Irradiated 3T3 Cells Research Articles

Geldanamycin Enhances the Radioprotective Effect of Peroxyredoxin 6 in Irradiated 3T3 Fibroblasts.

The aim of the study was to evaluate the possibility of increasing the radioprotective potential of peroxiredoxin 6 (Prdx6) and its mutant form S32A by their combined use with geldanamycin (GA) for 3T3 fibroblasts irradiated with X-rays at a dose of 6 Gy. The mutant enzyme S32A, which does not have phospholipase activity, exhibits a more pronounced radioprotective activity when combined with GA. The use of this combination of radioprotective drugs completely abolishes the peak of NF-κB activity in irradiated 3T3 cells. Another transcription factor, p53, which is an indicator of the level of cell apoptosis and increases upon irradiation, is also reduced by S32A in combination with GA. The low-molecular-weight protein p21, which is a marker of cell senescence and whose production increases upon irradiation, is also normalized when S32A is used in combination with GA. In addition, the use of this combination of radioprotective drugs significantly reduces the stress response of 3T3 cells to X-ray irradiation.

Peroxiredoxin 6 Applied after Exposure Attenuates Damaging Effects of X-ray Radiation in 3T3 Mouse Fibroblasts.

Although many different classes of antioxidants have been evaluated as radioprotectors, none of them are in widespread clinical use because of their low efficiency. The goal of our study was to evaluate the potential of the antioxidant protein peroxiredoxin 6 (Prdx6) to increase the radioresistance of 3T3 fibroblasts when Prdx6 was applied after exposure to 6 Gy X-ray. In the present study, we analyzed the mRNA expression profiles of genes associated with proliferation, apoptosis, cellular stress, senescence, and the production of corresponding proteins from biological samples after exposure of 3T3 cells to X-ray radiation and application of Prdx6. Our results suggested that Prdx6 treatment normalized p53 and NF-κB/p65 expression, p21 levels, DNA repair-associated genes (XRCC4, XRCC5, H2AX, Apex1), TLR expression, cytokine production (TNF-α and IL-6), and apoptosis, as evidenced by decreased caspase 3 level in irradiated 3T3 cells. In addition, Prdx6 treatment reduced senescence, as evidenced by the decreased percentage of SA-β-Gal positive cells in cultured 3T3 fibroblasts. Importantly, the activity of the NRF2 gene, an important regulator of the antioxidant cellular machinery, was completely suppressed by irradiation but was restored by post-irradiation Prdx6 treatment. These data support the radioprotective therapeutic efficacy of Prdx6.

Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use

Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

Radiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2 inhibitor.

Background:Recent evidences support that radiation can promote the invasion of cancer cells. As interactions between cancer cells and surrounding stromal cells can have an important role in tumour progression, we determined whether an irradiation to fibroblasts can enhance the invasiveness of breast cancer cells. The role of cyclooxygenase-2 (COX-2), an inflammatory enzyme frequently induced by radiotherapy, was investigated.Methods:Irradiated 3T3 fibroblasts were plated in the lower compartment of invasion chambers and used as chemoattractant for non-irradiated human breast cancer cell MDA-MB-231, which are oestrogen receptor negative (ER(−)) and the oestrogen receptor positive (ER(+)) MCF-7 cells. Stimulation of COX-2 expression in irradiated 3T3 cells was measured by a semi-quantitative qPCR and western blot. Capacity of the major product of COX-2, the prostaglandin E2 (PGE2), to stimulate the production of the matrix metalloproteinase-2 (MMP-2) and cancer cell invasion were assessed with a zymography gel and invasion chambers.Results:Irradiation (5 Gy) of 3T3 fibroblasts increased COX-2 expression and enhanced by 5.8-fold the invasiveness of non-irradiated MDA-MB-231 cells, while their migration was not modified. Addition of the COX-2 inhibitor NS-398 completely prevented radiation-enhancement of cancer cell invasion. Further supporting the potential role of COX-2, addition of PGE2 has increased cancer cell invasion and release of MMP-2 from the MDA-MB-231 cells. This effect of radiation was dependant on the expression of membrane type 1 (MT1)–MMP, which is required to activate the MMP-2, but was not associated with the ER status. Although irradiated fibroblasts stimulated the invasiveness of MDA-MB-231 ER(−) cells, no enhancement was measured with the ER(+) cell line MCF-7.Conclusions:Radiation-enhancement of breast cancer cell invasion induced by irradiated 3T3 fibroblasts is not dependant on the ER status, but rather the expression of MT1–MMP. This adverse effect of radiation can be prevented by a specific COX-2 inhibitor.

