Interstitial interfaces show marked differences in regenerating tubules, matured tubules, and the renal stem/progenitor cell niche

Denk²

2013

Transplant Technol

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Stem/progenitor cells are in the focus of research as a therapeutic perspective to cure acute and chronic renal failure. Following this innovative strategy one has to consider that stem/progenitor cells are first kept in the beneficial atmosphere of a CO 2 -dependent culture medium, while after implantation they are exposed to unbalanced interstitial fluid of diseased parenchyma. However, this abrupt transition has consequences, since it limits survival of implanted stem/progenitor cells. To offer a constant environment during isolation, culture, surgical processing and initial seeding within diseased parenchyma, an improved concept is under current work. In present experiments stabilization of fluid environment was investigated by isolating renal stem/progenitor cells in chemically defined and CO 2 -stabilized culture media. Interstitial influences on spatial development of tubules were tested by mounting stem/progenitor cells in a polyester fleece as a substitute for extracellular matrix and by transporting always fresh medium in perfusion culture for 13 days. Finally, morphological quality of raised constructs was analyzed by histochemistry, light and transmission electron microscopy. The present results demonstrate that three different culture media can be short-listed for protecting stem/progenitor cells during the process of implantation. Earlier approved Iscove´s Modified Dulbecco´s Medium buffered by HEPES revealed formation of numerous tubules. Also application of Leibovitz's L-15 Medium and CO 2 Independent Medium demonstrated unexpected promoting effects on the generation of tubules. In all series typical features of transporting tubule cells were registered. Formation of an excess of vacuoles as an indicator for cytotoxicity was not observed. In conclusion, although very different in chemical composition the tested culture media reflect an advantageous microenvironment for isolation, implantation and development of stem/ progenitor cells.

Section: Fluorescence Microscopy On Generated Tubulesmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Isolation Of Renal Stem/progenitor Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Initial steps to stabilize the microenvironment for implantation of stem/progenitor cells in diseased renal parenchyma

Denk²

2013

Transplant Technol

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“…Kidneys from one-day old New Zealand rabbits (Seidl, Oberndorf, Germany) were isolated under sterile conditions and cut into a ventral and dorsal half as earlier described [14]. Then the fibrous organ capsule was stripped off by fine forceps to obtain a constantly thin layer of stem/progenitor cell niches adherent to the explant.…”

Section: Methodsmentioning

confidence: 99%

Tannic acid label indicates abnormal cell development coinciding with regeneration of renal tubules

Denk

2014

BMC Clin Pathol

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BackgroundStem/progenitor cells are in the focus of research as a future therapeutic option to stimulate regeneration in diseased renal parenchyma. However, current data indicate that successful seeding of implanted stem/progenitor cells is prevented by harmful interstitial fluid and altered extracellular matrix. To find out possible parameters for cell adaptation, the present investigation was performed.MethodsRenal stem/progenitor cells were mounted in an artificial interstitium for perfusion culture. Exposure to chemically defined but CO2-independent culture media was tested during 13 days. Cell biological features were then analyzed by histochemistry, while structural details were investigated by transmission electron microscopy after conventional and improved fixation of specimens.ResultsCulture of renal stem/progenitor cells as well in Leibovitz’s L-15 Medium as CO2 Independent Medium shows in fluorescence microscopy spatial development of numerous tubules. Specimens of both media fixed by conventional glutaraldehyde exhibit in electron microscopy a homogeneous cell population in developed tubules. In contrast, fixation by glutaraldehyde including tannic acid illuminates that dispersed dark marked cells of unknown function are present. The screening further demonstrates that the dark cell type does not comply with cells found in embryonic, maturing or matured renal parenchyma.ConclusionsThe actual data show that development of abnormal cell features must be taken into account, when regeneration of renal tubules is simulated under in vitro conditions.

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

Denk

2015

Cytotechnology

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To meet specific requirements of developing tissues urgently needed in tissue engineering, biomaterial research and drug toxicity testing, a versatile perfusion culture system was developed. First an individual biomaterial is selected and then mounted in a MINUSHEET Ò tissue carrier. After sterilization the assembly is transferred by fine forceps to a 24 well culture plate for seeding cells or mounting tissue on it. To support spatial (3D) development a carrier can be placed in various types of perfusion culture containers. In the basic version a constant flow of culture medium provides contained tissue with always fresh nutrition and respiratory gas. For example, epithelia can be transferred to a gradient container, where they are exposed to different fluids at the luminal and basal side. To observe development of tissue under the microscope, in a different type of container a transparent lid and base are integrated. Finally, stem/progenitor cells are incubated in a container filled by an artificial interstitium to support spatial development. In the past years the described system was applied in numerous own and external investigations. To present an actual overview of resulting experimental data, the present paper was written.