Nicki Panoskaltsis

Stem cell bioprocessing: fundamentals and principles

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the ‘omics’ technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical—failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.

Quantification and cytokine production of circulating lymphoid and myeloid cells in acute myelogenous leukaemia

A simple assay was developed to assess the potential of patients with acute myelogenous leukaemia (AML) to respond to immunotherapy. Lymphocytes, monocytes and leukaemic blasts with their corresponding intracellular cytokine profiles were evaluated by four-colour flow cytometry. In 50 μl samples of whole blood, surface labelling for CD45, CD8 and CD3 was used for cell identification prior to intracellular staining for interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-γ. Absolute numbers of CD8+ and CD8− (putative CD4+) T-cells, NK cells (CD8+/CD3−) and monocytes were determined by reference to a fixed number of added fluorescent beads. The absolute numbers of CD8− and CD8+ T-cells in the blood of patients with AML were similar to those of normal controls. More of the lymphocytes in the blood of leukaemic patients spontaneously produced cytokines compared with those of controls. Furthermore, primary AML blasts secreted predominantly IFN-γ. After recovery from chemotherapy, lymphocyte counts tended to be lower than in normals and reduction of NK cells reached significance after the second chemotherapy (P=0.01). A prominent CD8lo/CD3lo-int lymphocyte subset appeared after recovery in some patients. This laboratory application of the study of cell subsets and intracellular cytokines in patients undergoing treatment may be helpful in monitoring immunological responses in AML.

Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds.

Cord blood expansion ex vivo can be achieved in liquid suspension through the addition of cytokines at the expense of often undesirable cell differentiation. In order to derive a cytokine-free dynamic culture system, we hypothesised that a three-dimensional (3D) environment in the form of highly porous scaffolds made of poly (D,L-lactide-co-glycolide) (PLGA) or polyurethane (PU) for the biomimetic growth of cord blood mononuclear cells (CBMNCs), would facilitate expansion of hematopoietic cells without exogenous cytokines. Both scaffolds supported cellular expansion ex vivo. Cytokine-free, long-term culture was best in PU coated with collagen type I (54-fold expansion). In contrast, traditional 2D well-plate cultures collapsed within 4 days in the absence of cytokines. CBMNCs cultured in the scaffolds were visualised by scanning electron microscopy and immunophenotypic/immunostaining analysis and the studies validated the presence of a dynamic culture containing erythroid precursors (CD45(-)/CD71(+)/CD235a(+)), hematopoietic stem/progenitor cells (CD38(-)CD34(+), CD117(+)), maturing myeloid cells (CD38(+), MPO(+)), CD4(+) and CD8(+) T-lymphocytes and megakaryocytes (FVIII(+)). Colony forming unit (CFU) assays indicated that BFU-E and CFU-GM increased (p < 0.05) whereas CFU-GEMM were maintained at week 4. In conclusion, this 3D culture system is capable of long-term, cytokine-free expansion of CBMNCs, enabling the study of hematopoiesis and providing a potential platform for drug discovery and therapeutic applications ex vivo.

The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a cancer of haematopoietic cells that develops in three-dimensional (3-D) bone marrow niches in vivo. The study of AML has been hampered by lack of appropriate ex vivo models that mimic this microenvironment. We hypothesised that fabrication and optimisation of suitable biomimetic scaffolds for culturing leukaemic cells ex vivo might facilitate the study of AML in its native 3-D niche. We evaluated the growth of three leukaemia subtype-specific cell lines, K-562, HL60 and Kasumi-6, on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials, such as poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate), poly (D, L-lactade), poly (caprolactone), and polystyrene. Our results show that PLGA and PU supported the best seeding efficiency and leukaemic growth. Furthermore, the PLGA and PU scaffolds were coated with extracellular matrix (ECM) proteins, collagen type I (62.5 or 125 microg/ml) and fibronectin (25 or 50 microg/ml) to provide biorecognition signals. The 3 leukaemia subtype-specific lines grew best on PU scaffolds coated with 62.5 microg/ml collagen type I over 6 weeks in the absence of exogenous growth factors. In conclusion, PU-collagen scaffolds may provide a practical model to study the biology and treatment of primary AML in an ex vivo mimicry.