High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60Co-gamma-rays. NIH 3T3 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 60Co-gamma-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 +/- 6.51 vs 66.0 +/- 3.51 and 67.7 +/- 7.37; transfection: 121.7 +/- 6.50 vs 65.3 +/- 3.51 and 69.0 +/- 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, c-myc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes.

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.

The Feeder layer‐mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3

Primary cultured epithelial cells that are used for basic research are often cultivated on plastic whereas those used for clinical purposes are usually cultured in the presence of a feeder layer. Here, we examined the influence of a feeder layer on the expression, affinity and DNA binding ability of the transcription factors, Sp1 and Sp3 in primary cultures of human skin keratinocytes. Co-culturing both newborn and adult skin keratinocytes with lethally irradiated 3T3 cells as a feeder layer contributed to maintain the cell's morphological and growth characteristics and delayed terminal differentiation in vitro. 3T3 also stabilized the DNA binding properties of Sp1 without altering its transcription. Stimulation of Sp1/Sp3 expression appears to be mediated through cell-cell interactions and by factors secreted by 3T3. Thus, we propose that the feeder layer delay terminal differentiation of primary cultured skin keratinocytes by preventing extinction of transcription factors, like Sp1 and Sp3, which play pivotal functions in the cell cycle.

An Improved Organ Culture for Regeneration of Pure Autologous Keratinocytes from Small Split-Thickness Skin Specimens

Background: Failure of autologous keratinocyte culture from small split-thickness skin specimens or contamination of the keratinocyte culture by melanocytes represents practical problems in basic medical research and clinical studies. Purpose: To establish a simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin specimen. Methods: Split-thickness (0.3 mm) skin explants (1 × 2 mm) were firstly cultured in DMEM containing 10% FCS till formation of keratinocyte strips, then cultured in serum-free keratinocyte growth medium or cocultured with lethally irradiated 3T3 fibroblasts (J₂) in a mixture of DMEM and Ham’s F₁₂ (DF) medium. Results: Pure autologous keratinocyte culture is easily and reliably established by this organ culture technique. Conclusion: Culturing of skin explants in serum-free keratinocyte growth medium or coculturing of the skin explants with lethally irradiated 3T3 cells in DF medium is proved to be a useful, simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin biopsy.

Gas6 enhances human oligodendrocyte viability and maturation

Oligodendrocyte development and survival occurs in response to signalling pathways activated by protein trophic factors. To identify novel factors that regulate human oligodendrocyte maturation/survival, we performed microarray analysis and identified genes expressed in moderate amounts in oligodendrocytes at 18–22 gestational weeks (gw). Among those characterized were the genes encoding the receptor tyrosine kinases, Axl and Mer. These receptors belong to the mammalian Axl/Mer/Rse receptor tyrosine kinase family and are developmentally regulated. The ligand for this family is the growth arrest‐specific 6 gene product (Gas6). Gas6 stimulates growth and prevents apoptosis in a variety of cell types. Oligodendrocytes isolated from 20 to 23 gw human fetal spinal cord were grown for 6 days on NIH 3T3 fibroblasts engineered to stably express Gas6 or on unmodified 3T3 cells. Oligodendrocytes were identified by immunostaining for proteins expressed by mature oligodendrocytes, CNPaseI and MBP. An increase in the numbers of CNPaseI+ and MBP+ oligodendrocytes, and the extent of process branching and extension, was observed when grown on irradiated 3T3‐cells secreting Gas6, but not on irradiated 3T3 cells. The effect of Gas6 was abolished in the presence of the Axl‐Fc receptor, but remained unchanged in the presence of TrkA‐Fc receptor. Our results suggest a role for the Gas6‐Axl signalling in regulating oligodendrocyte viability and maturation.

Large expansion of morphologically heterogeneous mammary epithelial cells, including the luminal phenotype, from human breast tumours.

