Quality control of the biopolymer and cytotoxicity testing
Three different batches (a-c) of biocompatible alginate isolated from freshly collected Laminaria pallida were tested in eight replicates, using three different cell lines (mouse, ape, human): L-929 (DSMZ # ACC 2), VERO-B4 (DSMZ ACC # 33) and NHDF (CellSystems, St. Katharinen, Germany). Experiments were repeated five times.
For cytotoxicity tests 100 ml of cell suspensions were plated at a density of 2×105 cells/ml in 96-well plates. Cells were incubated overnight in an incubator at 37°C in 5% CO2/95% air humidified atmosphere. A 0.1% (w/v) suspension of the biopolymer was prepared in cell culture medium (RPMI 1640, supplemented with 2 mM L-glutamine and 10% fetal calf serum). Culture medium was used as negative control and CuSO4 ( 5 0 ng/ml) in culture medium was used as positive control. Biopolymer suspension and control media were mixed overnight on an overhead shaker at room temperature. Cultured cells were checked for confluency and morphology after 24-hr incubation, when cell culture medium was removed and replaced by 100 mL of the biopolymer suspension. Positive and negative controls were tested in every dish. Cells were incubated for three days in the incubator. At day three 25 mL of Alamar-Blue solution (Biozol, Echingen, Germany) were added to each well and incubated. Fluorescence (excitation wavelength 535 nm, emission wavelength 590 nm) was measured after three hours. Metabolic activity of the cells was calculated versus baseline (negative controls) and expressed as percent difference.
Culture and encapsulation of human parathyroid tissue
Fresh human parathyroid tissue was obtained from six different patients undergoing surgery for parathyroid hyperplasia due to secondary hyperparathyroidism. Parathyroid tissue was transported to the laboratory under sterile conditions and then cut into fragments of about 2 mm3. Tissue particles were then kept either in RPMI 1640 medium supplemented with 2 mM L-gluta- mine and 10% AB-serum or in serum-free culture medium in the incubator for three days with daily changes of culture medium. Thereafter, tissue particles were microencapsulated using the CellBeads® Biopolymer, produced according to GMP requirements (CellMed AG, Alzenau, Germany), through suspension in the biopolymer solution. The solution was passed through a spray nozzle and the resulting microcapsules were transferred into culture medium and further cultured for 10 days. Buy generic viagra online to cure impotence
In vitro release of PTH from human encapsulated parathyroid tissue
Intact PTH release was daily measured during a period varying from seven to eight days. For the measurement of PTH release five microcapsules were transferred into 5 mL of fresh serum- free medium and incubated for two hours, when 500 ml of the supernatant were removed and immediately frozen at -20°C. After thawing human PTH 1-84 concentrations were measured by an ELISA method (DSL, Webster, USA) and the median PTH release of one capsule was derived.
At day seven encapsulated tissue particles were fixed in 4% formalin, Hematoxylin-Eosin (H&E) stained and histologically analyzed.