It is known that successful in vitro culturing of differentiated cells remains especially difficult and elusive when utilizing current cell culture techniques. The lack of established and stable, normal and malignant cultures of differentiated cells has severely hampered certain fields of research, especially cancer studies and treatment.
Currently, the culturing of cells is done primarily with one of three methods:
Organ culture: maintenance of organs separated from their central vascular supply but with the organ, as an entity, left intact.
Tissue culture: culture of tissues or fragmented organs. The "sociocellular" relationships of the tissue architecture are preserved.
Cell culture: culture of an individual cell type divorced from other cell types.
A marked difference in response of explanted cells occurs when they are cultured by cell rather than organ or tissue culture techniques. Methods which retain tissue architecture permit retention of tissue-specific functions including hormonal and pharmacological responses. However, the tissue normally degenerates within a few weeks, due primarily to difficulties in nutrient and waste products exchange. Cell culture procedures overcome this limitation since they use explanted tissues which are disaggregated into single cells. The cells are adpated to grow as a monolayer on a solid-state support such as, treated plastic, or as a cell suspension, and can be maintained in culture for extended periods. Nutrients are supplied by a liquid medium of defined basal composition supplemented with one of various sera. Ideally, one isolates a clonal cell line i.e., a single cell whose progeny are maintained in continuous cell culture. There is genetic uniformity, easier maintenance of the cells, and reduction of variables associated with a multicell culture system. Nevertheless, cell culture procedure usually result in distortion of cellular phenotype and karyotype; normal cells rarely adapt as permanent cell lines without developing abnormal karyotypes or losing tissue-specific functions; malignant cells adapt more easily than do benign tumor cells or normal cells; and fibroblasts or stromal components become established preferentially over epithelial cells. The difficulties of establishing differentiated cells in cell culture have been attributed to many causes. These include an inadequately defined basal medium; inadequately defined hormone requirements; the static conditions of cell culture in which nutritional and oxygen gradients develop and limit the growth and functioning of cells with strict nutritional and oxygen requirements; and loss of or damage of cell-cell junctions, perhaps essential in growth and/or differentiation or both by the cell culture procedures of mechanical and enzymic dissociation into single cell suspensions. Undoubtedly all of these have contributed to the impasse in maintaining differentiated cells in cell culture. Yet despite progress on these various fronts, the goal of routinely culturing normal differentiated cells remains elusive.
In a published article, namely, vol. LVIII of the Methods in Enzymology, entitled "New Techniques for Culturing Differentiated Cells: Reconstituted Basement Membrane Rafts" by L. M. Reid and M. Rojkind, cell culture techniques were set forth which were, in essence, attempts to simulate some of the cell-cell relationships and of the tissue matrix relevant to epithelial cells. The techniques described involved the culturing of epithelial cells on substrates of reconstituted basement membrane and in medium supplemented with hormones, serum, and with conditioned medium from feeder layers. That technique, more specifically, involved utilization of reconstituted basement membrane rafts on which were floated epithelial cells over primary cultures of mesenchymal cells normally in association with the epithelial cells. This is, therefore, not applicable to the disclosed technique which involves connective tissue-derived fibers as a substrate for body cells that are normally in contact with basal lamina material, especially differentiated epithelial cells, in cell cultures. Basal lamina material as used herein is a substance found on the surface of differentiated cells, on the basal side or surrounding said cells, and is composed of collagens, carbohydrates including glycosaminoglycans and non-collagenous proteins.
In a published article, namely, Vol. 2d. 17 of the Life Sciences, entitled "A Simple, Versatile, Nondisruptive Method for the Isolation of Morphologically and Chemically Pure Basement Membranes from Several Tissues" by Elias Meezan, J. Thomas Hjelle and Klause Brendel, a procedure was set forth for the isolation of intact basement membranes from bovine retinal and brain blood vessels, rabbit renal tubules and rat renal glomeruli. The techniques described involved a seven step procedure, with several steps utilizing primarily high concentrations of a Sodium Deoxycholate solution, concentrations much higher than in the disclosed technique. Other solutions used in the Meezan procedure contained sodium azide, sodium chloride and DNase. This procedure differs significantly from the disclosed technique is that using the Meezan procedure the resultant isolated membranes are partially denatured and not in a form usable by cells in culture. The disclosed technique, however, requires the use of functionally active, non-denatured, connective tissue-derived fibers and these, may only be acquired using the disclosed technique with its different steps, compounds and concentrations. Another significant distinction is that the disclosed method involves the use of ribonuclease in addition to DNase. The use of ribonuclease and DNase enables the production of truely pure connective tissue derived fibers, without any contaminating DNA or RNA, and this is essential for use as a culture substrate. The Meezan procedure does not use Ribonuclease. The use of DNase in the Meezan article to prevent "viscous gel" like DNA from interfering with the isolation procedure would not result in a pure enough or functional product required when using connective tissue-derived fibers as a culture substrate.
It is therefore an object of this invention to provide a method for the establishment of in vitro cultures of human or animal differentiated cells.
It is another object of this invention to provide a method as aforesaid in which human or animal differentiated cells are cultured using an extracellular matrix, more specifically, connective tissue-derived fibers, as a substrate.
It is a further object of this invention to provide a method which is applicable to both normal and malignant cells whatever their degree of differentiation.
It is still a further object of this invention to provide a method which significantly improves the survival and attachment rates for in vitro cultures of differentiated cells.
It is still another object of this invention to provide a method which significantly improves the growth properties of in vitro cultures of differentiated cells which are capable of growth in vitro.
It is still a further object of this invention to provide a method which will permit differentiated cells to retain a significant degree of their differentiated state.
It is still another object of this invention to provide a method which permits certain differentiated cells to retain much of their normal enzymatic activities.
It is still a further object of this invention to provide a method which permits certain differentiated cells to retain to a high degree, their ability to secrete substances such as hormones.
It is also an object of this invention to provide a method for the preparation and isolation of a novel culture substrate.
It is another object of this invention to provide tissue-specific connective tissue-derived fibers for the culturing of the related tissue-specific differentiated cells, both normal and malignant.
It is still a further object of this invention to provide a cell culture environment comprising a plurality of fibers as support for the cells with or without other solid state support(s) such as a petri dish or test tube, wherein differentiated cells would have an environment more nearly approximating normal tissue conditions.
The aforesaid objects as well as the other objects and advantages will be made more apparent in reading the following description and the adjoined claims.