Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.     

Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude

Although cardiac myocytes adherent to tissue culture polystyrene (TCPS) dishes retain the spontaneous beating, the pulsatile amplitude is highly limited compared to that in vivo. One of the main reasons for the limited pulsation may be the interface between the cells and the TCPS surfaces. Release of these cells from rigid TCPS surfaces may augment their pulsatile amplitude. With this perspective, we have developed a novel cell manipulation technique to detach cultured cardiac myocytes from rigid surfaces and to rescue higher pulsatile amplitude of the cells using temperature-responsive culture dishes and discuss the possibility of improving this heart tissue model. Primary cardiac myocytes were cultured on the slightly hydrophobic dish surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide). Cells adhered and proliferated, forming confluent cardiac myocyte sheets in a fashion similar to those on ungrafted TCPS dishes. Decrease in culture temperature resulted in surface change of the polymer from slight hydrophobic to highly hydrophilic due to extensive hydration of the grafted polymer on the dishes. This results in release of cardiac myocyte sheets from the dishes without enzymatic or EDTA treatment. When no support was used, the detached cardiac myocyte sheets shrank to one-tenth size, which ceased their pulsation. When chitin membranes were used to support the confluent sheets to prevent cell shrinkage, the detached cell sheets could be transferred and readily adhered onto another virgin TCPS dishes. These transferred cell sheets preserved the similar cell morphology and pulsation to those before the detachment. When polyethylene meshes were used to support cell sheet transfer, detached cardiac myocyte sheets partially attached to the mesh threads. Then, the constructs were inverted and placed in another culture dish to prevent direct association to dish surfaces. Moreover, the cardiac myocyte sheets were reorganized to heart tissue-like structures by the unisotropic contraction orientated by the mesh threads, and the pulsatile amplitude increased more than 10 times higher. This technique would bring about new insight in tissue engineering as well as cultured heart model.     

Two-dimensional cell sheet manipulation of heterotypically co-cultured lung cells utilizing temperature-responsive culture dishes results in long-term maintenance of differentiated epithelial cell functions.

Here we report two-dimensional cell sheet manipulation (2D CSM) of heterotypically co-cultured lung cell sheets and the maintenance of differentiated phenotypes of lung epithelial cells over prolonged periods of up to 70 days. This was facilitated by poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture dishes. PIPAAm-grafted dishes are responsive to temperature changes and offer a unique surface on which cells adhere and multiply like on ordinary tissue culture dishes under the permissive temperature of 37 degrees C, but on lowering of temperature resulting in changes in hydration of the polymer the cells spontaneously detach from the surface without use of enzymes like trypsin which is the common procedure. It has been well documented that type II pneumocytes of the lung lose many of their special features rapidly in culture. The culture system detailed here comprises random co-culture of epithelial and mesenchymal cells of lung. The heterotypic cell culture system promotes cell-cell interactions maintaining a harmonized physiology. When this heterotypic monolayer on PIPAAm-grafted dishes was subjected to lower temperature of 20 degrees C and 2D CSM we were able to transfer the monolayer as a single contiguous sheet with cell-cell connections intact to other surfaces. This non-invasive transfer of cell sheet resulted in shrinkage of the monolayer, enabling the type II cells to regain their cuboidal morphology and specialized characters like Maclura pomifera lectin binding and surfactant protein A (SP-A) expression. The active dome formation also observed subsequent to transfer reaffirms the uniqueness of the culture conditions and 2D CSM in future for developing tissue like architecture in vitro.     

Immobilization of cell-adhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest

We have developed temperature-responsive cell culture surfaces to harvest intact cell sheets for tissue-engineering applications. Both cost and safety issues (e.g., prions, bovine spongiform encephalopathy) are compelling reasons to avoid use of animal-derived materials, including serum, in such culture. In the present study, synthetic cell-adhesive peptides are immobilized onto temperature-responsive polymer-grafted surfaces, and cell adhesion and detachment under serum-free conditions were examined. The temperature-responsive polymer poly(N-isopropylacrylamide) (PI-PAAm) was functionalized by copolymerization with a reactive comonomer having both a carboxyl group and an isopropylacrylamide group. These copolymers were covalently grafted onto tissue culture-grade polystyrene dishes. Synthetic cell-adhesive peptides were then immobilized onto these surfaces via carboxyl groups. Bovine aortic endothelial cells both adhered and spread on these surfaces even under serum-free conditions at 37 degrees C, similar to those in 10% serum-supplemented culture. Spread cells promptly detached from the surfaces on lowering culture temperatures below the lower critical solution temperature of the polymer, 32 degrees C. These surfaces would be useful for serumfree culture for tissue-engineering applications.     

Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacrylamide)-grafted surfaces

Temperature-responsive hydration/dehydration changes in surface-grafted poly(N-isopropylacrylamide) (PIPAAm) were utilized for hydrophilic/hydrophobic surface property alterations in cell culture. In this report, we utilized PIPAAm-grafted surfaces to recover confluently-cultured vascular endothelial cells as coherent monolayers from this cell culture substrate and to transfer to new cell culture substrates. For this purpose, we used two different methods to recover and transfer cell monolayer cultures: (1) chitin membranes used as an apical side cell support during cultured cell transfer, allowing cell basal side reattachment to new culture substrates after transfer; and (2) a cell culture insert? (porous PET) used as both a support as well as new substrate, allowing basal surfaces of cultured cells to be exposed to the medium after transfer. In both cases, all cells grown on PIPAAm-grafted surfaces detach completely with maintenance of basement membrane-like structure. Recovered cells attach to the second culture surfaces, covering more than 60% of the new substrate, and retain approximately 90% viability and their original function as judged from tissue-type plasminogen activator secretion. This technique could be utilized to prepare novel bioartificial organs as well as cell co-culture systems by multi-layering different cell types to mimic tissue structures for tissue engineering.     

Mechanisms of dopamine effects on Na-K-ATPase activity in Madin-Darby canine kidney (MDCK) epithelial cells

Dopamine decreases tubular sodium reabsorption, attributed in part to Na-K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where specific dopamine DA1 binding sites have been demonstrated, we examined the effects of dopamine, as well as of DA1 and DA2 receptor agonists on Na-K-ATPase activity and on the number of units in Madin-Darby canine kidney (MDCK) cells, which retain differentiated properties of the renal cortical collecting tubule epithelium. Dopamine (10^–5 M) inhibited pump activity (by 50%) and reduced the number of units. This effect was reproduced by the DA1 agonist SKF 38393, which inhibited pump activity in a dose- and time-dependent manner (maximum, 10^–5 M). The DA2 agonist quinpirole hydrochloride was without effect, either alone or in combination with SKF 38393. Inhibition of pump activity by dopamine was totally abolished by H7 (100 M), an inhibitor of protein kinase (PK), but partially by 2, 5-dideoxyadenosine (DDA) and H4, respective inhibitors of cAMP production and PKA, which suggests that the dopamine effect on Na-K-ATPase activity may be linked to activation of both PKC and PKA. In these cells, amiloride addition during preincubation did not alter the effect of dopamine on Na-K-ATPase activity; in contrast, furosemide increased further the inhibitory effect of dopamine on the enzyme activity. Monensin addition (10^–3 M) reversed the inhibitory effect of dopamine after a 30-min preincubation. These results indicate that dopamine inhibits Na-K-ATPase activity in MDCK cells and that this inhibition is mediated by activation of the DA1 receptor, they also suggest that PKC and PKA activation inhibits apical sodium entry.     

Growth and differentiated properties of a kidney epithelial cell line (MDCK)

The MDCK dog kidney epithelial cell line has been shown to retain the capacity for vectorial salt and fluid transport, sensitivity to growth regulation, and the ability to regenerate kidney tubular-like structures when injected into athymic nude mice. MDCK cells grown in tissue culture or in baby nude mice have the morphological properties of distal tubular cells, form tight and gap junctions, lack proximal tubular enzyme markers, and possess appreciable activities of Na+-K4-ATPase, ectoleucine aminopeptidase, and ectoalkaline phosphatase. Adenylate cyclase in intact cells is responsive to vasopressin, prostaglandins E1 and E2, and glucagon. Two Na+ transport systems have been characterized: a Na+-H+ antiport system, sensitive to amiloride inhibition, and a NaCl-KCl cotransport system, dependent on metabolic energy and sensitive to furosemide inhibition. Genetic techniques have been used to modify the properties of the cells. The results suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cells of origin and that a clonally isolated cell possesses the receptor, transmission, and target enzyme systems necessary for the regulation of transcellular salt and fluid transport.     