A Real-Time Multi-Channel Monitoring System for Stem Cell Culture Process

A novel, up to 128 channels, multi-parametric physiological measurement system suitable for monitoring hematopoietic stem cell culture processes and cell cultures in general is presented in this paper. The system aims to measure in real-time the most important physical and chemical culture parameters of hematopoietic stem cells, including physicochemical parameters, nutrients, and metabolites, in a long-term culture process. The overarching scope of this research effort is to control and optimize the whole bioprocess by means of the acquisition of real-time quantitative physiological information from the culture. The system is designed in a modular manner. Each hardware module can operate as an independent gain programmable, level shift adjustable, 16 channel data acquisition system specific to a sensor type. Up to eight such data acquisition modules can be combined and connected to the host PC to realize the whole system hardware. The control of data acquisition and the subsequent management of data is performed by the systems software which is coded in LabVIEW. Preliminary experimental results presented here show that the system not only has the ability to interface to various types of sensors allowing the monitoring of different types of culture parameters. Moreover, it can capture dynamic variations of culture parameters by means of real-time multi-channel measurements thus providing additional information on both temporal and spatial profiles of these parameters within a bioreactor. The system is by no means constrained in the hematopoietic stem cell culture field only. It is suitable for cell growth monitoring applications in general.

Modeling and Analysis of Individualized Pharmacokinetics and Pharmacodynamics for Volatile Anesthesia

The presented procedure aims to establish an in-depth understanding of a derived mathematical model for drug distribution, pharmacokinetics, and drug effect, pharmacodynamics, during volatile anesthesia. A physiologically based, patient-specific model is derived, where the pharmacokinetic (PK) part consists of multiple blood and tissue compartmental models, each adjusted to the weight, height, gender, and age of the patient. The pharmacodynamic (PD) part is described by an effect site compartment and the Hill equation both linking the hypnotic effect measured by the Bispectral Index (BIS) to the arterial anesthetic concentration. Via a global sensitivity analysis the patient-specific PK and PD variables and parameters are analyzed regarding their influence on the measurable outputs, which are the end-tidal concentration of the volatile anesthetic and the BIS. Via this analysis, the uncertainty introduced by PD variability is identified to be more significant than the uncertainty introduced by PK variability. A case study of isoflurane-based anesthesia shows that the simulation results of the individualized PK variables are in good accordance with the measured end-tidal concentration. However, the PD parameters need to be estimated online to predict the hypnotic depth, measured by the BIS, correctly. As a result of this study, the aim should be to focus on the individual identification of the PD parameters before and during anesthesia with future application in safe and robust model predictive control.

A randomised comparison of the novel nucleoside analogue sapacitabine with low-dose cytarabine in older patients with acute myeloid leukaemia

The development of new treatments for older patients with acute myeloid leukaemia (AML) is an active area, but has met with limited success. Sapacitabine is a novel orally administered nucleoside analogue that has shown encouraging activity in unrandomised early-stage trials. We randomised 143 untreated patients with AML or with high-risk myelodysplastic syndrome (>10% marrow blasts) between sapacitibine and low-dose ara-C (LDAC) in our ‘Pick a Winner’ trial design. At the planned interim analysis there was no difference between LDAC and sapacitibine in terms of remission rate (CR/CRi, 27% vs 16% hazard ratio (HR) 1.98(0.90–4.39) P=0.09), relapse-free survival (10% vs 14% at 2 years, HR 0.73(0.33–1.61) P=0.4) or overall survival (OS; 12% vs 11% at 2 years, HR 1.24(0.86–1.78) P=0.2). Sapacitibine was well tolerated, apart from more grade 3/4 diarrhoea. On the basis of these findings sapacitibine did not show sufficient evidence of benefit over LDAC for the trial to be continued.

Chemotherapy Drug Scheduling for the Induction Treatment of Patients With Acute Myeloid Leukemia

Leukemia is an immediately life-threatening cancer wherein immature blood cells are overproduced, accumulate in the bone marrow (BM) and blood and causes immune and blood system failure. Treatment with chemotherapy can be intensive or nonintensive and can also be life-threatening since only relatively few patient-specific and leukemia-specific factors are considered in current protocols. We have already presented a mathematical model for one intensive chemotherapy cycle with intravenous (IV) daunorubicin (DNR), and cytarabine (Ara-C) [1]. This model is now extended to nonintensive subcutaneous (SC) Ara-C and for a standard intensive chemotherapy course (four cycles), consistent with clinical practice. Model parameters mainly consist of physiological patient data, indicators of tumor burden and characteristics of cell cycle kinetics. A sensitivity analysis problem is solved and cell cycle parameters are identified to control treatment outcome. Simulation results using published cell cycle data from two acute myeloid leukemia patients [2] are presented for a course of standard treatment using intensive and nonintensive protocols. The aim of remission-induction therapy is to debulk the tumor and achieve normal BM function; by treatment completion, the total leukemic population should be reduced to at most 10

Thrombotic thrombocytopenic purpura in pulmonary-renal syndromes.

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Design of optimal patient-specific chemotherapy protocols for the treatment of acute myeloid leukemia (AML)

cells, at which point BM hypoplasia is achieved. The normal cell number should be higher than that of the leukemic, and a 3-log reduction is the maximum permissible level of population reduction. This optimization problem is formulated and solved for the two patient case studies. The results clearly present the benefits from the use of optimization as an advisory tool for treatment design.