EP0929661A1 - Nouveaux procedes de mise en culture de cellules epitheliales humaines pour l'identification d'une therapeutique et d'un diagnostic du cancer - Google Patents

Nouveaux procedes de mise en culture de cellules epitheliales humaines pour l'identification d'une therapeutique et d'un diagnostic du cancer

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EP0929661A1
EP0929661A1 EP96940533A EP96940533A EP0929661A1 EP 0929661 A1 EP0929661 A1 EP 0929661A1 EP 96940533 A EP96940533 A EP 96940533A EP 96940533 A EP96940533 A EP 96940533A EP 0929661 A1 EP0929661 A1 EP 0929661A1
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cells
cell
epithelial
culture
human
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EP0929661A4 (fr
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Raymond L. White
Stephen Prescott
Leslie Jerominski
Norisada Matsunami
Christine B. Anderson
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University of Utah
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University of Utah
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0631—Mammary cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
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    • C12N2501/39—Steroid hormones
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
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    • C12N2503/00—Use of cells in diagnostics
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    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • the present invention relates to novel methods of obtaining human breast, colonic and prostate epithelial cell cultures, the cultures and/or cell lines thereby obtained and their biological and biochemical applications.
  • Neoplastic disease constitutes a complex illness of multifactorial origin that defies simple categorization.
  • the initiation of the cancer process may involve interactions between various factors, such as: environmental carcinogens or pollutants; diet; radiation; oncogenic viruses; chronic mechanical or thermal trauma; infection; genetic predisposition and the aging process.
  • Cancer remains a leading cause of death in Western societies. Cancer can occur in any organ of the body, in tissues of ectoder al, mesodermal, or endodermal origin. Despite the identification of a number of relatively effective therapies such as surgery, radiotherapy and chemotherapy in the treatment of neoplastic disease, the annual morbidity and mortality caused by the most common cancers, such as mammary (breast) cancer, colon cancer and prostate cancer is staggering. Thus a considerable amount of
  • cancer cells develop from normal tissue cells through a series of events which lead to malignancies. Such malignant cells grow without the restraints that regulate normal tissue growth (e.g., differentiation, organ size limitation or hormonal regulation) . In this manner, cancer can occur as a result of genetic mutation in a cell's regulatory genes which normally encode proteins that control cell division, differentiation or initiate cell death. There is a significant overlap in the pattern of mutated genes carried by different tumor cell types, such as between bladder and colon cancer cells, and among individual tumors of the same type. Mutations in the p53 gene, for example, are found in over half of all human tumors. See, e.g., Harris,
  • Tissue and/or cell culture techniques offer a way to explore the molecular basis of cancer, to identify potential genotypic and/or phenotypic cell markers which can be used as indicators of cancerous cells in clinical cancer screening, and to assay the effect of cancer therapeutics against neoplastic tissue or cells.
  • U.S. Patent No. 4,423,145 issued 27 December 1983 to Stampfer et al., describes a method of isolating and culturing human mammary epithelial cells to provide a clonal growth of cells suitable for quantitative assessment of the cytotoxicity of selected compounds.
  • HPV6b HPV6b and HPV16 in the immortalization of primary human foreskin epithelial cells.
  • Human Mammary Epithelial Cells Cul ture of Epithelial Cells , Eds. Wiley-Liss (1992) describe materials and methods for the culture of human mammary epithelial cells, and criteria for identification of epithelial cells in culture.
  • the art fails to disclose methods of providing a series of normal, precancerous and immortalized epithelial cell cultures wherein the cultures are all derived from the same tissue source or individual.
  • the present invention provides novel cell culturing systems that allow for the establishment of human breast, colonic and prostate epithelial cell cultures comprising normal, precancerous, immortalized or malignant cells. These systems are used to provide cell lines useful for screening agents which are specifically toxic to precancerous or malignant cells relative to normal cells, and for identifying agents capable of differentiating between precancerous or malignant cells and normal cells in clinical diagnosis of tissue or biological fluid samples.
  • a method is provided for establishing a primary culture of human epithelial cells using a fibroblast co-culture system. The method entails first isolating epithelial cell masses from a tissue sample obtained from a human subject.
  • the cell masses are then generally physically and enzymatically treated to yield epithelial cell clusters substantially free from stromal and other cellular material.
  • the epithelial cell clusters are applied to a membranous support arranged over the feeder cell population such that the membrane prevents co-mingling of the two cell types while allowing for the free diffusion of soluble moieties between the cell populations such as growth factors produced by the feeder cells and the like.
  • Outgrowth from the cell clusters onto the support provides a cell culture system capable of supporting the continued in vitro growth of the epithelial cells.
  • a method for passaging human epithelial cells from a primary co-culture system as described above to obtain a substantially pure epithelial cell culture.
  • the method entails initiating a fresh feeder cell population of human skin fibroblasts on a second substrate and in a suitable medium.
  • the epithelial cells from the primary co-culture are then passaged by first selectively detaching non-epithelial cells from the membranous support and then transferring the support having the outgrowth of epithelial cells from the primary co-culture by arranging the substrate over the fresh feeder cell population whereby a substantially pure epithelial cell culture is maintained on the support.
  • a passaged epithelial cell strain can be genetically manipulated to construct precursor cells of human cancers.
  • passaged cell strains can be subjected to homologous recombination in order to introduce mutations into target genes such as tumor suppressor genes or the like.
  • Transfection techniques are used to introduce various exogenous nucleotide sequences into the cells which can further carry any number of distinct mutations.
  • Introduction of DNA tumor virus genes, or portions thereof, can be used to selectively inactivate gene products of tumor suppressors, or to immortalize the cells to provide cell lines.
  • a method whereby in vitro assays can be conveniently carried out to determine the toxicity of an agent toward human epithelial cells of normal, precancerous, immortalized and malignant phenotype.
  • the method initially entails providing a population of human epithelial cells grown in a co- culture system constructed according to the invention.
  • healthy, growing epithelial cells are removed from the support and re-suspended in a volume of fresh medium.
  • a volume corresponding to a particular cell population is repetitively plated on fresh supports in a suitable substrate. A portion of the freshly plated cells are then exposed to an agent of interest for a selected period of time.
  • the exposure can generally be terminated by aspirating off the medium containing the agent, washing the cells and adding fresh media. After a sufficient time has passed to allow the cells to proliferate to a point where it is possible to distinguish those cells which continue to grow from those which have ceased to grow, agent toxicity is calculated by comparing the number of viable cells in a treated group with a control group of untreated cells.
  • the above described assay method is carried out using genetically altered cells having mutations in selected genes such as tumor suppressor genes or the like.
  • Such cells are constructed by transfecting DNA into the cells to provide mutations in target genes (tumor suppressors) by homologous recombination events, or by other transfection techniques wherein various exogenous nucleotide sequences carrying genetic mutations are introduced into the cells (e.g., inactivating mutations or selected mutations corresponding to those found in human tumors) .
  • a conditional immortalization system is constructed wherein portions of a population of epithelial cells cultured under the invention are assayed as described above prior to immortalization, while actively immortalized and when the immortalization system is silent or inactivated.
  • cell culture and “tissue culture” may be used interchangeably and denote the maintenance of cells in vitro , in suspension culture in a liquid medium or on a surface such as glass, plastic or agar provided with a liquid medium.
  • cell culture necessitates a medium that is buffered to maintain a con ⁇ tant suitable pH.
  • Media used in cell culture are generally formulated to include an adequate supply of necessary nutrients and can be osmotically tailored to the particular cells being maintained, with temperature and gas phase also being controlled within suitable limits.
