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A new field in biomedical science has been established. Cell biologists, engineers, and surgeons now work within a team. Artificial connective, epithelial, or neuronal tissues are being constructed using living cells and different kinds of biomaterials. Numerous companies and laboratories are presenting dynamic developments in this field. Prognoses predict that, at the beginning of the coming century, the industry of tissue engineering will reach the importance of the present genetic technology. An enormous demand for organ and tissue transplants motivates research activities and drives the acquisition of innovative techniques and creative solutions. At the front of this development is the creation of artificial skin for severely burned patients and the generation of artificial cartilage for implantation in articular joint diseases. Future challenges are the construction of liver organoids and the development of an artificial kidney on the basis of cultured cells. In this paper we show strategies, needs, tools, and equipment for tissue engineering. The presupposition for all projects is the induction, development, and maintenance of differentiation within the tissue under in vitro conditions. As experiments in conventional culture dishes continued to fail, new cell and tissue culture methods had to be developed. Tissues are cultured under conditions as close as possible to their natural environment. To optimize adherence or embedding, cells are grown on novel tissue carriers and on individually selected biomatrices or scaffolds. The tissues are subsequently transferred into different types of containers for permanent perfusion with fresh culture medium. This guarantees constant nutrition of the developing tissue and prevents the accumulation of harmful metabolites. An organo-typical environment for epithelial cells, for example, is obtained in gradient containers, which are permanently superfused at the apical and basal sides with different media. Long term experiments result in cultured tissues in a quality thus far unreached.

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Replacement of injured or diseased skeletal tissues by either autograft or allograft cartilage has increased steadily during recent decades. The ideal method is to use autologous cartilage; however, this is extremely limited due to the scarcity of donor sites. We present a new approach to the in vitro formation of cartilage grafts for autologous grafting in reconstructive surgery. Bioresorbable polymer fleeces of polylactic acid were used as temporary cell carrier matrices to establish three-dimensional cultures of human chondrocytes. The polymer surface was coated with poly-L-lysine before cell integration. These cell-polymer tissue constructs were encapsulated with low melting point agarose and then placed in perfusion culture chambers to provide a constant supply of nutrients into the cultures. The culture medium consisted of Ham's F12 supplemented with 2% fetal calf serum and 50 micrograms/ml ascorbic acid. The cell-polymer tissues were harvested and frozen for toloudine and alcian blue staining as well as electron microscopic examination after different periods of time in culture. A monoclonal antibody specific for collagen type II was used to characterize the cell phenotype. With this culture procedure chondrocytes maintained a differentiated phenotype with synthesis of collagen and proteoglycan. Collagen fibrils with clear cross-striation were evident in electron microscopic images. The results show that our organotypic cell culture method allows the in vitro production of bioartificial cartilage for transplantation.

The efficiency of cell or tissue cultures is usually judged by how quickly confluence is reached within a Petri dish or on a scaffold. Growth factors and fetal bovine serum are employed to drive cultured cells from one mitosis to the next as quickly as possible. The tissue specific interphase is extremely short under these conditions, so that the degree of differentiation desired in tissue engineering cannot be achieved. To reach the goal of functional differentiation in vitro mitosis and interphase must be separated experimentally and tailored to the specific requirements of the cell-type used. This could be achieved by a three step concept for tissue-engineering in vitro as we present here. The expansion phase is followed by a phase in which tissue differentiation is initiated. The final phase serves to express and maintain histotypical differentiation of the generated tissue.

