded in serum free media with or without the lipid microemulsion of Example 3, and cell growth monitored. It is apparent from figure 1 that the microemulsion is required for Sf9 cell growth.
  • Example 5 A second experiment was conducted to show the effect of the liquid supplement on the growth of Sf9 cells in ISFM-4 serum free media. After two passages in serum free media, the cells were seeded in serum free media with or without the lipid microemulsion of Example 3, and cell growth monitored. It is apparent from
  • An additional medium to those used in Example 4, that is, SFM2M without BSA as described in the Detailed Description above was also used in this example.
  • the recombinant baculovirus AcM4 was used to infect exponentially growth cultures at a multiplicity of infection of 1. As shown in Table 3, below, the serum containing culture and the serum free cultures which contain lipid microemulsions of this invention produced similar levels of rCSF-1 (about 10 6 U/ml) as estimated by RIA.
  • Sf9 cells maintained in 10% serum containing medium, mat is, IPL-41 complete medium, readily adapted to SFM2M without BSA, ISFM-3 and ISFM-4 upon the first and second passages.
  • Subsequent passages and infections with the recombinant baculovirus AcM4 and a second recombinant baculovirus AcM6 have confirmed that such media containing lipid microemulsions are suitable media for the large-scale growth of insect cells and for the production of recombinant protein products via a recombinant baculovirus expression vector system.
  • the lipid microemulsions can be formulated in a wide variety of media, and are not restricted to insect cell media nor to culture of insect cells.
  • the lipid microemulsion shown in Example 1 was used to supplement media consisting of a 1:1 mixture of MCDB 301, described by Hamilton and Ham, in In Vitro 12:(9) 537, 1977; and DME-spinners, plus 4 mM glutamine, 10 ⁇ g/ml insulin, 10 ⁇ g/ml transferrin, 10 mg/ml ethanolamine, and 400 ⁇ g/ml bovine serum albumin.
  • Cells were grown both in serum free media, or media supplement with 1% fetal bovine serum.
  • Table 4 shows that in serum containing media, CHO cells attain a density of about 1.3 x 10 6 cells/ml, and expreses m-CSF at a level of about 310,000 U/ml. In serum free media, a cell density of about 0.9 x 10 6 cells/ml is reached, and CHO cells in this media express about 240,000 U/ml of m-CSF.
  • lipid microemulsions shown herein support the growth and product expression of other mammalian cell types
  • several cell lines of immunologic origin, as well as the Namalwa cell line were cultured.
  • Table 4 shows that the cell lines MOPC21 and Namalwa respond favorable to media supplement with the A7 Kpid microemulsion.
  • Hie medium consisted of a 1:1 mixture of DM8 and IPL/41, plus the A7 lipid microemulsion of Example 2.
  • DM8 was formulated from sDME-HP, RPMI 1640, and IPL/41 (4:4:1). Also added were insulin (5 ⁇ g/ml), transfenin (5 ng/ml), selenium (5 ⁇ g/ml), glutamine (8 mM), and 10 mg/ml ethanolamine.
  • An ELISA assay to determine antibody levels was run.
  • lipid microemulsions are filter sterilizable alone or when combined with media. This is not true of prior art lipid compositions which are not readily filter sterilized with culture media because an indeterminate amount of lipid is not filtered, and remains trapped on the filter. Thus experiments were done to determine the particle size properties of the microemulsions which favor their filtration.
  • Low angle laser light scattering measurements were made using a Coulter model N4M laster light scattering instrument and version 10.0W software.
  • A7 lipid mix was prepared using standard prodedures and was then diluted (1:5) in DBM medium (dilution to this concentration was required to be in the range appropriate for the sensitivity of the instrument). Measuring intensity at a scattering angle of 90°, a major peak corresponding to particles of size about 95 nm was identified, with less intense peaks at about 9 and 890 nm ( Figure 2).
  • the A7 lipid mix was then filter sterilized through a 0.45 um filter (Sybron Nalge, Rochester NY, No. 120-0045) and the measurement repeated.
  • Peaks corresponding to similar particle sizes (78, 6 and 860 nm) were again detected. Because one does not expect lipids with the particle size distribution observed, particularly those over 800 nm, to readily filter through an 0.45 ⁇ M filter, it is thought that the microemulsion distributes on filtration into smaller particles which are filterable and then reassociate.
  • the present invention is not to be considered limited in scope by the deposited recombinant transfer vectors, since the deposited vectors are intended only to be illustrative of particular aspects of the invention. Any recombinant

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Abstract

Des micro-émulsions de lipides pouvant être ajoutées à des milieux de culture de cellules, pour obtenir des lipides essentiels sous une forme bio-disponible, ainsi que leurs composants, sont décrits. Des procédés de dispersion de lipides dans des milieux de culture en utilisant un ou plusieurs émulsifiants sont décrits. Sont également décrits des milieux qui supportent la croissance de cellules et la production de produits recombinants, viraux et/ou naturels où l'alimentation en lipides s'effectue sous la forme d'une micro-émulsion.
PCT/US1989/003985 1988-09-23 1989-09-13 Micro-emulsions de lipides pour milieux de culture Ceased WO1990003429A1 (fr)