  • Cell culture techniques are well known in the art. See, e . g . , Morgan et al., Animal Cell Culture , BIOS Scientific Publishers, Oxford, UK (1993) , and Adams, R.L.P. Cell Culture for
  • co-culture is used herein to refer to a cell culture system wherein a population of cells, termed “feeder cells,” is established in a cell culture in order to facilitate the growth of a subsequently added cell population of interest.
  • Feeder cells can be previously irradiated or otherwise treated to prevent their division in the culture.
  • mechanisms by which feeder cells assist in the growth of the cells of interest include: metabolic cooperation, wherein nucleotides, nutrients and hormonal moieties such as growth factors are passed to the cell culture via gap junctions; digestion of potentially toxic cell debris; provision of initial stimulus to cells via cell-to- cell contact; or combinations thereof.
  • fibroblast cells or other mesenchymal cells are used as feeder cells to maintain an epithelial cell culture.
  • epithelial co-culture systems have been described in the art. See, e . g. , Taylor- Papadi itriou et al. (1977) Int . J . Cancer 20:903-908 , Armstrong et al. (1978) Cancer Res . 3_8:984-998, and Kirkland et al. (1979) J . of Nat . Cancer Inst . 63:29- 42.
  • epithelial cell is used herein to denote differentiated cells derived from epithelial tissue such as breast, colon and prostate tissue.
  • Major epithelial cell types include basal and luminal cells and are isolated from the continuous layers of cells forming both internal and external tissue surfaces.
  • Epithelial cells derive from both the ectodermal and endodermal embryonic cell layers.
  • Cultured epithelial cells can be grown on glass or plastic substrates to which they adhere due to the secretion of proteins such as laminin and collagen.
  • fibroblast and “fibroblast cell” denote a type of stellate connective cell found in fibrous tissue that is responsible for collagen synthesis in cartilage, tendon, cornea, etc.
  • Cultured fibroblast cells are adherent cells and are normally grown on glass or plastic substrates. Cultured fibroblasts have the general morphology of tissue fibroblasts (flat, elongated or triangular-shaped cells) , however they are not as differentiated as true fibroblasts. Fibroblasts derive from embryonic mesoderm and the cells that grow in culture appear to be mesodermal stem cells. A number of continuous murine fibroblast cell lines have been described. Todaro et al. (1963) J . Cell Biol . r7:299, Aaronson et al. (1968) J. Cell Physiol . 72 . : 141, and Jainchill et al. (1969) J . Virol . 4_:549.
  • normal cell intends to refer to any cell directly derived from essentially normal cells or tissue.
  • normal epithelial cells are epithelial cells which are not of tumor cell origin, are not transformed in any detectable way and have not been i morta1ized.
  • a "malignant cell” refers to a cell directly derived from cancerous cells (or tissue) which have undergone phenotypic transformation, such as but not limited to, transformation by oncogenes, protooncogenes, TS mutations, or by other such mechanisms.
  • phenotypic transformation such as but not limited to, transformation by oncogenes, protooncogenes, TS mutations, or by other such mechanisms.
  • Malignant cells are generally characterized by their capacity for invasive, unregulated growth.
  • Such cells have one or more phenotypic derangements which can be expressed as alterations in cellular membranes, in the levels of certain cellular enzymes (e.g., enzymes involved in nucleic acid synthesis and metabolism) , or by the appearance of inappropriate gene products.
  • phenotypic derangements which can be expressed as alterations in cellular membranes, in the levels of certain cellular enzymes (e.g., enzymes involved in nucleic acid synthesis and metabolism) , or by the appearance of inappropriate gene products.
  • a "precancerous cell” refers to a cell directly derived from cells or tissue having a pathological condition which is likely to develop into cancer. "Precancerous cells” have undergone genetic damage but are not yet capable of invasive, unregulated growth, metastasis or the like. Under the invention, such cells can exhibit a precancerous phenotype or relate to an inherited (genetic) cancer predisposition such as genetic polymorphism of enzymes involved in activation and detoxification of carcinogens, or germline mutations in tumor suppressor genes (e.g., tumor suppressor genes which are defective in retinoblastoma (Rb) and adenomatous polyposis coli (APC) ) .
  • Rb retinoblastoma
  • APC adenomatous polyposis coli
  • primary culture refers to a culture of cells that are directly derived from cells or tissue taken from an organism without intermediate culture.
  • the cells or tissue from which a primary culture is derived is termed an explant.
  • Cells are generally considered to be “primary cells” until they are subcultured.
  • Primary cells will grow for a variable but finite length of time in culture, after which time they senesce and eventually die.
  • primary cultures can be derived from a variety of tissue sources and a number of techniques for their isolation from human tissue are known in the art. See, e . g . , Whitley et al. (1987) Mol . Cell . Endocrinol . 52.:279 and Fickling et al. (1992) Exp . Cell Res . 201:517.
  • Passage refers to the act of subculturing a cell population.
  • a “subculture” intends to refer to a cell culture established by the inoculation of fresh sterile medium with a sample from a previous culture. Each repeated subculture is counted as one passaging event.
  • a "cell strain” is a population of cells derived from a primary culture using subcultivation techniques. Thus, a primary culture can be subcultured into two or more new cultures and the subculturing repeated at periodic intervals for several months to maintain the cell strain.
  • a "cell line” refers to a population of cells derived from a single explant which are characterized as having the potential for unlimited growth in vitro .
  • a cell line can be isolated from a primary culture based on its ability to survive and continue to grow in culture.
  • Cell lines are frequently aneuploid due to an in vitro transformation event, and the capacity of cell lines to grow and divide indefinitely in culture is generally associated with an aneuploid karyotype.
  • Cell lines which have been derived originally from tumor tissue may have been transformed in vivo , although not all neoplastic cell populations have the capacity to grow indefinitely in vitro . Further, cell lines generally retain their differentiated character through many rounds of division.
  • a “clonal cell line” denotes a population of apparently genetically identical cells grown in culture from a single, isolated cell—provided that the cells have not undergone further differentiation in culture. Thus, a “clonal cell line” refers to the propagation of apparently identical daughter cells from a single parent cell.
  • Immortalized cell is a cell which by virtue of a transformation event, e.g. , infection with virus, becomes capable of indefinite growth and division.
  • Immortalized cell lines are genetically altered cells which have been derived from a primary culture or cell strain to produce a continuously growing cell line.
  • a cell line can be immortalized using various transfection techniques known in the art. Exemplary techniques are described further below.
  • a “transformed cell,” or a “transformed cell line” is a cell or cell line, respectively, which is either derived from a tumor cell or has been manipulated in some way (e.g., by transfection with oncogenes or treatment with carcinogens) to produce a cell or cell line that expresses a novel transformed phenotype. See, e . g . , Moore et al. (1966) J. Natl . Cancer Inst . , 3_6:405.
  • the transformed phenotype may manifest itself in a number of ways, such as an acquired capacity for unregulated growth (resembling the growth of cancer cells) , a newly acquired anchorage independence in adherent cells or by reduced serum and growth factor requirements.
  • a cell can be transformed following infection with a virus such as by SV40 or polyoma virus.
  • a transformed cell also has the capacity for unlimited growth in culture.
  • Transfection refers to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • the exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell.
  • the exogenous DNA can be maintained on an episomal element, such as a plasmid.
  • a eucaryotic cell is “stably transfected” when exogenous DNA has become integrated into the cellular genome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
  • Transient transfection refers to cases where exogenous DNA does not remain in the cells for an extended period of time, e.g., where plasmid DNA is transcribed into mRNA and translated into protein without integration into the host cell genome. A number of transfection techniques are known in the art. See, e.g., Graham et al. (1973) Virology,
  • a "host cell” is a cell which has been transfected, or is capable of transfection, by an exogenous DNA sequence.
