Abstract
Stem/progenitor cells are promising candidates for the regeneration of parenchyma in acute and chronic renal failure. However, recent data exhibit that survival of stem/progenitor cells after implantation in diseased renal parenchyma is restricted. To elaborate basic parameters improving survival, cell seeding was simulated under advanced in vitro conditions. After isolation, renal stem/progenitor cells were mounted in a polyester interstitium for perfusion culture. During generation of tubules, chemically defined CO2 Independent Medium or Leibovitz’s L-15 Medium was applied. Specimens were then fixed for transmission electron microscopy to analyze morphological features in generated tubules. Fixation in conventional glutaraldehyde (GA) solution shows development of tubules each exhibiting a polarized epithelium, an intact basal lamina and an inconspicuous interstitium. In contrast, special fixation of specimens in GA solution containing cupromeronic blue, ruthenium red or tannic acid unveils previously not visible extracellular matrix. Control experiments elucidate that a comparable extracellular matrix is not present in the interstitium of the matured kidney. Thus, generation of renal tubules in combination with advanced fixation of specimens for electron microscopy demonstrates that development of abnormal features in the newly developed interstitium has to be considered, when repair of renal parenchyma is performed by implantation of stem/progenitor cells.
Highlights
In the last few years, numerous investigations were performed dealing with an implantation of stem/progenitor cells for the repair of diseased renal parenchyma [1,2,3]
Generation of renal tubules in combination with advanced fixation of specimens for electron microscopy demonstrates that development of abnormal features in the newly developed interstitium has to be considered, when repair of renal parenchyma is performed by implantation of stem/progenitor cells
After 13 days of perfusion culture, the artificial interstitium was opened by tearing off the fleece layers so that the threedimensional pattern of generated tubules could be analyzed by confocal fluorescence microscopy (Figure 1)
Summary
In the last few years, numerous investigations were performed dealing with an implantation of stem/progenitor cells for the repair of diseased renal parenchyma [1,2,3]. One of the crucial points is the period between a performed implantation and the initial repair within diseased parenchyma During this phase of seeding, the promoting atmosphere for stem/progenitor cells co-implanted with a culture medium is replacing against harmful interstitial fluid of surrounding renal parenchyma [7,8]. The actual data show that fixation of specimens in glutaraldehyde solution containing cupromeronic blue, ruthenium red or tannic acid for transmission electron microscopy unmasks previously not visible structures [13] Based on these observations, for the first time causes for abnormal extracellular matrix in the interstitium of generated tubules can be systematically investigated
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