Regular expansion of heterogeneous populations of epithelial cells, including the luminal phenotype, was achieved from small biopsies of human breast tumours and cutaneous metastases by optimized feeder layer technique based on irradiated NIH 3T3 cells. Forty-one out of 47 primary tumour specimens and all three cutaneous metastases grew successfully for two to 10 passages in vitro. The main phenotypes of cultured cells and their changes in subcultures were characterized using immunocytochemistry and phase contrast microscopy (in few cases also time-lapse recording). In the majority of cultured cell populations a fraction of cells positive for keratin 19 (K19+), typical for the luminal phenotype, was detected. This is the cell type from which breast carcinoma is supposed to arise. While in cultures derived from benign lesions only basic phenotypes of luminal and myoepithelial cells were found, in cultures derived from malignant tumours unusual phenotypes of epithelial cells, in their majority K19+, were detected. The growth properties of cells from six benign and seven malignant samples were analyzed in detail. In the analyzed cell populations the culture lifetime - related to the number of colony-forming cells varied for cells from malignant tumours between 21 and 51 and from benign tumours between 22 and 40 cell generations. The total number of passages achieved was three to seven for malignant or four to nine for benign cultures. In spite of negative results of tumourigenicity testing in immunologically compromised Nu/nu mice the potential to culture apparently neoplastic cells was indicated by positive immunostaining for the p53 oncoprotein (seven of 23 tested malignant cases), the src oncoprotein (five of eight), and overexpression of the c-erbB-2 protein (five of 26). This was further confirmed by successful cultivation of malignant cells from cutaneous metastases. Two of the three metastasis-derived cultures were nearly homogeneously positive for K19 while the third was almost negative. The results proved the optimized feeder layer technique to be useful for regular yielding of large amounts of epithelial cells from small tumour biopsies and for supporting the majority of cell phenotypes present in the original tumour. Therefore, it appeared to be a promising tool for further analysis of interactions between luminal and myoepithelial cells in the development of human breast carcinoma and for the study of individual tumours.

Complementary antitumor immunity induced by plasmid DNA encoding secreted and cytoplasmic human ErbB-2.

A plasmid DNA was constructed to encode the N-terminal 505 aa of human ErbB-2 (E2, HER-2/neu) and designated as secreted ErbB-2 (secE2). Recombinant secE2 protein was detected in the transfected cells and was secreted as an 80-kDa glycoprotein. Vaccination of BALB/c mice with secE2 DNA induced both IgG1 and IgG2a ErbB-2-specific Abs and protected approximately 90% of mice against mouse mammary tumor D2F2, which expressed human ErbB-2 (D2F2/E2). The efficacy of secE2 vaccine was comparable with that of wild-type ErbB-2 DNA, which encodes the entire 1258 aa of ErbB-2 protein, induced only IgG2a E2-specific Abs, and stimulated greater CTL activity. Immune lymphocytes were stimulated in vitro with irradiated 3T3 cells, which expressed ErbB-2, K(d), and B7.1. CTL activity was measured by the lysis of E2-positive target cells and by intracellular IFN-gamma production. To enhance CTL activation, mice were immunized with a combination of secE2 and cytoplasmic E2 (cytE2); the latter encodes the 1258-aa ErbB-2 protein that was released into the cytoplasm upon synthesis. Significant increase in CTL activity was demonstrated after mice were immunized with the combined vaccines and all mice were protected from D2F2/E2 tumor growth. Therefore, secE2, which induced Th2 Ab and weak CTL, conferred similar protection as E2, which induced Th1 Ab and strong CTL. Combined vaccination with secE2 and cytE2 resulted in Th2 Ab, strong CTL, and the most effective protection against tumor growth. The strategy of coimmunization with DNA that direct Ags to different subcellular compartments may be adapted as appropriate to optimize immune outcome.

Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer.

The main drawback of the selective culture of human mammary epithelial cells from primary breast cancer is the overgrowth of tumor-associated stromal fibroblasts. This drawback may be overcome by using, in primary culture, lethally irradiated 3T3 cells which act as a feeder layer to maintain tumor-derived epithelial cell proliferation. These 3T3 cells, exposed to 60 Gy at confluence, form a specific cellular substrate which constitutes an obstacle to fibroblast attachment. Enzyme-disaggregated breast cells from six primary breast carcinomas were cocultured over lethally irradiated but living 3T3 cells. The method led to the purification of tumor-derived epithelial cells from all six cancer samples, and long-term culture was obtained in one. The epithelial nature of these purified tumor-derived epithelial cells was demonstrated by their general morphology and by the expression of cytokeratins and Epithelial Membrane Antigen. These results confirm the stimulatory effect of a this stromal feeder layer on breast epithelial cell growth and show that this stromal feeder layer can also control the fibroblast overgrowth. Our results provide an alternative approach in the selective culture of epithelial cells from primary breast carcinoma.