  • control sequences refer collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present so long as the desired gene is capable of being replicated, transcribed and translated in an appropriate packaging cell.
  • a control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • DNA or polypeptide sequences are "substantially homologous" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, vol ⁇ J & TJ, supra; Nucleic Acid Hybridization , supra.
  • a novel method of establishing a primary culture of human epithelial cell ⁇ using a fibroblast co-culture system is provided.
  • An explant taken from a human breast, colon or prostate tissue sample is initially treated to isolate epithelial cell clumps substantially free of stromal and other cellular material.
  • Treatment of the tissue sample to remove epithelial cell clumps from the stromal matrix and other tissue cells involves physical separation techniques such as laceration and scraping, enzymatic separation techniques such a ⁇ u ⁇ ing a dige ⁇ tion medium containing proteolytic enzymes, and combinations thereof.
  • a feeder cell population is initiated by plating a population of human skin fibroblasts onto a suitable substrate and growing those cell ⁇ in a ti ⁇ sue culture medium under suitable tissue culture conditions.
  • the feeder cells can be selected from an established cell strain of human or murine fibroblasts such as, but not limited to cell types Detroit 551, MRC-5, PH140SK, 3T3 or NIH/3T3. See, e.g., U.S.
  • the fibroblasts are obtained from the same tissue sample as the epithelial cells of interest.
  • an explant of connective tissue derived from the same tissue sample used to isolate the epithelial clumps can be plated under suitable tissue culture conditions to provide a primary culture of human fibroblasts using techniques well known in the art.
  • Suitable cell culture substrates are generally a container that can be sterilized, does not leach toxic factors and does not distort microscopy images.
  • plates formed from glass and plastic are suitable substrates under the invention.
  • Plastic containers may further be treated to encourage cell attachment using techniques known in the art. Ramsey et al. (1984) In Vitro ⁇ 0:802.
  • Suitable tissue culture media generally consist of an isotonic, buffered, basal nutrient medium which provides an energy source, coupled with inorganic salts, amino acids, vitamin ⁇ and various supplements. Supplements may include serum (e.g. , fetal calf serum, or the like) various antibiotics to prevent contamination or to provide selective conditions, attachment and growth factors, or the like.
  • serum e.g. , fetal calf serum, or the like
  • a number of media formulations are known in the art, such as, but not limited to, minimal essential medium (MEM) , Rosewell Park Memorial Institute (RPMI) 1640 or Dulbecco's modified Eagle's medium (DMEM) .
  • Suitable tissue culture conditions are also known in the art. See, e.g., Morgan et al.,
  • Suitable membranous supports under the invention include microporous, permeable films developed for tissue culture which generally permit the free permeation of substances such as soluble nutrients, metabolites and hormonal factors through the membrane while preventing cell migration therethrough.
  • Such supports are available from, for example, Costar® (Cambridge, MA) and include TranswellTM supports (e.g., Transwell-Col membranes having a membrane thickness of 25-50 ⁇ m, pore sizes from 0.4-3.0 ⁇ m and which are treated with Types I and II collagen derived from bovine placentae) .
  • TranswellTM supports e.g., Transwell-Col membranes having a membrane thickness of 25-50 ⁇ m, pore sizes from 0.4-3.0 ⁇ m and which are treated with Types I and II collagen derived from bovine placentae
  • Other suitable supports include fibronectin-, collagen Type I- and laminin-treated membranes, MatragelTM membranes, or the like.
  • the clumps are finely divided to provide epithelial cell clusters that are as small as possible.
  • the cell clusters (explants) are then brought into ⁇ uspension in a suitable medium and applied to the membranous support arranged over the culture of human skin fibroblast feeder cells.
  • the co-culture system thus provided is incubated under suitable tissue culture conditions. After a suitable time (generally about 2- 3 days) , outgrowth from the explant to the support occurs, whereby a primary culture of human epithelial cells is formed capable of further in vitro growth.
  • the above described methods can be used to produce primary cultures of normal human epithelial cells derived from samples of human breast, colon or prostate tissue.
  • primary cell culture systems derived from excised cancerous or malignant breast, colon or prostate tissue can also be produced. These system ⁇ have, for the most part, not been obtainable in the prior art, primarily due to the lack of suitable in vitro tissue culture methods for establishing well- defined, malignant epithelial cells.
  • human subjects are identified which expres ⁇ precancerous phenotypes, such as those individual ⁇ carrying mutated tumor suppressor genes or the like.
  • Tissue samples can be biopsied from appropriate organs from those individuals, and the above described methods used to establish primary epithelial cell cultures of precancerous cells.
  • tumor suppressor ⁇ proteins which are encoded by these genes are essential in the control of normal cell growth.
  • inherited mutation of a tumor suppressor gene may render an individual at high risk for certain types of cancer.
  • mutational damage to such genes the internal metabolism of the cell may be altered, leading to abnormal growth characteristics.
  • genes which encode growth-stimulating products can be induced by the inherited mutation, leading to a condition in which a higher incidence of genetic mutation is likely to occur (“hypermutable state”) .
  • hypermutable state accelerates emergence of mutationally damaged cells that are capable of unregulated growth, leading to cancer.
  • NF1 neurofibromatosis NF1
  • von Recklinghausen NF affect ⁇ one in 4000 children, half of whom carry germ line mutations in the NF1 gene.
  • NF1 is generally recognized as a tumor suppressor gene that may be involved in the regulation of the p21ras signal transduction pathway. Inactivating mutations in NF1 have been connected with increased intracellular levels of p21ras-GTP, suggesting that inactivation of NF1 leads to abnormal cell growth.
  • Neurofibromatosis is characterized by the variable expression of features such as neurofibromas, cafe-au- lait mascules, predisposition to certain malignant tumors such as schwannoma, glioma, pheochromocytoma, and, at low frequency, leukemia and rhabdomyosarcoma.
  • Thu ⁇ it i ⁇ a particular object of the invention to provide breast epithelial cell cultures derived from tissue samples taken from individuals expressing mutations in one or more of these genes.
  • Such culture system ⁇ provide valuable in vitro models for the analysis and characterization of tumor suppressor genes and their participation in the cancer process.
  • human epithelial cell culture ⁇ are initiated from colon tissue samples taken from individuals expressing mutations in those genes. Once primary cultures have been obtained as described above, epithelial cells from those cultures can be passaged using tissue culture techniques known in the art. After several pas ⁇ ages, an outgrowth of epithelial cells capable of many population doublings, and even growth as individual clones is obtainable.
  • a method for passaging human breast, colon and prostate epithelial cells from the primary cultures establi ⁇ hed above In the maintenance of the primary culture ⁇ , epithelial cells are not permitted to reach confluence on the membranous supports in order to avoid diminished growth potential upon subculture. Thus, when the cells approach confluence, they are pas ⁇ aged to a fresh co-culture system. Pas ⁇ aging technique ⁇ are used under the invention to obtain substantially pure epithelial cell cultures and to establish subcultures thereof. In order to obtain a substantially pure epithelial cell culture from the primary cultures initiated above, a fresh culture of fibroblast feeder cells is establi ⁇ hed u ⁇ ing technique ⁇ a ⁇ previously described.
  • the feeder cells are allowed to adhere to the fresh substrate and grow for at least about 12 hours. In this regard, it is generally preferable to use the same fibroblast line with which the co-culture ⁇ ystem was started. However, if a fibroblast line has also been established from the same source (e.g., the same tissue sample) from which the primary epithelial culture was derived, those cells can be used in place of an existing fibroblast cell line.
