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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
• • 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

• the present invention involves the field of cellular biology and it is about a method for culturing cells adhering to surfaces, (i.e., culture vessels, bottles, etc.) that allows a simple detachment of cells and their easy recovery once they reached a particular growth state (i.e. confluence) without the use of enzymatic reagents or dedicated mechanical devices, simplifying the cell collection process.
• surfaces i.e., culture vessels, bottles, etc.
• Cell cultures may be subdivided into two different culture typologies depending if cells grow in the culture medium (i.e., lymphocytes), or adherent to a substrate (i.e., glia, epathocytes, epithelial cells, etc.). In the latter case, cells generally grow adherent to the inner surface of the container where they were placed, up to the formation of a uniform cell monolayer. Once allowed to grow in flasks, dishes, roller bottles, etc., cells rapidly reach confluence: the available space for further growth is limited by other cells. In this case, replacement of cell culture media is not useful and a cell detachment step followed by a splitting of the culture in another (or bigger) container is generally required.
• the culture medium i.e., lymphocytes
• a substrate i.e., glia, epathocytes, epithelial cells, etc.
• the detachment step of adhering cells from the support is also required when cytometry, classical or fluorescence microscopy should be performed.
• Methods for cell detachment from culture vessels may be divided into enzymatic and non-enzymatic methods, further subdivided into chemical and physicochemical methods.
• the most used enzymatic methods require several working steps: 1) remove the culture medium from the container, 2) wash cells several times with phosphate buffer (PBS), 3) remove PBS from the container, 4) addition of an enzymatic lysis solution (i.e., tripsin, tripsin/EDTA (0.25% w/v) prewarmed to 37°C, collagenase or other enzymes).
• Residual cell culture medium is removed prior to the addition of lysis solution also to prevent inhibitions by serum proteins.
• the container once the lysis solution is added, is firstly incubated for few minutes then gently shaken to facilitate cells detachment. In the hardest conditions where cells are firmly adherent, also a scraper is used in order to remove the cellular monolayer.
• trypsin solution 0.05-0.12%
• Non enzymatic detachment steps are based on a similar concept but generally require the use of buffers that after an incubation period (5-10 minutes) allow for a moderate detachment under vigorous shaking.
• This method requires the elimination of the culture medium once cells reached confluence, a washing step with PBS, waste of laboratory plasticware to dispense solutions and some time for incubation.
• this method is only applicable to cell culture moderately adhering to the substrate.
• Non- enzymatic methods are the physical ones, that allow the detachment with the use of ultrasounds, shock waves that generate bubbles, termoresponsive polymers like the poly-N-isopropylacrylamide (PIPAAm) where cells were allowed to grow.
• PIPAAm poly-N-isopropylacrylamide
• the detachment step from supports covered with such polymers is accomplished by allowing the culture to stay at 10-20 0 C up to complete detachment from the container (Okano T. et al. Biomaterials. 1995 Mar; 16(4):297-303). Following this procedure several morphological changes were usually observed in cells, indicating an alteration (activation or deactivation) of particular methabolic processes.
• the present invention refers to a method for culturing cells adhering to surfaces, comprising the culture of such cells up to the desired period of time or confluence, in a suitable container containing culture medium additioned with one or more non-ionic surfactant molecules up to a final surfactant concentration ranging from 0,001% to 1% w/v with respect to the total volume of culture medium, followed by a detachment step consisting in a gentle shaking of the container where adherent cells were allowed to grow.
• Cell lines used in the method of the invention comprise mammals, mouse, rat, vegetal and human (normal, tumor, stem) cells (i.e., CHO, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, C6, HEK-293, GP293, U87-MG, lymphoblastoids and stem cells).
• Non-ionic surfactant molecules like sorbate or polysorbate esters, alcohol, amines, amides, acid etoxilates, etc.
• non-ionic surfactants in the cell culture method according to the invention.
• the non-ionic surfactant additioned to the culture medium is choosen among the group of compounds of formula (I):
• R 1 , R 2 , R 3 e R 4 equal or different between them, are choosen among H, alkylic chains, branched or linear with a number of C atoms from 10 to 20, fatty acids residues, saturated or insaturated, with a number of C atoms from 10 to 20, and X representing an ethylene, propylene or higher chain, compounds of formula (II)
• fatty acid residue is an acid residue choosen among decanoic, lauric, myristic, palmitic, stearic, arachidic, oleic and arachidonic acid.
