METHOD FOR CULTURING AND DETACHING CELLS ADHERING TO SURFACES

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TECHNICAL FIELD

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. BACKGROUND ART

Cell cultures are widely used in various field of research: in biomedecine and in cellular biology human cells (i.e., primary, tumor, stem cells) are cultured in vitro in order to produce recombinant proteins requiring post-transcriptional modifications and not easily produced by host organisms like procatiotes; in the field of gene therapy cells are cultured for preliminary studies concerning toxicities and/or transfection abilities of DNA complexes, and in biochemistry are currently used for other evaluations (i.e., cytometry, identification of stem cells markers, etc.)

Cell cultures, cultivated in sterility conditions, 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 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. Generally, 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. In the literature is known the use of trypsin solution (0.05-0.12%) additioned with 0.2-0.5 mM EDTA and 0,5-0,8 mM Ca(II) for a moderate and selective detachment of microglia instead of other types of adherent cells like astrocytes (Saura J. et al. GHa. 2003 Dec; 44(3): 183-9). These detachment procedures require the use of sterile enzymatic solutions after filtration through 0.22 μm filters, that however do not prevent from the contamination by viruses, mycoplasma, some kinds of Pseudomonas and other bacteria, or toxins produced by bacteria themselves (Melnick, J. L. et al. Developments in biological standardization (1976) Dec 13-15, 3777-82). Moreover, the use of such procedures increases the waste of reagents, containers, plasticware and other equipments and also the risk of contaminations is increased. Other negative aspects connected with the use of enzymatic solutions for cell detachment are their hydrolytic effect toward proteins

present in the culture solution, an eventual adsorption of other proteins, degradation of cell membrane proteins, receptors and other similar molecules. The use of non- enzymatic methods are hence particularly useful once studies dependent on membrane proteins integrity are required or when antibodies directed to them should be used. 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. However, this method is only applicable to cell culture moderately adhering to the substrate. Other 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. 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.

Nowadays, is hence required to have a method that allows a simple detachment of adherent cells without the use of enzymatic reagents or dedicated mechanical devices and without the above reported disadvantages. DISCLOSURE OF INVENTION

Inventors have found that in the case of cells adhering to substrates, (i.e. culture flasks), the addition of low concentration of non-ionic surfactants to the culture medium, allows

for the easy detachment of cells from the containers where they were cultured. 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. Other objects of the present invention are: - the use of one or more non-ionic surfactants, in culture media used for cell cultures, - the culture media additioned with one or more non-ionic surfactants. Characteristics and advantages of the present invention will be described in the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

The aim of the present inviention is to provide a new method for culturing cells adhering to surfaces, that allows a simple detachment of cells and their easy recovery without the use of enzymatic reagents or dedicated mechanical devices. 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. or more complex like block copolymers formed by mixtures of polyoxyethylene and polyoxypropilene, polyalcohols, etc. or thei mixtures, may be used as non-ionic surfactants in the cell culture method according to the invention. According to a form of realization, the non-ionic surfactant additioned to the culture medium is choosen among the group of compounds of formula (I):

where w, x, y e z, equal or different between them, are integers ranging from O to 100 but not all 0 at the same time, 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)

(H) where R is choosen among alkylic groups, 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. compounds of formula (III)

R(OCH ₂ CH ₂ )nOH (III) where R is defined as above for compounds of formula (II) and n is an integer number

ranging from 1 to 100, compounds of formula (IV)

RO(CH ₂ CH ₂ O) _n (IV) where R is defined as above for compounds of formula (III), and their mixtures.

For "fatty acid residue" according to the invention, is an acid residue choosen among decanoic, lauric, myristic, palmitic, stearic, arachidic, oleic and arachidonic acid. Preferred are compounds of formula (I) reported above, where w+x+y+z is greater than 20.

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 polyoxiethylen (20) sorbitan trioleate (Tween ^® 85); preferred is polyetoxilene (20) sorbitan monooleate (Tween ^® 80).

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 ^® 97), polyoxyethylen (20) oleyl ether (Brij ^® 98) and polyoxyethylen (21 ) stearyl ether (Brij ^® 721).

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).

Other non ionic surfactants not cited above, able to give equivalent results in terms of adherent cell detachment once added to the culture medium are to be considered included into the scope of the present invention.

Applicants have interestingly found that the addition of one or more of the cited surfactants to the culture medium, allow for an easy detachment of the cellular monolayer by a gentle shaking or edge-beating of the flask (or other containers). With this method, it is possible to use a non-enzymatic method without the disadvantages described above (lysis of antigens and other membrane proteins). The behaviour of these non ionic surfactants is surprising because it is known in the literature that the addition of surfactants to cell cultures leads to disrupt the cellular membrane, emulsifying membrane proteins, cholesterol and other phospholipids, and, as a consequence, to diffuse the protein content into the culture medium. On the contrary,

the applicants have found that at moderately low concentration (hence, non cytotoxic) 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. According to the invention, 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. In terms of results and quality of cell culture, the two methods are equally valid, in both cases cells grow uniformly up to confluence without visible collateral effects and are easily detached from the containers. The detachment step may be performed also manually, by shaking the flask or beating its external edge without the use of other reagents or other mechanical devices. 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. Optimal results were obtained by the inventors using 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.

EXAMPLE 1

Preparation of the culture medium

One liter of DMEM (Dulbecco's Modified Eagle's Medium) supplemented with FCS serum (10%), 2mM L-glutamine and 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 μm sterile filters bifore using it.

EXEMPLE 2

Hnuman glioma U87-MG cell culture using the medium of the invention.

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%

CO ₂ . 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.

EXAMPLE 3

Citotoxicitv evaluation of the culture medium prepared accordino to the invention.

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

10 days (Fig. 1B).

EXAMPLE 4

Cell proliferation assay and evaluation of proteins production activity of cells cultured in the medium of the invention.

In order to verify if the addition of non ionic surfactants to the culture medium may interfere with cellular processes like gene expression, mRNA translation, etc., the eventual alteration of membrane protein expression on the surface of lymphocytes cultured with the medium of the invention with respect to cells cultured following a classical procedure, was evaluated by cytofluorimetry. This difference may be considered as an indirect validation of different expression levels of those genes involved in the production of those receptors. With a good approximation, this data may be extended also to the expression of other proteins hence it may be considered as a good marker for the "vitality state" of the considered cell cultures. With a Beckman- Coulter (Cytomics FC 500) cytofluorimeter it was obtained that cells cultured in the presence of surfactants in the medium of the invention as described in the Example 2, do not show appreciable morphologic or functional alterations with respect of an analogous cell culture grown in a medium not additioned with surfactants. In particular, cells were analyzed in the lymphocyte gate positive for CD45; lymphocytes were analyzed for CD4 (helper/inducer), CD8 (cytotoxic) and CD 14 (macrophages and monocytes). Population of lymphocytes CD4 equals 34.2% for the medium additioned with the surfactant while 32% for the non additioned medium (Figures 2A and 2B). Population of 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.