Development of an Ex Vivo Three Dimensional (3-D) Model of Acute Myeloid Leukaemia (AML)

Acute Myeloid Leukaemia (AML) is a cancer of hematopoietic stem cells that develops in the three-dimensional (3-D) niches provided by the bone marrow microenvironment in vivo. The study of AML has been hampered by the lack of appropriate ex vivo models, which can mimic this microenvironment. It was hypothesised that the fabrication of scaffolds for the biomimetic growth of leukemic cells ex vivo could facilitate the study of the disease in its native 3-D niche. The growth of different leukemic cell lines was first evaluated, namely K- 562, HL-60 and Kasumi-6 on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials: poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate) (PMMA), poly (D, L-lactade) (PDLLA), poly (caprolactone) (PCL), and polystyrene (PS). These results were compared with two commercially available scaffolds from BD™ Biosciences. Overall, out of all the scaffolds, PLGA and PU displayed the best seeding efficiency and leukemic cellular growth, assessed by MTS assay, scanning electron microscopy and immunohistochemistry. In order to improve the ex vivo 3-D leukemic cell culture, PLGA and PU scaffolds were coated with bone marrow extracellular matrix (ECM) proteins, collagen (62.5 or 125 μg/ml) and fibronectin (25 or 50 μg/ml) and a combination of both proteins: collagen + fibronectin (62.5 + 25 μg/ml) respectively. Once the abnormal hematopoietic 3-D model was established, a new model to culture normal hematopoietic cord blood mononuclear cells was studied and compared. All 3 leukemic cell lines and cord blood cells grew better in PU scaffolds coated with collagen type I using the low concentration and sustained growth in the absence of exogenous cytokines. As a result, it was concluded that PU-collagen scaffold could provide a practical model with which to study the biology and treatment of primary AML in an ex vivo mimicry without the use of animal models

Altering crystal growth and annealing in ice-templated scaffolds.

The potential applications of ice-templating porous materials are constantly expanding, especially as scaffolds for tissue engineering. Ice-templating, a process utilizing ice nucleation and growth within an aqueous solution, consists of a cooling stage (before ice nucleation) and a freezing stage (during ice formation). While heat release during cooling can change scaffold isotropy, the freezing stage, where ice crystals grow and anneal, determines the final size of scaffold features. To investigate the path of heat flow within collagen slurries during solidification, a series of ice-templating molds were designed with varying the contact area with the heat sink, in the form of the freeze drier shelf. Contact with the heat sink was found to be critical in determining the efficiency of the release of latent heat within the perspex molds. Isotropic collagen scaffolds were produced with pores which ranged from 90 μm up to 180 μm as the contact area decreased. In addition, low-temperature ice annealing was observed within the structures. After 20 h at -30 °C, conditions which mimic storage prior to lyophilization, scaffold architecture was observed to coarsen significantly. In future, ice-templating molds should consider not only heat conduction during the cooling phase of solidification, but the effects of heat flow during ice growth and annealing.The authors gratefully acknowledge the financial support of the Gates Cambridge Trust, the Newton Trust, and ERC Advanced Grant 320598 3D-E. A.H. held a Daphne Jackson Fellowship funded by the University of Cambridge.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s10853-015-9343-

Development of an ex vivo three dimensional ( 3-D) model of acute myeloid leukemia ( AML)

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of an ex vivo three dimensional ( 3-D) model of acute myeloid leukemia ( AML)

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Robust superstructure optimisation of a bioreactor that produces red blood cells

Recent work developed a novel, biomimetic, cost effective 3D hollow fibre bioreactor for growing healthy red blood cells ex vivo (Panoskaltsis et al., 2012). This promising bioreactor recapitulates the architectural and functional properties of erythrocyte formation and thereby reduces the need for expensive growth factors by more than an order of magnitude. The optimal bioreactor configuration has not been defined; design choices include: number of bioreactors run in parallel, number of hollow fibres in each reactor, size and aspect ratio of each bioreactor. Individual experiments on the bioreactor are cost- and labour-intensive, so we propose global, robust, superstructure optimisation for designing and operating the bioreactor. Beyond this individual bioreactor, robust superstructure design has the potential to more generally enable bioprocess optimisation. © 2014 Elsevier B.V

A framework for the design, modeling and optimization of biomedical systems

We present an overview of the key building blocks of a design framework for modeling and optimization of biomedical systems with main focus on leukemia, that we have been developing in the Biological Systems Engineering Laboratory and the Centre for Process Systems Engineering at Imperial College. The framework features the following areas: (i) a three-dimensional, biomimetic, in vitro platform for culturing both healthy and diseased blood; (ii) a novel, hollow fiber bioreactor that upgrades this in vitro platform to enable expansion and continuous harvesting of healthy and diseased blood; (iii) a global optimization-based approach for the design and operation of the aforementioned bioreactor; (iv) a pharmacokinetic / pharmacodynamic model representing patient response to Acute Myeloid Leukemia treatment; (v) an experimental framework for cell cycle modeling and quantitative analysis of environmental stress. This manuscript recapitulates the progress made in the different areas as well as the way in which these areas are connected, finally leading to a hybrid in vitro/in silico platform which allows the optimization of the ex vivo expansion of healthy and diseased blood. © 2014 Elsevier B.V

Perturbed hematopoietic stem and progenitor cell hierarchy in myelodysplastic syndromes patients with monosomy 7 as the sole cytogenetic abnormality.

The stem and progenitor cell compartments in low and intermediate-risk myelodysplastic syndromes (MDS) have recently been described, and shown to be highly conserved when compared to those in acute myeloid leukemia (AML). Much less is known about the characteristics of the hematopoietic hierarchy of subgroups of MDS with a high risk of transforming to AML. Immunophenotypic analysis of immature stem and progenitor cell compartments from patients with an isolated loss of the entire chromosome 7 (isolated -7), an independent high-risk genetic event in MDS, showed expansion and dominance of the malignant -7 clone in the granulocyte and macrophage progenitors (GMP), and other CD45RA+ progenitor compartments, and a significant reduction of the LIN-CD34+CD38low/-CD90+CD45RA- hematopoietic stem cell (HSC) compartment, highly reminiscent of what is typically seen in AML, and distinct from low-risk MDS. Established functional in vitro and in vivo stem cell assays showed a poor readout for -7 MDS patients irrespective of marrow blast counts. Moreover, while the -7 clone dominated at all stages of GM differentiation, the -7 clone had a competitive disadvantage in erythroid differentiation. In azacitidine-treated -7 MDS patients with a clinical response, the decreased clonal involvement in mononuclear bone marrow cells was not accompanied by a parallel reduced clonal involvement in the dominant CD45RA+ progenitor populations, suggesting a selective azacitidine-resistance of these distinct -7 progenitor compartments. Our data demonstrate, in a subgroup of high risk MDS with monosomy 7, that the perturbed stem and progenitor cell compartments resemble more that of AML than low-risk MDS