Infection of cultured human airway epithelial cells by influenza A virus

The lack of an adequate in vitro model has hampered study of the cellular basis by which influenza A virus causes disease in the human airway. We report in vitro infection of human airway epithelial cells by influenza A virus. Fetal and adult human tracheal and bronchial epithelial cells cultured from explants and SV40 transformed adult human tracheal epithelial cells were exposed to a recently isolated strain of influenza A virus (H1N1) and a laboratory passaged strain (WSN) of influenza A virus at similar multiplicity of infection. All cultures derived from explants showed hemadsorption (approximately 30% of the cells) with the H1N1 virus. No hemadsorption was detected with the WSN virus. One of two transformed cell lines showed a 5-10% hemadsorption to cells after H1N1 exposure and none following exposure to WSN. Immunofluorescent staining for influenza A-specific antigens in virus-exposed, explant-derived cells indicated viral infection and replication in these cells. Hemagglutinating material in the growth medium of infected, explant-derived cell lines, increased as a function of time, indicating the production of virus proteins. Exposure of rhesus monkey kidney cells and new human tracheal epithelial cultures to supernatant from these cells resulted in hemadsorption, indicating the presence of infectious virus in the supernatant. Light microscopic examination of virally infected bronchial epithelial cells demonstrated that the common types of cytopathic changes were rarely seen while cell proliferation continued over time. The data indicate that influenza A virus can infect, replicate, and produce infectious virus in cultured human tracheal and bronchial epithelial cells.     

Production of tissue-engineered three-dimensional human bronchial models

We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro.     

A biphasic chamber system for maintaining polarity of differentiation of culture respiratory tract epithelial cells

Summary A simple, disposable, biphasic cultivation chamber has been developed for respiratory tract epithelial cells. This chamber, the Whicutt chamber, contains a movable, transparent, permeable gelatin membrane that can be employed either submerged in the culture medium, thereby feeding the cells by the traditional immersion method, or raised to the surface of the culture medium, to bring the apical surfaces of the cells into contact with air and provide nutrients only from below (basal feeding). The effects of biphasic cultivation on the growth and differentiation of respiratory tract epithelial cells from different sources have been studied in Whitcutt chambers. Primary hamster tracheal epithelial (HTE) cells grown to confluence with basal feeding developed a ciliated columnar morphology, with differentiated features (cilia and mucous granules) located in the apical region of the epithelial layer. These cells secreted mucinlike molecules from the apical surface (i.e. the surface in contact with air). Although the apical localization of differentiation features was greater, mucous cell differentiation achieved by basal feeding was quantitatively not greater than that achieved by continuous immersion feeding. Similarly, basal feeding did not alter the degree of epithelial cell differentiation in cultures derived from rat, rabbit, and monkey tracheas or from human bronchial and nasal tissues. In contrast, the differentiation of guinea pig tracheal epithelial cells in culture was significantly influenced by the feeding method employed. When fed basally, guinea pig tracheal epithelial cell cultures expressed various mucociliary functions with resemblance to mucociliary layers in vivo, whereas constantly immersed cultures seemed stratified and squamous. These results suggest that, at least for guinea pigs, the combination of feeding methods provided by the Whitcutt chamber can be used to achieve differentiated cultures of tracheal epithelial cells with a polarity of differentiation that is similar to that observed in intact airways in vivo. Editor's statement Supported in part by grants from NCI (CA42097) and American Cancer Society (BC-465) to R. W., and grants from The Council for Tobacco Research-USA, and Cystic Fibrosis Foundation to K.B.A.     

Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGF beta

In this paper we examined the effects of transforming growth factor beta (TGF beta) on the proliferation and differentiation of rabbit tracheal epithelial cells in primary culture. Treatment of these cells with TGF beta inhibits cell proliferation in a time- and dose-dependent manner; concentrations as low as 1 pM are able to inhibit cell growth. Concomitantly, TGF beta causes cells to accumulate in the G0/G1 phase of the cell cycle and a sharp reduction in the ability of the cells to form colonies after subculture at clonal density. These results indicate that TGF beta induces terminal cell division in these cells. The inhibition of cell growth is accompanied by changes in cell morphology and a stimulation of the formation of cross-linked envelopes. TGF beta enhances the levels of transglutaminase activity and cholesterol sulfate, two markers of squamous differentiation. Our results indicate that TGF beta induces terminal squamous cell differentiation in rabbit tracheal epithelial cells. Retinoic acid (RA) does not affect the commitment to terminal cell division induced by TGF beta, but inhibits the expression of the squamous phenotype. Growth of normal human bronchial epithelial cells was affected by TGF beta in a way similar to that of rabbit tracheal epithelial cells. Several carcinoma cell lines tested were quite resistant to TGF beta, whereas growth of one carcinoma cell line was stimulated by TGF beta. These results indicate that a modified response to TGF beta could be one mechanism involved in the aberrant growth control of malignant cells.     

