We hypothesise that, using the 3-D perfused culture system, a good representation of the original leukaemic bone marrow architecture will be reproduced ex vivo and that a leukaemic cell culture will be maintained with its original phenotypic features, independent of exogenous cytokines. It is anticipated that this 3-D culture system, once established, will be used to study and test models of leukaemogenesis. In addition, it could be used to develop and test novel strategies (chemotherapeutic, immunologic, and targeted) in treatment of the disease, possibly replacing some of the current animal models in use.
We have developed three-dimensional culture systems based on the hypothesis that growth in 3-D replicates the in vivo marrow environment more faithfully. The scaffolds used for bone marrow tissue engineering applications are either naturally derived materials, such as macroporous collagen carriers, or synthetic polymers that permit the creation of complex architectures and shapes. Scaffolds were prepared by thermally induced solid-liquid phase separation from poly (D,L-lactide) (PDLLA), poly(D,L-lactide-co-glycolide) (PLGA, 53:47), polystyrene (PS), poly(methyl methacrylate) (PMMA), polycaprolactone (PCL), and polyurethane (PU). These scaffolds were selected because they are bio-inert, biocompatible, and FDA approved. The scaffolds have been surface characterised using zeta-potential (z) and contact angle measurements in order to assess their wettability and surface chemistry. Biomaterial surface properties such as morphology, hydrophilicity, surface charge and energy are critical parameters for consideration in designing biomimetic scaffolds. Surface modification confers favourable properties that assist in overcoming surface limitations and support cell adhesion, differentiation, and ECM protein production. The chemical composition of the samples has been identified by Fourier Transform Infra-Red spectroscopy. Scanning electron microscopy and gas adsorption technique will also be utilised to observe the physical properties of the scaffolds essential for cell attachment, such as internal pore morphology and surface area. Surface modification using plasma treatment in a radio-frequency discharge of anhydrous ammonia or air are being used to etch, cross-link, or activate the polymer surfaces, thus modifying their surface chemistry within the pores of the scaffolds. In particular, surface coating with ECM components, such as human plasma fibronectin and collagen, is being investigated.
Leukaemic cell lines of different subtypes, specifically HL-60, K562 and Kasumi-6, were used to initially establish the 3-D cultures. In addition to assessing morphology, cell number and kinetics of the cells in the non-adherent portion of the cultures, surface phenotyping using flow cytometry and molecular expression of genes (such as early and late expression genes including Wnt, ƒ"-catenin, and PU.1) will be assessed during the culture period and compared with that of cells cultivated in 2-D flask cultures. Immunocytochemistry in order to evaluate ECM proteins such as collagen and fibronectin, vasculature (if it develops), cell line-specific surface markers, and integrins will also be done so that a virtual model of the leukaemic cell growth within the bioreactor cultures will be generated using the 3-D reconstruction feature of the image analysis software. The 3-D cultures of the leukaemic cell lines will be utilised to determine the optimal culture parameters of the perfused bioreactors, such as perfusion rate, feeding schedule, oxygenation, and control of pH.
Therefore it is anticipated that this 3-D culture system with primary leukemic cells, once established, will be used to study and test models of leukaemogenesis. In addition, it could be used to develop and test novel strategies (chemotherapeutic, immunologic, and targeted) in treatment of the disease, possibly replacing some of the current animal models in use.
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