A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver.

"Upcyte (R) hUman hepatocytes" have the unique property of combining proliferation with the expression of drug metabolising activities. In our current study, we evaluated whether these cells would be suitable for early in vitro micronucleus (MN) tests. A treatment period of 96h without a recovery period was most reliable for detecting MN formation in upcyte (R) hepatocytes from Donor 740. The basal MN rate in upcyte (R) hepatocytes varied considerably between donors (7-28%); therefore, modifications to the assay medium were tested to determine whether they could decrease inherent MN formation. Optimal medium supplements were 10 ng/ml oncostatin M for the pre-culture and recovery periods and 25 ng/ml epidermal growth factor and 10 ng/ml oncostatin M for the treatment period. Using the optimised conditions and outcome criteria, the upcyte (R) hepatocyte MN assay could correctly identify directly acting (e.g. mitomycin C, etoposide) and metabolically activated genotoxins (e.g. benzo[a]pyrene, cyclophosphamide). "True negative" and "false positive" compounds were also correctly identified as negative. The basal %MN in upcyte (R) hepatocytes from Donor 740 treated with DMSO, cyclophosphamide or MMC, was essentially unaffected by the growth stage ranging from population doublings of 14-61, suggesting that billions of cells could be produced from a single donor for standardised drug toxicity testing.In conclusion, we have established and optimised an in vitro MN test by using upcyte (R) hepatocytes to correctly identify known direct and metabolically activated genotoxicants as well as "false positives" and true negative compounds. The almost unlimited supply of cells from a single donor and optimised test conditions increase reproducibility in early and more predictive in vitro MN tests. (C) 2013 Elsevier B.V. All rights reserved.

We have developed a new technology to allow the proliferation of differentiated primary cells without inducing permanent immortalization, uncontrolled cell growth, or loss of phenotype. The Upcyte® (“upregulated”) technology involves a viral gene transfer system to introduce a unique combination of genes that induce and maintain cell proliferation until the cells reach confluence. This allows the primary cells to be passaged many times with the generation of billions of cells. Upcyte® technology can be applied to different cell types such as hepatocytes. Here, we summarise some of the comparisons we have made between primary hepatocytes and the upcyte® hepatocytes derived thereof. Metabolic enzymes: CYP activities e.g. CYP3A4, CYP2C8 and CYP2B6, are expressed in significant abundance and, moreover, can be induced by prototypical inducers. For example, rifampicin mediated CYP3A4 induction responses of upcyte® hepatocytes from over 5 donors by now were in line with the FDA guideline pass criteria. Furthermore induction potential was stable over the course of expansion. Application of upcyte® cells as optimal alternatives to current in vitro screening assays: Upcyte® technology allows for the mass production of primary cells from different organs and donors for use in multiple in vitro ADME-Tox assays. The flexibility of the application of upcyte® cells to different cellular-based assays, together their abundant availability from different donors for routine testing means models are now available with sustained quality and sufficient quantities to allow for reproducible and reliable in vitro ADMET studies. Since upcyte® technology allows for the generation of hepatocytes with differentiated function, they offer a unique advantage of combining the phenotype of primary hepatocytes with the virtually unlimited availability and ease of handling human tumour cells and cell lines.

Major disadvantages of using human primary hepatocytes are their lack of proliferation and dedifferentiation in culture. Therefore, proliferating hepatocytes in combination with sophisticated 3D-culture systems capable of sustaining long-term functional cultures are highly desirable.Medicyte has developed a new technology that drives differentiated primary cells into proliferation in vitro, without uncontrolled cell growth or loss of phenotype. Upcyte® technology is based on targeted genetic modification of human primary cells allowing multiple passaging and the generation of billions of quasi primary cells.