Long-term culture of normal human colonic epithelial cells in vitro.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.     

Isolation and characterization of guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture

Summary A simple 30-min enzyme digestion procedure has been used to release guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture. Primary cell cultures initially consist of numerous epithelial colonies and 70–1000 morphologically differentiated smooth muscle cells per 600 mg (wet weight) tracheal tissue depending on the age of the animal. Both cell types proliferate to form a confluent monolayer within 5–17 days. Pure subcultures of tracheal smooth muscle cells are obtained by limited trypsin digestion of the primary culture. Eighty percent of these subcultured smooth muscle cells retain the ability to contract in response to histamine (10^-6 M) and to form reaggregates even after 20 or more passages. Examination of these cells by electron microscopy reveals both biosynthetic and contractile components of smooth muscle. Analysis of this dual phenotype may provide valuable information about the regulation of tracheal smooth muscle cell growth and differentiation.This research was supported in part by a grant from the Foundation for Research in Bronchial Asthma and Related Diseases, Worcester, Massachusetts 01604, USA     

维胺酸对体外转化人支气管上皮细胞及体内构建人支气管上皮组织的抑制作用

癌前改变是肿瘤演变过程中的关键阶段。许多研究显示维甲类化合物对动物肿瘤及体外恶性细胞系具有抑制作用,但尚未见其对肺癌前病变作用的实验室研究报道。人类肺癌的绝大部分起源于支气管上皮,为研究维胺酸对体外转化人支气管上皮M细胞系以及在大鼠气管构建后移植到裸鼠体内生长的具有癌前病变特点的人支气管上皮组织的抑制作用,采用上皮细胞无血清培养技术,人支气管上皮组织大鼠气管内构建／裸鼠皮下移植生长技术,流式细胞学分析,免疫组化、凋亡细胞原位末端标记以及病理学检查等研究方法发现,维胺酸可抑制体外培养的转化人支气管上皮细胞的增殖,使S期细胞比例下降,以及细胞增殖标志Ki-67、mpm-2阳性反应细胞比例下降;明显诱导细胞凋亡。裸鼠腹腔注射给予维胺酸也可使大鼠气管内构建后移植到裸鼠体内生长的癌前期人支气管上皮组织的生长率明显降低,病变程度明显减轻;同样可以诱导细胞凋亡。研究结果提示,维胺酸对体外培养的转化人支气管上皮细胞系及大鼠气管构建／裸鼠体内移植生长的人支气管上皮组织均有明显的抑制作用,是有希望的肺癌化学预防药物。     

维胺酸对体外转化人支气管上皮细胞及体内构建人支气管上皮组 …

Precancerous lesion is one of most important steps in tumorigenesis. It has been shown that retinoids have reliable effects on controlling many kinds of animal tumor and malignant tumor cell lines in vitro, but there is no laboratory report on the biological effect of retinoids on the precancerous lesion of human lung cancer. In this study the methods including of cell serum-free culture, precancerous model of human bronchial epithelium reconstructed in rat trachea/xenotransplanted in nude mice, flowcytometry, immunohistochemistry, TUNEL and pathological observation have been used to study the biological effects of N-(4-hydroxylphenol) retinamide (4-HPR), one new kind of retinoids, on transformed human bronchial epithelial cells in vitro and premalignant human bronchial epithelium in vivo. The results showed that in the study in vitro, the proliferation of transformed human bronchial epithelial cells, the ratio of cells in S phase, and the percentage of cells that positively react to antibody Ki-67 and mpm-2 were inhibited, but apoptotic cells were induced significantly by 4-HPR exposure. At the experiment in vivo, both growth rates and precancerous grades of the reconstructed human bronchial epithelium were reduced, and apoptotic cells were also observed in epithelium after 4-HPR treatment. The results suggested that 4-HPR is one of hopeful chemopreventive medicines to lung cancer.     

Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ

In this paper we examined the effects of transforming growth factor β (TGFβ) on the proliferation and differentiation of rabbit tracheal epithelial cells in primary culture. Treatment of these cells with TGFβ inhibits cell proliferation in a time- and dose-dependent manner; concentrations as low as 1 pM are able to inhibit cell growth. Concomitantly, TGFβ causes cells to accumulate in the G0/G1 phase of the cell cycle and a sharp reduction in the ability of the cells to form colonies after subculture at clonal density. These results indicate that TGFβ induces terminal cell division in these cells. The inhibition of cell growth is accompanied by changes in cell morphology and a stimulation of the formation of cross-linked envelopes. TGFβ enhances the levels of transglutaminase activity and cholesterol sulfate, two markers of squamous differentiation. Our results indicate that TGFβ induces terminal squamous cell differentiation in rabbit tracheal epithelial cells. Retinoic acid (RA) does not affect the commitment to terminal cell division induced by TGFβ, but inhibits the expression of the squamous phenotype. Growth of normal human bronchial epithelial cells was affected by TGFβ in a way similar to that of rabbit tracheal epithelial cells. Several carcinoma cell lines tested were quite resistant to TGFβ, whereas growth of one carcinoma cell line was stimulated by TGFβ. These results indicate that a modified response to TGFβ could be one mechanism involved in the aberrant growth control of malignant cells.     

Bronchial epithelial cells release IL-6, CXCL1 and CXCL8 upon mast cell interaction

Although mast cells have been found in increased numbers in bronchial epithelium in asthma patients, the pathogenic role of the interaction of mast cells with bronchial epithelial cells in the development of local inflammation in asthma is not well understood. In this study, primary human bronchial epithelial cells and a human mast cell line (HMC-1) were cultured either together or separately in the presence or absence of various signaling molecule inhibitors or dexamethasone. Cytokine IL-6, and chemokines including CXCL1 and CXCL8 in cell culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA), and the activity of mitogen-activated protein kinases (MAPKs), or nuclear factor-κB (NF-κB) in co-culture system was analyzed by ELISA. Co-culture of bronchial epithelial cells and mast cells induced a significant elevation of IL-6, CXCL1 and CXCL8 in bronchial epithelial cells, and both IL-17A and IL-17F could further enhance the release of these inflammatory mediators from co-culture. The induction of IL-6, CXCL1 and CXCL8 upon the interaction of bronchial epithelial cells with mast cells was mediated by MAPKs and NF-κB signaling pathways. These data indicate that the interaction of mast cells with bronchial epithelial cells may represent a crucial mechanism of regulating local inflammatory response in allergic asthma.     

Characterization of epithelial cell cultures derived from human tracheal glands

Summary Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 μg/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 μg/ml), and bovine pituitary extract (25 μg/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide β-d-galactose-(l→ 3)N-acetyld-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl⁻ channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established. This research was supported by USPHS grants HL 41979 and HL 33142 from the National Heart, Lung and Blood Institutes.     

Human bronchial epithelial cells synthesize cholesterol sulfate during squamous differentiation in vitro

Epithelial cells of the airways can, under pathological conditions, undergo squamous metaplasia. The accumulation of cholesterol sulfate has recently been described as a new marker for squamous cell differentiation in rabbit tracheal epithelial cells. We now report that normal human bronchial epithelial cells in culture metabolically incorporated [35S]-sulfate and [3H]-mevalonate into material indistinguishable from cholesterol sulfate by the criteria of solubility in organic solvents, behavior on ion-exchange chromatography, susceptibility to solvolysis, and behavior on thin-layer chromatography before and after solvolysis. The accumulation of cholesterol [35S]-sulfate correlated well with squamous cell differentiation (as measured by cross-linked envelope formation), which occurred when the cells reached confluency. The increase in the level of cholesterol sulfate could be inhibited by the inclusion of retinoic acid in the cell-culture medium. The addition of phorbol-12-myristate-13-acetate or the presence of high Ca2+ concentration in the medium stimulated the accumulation of cholesterol sulfate. An increased activity of cholesterol sulfotransferase seems to account for the cholesterol sulfate accumulation. The original observation of cholesterol sulfate accumulation during squamous differentiation thus extends across species lines and strengthens the suggestion that the cholesterol sulfate may play an important role in this type of differentiation. Moreover, cholesterol sulfate provides a sensitive biochemical marker to study this pathway of differentiation of human bronchial epithelial cells.     