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DE4038385A1 (de) * 1990-12-01 1992-06-04 Roecar Holdings Nv Sitosterol und seine glykoside mit verbesserter bioverfuegbarkeit
US5378612A (en) * 1990-05-11 1995-01-03 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Culture medium for production of recombinant protein
EP0573107A3 (fr) * 1992-06-04 1995-04-26 Merck & Co Inc Procédé de production de vaccin de virus de Varicella Zoster atténué.
WO1998024883A3 (fr) * 1996-12-04 1998-10-01 Medi Cult As Milieux de cultures cellulaires sans serum
WO2001019354A3 (fr) * 1999-09-16 2001-06-14 Novo Nordisk As Composition pour fecondation in vitro
WO2001059069A1 (fr) * 2000-02-11 2001-08-16 Medi-Cult A/S Milieux de culture cellulaire
WO2002062287A1 (fr) * 2001-02-06 2002-08-15 Novo Nordisk A/S Procede et contenant a faible teneur en oxygene et renfermant des substances activant la meiose
WO2004076561A1 (fr) * 2003-02-27 2004-09-10 Novasel Australia Pty Ltd Preparations sous forme d'emulsions a base de poloxameres
US6833271B2 (en) 1996-12-04 2004-12-21 Medi-Cult A/S Serum-free cell culture media
US6988613B2 (en) 2001-02-06 2006-01-24 Novo Nordisk A/S Composition for IVF
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USRE41974E1 (en) 1990-10-17 2010-11-30 Glaxosmithkline Llc Method for culturing Chinese hamster ovary cells
US8802116B2 (en) 2003-02-27 2014-08-12 Novasel Australia Pty. Ltd. Poloxamer emulsion preparations
US20210030806A1 (en) * 2019-03-28 2021-02-04 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Intraperitoneal injection of human placenta stem cells protect the brain from stroke injury via exosome/microparticle formation and ace2 maintenance of brain perfusion
CN116445577A (zh) * 2023-06-13 2023-07-18 中国医学科学院北京协和医院 用于霉菌酵母菌微生物限度检查的沙氏葡萄糖温敏凝胶培养基及应用

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Cited By (22)

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Publication number Priority date Publication date Assignee Title
US5378612A (en) * 1990-05-11 1995-01-03 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Culture medium for production of recombinant protein
USRE41974E1 (en) 1990-10-17 2010-11-30 Glaxosmithkline Llc Method for culturing Chinese hamster ovary cells
DE4038385A1 (de) * 1990-12-01 1992-06-04 Roecar Holdings Nv Sitosterol und seine glykoside mit verbesserter bioverfuegbarkeit
DE4038385C2 (de) * 1990-12-01 1998-09-17 Hoyer Gmbh & Co Kg Sitosterol und seine Glykoside mit verbesserter Bioverfügbarkeit
EP0573107A3 (fr) * 1992-06-04 1995-04-26 Merck & Co Inc Procédé de production de vaccin de virus de Varicella Zoster atténué.
EP1097988A1 (fr) * 1992-06-04 2001-05-09 Merck & Co., Inc. Procédé de culture des cellules dans une monocouche
US6833271B2 (en) 1996-12-04 2004-12-21 Medi-Cult A/S Serum-free cell culture media
WO1998024883A3 (fr) * 1996-12-04 1998-10-01 Medi Cult As Milieux de cultures cellulaires sans serum
WO2001019354A3 (fr) * 1999-09-16 2001-06-14 Novo Nordisk As Composition pour fecondation in vitro
US6844313B1 (en) 1999-09-16 2005-01-18 Novo Nordisk A/S Composition containing a meiosis activating substance
AU781994B2 (en) * 1999-09-16 2005-06-23 Novo Nordisk A/S Composition for IVF
WO2001059069A1 (fr) * 2000-02-11 2001-08-16 Medi-Cult A/S Milieux de culture cellulaire
WO2002062287A1 (fr) * 2001-02-06 2002-08-15 Novo Nordisk A/S Procede et contenant a faible teneur en oxygene et renfermant des substances activant la meiose
US6988613B2 (en) 2001-02-06 2006-01-24 Novo Nordisk A/S Composition for IVF
EA017434B1 (ru) * 2003-02-27 2012-12-28 НОУВАСЕЛ ОСТРЕЙЛЮЭ ПиТиУай эЛТиДи Стабильная местная и/или мукозальная эмульсионная композиция (варианты), способ ее приготовления (варианты) и ее применение (варианты)
EP1597318A4 (fr) * 2003-02-27 2006-06-14 Novasel Australia Pty Ltd Preparations sous forme d'emulsions a base de poloxameres
AU2009202265B2 (en) * 2003-02-27 2012-02-16 Novasel Australia Pty Ltd Poloxamer Emulsion Preparations
WO2004076561A1 (fr) * 2003-02-27 2004-09-10 Novasel Australia Pty Ltd Preparations sous forme d'emulsions a base de poloxameres
US8802116B2 (en) 2003-02-27 2014-08-12 Novasel Australia Pty. Ltd. Poloxamer emulsion preparations
US7754484B2 (en) * 2005-11-16 2010-07-13 Cognis Ip Management Gmbh Use of esters of unsaturated, physiologically active fatty acids as nutrient media for cell cultures
US20210030806A1 (en) * 2019-03-28 2021-02-04 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Intraperitoneal injection of human placenta stem cells protect the brain from stroke injury via exosome/microparticle formation and ace2 maintenance of brain perfusion
CN116445577A (zh) * 2023-06-13 2023-07-18 中国医学科学院北京协和医院 用于霉菌酵母菌微生物限度检查的沙氏葡萄糖温敏凝胶培养基及应用

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