Culture of embryonic renal collecting duct epithelia in a gradient container

Abstract

During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.

Modulation of Cell Differentiation in Perfusion Culture

“…Under these conditions the tissue receives constant nutrition, harmful metabolic products do not accumulate and paracrine factors remain at a physiological level. Previous experiments have shown that embryonic CD epithelia can be cultured in a container for weeks and months exhibiting a morphology comparable to the situation within the maturing kidney [6, 7, 20]. …”

“…Tissue culture experiments with embryonic CD epithelia revealed that the development of individual CD cell features can be triggered by aldosterone [19], amiloride [20]and the extracellular electrolyte environment [21, 22]. Under static culture conditions in medium containing serum, aldosterone is known to generate a P cell-like epithelium with an amiloride-sensitive Na + transport and tight characteristics [23].…”

Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system

“…Application of a gradient perfusion culture container made it further possible to investigate the influence on differentiation of different fluid composition at the luminal and basal sides of embryonic renal collecting duct (CD) epithelia (Minuth et al 1992b(Minuth et al , 1997a(Minuth et al , b, 1999(Minuth et al , 2001(Minuth et al , 2005aSteiner et al 1997, Schumacher et al 2002aMinuth et al 2009a). In the course of performed experiments it was detected that development of a CD epithelium starts with an unexpected long latent period of three days and needs at least 10 days for up-regulation of typical signs of differentiation.…”

Embryonic Renal Collecting Duct Cell Differentiation Is Influenced in a Concentration-Dependent Manner by the Electrolyte Environment

“…1A) were transferred to a gradient perfusion culture container ( fig. 1B; Minucells and Minutissue) to create optimized culture conditions [16]. Fresh medium was continuously superfused on the basal and luminal sides of the epithelia for the following culture period of 13 days at a rate of 1 ml/h with an IPC N8 peristaltic pump (Ismatec, Wertheim, Germany; fig.…”

“…Epithelia cultured in media with a low Na content showed a completely different developmental pattern as compared with epithelia cultured in media with a high Na content. Evidence for a definitive electrolyte-sensitive reaction during CD development was finally obtained by culturing the embryonic epithelia in the presence of aldosterone and under serum-free conditions in a gradient container [16]. During a culture period of 14 days, epithelia cultured with the same medium on luminal and basal sides develop completely different features as compared with epithelia cultured with different media on both sides [8,11].…”