Enhanced Growth Potential of Cultured Rahbit Tracheal Epithelial Cells Following Exposure to N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine

To establish a standardized model for the transformation of rabbit airway epithelial cells, we attempted to transform rabbit tracheal epithelial (RbTE) cells in culture with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). RbTE cells, harvested by enzymatic digestion from male New Zealand white rabbits, were plated onto feeder layers of irradiated 3T3 cells. Control cells proliferated exponentially during the 2nd week of culture and reached the plateau phase by the 3rd week. Cells exposed to MNNG (0.1 γg/ml) proliferated in a fashion similar to the control cells, except that there was some delay before proliferation began. The clonogenic activity of RbTE cells rapidly decreased in parallel with the increase in cell population equally in the control and MNNG groups. During the late plateau phase, cells exposed to MNNG regained clonogenic activity, and this compartment size expanded with time, whereas the clonogenic activity in control cultures remained below the detectable level. In RbTE cell cultures exposed three times to 0.1 γg/ml MNNG, large, persistent and proliferating colonies emerged at a frequency of 1–3 × 10 x−2 among the surviving clones, whereas all the control cultures eventually became senescent. The MNNG‐indnced alteration in the growth potential of RbTE cells, i.e., the extended lifespan, and the maintenance and even expansion of clonogenic activity, was similar to that of transformed rat tracheal epithelial cells. However, no immortal cell line could be established from these growth‐altered RbTE cells. We therefore concluded that the growth‐altered RbTE cells were partially transformed.

A recombined skin composed of human keratinocytes cultured on cell-free pig dermis

Treatment of full skin thickness burns requires replacement of both the dermal and the epidermal components of the skin. We describe a method of preparing recombined human/pig skin (RHPS) by cultivating human keratinocytes on dried cell-free pig dermis (CFPD). CFPD dried on a tissue culture dish forms a thin collagen film which behaves like a firm substrate for cell cultures. HK were grown on the epidermal side of the CFPD using lethally irradiated 3T3 cells as feeders. After reaching confluency of human keratinocytes, human fibroblasts can be cultured on the dermal side of the RHPS. It was possible to obtain approximately 500 cm 2 of the RHPS from 1 cm 2 human split-skin graft in 3 weeks. RHPS is easy to handle, is similar in structural, mechanical and adhesive properties to the normal skin, and can be meshed. This RHPS might be advantageous for permanent covering of wounds in major burns.

Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures.

Growth of hematopoietic stem and progenitor cells found in the MNC fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Reduced oxygen tension increased total cell numbers by as much as 5-fold in cord blood suspension cultures, but this effect was less pronounced in cultures containing an irradiated bone marrow stromal cell layer. However, reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in both types of LTHC studied. Under low oxygen, CFU-C progenitor cell numbers were as much as 10-fold higher. Finally, reduced oxygen tension slowed the rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of human stromal and progenitor cells in vitro. These low oxygen findings were then applied to a murine model LTHC perfusion system. In this system, irradiated 3T3 stromal layer integrity was improved under low oxygen and was substantially further improved with continuous medium perfusion. Cell counts and flow cytometry analysis indicated that the total cell production and the production of immature cells from murine bone marrow MNC on irradiated 3T3 cells were significantly enhanced under low oxygen with perfusion. After three weeks of culture, a 24-fold higher number of Thy1.2lo F4/80- MAC1- cells (indicative of murine stem and progenitor cells) was observed in the perfusion system as compared with static culture under ambient oxygen.

Serial Culture and Characterization of the Chinchilla Middle Ear Epithelium

We have successfully cultured a fibroblast-free chinchilla middle ear epithelium up to the 10th passage by using conditioned medium or using irradiated 3T3 cells as feeder cells. The cultured epithelial cells assumed a polygonal shape with a cobblestone appearance, indicating tight junction formation. A small number of the cells began to show abnormal morphology, such as indistinct cell boundaries, fibroid appearance, or giant cell formation, as the passage increased, particularly after the 5th passage. These morphologically transformed cells showed positive labeling with an anticytokeratin antibody, which indicated the epithelial origin of these cells. Neither ciliated nor secretory cells were observed in the serially cultured cells. The rate of cell growth slowed after the 7th passage, and after the 11th passage the cells no longer proliferated. Even with the above limitations, these cultured cells can be used for a number of in vitro experiments.