  • a selective detaching step is performed whereby non-epithelial cells are detached from the support.
  • One particular technique which i ⁇ not limiting in the invention, entails differential trypsinization techniques that are known in the art. Taylor-Papadimitriou et al., "Culture of Human Mammary Epithelial Cells,” in Culture of Epithelial Cells , Eds. Wiley-Lis ⁇ (1992) .
  • the ⁇ upport-bound tissue explant and any cell outgrowth can be washed in a saline-trypsin-versene (STV) ⁇ olution and allowed sit for about 1-2 minutes. Once fibroblast cells are observed to detach from the support while the epithelial cells remain adherent, the STV medium is removed, the cells wa ⁇ hed and fed with fre ⁇ h tissue culture medium.
  • STV saline-trypsin-versene
  • the support After selectively detaching non-epithelial cells from the support, the support is positioned over the fresh fibroblast feeder cells and the epithelial culture maintained on the support. The process can be repeated a number of times to provide a substantially pure epithelial cell culture. Generally, the epithelial cells will grow actively through several passages, after which the cell population will gradually change morphology. At this time, breast epithelial cells begin to expres ⁇ fibrillar fibronectin, high levels of keratins and vimentin. Taylor-Papadimitriou et al. (1989) J . Cell . Sci . 94 . :403-413.
  • epithelial cells undergo their morphological change, actively growing cells can be selected from the background of senescing cells. Such cells are then subcultured to establish an epithelial cell strain. Subculturing under the invention can be carried out using established tissue culture techniques. Generally, a fresh fibroblast cell culture is established as described above. The support is removed from the co-culture system and the remaining "conditioned" media is transferred to the fresh fibroblast culture. Epithelial cells are detached from the support using techniques known in the art. At this point, the cells may be counted and known populations seeded onto fresh ⁇ upport ⁇ arranged over a fresh fibroblast feeder culture to provide epithelial cell strains.
  • precursor cells of human cancers can be constructed from the breast, colon and prostate epithelial cell strain ⁇ described above. Mutations to inactivate the gene products of suspected tumor suppressors can be introduced into those cell strains using homologous recombination or transfection techniques (e.g., using DNA tumor virus genes or potions thereof) .
  • the tumor suppre ⁇ sor genes p53 and Rb, and the putative antioncogene BRCA1 are each believed to play a role in human breast cancer carcinogenesi ⁇ .
  • Family studies indicate that p53 can be rate-limiting in breast cancer formation. Martin et al. (1990) Science 250: 1233-1238.
  • mutations in the p53 gene have either been found directly, or implicitly e.g., by ⁇ pecific lo ⁇ of heterozygosity. See e . g . , Harris, CC. (1994) Science 262: 1980, Harris et al. (1993) N . Engl . J . Med . 329:1315, Harper et al.
  • mutations in the putative antioncogene BRCA1 is indicated in approximately 45% of families with high breast cancer incidence, and at least about 80% of families with increased incidence of both early-onset breast cancer and ovarian cancer.
  • Precursor cells of human cancers are constructed from the human breast, colon and pro ⁇ tate epithelial cell ⁇ trains under the invention using transfection techniques to introduce exogenous DNA capable of inactivating tumor suppressor genes into the cells.
  • transfection techniques There are generally two major steps in transfection: first, the exogenous DNA must traverse the recipient cell plasma membrane in order to be exposed to the cell's transcription and replication machinery; and second, the DNA must either become stably integrated into the host cell genome, or exist as an episomal moiety that is capable of extra- chromosomal replication at a sufficient rate.
  • transfection methods have been described in the art, such as calcium phosphate co-precipitation
  • homologous recombination can be used to inactivate cellular genes such as p53 or Rb in the epithelial cell strains of the invention.
  • a nucleotide sequence (or a gene) homologous to a gene of interest, or portion thereof, is introduced to the cell using transfection techniques.
  • the sequences of p53 and Rb have been described. Lamb et al. (1986) Mol . Cell . Biol . 6 : 1379-1385, Ewen et al. (1992) Science 255:85-87 f Ewen et al. (1991) Cell j56_: 1155-1164, and Hu et al.
  • Cell ⁇ in which successful homologous recombination events have occurred can be enriched for using selectable marker genes.
  • epithelial cells can be transfected with a cloned p53 gene which has been disrupted by insertion of an aph gene (encoding resistance to geneticin) , rendering it nonfunctional.
  • the aph gene lacks a promoter of its own such that random integration events do not lead to transcription of the gene, rendering those cells geneticin- sensitive.
  • the aph genes becomes part of the targeted gene's mRNA, leading to a geneticin-resistant cell in conjunction with the disruption of the targeted gene. Culture of the cells may thus be carried out in media containing geneticin to select successful recombinants having a disrupted p53 gene.
  • transfection techniques are employed to introduce genes which encode proteins that act in trans to inactivate tumor suppressor genes.
  • Such cells can also be stably transformed using known techniques. In this manner, transcription and translation of the transfected E6 and/or E7 genes provides a basis for inactivation of either or both of the p53 or Rb gene products in transfected epithelial cell ⁇ .
  • epithelial cell strains derived from prostate, breast or colon tis ⁇ ue are immortalized to provide cell line ⁇ .
  • the u ⁇ e of tran ⁇ fection techniques to introduce "immortalization genes" into primary cells in vitro has been described. See, e . g . , Fickling et al. (1992) Exp . Cell Res . 201:517, and Ambesi-Impiombato et al. (1980) Proc . Natl . Acad . Sci . USA 22:3455.
  • These techniques allow cells to proliferate as immortal cell lines without suffering a concomitant loss of function.
  • Use of immortalized cells constructed under the invention in, for example, cancer research avoids the problems associated with having to use cell line ⁇ derived from malignant ti ⁇ ue which express a transformed phenotype.
  • SV40 Large T gene
  • SV40 is a small (5kb genome) DNA virus of the Papovavirus family which i ⁇ capable of amphotropic infection. It is generally recognized that infection of quie ⁇ cent cell ⁇ by SV40 induce ⁇ synthesis of cellular DNA and cell replication.
  • SV40 T-antigen can induce proliferation in cells without changing their morphology, thereby possibly inducing immortality rather than transformation.
  • Plasmids expressing SV40 large T protein have been used to immortalize a number of different primary cell types. See, e.g., Fickling et al. (1992) Exp . Cell Res . 201: 517 , and Ambesi- Impiombato et al. (1980) Proc . Natl . Acad . Sci . USA 77:3455.
  • human breast, colon and prostate epithelial cell strains are immortalized using amphotropic recombinant virus tran ⁇ fection to introduce the E6 and/or E7 genes of human papilloma virus 16 (HPV16) into the cells.
  • Recombinant retrovirus vectors containing the E6 and/or E7 genes from HPV16 can be transfected into an ecotropic packaging cell line, and viruses produced by those cells used to infect an amphotropic packaging cell line as previously described.
  • the retrovirus vectors are constructed to contain a selectable marker, whereby successive ⁇ fully immortalized cells can be selected for using an appropriate medium.
  • the primary cell cultures, passaged cell strain ⁇ , genetically altered cell strains and immortalized cell lines can be characterized by their expression of specific functional markers such as keratinocytes, hormonal and growth factor receptors and the like.
  • specific functional markers such as keratinocytes, hormonal and growth factor receptors and the like.
  • human breast epithelial cells may be phenotypically characterized by their expres ⁇ ion of keratin 19 marker (luminal cell phenotype) , keratin 14 marker (ba ⁇ al cell phenotype), keratin 18, Vimentin and Fibronectin u ⁇ ing immunoaffinity and detection technique ⁇ known in the art. See, e.g., Hermanson et al.
