Reduced thrombocyte adhesion to endothelialized poly 4-methyl-1-pentene gas exchange membranes­a first step toward bioartificial lung development.

Polymeric materials used in biomedical devices, bioartificial organs, or for the fabrication of tissue engineering scaffolds should completely prevent the activation of the coagulation system and subsequent clot formation. Surface endothelialization is considered an important tool to optimize the blood compatibility of synthetic materials, as a functional endothelial cell layer on an artificial material may help control hemostasis and, therefore, provide a solution to improve the biocompatibility of these materials. Here we report on the endothelialization of poly 4-methyl-1-pentene (PMP) gas exchange membranes using human cord blood-derived late outgrowth endothelial colony forming cells. We achieved complete endothelialization of PMP membranes; and when seeded and cultivated on the membrane, cord blood-derived late outgrowth endothelial colony forming cells maintained both endothelial characteristics and functionality. Endothelialization resulted in significantly lower platelet adhesion and activation compared with unseeded membranes. Of importance, the endothelial layer had no major impact on gas permeability of PMP membranes. This study is a first promising step toward the development of a biofunctionalized surface for the use in gas exchange devices with blood contacting surfaces and a straightforward approach toward a long-term bio-hybrid lung replacement system.

Cell adhesion profiling using extracellular matrix protein microarrays.

We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.

Development of an 18S rRNA gene-targeted PCR-based diagnostic for the blue crab parasite Hematodinium sp..

The 18S rRNA gene from Hematodinum sp., a parasitic dinoflagellate that infects blue crabs, was amplified, cloned, and sequenced. The sequence showed a high similarity (95% at the nucleotide level) to sequences obtained from other dinoflagellate species, including both free-living and symbiotic species. Sequence similarity was much lower when compared with parasites of other marine invertebrates with similar life histories and with the 18S rRNA gene from the blue crab. Based on comparison of sequence alignments between Hematodinium, other dinoflagellate species, protozoan pathogens of oysters, and blue crab 18S rRNA gene sequences, 2 sets of PCR primers that specifically amplified fragments of the Hematodinium 18S rRNA gene were developed and tested. One of these primer sets (Hemat-F-1487 and Hemat-R-1654) amplified a 187 bp fragment that could be used routinely as a diagnostic test for the presence of Hematodinium in hemolymph from blue crabs. This fragment was consistently amplified from genomic DNA extracted from hemolymph of Hematodinium infected blue crabs. Comparison between the PCR technique and standard histological examination indicated that the PCR technique was reliable and provided 1000 times more sensitivity than the histological methods. The sensitivity of the PCR diagnostic was estimated to be one parasite cell among 300,000 crab hemocytes. Preliminary studies using the PCR diagnostic technique suggest that Hematodinium sp. is absent in crabs collected from waters with low salinity (5 to 10 ppt), but common in crabs from higher salinity environments in estuarine waters from southeastern Georgia (USA).