Tissue culture and cutaneous amyloidoses

Tissue culture refers to the propagation of living tissue cells in special media conducive to their growth. Initially devised by Harrison 1 to study frog nerve fiber at the beginning of this century, tissue culture technology has now become an indispensable tool in almost every discipline of biomedical research. Dermatology is by no means an exception. Ljunggren 2 described the growth of skin in amniotic fluid early in 1898, and Lewis 3 observed human epidermal cells in tissue culture with phase-contrast microscopy in 1949. The development of modern methodology for keratinocyte culture did not start, however, until about two decades ago. In 1967, Briggaman et al. 4 prepared a viable suspension of postembryonic human epidermal cells and described their growth characteristics in cell culture. A cell line derived from mouse embryo fibroblasts by Todaro and Green, 5 3T3, was also utilized to promote growth of keratinocytes in culture. Rheinwald and Green 6 showed in 1975 that human epidermal keratinocytes could be cultured selectively on feeder layers of lethally irradiated 3T3 cells. Addition of hydrocortisone, cholera toxin, and epidermal growth factor 7 to the medium has made continued serial subculture possible over many cell generations. By altering the constituents of the medium, Peehl and Ham 8 further cultured keratinocytes from human foreskin successfully without a feeder layer. Various models proposed in recent years 9,10 for growing epidermal cells on a dermal equivalent at the air-liquid interface reflect attempts to more closely approximate physiological conditions in vitro.

Long-term transplantation of canine keratinocytes made resistant to G418 through retrovirus-mediated gene transfer.

We studied cultured canine keratinocytes to determine whether they could serve as targets for retrovirus-mediated gene transfer and whether infected cells could persist after transplantation into dogs, a large random-bred model for gene transfer studies. Canine keratinocytes obtained from skin biopsy samples were cultured in vitro with lethally irradiated NIH 3T3 cells used as a feeder layer. The keratinocyte colonies consisted of squamous epithelium with numerous desmosomes, tonofilaments, and keratohyalin granules. In addition, the cells were strongly reactive with monoclonal antibodies to cytokeratin intermediate filament proteins. For the infection studies, we grew the keratinocytes on a feeder layer of lethally irradiated PA317 retrovirus packaging cells, which produced a helper-free amphotropic retroviral vector containing the neomycin phosphotransferase (neo) gene. After cocultivation, 34% (range, 10-76%) of the keratinocytes were found to be resistant to the neomycin analogue G418. Infected keratinocytes were then transplanted into the dog of origin; 1% (range, less than 0.1-3%) of the keratinocytes obtained 27-130 days after transplantation from skin biopsy samples gave rise to G418-resistant colonies. We conclude that canine keratinocytes cultured in vitro can be infected efficiently with a neo gene-containing retroviral vector, and they show persistent G418 resistance for at least 130 days after transplantation into the skin donor.

Epithelial-stromal interactions in the adult bladder: Urothelial growth, differentiation, and maturation on culture fascsimiles of bladder stroma

Analysis of the responsiveness of isolated adult urothelium to a series of different stromal cell-extracellular matrix combinations demonstrated the capacity of stromal cells to induce and maintain normal patterns of urothelial growth, differentiation, and maturation in vitro. By incorporating embryonic mesenchymal derived (Swiss 3T3) cells into type I collagen matrices, simplified three-dimensional tissue-like facsimiles of bladder stroma were derived. When recombined with sheets of isolated urothelium these facsimiles could approximately reproduce the capacity of natural stromal tissue to support the expression of normal urothelial tissue specific characteristics. In contrast cocultures between urothelia and monolayers of 3T3 cells, applied to the surface of planar collagen substrata could only permit urothelial cell attachment but not growth or differentiation whereas lethally irradiated 3T3 (feeder) cells, under similar experimental conditions, could support the maintenance of an immature or incompletely differentiated urothelium. Conditioned medium elaborated by cultured 3T3 cells could not stimulate further differentiation in urothelia cultured alone on planar collagen substrata. These studies indicate that a significant portion of the regulatory capacity of the stroma in stromal-urothelial interactions can be accounted for by the activities of a closely applied population of stromal cells, provided the cells are viable and presented to the urothelium in a three-dimensional context in combination with collagen. The capacity of embryonic mesenchymal cells to express properties appropriate to the development of a multilayered terminally differentiated urothelium suggests that normal interactions between adult urothelium and stroma are of limited specificity with the urothelium requiring an essential input of permissive signals only.