  • Anti-keratin 19 monoclonal antibodies e.g. BA16 and BA17, Bartek et al. (1985) J. Cell Sci . 2 . 5:17-33
  • anti-keratin 14 monoclonal antibodies e.g., LL001, Taylor- Papadimitriou et al. (1989) J . Cell Sci . 94:403-413 , and 12C8-1, Dairkee et al. (1985) Proc . Natl .
  • anti-keratin 18 monoclonal antibodies e.g., C04, Bartek et al. (1989) : in "Monoclonal Antibodies to Tumor Associated Antigens and Their Clinical Application , " Abelev G.I. (ed) , Akademai Kiado, Budapest, DA7, Lauerova et al. (1988) Hybridoma 2:495-504, and LE61, Lane, (1982) J . Cell . Biol . 9_2.:665-673) , anti-Vimentin monoclonal antibodies (e.g., V9, Osborn et al.
  • monoclonal antibodies specific for a selected cell marker can be labeled with a suitable detectable chemical group, such as an enzyme, radioisotope, fluore ⁇ cer, chromophore, luminescer or ligand for rapid and sensitive detection using techniques known in the art.
  • a suitable detectable chemical group such as an enzyme, radioisotope, fluore ⁇ cer, chromophore, luminescer or ligand for rapid and sensitive detection using techniques known in the art.
  • a number of techniques to detect the presence of the label are known, e.g., fluorometric, spectrophotometric, autoradiography, scintillation counting, and visual (e.g., colorimetric or chemiluminescence) techniques.
  • estrogen is important in vivo in the regulation of growth and function of the mammary gland, and is indicated in promotion of brea ⁇ t epithelial cell proliferation.
  • Hormone receptors for estrogen (ER) are present in breast epithelial cells, and ER concentration varies depending on the developmental state of the mammary gland.
  • ER concentration varies depending on the developmental state of the mammary gland.
  • breast epithelial cells can be further characterized by assay of ER wherein the cells are incubated with [ 3 H] estradiol with or without radioinert estradiol, processed as previously described (Haslam, S.Z.
  • polyclonal antibodies can be used as an antigen to produce antibodie ⁇ , either polyclonal, monoclonal, or both, by methods which are well known in the art.
  • a selected mammal e.g., mouse, rabbit, goat, hor ⁇ e, pig, etc.
  • Serum from the immunized animal is collected and treated according to known procedures.
  • serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.
  • Monoclonal antibodies to a selected cell marker can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies using hybridoma technology is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other technique ⁇ ⁇ uch a ⁇ direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al . , Hybridoma Techniques (1980); Hammerling et al . , Monoclonal Antibodies and T-cell Hybridomas (1981) ; Kennett et al . , Monoclonal Antibodies (1980); see also U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890.
  • a method whereby in vitro assays are carried out using human breast, colon or prostate epithelial cell cultures prepared according to the invention.
  • phenotypically normal, precancerous, and/or malignant cells can be screened to identify compounds with potential diagnostic efficacy.
  • Such compounds include, but are not limited to, differential stains which allow detection of cancerous cells in tissue or fluid samples, or signal enhancers for radiographic images (e.g. , X-ray or MRI) .
  • in vitro as ⁇ ays are carried out under the invention using human breast, colon or prostate epithelial cell cultures, wherein the effect of noxious agents (e.g., as chemicals, chemotherapy drugs, radiation and the like) on phenotypically normal, precancerous and malignant cells can be determined.
  • agents which are capable of selectively killing, inhibiting the growth of, or inducing apoptosi ⁇ in cancerous cells or precancerous cells can be identified by screening agents in tandem against normal, precancerous and/or cancerous cells derived from the same tissue source.
  • a method wherein a population of human epithelial cell ⁇ is established using the co-culture sy ⁇ tem a ⁇ de ⁇ cribed above.
  • the cells are prepared by detaching growing epithelial cell ⁇ from a co-culture support using techniques known in the art.
  • the detached cells are then resuspended in a suitable medium and counted.
  • a particular volume of the cell ⁇ uspension corresponding to a particular cell population is then repetitively plated on fresh supports in a suitable sub ⁇ trate, e.g. , a multiwell tissue culture plate.
  • it is generally desired to establi ⁇ h a number of identical co-culture ⁇ from a single epithelial cell culture so as to provide an experimental memori ⁇ with control culture ⁇ (non- treatment groups) , or the like.
  • a portion of the cell ⁇ are exposed to an agent or treatment of interest for a selected period of time.
  • a chemical or drug in solution with tissue culture medium is added to the co-culture system in an effective amount.
  • the exposure to the agent or treatment is terminated by removing culture media containing the agent, washing with a suitable buffered wash solution (e.g. basal salts medium) and adding fresh growth media.
  • agent toxicity or treatment effectiveness can be calculated by comparing the number of viable cells in a treated group with a control group of untreated cells using techniques known in the art.
  • the ⁇ ubject in vitro assay method is practiced using the aforementioned genetically altered cells.
  • transfected epithelial cells having inactivating mutations in tumor ⁇ uppressor genes such as p53, Rb, BRCA1, APC,
  • MSH 2 and MSH 3 are used to screen for differential stains, chemicals or chemotherapeutic agents effective in the diagnosis or selective killing of precancerous cells.
  • epithelial cell lines which have been immortalized by transfection with SV40 T-antigen, or HPV16 E6 and/or E7 genes as described above are used in the in vitro as ⁇ ay method. Such cell lines can be selected to screen cells of a particular phenotype.
  • the in vitro assay method is practiced using immortalized human breast epithelial cells expres ⁇ ing either the keratin 14 or keratin 19 phenotype.
  • an in vitro a ⁇ ay method whereby a conditional immortalization system i ⁇ con ⁇ tructed in order to allow assay of a cell population prior to immortalization, while actively immortalized and when the immortalization system is inactive.
  • a human breast, colon or prostate epithelial cell strain is selected and portions of the strain subjected to immortalization using the temperature- ⁇ en ⁇ itive mutant SV40 Large T ⁇ ystem described herein.
  • the assay is carried out by subjecting cultures of the cell strain which have not been immortalized to the same experimental protocol as employed on cultures of the cell strain which have been immortalized.
  • One of the immortalized culture ⁇ is incubated at 33°C, the other at 37°C in order to provide cell populations under the effect of both active and inactive immortalization systems.
  • a conditional immortalization system is constructed using a tetracycline inducible transactivator (tTA) to control expression of transfected E6 and/or E7 genes in immortalized human breast, colon or prostate epithelial cells.
  • expression of E6 and/or E7 in transfected cells may be regulated using a tetracycline inducible transactivator (tTA) regulation sy ⁇ tem containing control element ⁇ of the tetracycline-re ⁇ istance operon encoded in TnIO of Escherichia coli fused with the activating domain of virion protein 16 of herpes simplex virus as previously described.
  • tTA tetracycline inducible transactivator
  • the tTA system allows differential control over expression of the subject genes, as well as reversible "on/off" ⁇ witching. Integration of a luciferase reporter gene controlled by a tTA-dependent promoter in the above- described tTA system further allows efficient monitoring of E6 and/or E7 expression in response to various tetracycline concentration ⁇ by assay of luciferase activity. Gossen et al., supra .
  • cultures of a cell strain which have not been immortalized are treated under the same experimental protocol which immortalized cells are subjected to, wherein the immortalized cells are divided into two groups and incubated in media with or without tetracycline to provide immortalized cell populations under the effect of both active and inactive immortalization systems.
  • the human breast, colon and prostate epithelial cell cultures of the invention can also be used in the context of in vitro as ⁇ ay ⁇ to screen for potential carcinogens.