Protein kinase C activation causes inhibition of Na/K-ATPase activity in Madin-Darby canine kidney epithelial (MDCK) cells

To evaluate the influence of protein kinase C (PKC) activation on Na/K-ATPase activity in MDCK cells, we studied the effect of phorbol myristate acetate (PMA) and two diacylglycerol analogues, oleoylacetylglycerol and dioctanoylglycerol, on the enzyme activity. Na/K-ATPase activity was determined by cytochemistry. PMA induced a time- and dose-dependent inhibition of Na/K-ATPase activity and at 100 ng/ml decreased the enzyme activity by 55% of the initial value. These effects were mimicked by oleoylacetylglycerol and dioctanoylglycerol, and were abolished by two inhibitors of PKC, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) and sphingosine. A phorbol ester that does not activate PKC, 4-phorbol 12, 13-didecanoate, did not inhibit Na/ K-ATPase activity. PMA inhibition persisted in the presence of cycloheximide and actinomycin D but not in the presence of amiloride. Dopamine (10 M) inhibition of Na/K-ATPase activity was abolished in a dose-dependent manner by sphingosine. Results suggest that in MDCK cells Na/K-ATPase is an effector protein for PKC and that dopamine inhibition of its activity may be mediated by PKC.     

Comparison of Madin-Darby canine kidney cells (MDCK) with a green monkey continuous cell line (Vero) and human lung embryonated cells (MRC-5) in the isolation of influenza A virus from nasopharyngeal aspirates by shell vial culture.

We report a comparative study of the MDCK, Vero, and MRC-5 cell lines in the isolation of the influenza A (IA) virus. We studied 746 samples in which 63 IA viruses were isolated. The MDCK line displayed 100% sensitivity, the Vero line displayed 71.4%, and the MRC-5 displayed 57.1%. The MDCK line showed a statistically significant difference with respect to the Vero line (P = 0.001) and the MRC-5 line (P = 0.001). The quantitative sensitivity analysis showed the MDCK line to be superior to the other lines. It seems that the MDCK line is still one of the most recommendable for the isolation of the IA virus from respiratory samples.     

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.     

Permanent canine kidney (MDCK) cells for isolation and plaque assay of influenza B viruses

A wide range of influenza B virus strains with various passage histories uniformly formed well-defined clear plaques with high efficiency in cultures of an established line of canine kidney cells (MDCK). PFU titers of the viruses assayed in MDCK exceeded the titers assayedin ovo. With recently isolated strains such as B/Hong Kong/5/72 and Gifu/2/73, the PFU/EID₅₀ ratios were as high as 100 to 400. MDCK cells have been successfully employed for primary isolation of influenza B viruses from throat washings of patients by direct plaquing.     

Adaptation of a Madin-Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce avian influenza virus to Vero and BHK21 cell lines

Madin-Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both α-2,6 and α-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the α-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production.     

Plaque assay and primary isolation of influenza a viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin

A wide variety of influenza A viruses, comprising human, equine, porcine, and avian strains, grew productively in an established line of canine kidney cells (MDCK) under an overlay medium containing trypsin, and formed well-defined plaques regardless of their prior passage history. Plaquing efficiency was comparable to the efficiency of infection in fertile eggs via allantoic route. MDCK cells have also been successfully employed for the primary isolation of influenza A virus from throat washings of patients. Parallel titration of several clinical specimens showed that the inoculation into MDCK cells followed by incubation in the presence of trypsin was an isolation procedure as sensitive as the amniotic inoculation into fertile eggs.     

