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Full Process Characterization of Hematopoietic Stem Cell Cultures Using Design of Experiments to Study Individualistic and Interactive Effects of Culture Parameters

Mayasari Lim1, Hua Ye1, Nicki Panoskaltsis2, and Athanasios Mantalaris1. (1) Chemical Engineering and Chemical Technology, Imperial College London, London, United Kingdom, (2) Department of Haematology, Northwick Park Hospital, Watford Road, Harrow, London, HA1 3U, United Kingdom

The study of hematopoietic stem cell culture processes is driven by the demand in stem cell therapy to cure many blood related diseases, metabolic disorders and as future potential treatments. In order to harvest the full potential of stem cells in an ex-vivo environment, process characterization providing quantitative representations of cellular activities is essential. Culture parameters such as pH, oxygen tension, glucose, and other metabolites, not only growth factors, play a vital and interactive role in driving various cell culture processes of interest. Our goal in this project is to be able to use design of experiment methods to identify and characterize the individualistic and interactive roles of key culture parameters on cellular activities such as proliferation and differentiation. The outcome of this investigation will provide us with the key in finding a balance amongst the various culture parameters that are crucial to process control. Our ability to orchestrate the symphony of cell culture parameters will allow us to replicate any desired hematopoietic process. The possibility of creating a homogeneous population of “designer cells” with unique and highly desirable characteristics can therefore be realized.

Design of experiments, a long-established methodology, utilized in various research and engineering applications, has proven to be a highly-effective method for process characterisation and optimization [1]. The application of these clever design methods to perform cell culture experiments will not only accelerate stem cell research but will help us gain a better understanding of the process picture [2, 3]. This is accomplished by providing a quantitative representation characterizing the process dynamics involved in culture processes of interest. Clearly, this task is not as easy to achieve due to the large number of culture parameters and complexity of their interactions. Our approach to this intricate problem is to use design of experiments in a step-wise manner through a process of screening, characterization, and optimization steps. Screening, as a first step to such process problems, will be crucial in helping us identify the key process parameters that play a significant role in manipulating the various culture processes. Screening experiments will be performed in several experimental blocks due to the complexity and numerousness of parameters that need to be investigated. Once the key parameters that facilitate the process have been identified, a full characterization can be obtained using response surface methods such as a central composite design. Initial screening of the process parameters will be performed mostly in static 2D culture systems such as a 24-well plate. However, the ultimate goal of this project is to demonstrate process performance in a fully enclosed, perfused 2D customized bioreactor. Isolated CD34+ cells from cryopreserved cord blood samples are used as a starting population, and the first target process is the process of erythropoiesis.

1. Montgomery, D.C., Design and Analysis of Experiments. 5th ed. 2001, Arizona: John Wiley and Sons, Inc. 696.

2. Zandstra, P.W., et al., Cytokine manipulation of primitive human hematopoietic cell self-renewal. Proceedings of the National Academy of Sciences, 1997. 94: p. 4698-4703.

3. Cortin, V., et al., Efficient in vitro megakaryocyte maturation using cytokine cocktails optimized by statistical experimental design. Experimental Hematology, 2005. 33: p. 1182-1191.