  • Epithelial cells provide a useful model sy ⁇ tem in carcinogen te ⁇ ting ⁇ ince they mimic more closely the usual situation in humans and animals, where carcinomas are typically derived from epithelial, not mesenchymal cells.
  • Epithelial cell cultures of normal or precancerous phenotypes that are produced using the co-culture system of the present invention can be plated, allowed to adjust, and a portion thereof exposed to an agent suspected of being a carcinogen for a suitable period of time.
  • Exposure to the agent is terminated by removal of culture media containing the agent, washing with a suitable buffered wash solution and adding fresh growth media. Transformation events which occur as a consequence of exposure to the agent can be identified by development of foci of transformed cells. Alternatively, transformation events can be monitored by injection of treated cell ⁇ (either sub-cutaneously or intraperitoneally) , into immunocompromised animal models (e.g., nude or SCID mice), wherein the ability of the treated cells to form solid tumors can be readily ascertained.
  • immunocompromised animal models e.g., nude or SCID mice
  • the epithelial cell cultures formed herein can be used in the context of in vitro assays to screen for agents capable of at least participating in inducing apoptotic events.
  • Apoptosi ⁇ or programmed cell death
  • apoptosis can be ascertained morphologically by appearance of membrane blebbing, cytoplasmic shrinking, chromatin condensation, and digestion of the genomic DNA into fragments (e.g., formation of a "DNA ladder") . See, e . g . , Ellis et al. (1991) Annu . Rev . Cell Biol .
  • the ability to identify agents that may trigger apoptotic events in cells that are refractive to an apoptosis-inducing signal provides a unique and promising approach to the identification of chemotherapeutics.
  • Epithelial cell strains comprising human cancer precursor cells that are formed a ⁇ described above can be provided, wherein the p53 gene has been mutated to yield cells that are refractive to DNA damage-induced apoptosis.
  • Such cell strains can be cultured, exposed to ultraviolet light, exposed to agents suspected of being able to trigger apoptosis, and then examined for signs of apoptosis.
  • one of the hallmarks of apoptosis is the digestion of the genomic DNA of the dying cell into small fragments of about 180 base pairs (bps) , or multiples thereof (e.g., formation of DNA ladders) .
  • the treated cells can be readily assayed for positive apoptotic events using gel electrophoresis techniques that are known in the art. It is to be under ⁇ tood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the description above as well a ⁇ the example ⁇ which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
  • Collagenase (Sigma, St Louis, MO) (1500 U/ml) was dis ⁇ olved in appropriate amount ⁇ of MCDB 170 complete medium at 37°C and ⁇ terilized by filtering through a 0.22 ⁇ m filter.
  • Hyaluronida ⁇ e (Sigma) (1000 U/ml) wa ⁇ di ⁇ olved in an appropriate amount of MCDB 170 complete medium at 37°C and sterilized by filtering through a 0.22 ⁇ m filter.
  • Equal volumes of the sterile collagenase and hyaluronidase solutions were then combined to yield a 5X solution.
  • the enzyme solution may be stored in aliquots at 80°C for up to one year.
  • tissue digestion media To make 50 mis of sterile tissue digestion media, 10 mis of 5X Collagenase and Hyaluronidase solution were added under ⁇ terile condition ⁇ to 5 mis of fetal calf serum (Hyclone, Logan, UT) and brought to volume with 35 mis of sterile complete MCDB 170 media.
  • fibroblast cells were plated into sterile six well di ⁇ hes containing DMEM (Sigma, St Louis, MO) plus 10% fetal calf serum at a concentration of at lea ⁇ t 5 X IO 4 cell ⁇ per well. The cell ⁇ were then allowed to attach to the plates and grow 12 to 24 hours before use thereof in a co-culture system.
  • DMEM Sigma, St Louis, MO
  • fibroblast cells for the primary co-culture system do not have to be from the same individual; however, for long term co-culture growth it is be ⁇ t to use fibroblasts derived from the same individual as the epithelial culture.
  • the breast tis ⁇ ue ⁇ ample was prepared by first cutting the tissue into small pieces in a sterile reaction ves ⁇ el such as a petri di ⁇ h.
  • the epithelial areas of the cut tissue were separated from the adipose tissue and stromal matrix by manipulating the tissue sample with sterile scalpel blades, scissors, forceps, or any combination thereof.
  • the remaining tissue was finely minced using opposing scalpels and transferred to a sterile 15 ml centrifuge tube. Again, colon and prostate tissue samples were handled in an analogous manner.
  • the minced ti ⁇ ue was then incubated for about 12 to 18 hours at 37°C in tis ⁇ ue digestion media, preferably using a tube rotator for gentle mixing. After incubation, the tube containing the digested tis ⁇ ue wa ⁇ ⁇ pun down at 1000 rpm for 10 minutes, and the supernatant (along with any floating adipose tissue) removed and discarded leaving a pellet of minced digested tissue. Digestion may be monitored at this point and is complete when microscope examination reveals clump ⁇ of cells with ductal, alveolar, or ductal-alveolar structures free from attached stroma. If necessary, further digestion may be carried out by resuspending the pellet in fresh tissue digestion media and reincubating as above.
  • the pellet of tis ⁇ ue thus obtained was then decanted into a sterile 100 mm petri dish where the larger pieces of tissue were further divided using opposing scalpels until all of the tissue was a ⁇ ⁇ mall as possible.
  • the tissue was then resuspended in 5 ml complete MCDB 170 media, and centrifuged quickly to pellet any large pieces of tissue in the bottom of the tube.
  • A.6 Setting up the co-culture system. Six well dishe ⁇ containing fibroblast cells which were plated 12 to 24 hours earlier were checked to ensure that the fibroblast ⁇ had adhered to the plate ⁇ and were growing. The cells should be about 40 to 60% confluent in the six well dishes. The fibroblast growth media was aspirated off and discarded, and 3 is of fresh MCDB 170 complete media added to each well. A collagen-coated microporous membrane, such as a Transwell-COL 0.4 ⁇ m support (available from COSTAR, Cambridge, MA) was placed within each well using sterile technique. After placing the supports in the wells, all air bubbles that became trapped under the support were removed, and about 0.5 mis of MCDB 170 complete medium added to each support to ensure that the entire surface area of the support was sufficiently moist.
  • a Transwell-COL 0.4 ⁇ m support available from COSTAR, Cambridge, MA
  • the growth media wa ⁇ partially replaced with fresh MCDB 170 complete media at least about every six to seven days.
  • the media was never completely removed from the wells, as it is important that conditioned media always remain on the co-culture system to maintain constant cell growth.
  • spent media on top of the support wa ⁇ carefully aspirated without removing any media present under the support.
  • each individual primary epithelial culture generally grows at a different rate.
  • the cultures were carefully monitored to avoid allowing an ⁇ upport to reach confluence which can cause cell death.
  • the cells reach 80% to 90% confluence, the cells were pas ⁇ aged to a new co-culture system.
  • a fresh culture of fibroblast cells was established using technique ⁇ a ⁇ de ⁇ cribed above. Thi ⁇ wa ⁇ carried out at lea ⁇ t about 12 hour ⁇ in advance of the pa ⁇ aging so that the fresh fibrobla ⁇ t cell ⁇ were able to properly adhere to the new plate ⁇ .
  • a fibroblast line wa ⁇ initiated from the same individual from which the primary epithelial cultures were derived, tho ⁇ e cell ⁇ can be used in place of an existing fibroblast line.
  • a fibroblast cell line which has been established from the same individual is preferred under the invention, but this is not always possible when primary cultures are initiated.