• Examples of compounds of formula (I) commercially available include compounds belonging to the family of Tween ® , that is polyoxiethylen derivatives of fatty acid esters with sorbitol, like polyoxiethylen (20) sorbitan monolaurate (Tween ® 20), polyoxiethylen (4) sorbitan monolaurate (Tween ® 21), polyoxiethylen (20) sorbitan monopalmitate (Tween ® 40), polyoxiethylen (20) sorbitan monostearate (Tween ® 60), polyoxiethylen (4) sorbitan monostearate (Tween ® 61), polyoxiethylen (20) sorbitan tristearate (Tween ® 65), polyoxiethylen (20) sorbitan monooleate (Tween ® 80), polyoxiethylen (5) sorbitan monooleate (Tween ® 81), and polyoxi
• Examples of compounds of formula (II) commercially available include compound belonging to the family of Span ® , that are fatty acid esters with sorbitol, like sorbitan monolaurate (Span ® 20), sorbitan monopalmitate (Span ® 40), sorbitan monostearate (Span ® 60), sorbitan tristearate (Span ® 65), sorbitan monooleate (Span ® 80), and sorbitan trioleate (Span ® 85).
• Examples of compounds of formula (III) commercially available include compound belonging to the family of Brij ® , that are polyoxyethylen ethers of fatty acid derived from laurylic, cetilic, stearic and oleic alcohols like polyoxyethylen (4) lauryl ether (Brij ® 30), polyoxyethylen (23) lauryl ether (Brij ® 35), polyoxyethylen (2) cetil ether (Brij ® 52), polyoxyethylen (20) cetil ether (Brij ® 58), polyoxyethylen (2) stearyl ether (Brij ® 72), polyoxyethylen (10) stearyl ether (Brij ® 76), polyoxyethylen (20) stearyl ether (Brij ® 78), polyoxyethylen (2) oleyl ether (Brij ® 93), polyoxyethylen (10) oleyl ether (Brij
• Examples of compounds of formula (IV) commercially available include compound belonging to the family of Myrj ® , that are polyoxyethylen derivatives of stearic acid, like polyoxyethylen (8) stearate (Myrj ® 45), polyoxyethylen (20) stearate (Myrj ® 49) and polyoxyethylen (100) stearate (Myrj ® 59).
• non ionic surfactants not only may be added from the beginning of the culture procedure into the culture medium, but help also the cell detachment step once these have reached the desired growth level or confluence.
• non ionic surfactants may be present from the beginning into the culture medium, acting as a simple additive into the medium, or may be added once the cell culture has reached the desired growth level or confluence.
• the first choice represent the best choice because it allows to start the experiment with a complete culture medium without the need of further manipulations or additions.
• culture media to which the additive constituted by one or more non ionic surfactants according to the invention, are basal media like Dulbecco's, Earle's, Hank's, DME/F-12 Ham, MEM 1 DMEM, GMEM, IMDM, L-15, McCoy 5A, MCDB, Medium 199, NCTC, RPMI, etc.
• DMEM Dulbecco's Modified Eagle's Medium
• the culture medium described in the present invention has the advantage to leave unaltered the production of extracellular proteins by the cell culture and may be used for the production of molecules of some interest for pharmaceutical industry like antibody fragments, engineered proteins, and so on.
• the following examples are reported to illustrate the invention and do not limit the application and scope of the invention.
• DMEM Dulbecco's Modified Eagle's Medium
• FCS serum 10%
• 2mM L-glutamine 100 mg/ml
• antibiotics 100 U/ml Penicyllin and 100 mg/ml
• Streptomycin was additioned with 50 mg of Tween ® 80, and the misture were mixed up to complete dissolution of the surfactant.
• the culture medium obtained with this procedure was filtered through 0.2 ⁇ m sterile filters bifore using it.
• Human glioma cell line U87-MG were cultured using the medium prepared as described above in the Example 1 , and incubated at 37°C in a hymidified atmosphere with 5%
• U87-MG cells were plated in a 75 ml flask containing 40 ml of the medium, at a density of 10 4 and allowed to grow up to confluence. The flask was firmly shaken against the open hand up to the complete detachment of cellular monolayer.
• Human glioma cell line U87-MG cultured according as reported above in the Example 1 were counted periodically (every 2 days) with an optical microscope in order to evaluate the ratio between living/death cells with a colorimetric assay (Trypan Blue). Cell counting was performed following standard procedures well known to people working in this field. It was possible to observe that the culture medium object of the invention is not cytotoxic and allow for the cells to grow esponentially as a function of time (Fig.1A) with a vitality (percentage of living cells/total cells x 100) was higher than 95% for up to
• lymphocytes CD8 equals 36.8% for the medium additioned with the surfactant while 37.2% for the non additioned medium.
• population of lymphocytes CD14 equals 12.3% for the medium additioned with the surfactant while 10.6% for the non additioned medium.

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• 2006
  • 2006-01-18 IT ITRM20060020 patent/ITRM20060020A1/it unknown
• 2007
  • 2007-01-18 WO PCT/IT2007/000038 patent/WO2007083344A1/en active Application Filing

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