Assessment of fibronectin conformation adsorbed to polytetrafluoroethylene surfaces from serum protein mixtures and correlation to support of cell attachment in culture

Surfaces of polytetrafluoroethylene (PTFE) were exposed to buffered aqueous solutions containing radio-labeled human fibronectin ([¹²⁵I]Fn), Fn/bovine serum albumin (BSA) binary mixtures of various ratios or whole human plasma dilutions for 1 h. Total adsorbed Fn and albumin adsorption following rinsing was quantified on this surface. ¹²⁵I-labeled monoclonal antibodies against either the tenth type-III Fn repeat unit (containing the cell-binding RGDS integrin recognition motif) or the Fn amino-terminal domain were used to probe the accessibility of each of these respective Fn regions post-adsorption. Human umbilical vein endothelial cells (HUVECs) were cultured on PTFE surfaces pre-exposed to each of these protein adsorption conditions and compared to identical conditions on tissue culture polystyrene (TCPS). Fn adsorption to PTFE is dependent upon the concentration of albumin co-adsorbing from solution: albumin out-competes Fn for PTFE surface sites even at non-physiological Fn/HSA ratios 10–100-fold biased in Fn. Antibodies against Fn do not readily recognize Fn adsorbed on PTFE as the HSA co-adsorption concentration in either binary mixtures or in plasma increases, indicating albumin masking of adsorbed Fn. At Fn/HSA ratios rich in Fn (1 : 1, 1 :100), albumin co-adsorption actually improves anti-Fn antibody recognition of adsorbed Fn. HUVEC attachment efficiency to PTFE after protein adsorption correlates with amounts of Fn adsorbed and levels of anti-Fn antibody recognition of Fn on PTFE, linking cell attachment to integrin recognition of both adsorbed Fn density and Fn adsorbed conformation on PTFE surfaces.     

Assessment of fibronectin conformation adsorbed to polytetrafluoroethylene surfaces from serum protein mixtures and correlation to support of cell attachment in culture

Surfaces of polytetrafluoroethylene (PTFE) were exposed to buffered aqueous solutions containing radio-labeled human fibronectin ([125I]Fn), Fn/bovine serum albumin (BSA) binary mixtures of various ratios or whole human plasma dilutions for 1 h. Total adsorbed Fn and albumin adsorption following rinsing was quantified on this surface. 125I-labeled monoclonal antibodies against either the tenth type-III Fn repeat unit (containing the cell-binding RGDS integrin recognition motif) or the Fn amino-terminal domain were used to probe the accessibility of each of these respective Fn regions post-adsorption. Human umbilical vein endothelial cells (HUVECs) were cultured on PTFE surfaces pre-exposed to each of these protein adsorption conditions and compared to identical conditions on tissue culture polystyrene (TCPS). Fn adsorption to PTFE is dependent upon the concentration of albumin co-adsorbing from solution: albumin out-competes Fn for PTFE surface sites even at non-physiological Fn/HSA ratios 10-100-fold biased in Fn. Antibodies against Fn do not readily recognize Fn adsorbed on PTFE as the HSA co-adsorption concentration in either binary mixtures or in plasma increases, indicating albumin masking of adsorbed Fn. At Fn/HSA ratios rich in Fn (1:1, 1:100), albumin co-adsorption actually improves anti-Fn antibody recognition of adsorbed Fn. HUVEC attachment efficiency to PTFE after protein adsorption correlates with amounts of Fn adsorbed and levels of anti-Fn antibody recognition of Fn on PTFE, linking cell attachment to integrin recognition of both adsorbed Fn density and Fn adsorbed conformation on PTFE surfaces.     

Expression of metastasis-associated mts1 gene is co-induced with membrane type-1 matrix metalloproteinase (MT1-MMP) during oncogenic transformation and tubular formation of Madin Darby canine kidney (MDCK) epithelial cells

Madin-Darby canine kidney (MDCK) epithelial cells form branching tubules in three-dimensional collagen gel in the presence of hepatocyte growth factor (HGF). Membrane type-1 matrix metalloproteinase (MT1-MMP), expression of which was induced by collagen-gel culture, was demonstrated to play an essential role in tubular formation (Y. Kadono et al. Biochem Biophys Res Commun 1998; 251: 681–7 [13]). Oncogenic transformation of MDCK cells by erbB2 and v-src induced expression of MT1-MMP, loss of cell-cell adhesion and scattered invasion into collagen gel. mRNA differential display and Northern hybridization identified metastasis-associated mts1 as one of the genes co-induced with MT1-MMP by oncogenic transformation or collagen-gel culture of MDCK cells. Expression of antisense RNA to mts1 in MDCK cells interfered with the extension of tubules into the collagen gel, however, it did not affect the morphological changes induced by HGF in culture on plastic dishes. ErbB2-transformant transfected with mts1 antisense construct, which showed unaltered morphology in culture on plastic dishes, did not scatter into collagen gel but formed aggregates. These results suggested that Mts1 contributes not only to tumor invasion but also to kidney tubulogenesis in cooperation with MT1-MMP. The coordinated action of MT1-MMP and Mts1, which is responsible for the highly invasive properties of mesenchymal cells, may be involved in epithelial tubulogenesis and invasion of malignant carcinoma cells.     