  • the epithelial cells were selectively transferred to a new co-culture system containing fresh fibrobla ⁇ t cells.
  • two steps were generally carried out at the same time, selective trypsinization of the epithelial cells from the support and preparation of the new fibroblast culture.
  • the media on top of the support was aspirated and discarded, and the support gently transferred under sterile conditions to a fresh, sterile dish that did not contain any fibroblast cells.
  • any remaining media from the old co-culture dish was transferred to the wells in the new plate (containing the freshly plated fibroblasts) .
  • Use of the remaining media in this manner is considered important since it is "conditioned" media which is thought to aid in the promotion of cell growth in the newly passaged cells.
  • the cell ⁇ may be split at a ratio of 1:2 or as high as 1:5 depending on the desired final cell density, however, they ⁇ hould not be split at too high of a ratio or they will not recover.
  • the cultures were generally checked for attachment and growth after 24 hours.
  • the passaged cell ⁇ were fed by partial replacement of the growth media at lea ⁇ t about every 4 to 5 days. Partial replacement was carried out by removing the media from above the support and replacing it with new media such that the media was never completely removed from the wells. Leaving conditioned media in the cultures helps to maintain constant growth in the co-culture sy ⁇ tem. The new media defuses through the support and mixes with the old media so that nutrients were constantly being replenished in the wells.
  • An appropriate cell-preservative media is MCDB 170 complete media with 20% fetal calf serum plu ⁇ 20% glycerin (Sigma, St Loui ⁇ , MO) . Any remaining dige ⁇ ted ti ⁇ sue prepared as described above may be preserved by freezing. Thus, remaining tissue was centrifuged at 1000 rpm for 10 minutes to form a pellet, all supernatant removed and discarded, and the remaining pellet resuspended in cell-preservative media.
  • the pellet was generally resuspended to provide a 1:2 ratio of tissue to media and about 1.2 mis of the resuspended tissue aliquoted into appropriate vials (e.g., 1.8 ml Nunc Cryovials®) .
  • appropriate vials e.g., 1.8 ml Nunc Cryovials®
  • the vials containing the tissue were frozen slowly (e.g. , at 1 degree per minute) and then transferred to storage at -135°C for future use.
  • a cell line can be established using methods known in the art.
  • Retroviral vector sy ⁇ tem ⁇ for gene transfer comprise two major components, the retroviral vector and the packaging cells.
  • the vector can be manipulated in its DNA form as part of a bacterial plasmid.
  • the vector does not encode viral proteins.
  • the retrovirus- packaging cells provide the viral proteins necessary for encapsidation of the vector RNA into virions and for subsequent infection, reverse transcription, and integration of the vector into the genomic DNA of the host cells.
  • Retrovirus vector pLXSN (available from Fred Hutchinson Cancer Research Center, Seattle, WA) , was obtained.
  • pLXSN is characterized as having the following major elements: L (long terminal repeat) ; X (cloning site) ; S (simian virus 40 (SV40) early promoter); and N (neo gene) .
  • L long terminal repeat
  • X cloning site
  • S simian virus 40 (SV40) early promoter
  • N neo gene
  • the vector pLXSN contains two promoters, one driving expression of the selectable marker (neo) and the other driving expre ⁇ ion of the inserted DNA.
  • DNA fragments containing the E6 or E7 gene or the contiguous region encoding E6 and E7 from HPV16 were isolated using appropriate restriction enzymes and exonuclease III digestions.
  • the fragments were cloned into the pLXSN vector to produce the following construct ⁇ : pLXSN16E6 (containing the E6 gene) ; pLXSN16E7 (containing the E7 gene) ; and pLXSN16E6E7 (containing the E6/E7 contiguou ⁇ gene region) using techniques known in the art. See, e . g. , Sambrook, supra .
  • the constructs pLXSN16E6, pLXSN16E7 and pLXSN16E6E7 were transfected by calcium phosphate precipitation into the Psi-2 ecotropic packaging cell line (Mann et al. (1983) Cell 3_3:153) .
  • Viruses produced from the Psi-2 cell ⁇ were then used to infect the amphotropic packaging line PA317 (available from the American Type Culture Collection, Rockville, MD, ATCC No. CRL 9078) which provide the following recombinant amphotropic viruse ⁇ : LXSN16E6, LXSN16E7 and LXSN16E6E7.
  • Recombinant amphotropic viruses LXSN16E6, LXSN16E7 and LXSN16E6E7 were then used to infect the human breast, colon and prostate epithelial cell strain ⁇ produced under the invention using techniques known in the art.
  • the epithelial cells were used when in the logarithmic growth phase.
  • Cell concentration wa ⁇ adju ⁇ ted to about 2X10 5 /ml, and 2ml placed in a suitable tissue culture flask.
  • the cells When infection was complete, the cells were centrifuged for 5 min at 200 rpm and resuspended in MCDB 170 + 20% (v/v) fetal calf serum (Hyclone, Logan, UT) before cloning by limiting dilution in the presence of geneticin G418® (GIBCO-BRL, Gaithersburg, MD) at an effective concentration of from about 50-300 ⁇ g/ml.
  • G418® geneticin G418®
  • I biopsy (possible pre ⁇ either MCDB 170 DFCl-1; BRCA-1 carrier) cancerous or DFCl-1 medium 5-7 days in plu ⁇ matragel MCDB 170 ⁇ upport
  • the results thus obtained indicate that the present co-culture methods are useful to provide primary cultures of human breast epithelial cells from a variety of sources.
  • primary cultures of normal epithelial cells were readily establi ⁇ hed within 2-7 days from normal breast tis ⁇ ue explant ⁇ (see, e.g., BE-20, BE-23, BE-27, BE-28, BE-29-N, BE- 31-E, BE-31-F and BE-32) .
  • Primary cultures of potentially precancerous cells were established (BE- 30-PC) from a subject identified as a possible BRCA-l carrier.
  • four primary cultures were obtained in co-culture systems using collagen type I, collagen type I & III, fibronectin and Matragel® membranous supports.
  • a primary culture of precancerous cells was established from a positively identified BRCA-l carrier.
  • a cell strain (BE-40) was derived from the primary culture, and has been deposited with the ATCC (ATCC Accession No. ATCC CRL 11981) .
  • ATCC Accession No. ATCC CRL 11981 ATCC Accession No. ATCC CRL 11981 .
  • Several primary cultures of cancerous cells were also obtained within about 2-3 days from cancerous tissue explants (see, e . g . , BE-29-T and BE- 30-T) .
  • Example 1 A number of the primary cultures obtained in Example 1 were passaged using the methods of the invention to obtain substantially pure human breast epithelial cell strains. The cells were further characterized to monitor differentiation of those cells after each passage. Particularly, keratins 14, 18 and 19, as well as estrogen receptor and epidermal growth factor receptor markers were assayed using the characterization methods described above.
  • the BE-31-E and BE-30-PC cells are further characterized as 87% and 16% keratin 19 positive, respectively, after their second passage.
  • the BE-28 (normal breast epithelial) cell strain is characterized as both keratin 14 and keratin 19 po ⁇ itive after it ⁇ fourth passage.
  • Example 3 In order to provide immortalized human mammary epithelial cell lines, the E6 and E7 genes of
  • HPV16 were introduced separately or in combination into the human mammary epithelial cell strain BE-31-E by transfection with amphotropic recombinant retroviruses using the methods described herein. Halbert et al (1991) J . Virology 65:473-478.
