Institute of Fundamental Technological Research

The primary hepatocytes culture is still one of the main challenges in toxicology studies in the drug discovery process, development of in vitro models to study liver function, and cell-based therapies. Isolated hepatocytes display a rapid decline in viability and liver-specific functions including albumin production, conversion of ammonia to urea, and activity of the drug metabolizing enzymes. A number of methods have been developed in order to maintain hepatocytes in their highly differentiated state in vitro. Optimization of culture conditions includes a variety of media formulations and supplements, growth surface coating with the components of extracellular matrix or with synthetic polymers, three-dimensional growth scaffolds and decellularized tissues, and coculture with other cell types required for the normal cell-cell interactions. Here we propose a new substratum for hepatic cells made by drying confluent human skin fibroblasts' culture. This growth surface coating, prepared using maximally simplified procedure, combines the advantages of the use of extracellular matrices and growth factors/cytokines secreted by the feeder layer cells. In comparison to the hepatoma cells grown on a regular tissue culture plastic, cells cultured on the dried fibroblasts were able to synthesize albumin in larger quantities and to form greater number of apical vacuoles. Unlike the coculture with the living feeder layer cells, the number of cells grown on the new substratum was not reduced after fourteen days of culture. This fact could make the dried fibroblasts coating an ideal candidate for the substrate for non-dividing human hepatocytes.

cocultures, culture substratum, dried fibroblasts, human skin fibroblasts, C3A cells

Albumin is rarely used for electrospinning because it does not form fibres in its native globular form. This paper presents a novel method for electrospinning human albumin from a solution containing pharmaceutical grade protein and 25% polyethylene oxide (PEO) used as the fibre-forming agent. After spontaneous cross-linking at body temperature, with no further chemicals added, the fibres become insoluble and the excess PEO can be washed out. Albumin deposited along the fibres retains its native characteristics, such as its non-adhesiveness to cells and its susceptibility for degradation by macrophages. To demonstrate this we evaluated the mechanical properties, biocompatibility and biodegradability of this novel product. After subcutaneous implantation in mice, albumin mats were completely resorbable within six days and elicited only a limited local inflammatory response. In vitro, the mats suppressed cell attachment and migration. As this product is inexpensive, produced from human pharmaceutical grade albumin without chemical modifications, retains its native protein properties and fulfils the specific requirements for anti-adhesive dressings, its clinical use can be expedited. We believe that it could specifically be used when treating paediatric patients with epidermolysis bullosa, in whom non-healing wounds occur after minor hand injuries which lead to rapid adhesions and devastating contractures.

albumin, nanofibers, wound dressing, biocompatibility, bioresorption

electrospinning, microfibers, nanofibers, in vivo, animal model

We used the principles of electrospinning to produce materials for applications in regenerative medicine of urinary bladder wall, ureter, wound dressing and potential applications in cancer therapy. Our research is based on biodegradable polymers produced by ring-opening polymerization. Scaffolds of poly(L-lactide-co-caprolactone) (PLCL) gradually degrade leaving no artificial material behind to be replaced by natural extracellular collagen matrix. We formed flat membranes of micro- and nanofibers to carry out regeneration of urinary bladder wall as animal model of cancer treatment. Grafts were tested for biocompatibility and aimed for guided cell growth, yet we were unsuccessful in mechanical compliance of nanomaterial and reconstructed tissue. We tested tubular scaffolds made of nanofibers aimed for ureter tissue engineering. We found stem cells seeding unnecessary. The results of nanomaterial implantation on animal model were better than for collagen matrices. Animal model was also tested for use of nanofibers of human serum albumin as wound dressing. The native structure of the protein was retained to maintain its anti-adhesive properties, despite poor mechanical characteristics. Nanomaterial caused no inflammation and was resorbed during 16 days. Last application of presented materials was targeted drug delivery system made of PLCL nanofibers. Release of anticancer drug complexed with nanoparticles is to be triggered by tumor cells. Such nanomaterial is potential drug delivery system. Acknowledgements: The authors wishes to thank for the cooperation: T. Chmielewski, P. Nakielski, K. Zembrzycki, G. Mikulowski and prof. T. A. Kowalewski from IPPT PAN. The project was partially supported by the National Centre for Research and Development. Grant No. STRATEGMED1/235368/8/NCBR/2014.

electrospinning, nanofibers, regenerative medicine, wound dressing, urology, biodegradable polymers, animal model.