Supportive development of functional tissues for biomedical research using the MINUSHEET(R) perfusion system

Functional tissues generated under in vitro conditions are urgently needed in biomedical research. However, the engineering of tissues is rather difficult, since their development is influenced by numerous parameters. In consequence, a versatile culture system was developed to respond the unmet needs.Optimal adhesion for cells in this system is reached by the selection of individual biomaterials. To protect cells during handling and culture, the biomaterial is mounted onto a MINUSHEET(R) tissue carrier. While adherence of cells takes place in the static environment of a 24 well culture plate, generation of tissues is accomplished in one of several available perfusion culture containers. In the basic version a continuous flow of always fresh culture medium is provided to the developing tissue. In a gradient perfusion culture container epithelia are exposed to different fluids at the luminal and basal sides. Another special container with a transparent lid and base enables microscopic visualization of ongoing tissue development. A further container exhibits a flexible silicone lid to apply force onto the developing tissue thereby mimicking mechanical load that is required for developing connective and muscular tissue. Finally, stem/progenitor cells are kept at the interface of an artificial polyester interstitium within a perfusion culture container offering for example an optimal environment for the spatial development of renal tubules.The system presented here was evaluated by various research groups. As a result a variety of publications including most interesting applications were published. In the present paper these data were reviewed and analyzed. All of the results point out that the cell biological profile of engineered tissues can be strongly improved, when the introduced perfusion culture technique is applied in combination with specific biomaterials supporting primary adhesion of cells

Atypical Features in Regenerating Tubules Point to a Risk for Implantation of Renal Stem/Progenitor Cells

Stem/progenitor cells are seen as a therapeutic option for repair of diseased renal parenchyma. However, actual data show that survival of implanted stem/progenitor cells is impeded by harmful interstitial environment. To learn about parameters for cell adaptation, renal stem/progenitor cells were mounted in a polyester (POSI-4) interstitium during perfusion culture. Controlled fluid environment was maintained by chemically defined CO2 - independent culture media for 13 days. Cell biological features were then analyzed by immunohistochemistry, while structural details were investigated by advanced fixation of specimens for microscopy. When stem/progenitor cells are kept in Leibovitz’s L-15 Medium or CO2 Independent Medium, spatial development of numerous tubules is observed. Immunolabel for TROMAIII, cingulin and laminin ɣ1 depicts that a homogenous cell population is contained. Semithin sections of specimens fixed in traditional glutaraldehyde (GA) solution reflect an unobtrusive morphology. In contrast, fixation by GA solution including ruthenium red unveils in tubules a thickened basal lamina. Fixation by GA solution including tannic acid illustrates atypical development of a heterogeneous epithelium consisting of bright and dark cells. Thus, advanced fixation of specimens makes pathological features visible, when regeneration is investigated by renal stem/ progenitor cells. To what extent a comparable risk lurks behind an implantation, has to be elaborated

Search for Chemically Defined Culture Medium to Assist Initial Regeneration of Diseased Renal Parenchyma After Stem/Progenitor Cell Implantation

Before an intended implantation stem/progenitor cells are usually kept in the beneficial atmosphere of a selected culture medium. However, after implantation the situation is drastically changing for them. Yet stem/progenitor cells must stand the harmful fluid environment within a diseased organ. In this coherence it is unknown, to which degree alterations in molecular composition of interstitial fluid can influence the initial regeneration of parenchyma. To obtain first insights in the sensitivity against changes in fluid composition, renal stem/progenitor cells were mounted within a polyester interstitium for perfusion culture. To model interstitial fluids different chemically defined culture media all including aldosterone were administered continuously for 13 days. Then morphological quality of generated tubules was registered by light and transmission electron microscopy. Culture of stem/progenitor cells in earlier approved Iscove´s Modified Dulbecco´s Medium served as internal standard. These experiments revealed generation of numerous tubules. In comparison, application of Williams’ E Medium, Basal Medium Eagle, McCoy’s 5A Modified Medium and Medium 199 produced only a lean quality of generated tubules, since contained cells exhibited numerous vacuoles. In contrast, administration of Leibovitz’s L-15 Medium and CO2 Independent Medium showed unexpected promoting effects on development of tubules. In this series numerous and intact tubules without formation of an excess of vacuoles were detected. In consequence, Leibovitz’s L-15 Medium and CO2 Independent Medium appear as challenging candidates to be tested in future for implantation in combination with stem/progenitor cells