The directed expansion and differentiation of haematopoietic stem cells (HSCs) into unique functional lineages not only requires lineage-specific growth factors, but also physio-chemical parameters such as pH, oxygen and CO2 tension and nutrients and metabolite concentrations which are unique to specific cell types. These parameters may change as the cultivated cells differentiate in phenotype and expand in number and even slight deviations in the culture parameters can affect the type and amount of final cell output.

Here we present the development of a novel culture system for HSCs, which controls and regulates stem cell self-renewal, expansion and differentiation and death. The novelty of the culture system lies upon the real-time, quantitative monitoring of bioactivity and the design of experiments to reveal the interactions between different factors. The perfused cell culture bioreactor unit, the single sensor devices (like oxygen, pH, glucose and ammonium sensors), sensor electronic and data acquisition software were developed separately.

For the measurements of pH and ammonium ions, conventional polymer membrane microelectrodes are applied. The concentration of main nutrient and metabolites (e.g. glucose) is measured using amperometric enzyme sensors which were obtained by “wiring” enzyme to cross-linked poly(vinylimidazole) polymer complexed with osmium (II/III)-(dimethoxy-bipyridine)2Cl. A prototype measurement platform of up to 128 channel was designed and produced to acquire real-time data to monitor the culture environments and therefore to achieve the optimize control for the whole culture process. The whole measurement system is PC-based and the user interface programmed with LabVIEW is easy to use for storing raw data and it has additional data management and data analysis functions.

DOE was used firstly to identify key factors in the culture process involving a series of screening experiments using fractional factorial designs. More in-depth process information was gained by a full factorial or response surface design and the analysis of the experimental data leaded to the final stage of the design – process optimisation.

In the case study, CD34+ stem cells were selected from cryopreserved cord blood units and cultured in the perfused bioreactor for up to 14 days. With the help of DOE and on-line monitoring, we were able to identify the optimal combination of growth factors and other culture parameters to drive the stem cells towards erythropoiesis. CFU assay and flowcytometry were used to identify stem/progenitor cell and differentiated cell population.