  • Example 4 Using the fibroblast co-culture system of the present invention, primary cultures of human colonic epithelial cells were succe ⁇ fully e ⁇ tablished and maintained for more than four months using the following procedure: normal human colon tissue was obtained immediately following surgery and placed into ice-cold culture media containing the following: 10% fetal calf serum (Hyclone, Logan, UT) , 2mM glutamine (Sigma, St Louis, MO) , 10 ⁇ g/L EGF (Sigma) , 5 ⁇ g/L hydrocortisone (Sigma) , 0.5 ⁇ g/L transferrin (Sigma) , 0.5 ⁇ g/L insulin (Sigma), 4.2 g/L bovine pituitary extract (Sigma) , and 200 mg/L gentamicin (Sigma) in Dulbecco's Modified Eagle's Media (D-MEM, GIBCO-BRL, Gaithersburg, MD) .
  • D-MEM Dulbecco's
  • Tissue was fragmented and digested a ⁇ previously described to obtain suitable tissue explants. Initially, small tissue pieces were placed on collagen-coated membrane ⁇ seated over human skin fibroblast cells. After approximately two week ⁇ , there wa ⁇ vi ⁇ ible outgrowth from the tissue pieces which were subsequently removed. One-half volume of media was removed from the cells and replaced with fresh media every week.
  • Epithelial cell ⁇ were selected from the other cell types that initially grew from the colon tissue by selective trypsin digestions (to provide a substantially pure epithelial cell strain) . Since epithelial cells are more firmly attached to the collagen substrate than the fibroblast cells, it was possible to remove the majority of contaminating fibroblasts while leaving the epithelial cells undisturbed. This selective trypsinization was performed approximately eight times, each time saving the resulting colon fibroblast ⁇ to culture independently. As soon as possible, these fibroblasts were used as the feeder layer below the colon epithelial cells.
  • the colon epithelial cells grow very slowly, doubling every week or so and di ⁇ play a rounded "cobblestone" morphology characteristic of epithelial cells.
  • the human colonic epithelial cell strain thus obtained was immortalized by transfection with an amphotropic recombinant retrovirus expressing the HPV16 E6 or E7 gene as described above.
  • fibroblast co-culture system of the present invention Using the fibroblast co-culture system of the present invention, a number of primary cultures of human prostate epithelial cell ⁇ were successive ⁇ fully established using the above-described procedures.
  • the cell cultures are depicted in Table 3 wherein the source of the prostate tissue, tis ⁇ ue type and co- culture conditions are reported.
  • PC-65 65 year old cf, normal epithelial MCDB 170 plus no tumor biopsy, left side collagen support tissue, prostate gland only
  • PC-65 65 year old cf, normal epithelial MCDB 170 plus no tumor biopsy, right side collagen support tis ⁇ ue, prostate gland only
  • Cells from the PC-59, PC-63, PC-64 , PC-65 and PC-66 primary cultures were passaged using the methods described above to provide cell ⁇ trains.
  • Cells from the PC-67, PC-68 and PC-69 primary cultures were immortalized at the time of first passage by transfection with an amphotropic recombinant retrovirus expres ⁇ ing the HPV16 E6 or E7 gene as described above.
  • a deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland.
  • ATCC American Type Culture Collection
  • the accession number indicated was a ⁇ igned after successful viability testing, and the requisite fees were paid.
  • the deposit ⁇ were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) . This assures maintenance of viable cultures for a period of thirty (30) years from the date of depo ⁇ it and at lea ⁇ t five (5) years after the most recent request for the furnishing of a sample of the deposit by the depository.

Abstract

L'invention concerne des procédés d'obtention de cultures primitives de cellules épithéliales du sein, colique et prostatique chez l'homme, à l'aide d'un système de culture tissulaire en co-culture avec du fibroblaste. On peut utiliser les procédés pour obtenir des cultures cellulaires épithéliales normales, précancéreuses et malignes. L'invention concerne également des procédés permettant de dériver des souches des cellules épithéliales sensiblement pures à partir des cultures primitives, et d'immortaliser les souches de cellules afin de produire des lignées cellulaires. Les cultures primitives, les souches de cellules et les lignées cellulaires sont utilisées dans des titrages de criblage et/ou de diagnostic in vitro biochimiques.
EP96940533A 1995-11-15 1996-11-14 Nouveaux procedes de mise en culture de cellules epitheliales humaines pour l'identification d'une therapeutique et d'un diagnostic du cancer Withdrawn EP0929661A4 (fr)

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US6074874A (en) * 1997-08-29 2000-06-13 University Of Pittsburgh Epithelial cell cultures for in vitro testing
ATE283477T1 (de) * 1998-07-05 2004-12-15 Fraunhofer Ges Forschung Luminometrische atp-bestimmung
AU2001244341A1 (en) 2000-04-01 2001-10-15 Onyvax Limited New prostate cell lines
DE60126815T2 (de) * 2000-12-08 2007-11-15 Xgene Corp., San Carlos Herstellung von geschichteten und nach zelltyp geordneten geweben in vitro
WO2003025157A1 (fr) * 2001-09-14 2003-03-27 The Genetics Company Systeme de coculture permettant d'identifier des proteines declenchant la redifferenciation de cellules tumorales
US20050019336A1 (en) 2003-07-23 2005-01-27 Dalgleish Angus George Human prostate cell lines in cancer treatment
DE102011008050A1 (de) * 2011-01-07 2012-07-12 Universitätsklinikum Schleswig-Holstein Verfahren zur Diagnose der familiären adenomatösen Polyposis (FAP)
US10329623B2 (en) 2015-08-28 2019-06-25 Slmp, Llc Synthetic tissue controls and synthetic tissue microarray controls
CN115322967B (zh) * 2022-06-13 2023-08-08 浙江省人民医院 一种永生化人甲状腺乳头状癌成纤维细胞株及其构建方法和应用

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EP0117486A2 (fr) * 1983-02-25 1984-09-05 Sloan-Kettering Institute For Cancer Research Méthode de croissance et de purification de lignées de cellules humaines à partir de populations de cellules hétérogènes de tissu en culture
WO1988008448A2 (fr) * 1987-04-22 1988-11-03 Michael Bay Procedes de culture de cellules, et produits
WO1990015862A1 (fr) * 1989-06-12 1990-12-27 Cornell Research Foundation, Inc. Culture in vitro de cellules epitheliales
WO1992013103A1 (fr) * 1991-01-16 1992-08-06 The Johns Hopkins University Mutations hereditaires et somatiques du gene apc dans les cancers recto-coliques chez l'homme
WO1993021529A1 (fr) * 1992-04-14 1993-10-28 Duke University Procede de detection de tumeurs contenant des complexes de p53 et de hsp70
WO1993021958A1 (fr) * 1992-04-27 1993-11-11 Georgetown University Genes du virus humain du papillome et leur utilisation en therapie genique

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EP0117486A2 (fr) * 1983-02-25 1984-09-05 Sloan-Kettering Institute For Cancer Research Méthode de croissance et de purification de lignées de cellules humaines à partir de populations de cellules hétérogènes de tissu en culture
WO1988008448A2 (fr) * 1987-04-22 1988-11-03 Michael Bay Procedes de culture de cellules, et produits
WO1990015862A1 (fr) * 1989-06-12 1990-12-27 Cornell Research Foundation, Inc. Culture in vitro de cellules epitheliales
WO1992013103A1 (fr) * 1991-01-16 1992-08-06 The Johns Hopkins University Mutations hereditaires et somatiques du gene apc dans les cancers recto-coliques chez l'homme
WO1993021529A1 (fr) * 1992-04-14 1993-10-28 Duke University Procede de detection de tumeurs contenant des complexes de p53 et de hsp70
WO1993021958A1 (fr) * 1992-04-27 1993-11-11 Georgetown University Genes du virus humain du papillome et leur utilisation en therapie genique

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