Aldosterone enhances synthesis and enzyme activity of Na/K-ATPase and has been found to stimulate sodium reabsorption by the renal cortical collecting duct. Moreover, chronic exposure to aldosterone is associated with a remarkable morphological-functional adaptation. This is seen as a magnification in basolateral membrane area of principal cells. In the present paper we investigated the acquistion of Na/K-ATPase alpha-subunit in renal tissue and examined whether aldosterone initiates functional and adaptive changes in cultured collecting duct cells similar to those observed in vivo. Using a monoclonal antibody, immunofluorescence microscopy demonstrated the acquisition of the alpha-subunit of Na/K-ATPase in the developing cortical collecting ducts of the kidney of neonatal rabbits. The mature collecting ducts in the medulla and papilla of the developing kidney were strongly labelled at the basolateral side, while in the cortical portion of the fetal collecting duct adjacent to the embryonic ampullae the immunolabel was found at the apical and the basolateral aspect of the epithelium. However, the embryonic collecting duct ampullae in the outer cortex did not show any reaction with the antibody. In the collecting duct cells cultured for 24 hours the alpha-subunit of Na/K-ATPase was found to be distributed at both the apical and basolateral aspect of the epithelium. After two to 16 days, the immunolabel was strictly found distributed at the basolateral side. Culturing collecting duct cells in the presence of aldosterone (10(-6) M), the hormone modulated the cellular shape of epithelia after five days by infolding the lateral plasma membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Schie␤l IM, Rosenauer A, Kattler V, Minuth WW, Oppermann M, Castrop H. Dietary salt intake modulates differential splicing of the Na-K-2Cl cotransporter NKCC2. Am J Physiol Renal Physiol 305: F1139 -F1148, 2013. First published August 14, 2013 doi:10.1152/ajprenal.00259.2013.-Both sodium reabsorption in the thick ascending limb of the loop of Henle (TAL) and macula densa salt sensing crucially depend on the function of the Na/K/2Cl cotransporter NKCC2. The NKCC2 gene gives rise to at least three different full-length NKCC2 isoforms derived from differential splicing. In the present study, we addressed the influence of dietary salt intake on the differential splicing of NKCC2. Mice were subjected to diets with low-salt, standard salt, and high-salt content for 7 days, and NKCC2 isoform mRNA abundance was determined. With decreasing salt intake, we found a reduced abundance of the low-affinity isoform NKCC2A and an increase in the high-affinity isoform NKCC2B in the renal cortex and the outer stripe of the outer medulla. This shift from NKCC2A to NKCC2B during a low-salt diet could be mimicked by furosemide in vivo and in cultured kidney slices. Furthermore, the changes in NKCC2 isoform abundance during a salt-restricted diet were partly mediated by the actions of angiotensin II on AT 1 receptors, as determined using chronic angiotensin II infusion. In contrast to changes in oral salt intake, water restriction (48 h) and water loading (8% sucrose solution) increased and suppressed the expression of all NKCC2 isoforms, without changing the distribution pattern of the single isoforms. In summary, the differential splicing of NKCC2 pre-mRNA is modulated by dietary salt intake, which may be mediated by changes in intracellular ion composition. Differential splicing of NKCC2 appears to contribute to the adaptive capacity of the kidney to cope with changes in reabsorptive needs. differential spacing; NKCC2; salt transport THE THICK ASCENDING LIMB OF the loop of Henle (TAL) contributes to 25-30% of total renal Na ϩ reabsorption. Apical Na uptake in cells of the TAL is primarily mediated by the bumetanide-sensitive Na-K-2Cl cotransporter NKCC2 (5). In addition to its function in TAL salt retrieval, the NKCC2-dependent transport activity of macula densa cells constitutes the initial step in the tubulovascular signaling pathways between the TAL in the juxtaglomerular region and the afferent arteriole (20,33). By detecting changes in luminal NaCl concentration, macula densa cells modulate the tone of the afferent arteriole and subsequently control the single-nephron glomerular filtration rate. This negative feedback loop is known as tubuloglomerular feedback (TGF; Ref. 38). Macula densa cells also control the secretion of renin from granular cells of the afferent arteriole (4).NKCC2 is encoded by the gene Slc12a1 (8, 16). In humans and all other examined mammalian species, differential splicing of Slc12a1 gives rise to at least three different full-length isoforms of NKCC2, known as NKCC2B, NKCC2A, and NKCC2F (3,16,28,40). ...