Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity.

PURPOSE: To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. METHODS: Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. RESULTS: The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and ß4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and ß1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. CONCLUSION: The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea.

Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage, blood outgrowth endothelial cells and cells from milk teeth.

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g., for children and minors) dental pulp cells from milk teeth represent a valuable alternative.

Preservation of human placenta facilitates multicenter studies on the local immune response in normal and aberrant pregnancies.

Our standard procedure for phenotypic and functional analysis of immune cells present in the placenta is to isolate leukocytes from the decidua within five hours of the delivery. However, this results in logistical problems with deliveries at night, weekends or in other medical centers. Collecting placentas after complicated pregnancies is even more difficult owing to the low prevalence and the often unscheduled delivery. The aim was to investigate the possibility of preserving the human placenta before phenotypic and functional analysis of decidual lymphocytes. Placentas were obtained after uncomplicated pregnancy. The tissue was divided into two equal parts: decidual lymphocytes from one part were isolated within five hours according to our standard procedure, whereas the other part was preserved in either Celsior(®), a storage solution for solid organ preservation, or phosphate-buffered saline (PBS) for 24h at 4°C before isolation. Overall, the phenotype and functional capacity of decidual lymphocytes isolated within five hours was comparable to decidual lymphocytes isolated after 24-h preservation in Celsior(®) or PBS. Minor differences were found between decidual lymphocytes isolated within five hours and decidual lymphocytes isolated after 24-h preservation in Celsior(®). The results indicate that PBS is sufficient to preserve the placenta for 24h for phenotypical and functional studies. The ability to preserve the placenta will simplify the procedure for the isolation of decidual lymphocytes and makes it easier to analyze tissue from women who deliver during the night, at weekends or in other hospitals, and possibly even women with complicated pregnancies.