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

Dose-controlled exposure of A549 epithelial cells at the air-liquid interface to airborne ultrafine carbonaceous particles.

The geometry of commercially available perfusion chambers designed for harbouring three membrane-based cell cultures was modified for reliable and dose-controlled air–liquid interface (ALI) exposures. Confluent A549 epithelial cells grown on membranes were integrated in the chamber system and supplied with medium from the chamber bottom. Cell viability was not impaired by the conditions of ALI exposure without particles. Expression of the inflammatory cytokines interleukin 6 and interleukin 8 by A549 cells during ALI exposure to filtered air for 6 h and subsequent stimulation with tumor necrosis factor was not altered compared to submersed controls, indicating that the cells maintained their functional integrity. Ultrafine carbonaceous model particles with a count median mobility diameter of about 95 ± 5 nm were produced by spark discharge at a stable concentration of about 2 × 106 cm−3 and continuously monitored for accurate determination of the exposure dose. Delivery to the ALI exposure system yielded a homogeneous particle deposition over the membranes with a deposition efficiency of 2%. Mid dose exposure of A549 cells to this aerosol for 6 h yielded a total particle deposition of (2.6 ± 0.4) × 108 cm−2 corresponding to (87 ± 23) ng cm−2. The 2.7-fold (p ⩽ 0.05) increased transcription of heme oxygenase-1 indicated a sensitive antioxidant and stress response, while cell viability did not reveal a toxic mechanism

Production of a human calcitonin precursor with Staphylococcus carnosus : secretory expression and single-step recovery by expanded bed adsorption

Recombinant human calcitonin (hCT) can be expressed by Staphylococcus carnosus as a precursor peptide being part of a secreted fusion protein which contains the propeptide of a Staphylococcus hyicus lipase as secretion carrier and multiple repeats of the hCT precursor. Fusion proteins containing two (PhCT2), four (PhCT4), or eight (PhCT8) hCT precursors were expressed. Due to the unfavourable biochemical properties of PhCT4 and PhCT8, PhCT2 was the most efficient fusion protein for a production process. Expression from a xylose-inducible promoter from Staphylococcus xylosus resulted in a maximum secretory production of PhCT2, since the amount of PhCT2 synthesized in total corresponded almost precisely to the upper limit of the capacity of the cellular protein secretion apparatus. An expanded bed adsorption procedure for the recovery of PhCT2 from unclarified culture broth was established using the cation exchanger STREAMLINE SP at pH 5. This integrated operation allows a single-step recovery of PhCT2 and was successfully transferred to a pilot scale (100 1 culture broth). (C) 2003 Elsevier Science Ltd. All rights reserved