Poorly differentiated (small cell) carcinoma of the ovary in young women: evidence supporting a germ cell origin

The clinical and pathologic features, including immunohistochemistry and electron microscopy, of six cases of poorly differentiated carcinoma of the ovary (small cell carcinoma) are presented. These tumors occurred in six young patients ranging in age from 10 to 24 years. Two patients had hypercalcemia. All tumors were unilateral, and four patients had advanced stage disease at presentation. Histologic features included sheets, nests, and cords of cells in a fibrous stroma, focal microcysts, and a dimorphic population of small and large cells. Eosinophilic, hyaline globules occurred in five cases, intercellular basement membrane-like substance in two cases, and glycogen in all cases. Five of six cases stained strongly for cytokeratin and vimentin; intracytoplasmic laminin was identified in three cases; and three cases were believed to show faint positivity for alpha-1-antitrypsin. Stains for alpha-fetoprotein were negative. Ultrastructural examination of two cases showed granular material in dilated rough endoplasmic reticulum, intermediate filaments, intracytoplasmic dense globules, maculae adherens, and extracellular basement membrane-like material. All of the cases proved rapidly fatal despite various therapies, as did a histologically similar testicular tumor that was admixed with seminoma and teratoma. We interpret these findings to indicate that this ovarian cancer is most likely of germ cell origin, and it may be related to yolk sac tumor, although it is clearly distinct from the classical yolk sac tumor.     

Determination of cell membrane resistance in cultured renal epithelioid (MDCK) cells: effects of cadmium and mercury ions

Previous studies have indicated that the cell membrane of Madin Darby Canine Kidney (MDCK) cells is hyperpolarized by a number of hormones and trace elements, in parallel with an enhancement of potassium selectivity. Without knowledge of the cell membrane resistance (R _m), however, any translation of potassium selectivity into potassium conductance remains equivocal. The present study was performed to determine the R _m of MDCK cells by cellular cable analysis. To this end, three microelectrodes were impaled into three different cells of a cell cluster; current was injected via one microelectrode and the corresponding voltage deflections measured by the other two microelectrodes. In order to extract the required specific resistances, the experimental data were analysed mathematically in terms of an electrodynamical model derived from Maxwell's equations. As a result, a mean R _m of 2.0±0.2 kcm² and an intercellular coupling resistance (R _c) of 6.1±0.8 M were obtained at a mean potential difference across the cell membrane of -47.0±0.6 mV. An increase of the extracellular K⁺ concentration from 5.4 to 20 mmol/l depolarized the cell membrane by 16.2±0.5 mV and decreased R _m by 30.6±3.0%; 1 mmol/l barium depolarized the cell membrane by 20.1±1.1 mV and increased R _m by 75.9±14.3%. Omission of extracellular bicarbonate and carbon dioxide at constant extracellular pH caused a transient hyperpolarization (up to –60.4±1.4 mV), a decrease of R _m (by 75±4.5%) and a decrease of R _c (by 23.1±8.4%). The changes in R _m and R _c were probably the result of intracellular alkalosis. Cadmium ions (1 mol/l) led to a sustained, reversible hyperpolarization (to –64.8±1.3 mV) and to a decrease of R _m (by 77.0±2.7%); mercury ions (1 mol/l) cause a sustained hyperpolarization (to –60.1±1.2 mV) and a decrease of R _m (by 76.3±3.9%). Neither manoeuvre significantly altered R _c. We have previously shown that both cadmium and mercury hyperpolarize the cell membrane potential and increase its potassium selectivity; the decrease of the R _m observed in the present study indicates that these effects are due to an increase of the potassium-selective conductance of the cell membrane.