Serial culturing of human bronchial epithelial cells derived from biopsies

Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

Signalling and cellular specificity of airway nitric oxide production in pertussis

Bordetella pertussis , the aetiological agent of whooping cough (pertussis), causes selective destruction of ciliated cells of the human airway mucosa. In a hamster tracheal organ culture model, B. pertussis causes identical cytopathology as does tracheal cytotoxin (TCT), a glycopeptide released by the bacterium. The damage caused by B. pertussis or TCT has been shown to be mediated via nitric oxide (NO·). Using immunofluorescence detection of the cytokine-inducible NO synthase (iNOS; NOS type II), we determined that B. pertussis induced epithelial NO· production exclusively within non-ciliated cells. This epithelial iNOS activation could be reproduced by the combination of TCT and endotoxin. However, neither TCT alone nor endotoxin alone was capable of inducing epithelial iNOS. This result mirrors the synergistic activity of TCT and endotoxin exhibited in monolayer cultures of tracheal epithelial cells. Therefore, TCT and endotoxin are both important virulence factors of B. pertussis , combining synergistically to cause the specific epithelial pathology of pertussis.     

In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique

Epithelial cells from tracheal mucosa offer significant potential as a cell source in development of tissue-engineered trachea. The purpose of this study was to investigate and optimize a suitable culture system for tracheal epithelial cells, including the methods of primary culture, passage, identification, and cryopreservation. Epithelial cells were isolated from rabbit tracheal mucosa using tissue explant technique and were subjected to immunohistochemistry, immunofluorescence, and cryopreservation after purification. Epithelial cells reached confluency at 14–15 d. Immunohistochemical staining for cytokeratin showed brown yellow-positive cytoplasm and blue-counterstained nuclei, while immunofluorescence staining for cytokeratin showed green-positive cytoplasm and clear cell outline, indicating that the cultured cells had properties of epithelial cells. After recovery, epithelial cells exhibited high survival and viability. The results demonstrated that in vitro isolation and cultivation model was successfully established to provide high proliferative capacity, typical morphology and characteristics of tracheal epithelial cells from trachea mucosa by the use of the tissue explant technique.     

Serial propagation of adult human prostatic epithelial cells with cholera toxin

Summary Reproducible subculture of adult human prostatic epithelial cells from normal, benign hyperplastic and malignant tissue has been achieved. Cholera toxin is the key component in the culture system, but use of an optimal basal medium (PFMR-4) supplemented with a high level of serum in collagen-coated dishes also improves growth and serial propagation. Editor’s statement The critical first step in the development of an optimized culture system for any cell type is to obtain enough growth to perform detailed growth-response studies. Multiplication of human prostatic epithelial cells in primary culture has been possible for several years, but scarcity of tissue specimens and inability to subculture have severely limited the usefulness of such cultures. The procedures described by Peehl and Stamey in this communication make it possible to expand the original inoculum substantially and to use subcultures to perform precisely controlled replicate experiments. This will open the way for rapid progress in the development of practical cell culture model systems for research on normal, hypertrophic, and malignant human prostatic cells. Richard G. Ham     

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed “enteroids” when derived from small intestine and “colonoids” when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.     

利用气液界面无血清培养原代兔气管上皮细胞的研究

用低温酶消化法分离兔气管上皮细胞,具有细胞损伤小,活力及纯度高的优点,成纤维细胞污染极低。人胎盘胶原提高了气管上皮细胞贴壁性。无血清培养基能促进细胞增殖,分化和成熟。气液界面培养方式更好地模拟了气管上皮细胞的天然生长环境,在膜上呈复层生长,有利于细胞的分化成熟及功能表达。光镜下细胞形态及免疫组化细胞角蛋白染色阳性证实培养细胞为气管上皮细胞。本文所建立的兔气管上皮细胞体外气液界面无血清培养方法为研究气 管上皮细胞的生理和病理提供了一个十分有用的模型。     

Serial propagation of adult human prostatic epithelial cells with cholera toxin

Reproducible subculture of adult human prostatic epithelial cells from normal, benign hyperplastic and malignant tissue has been achieved. Cholera toxin is the key component in the culture system, but use of an optimal basal medium (PFMR-4) supplemented with a high level of serum in collagen-coated dishes also improves growth and serial propagation.