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Realtime Monitoring and Control of Soluble Signaling Factors Enables COMMUNICATION TO THE EDITOR Real-Time Monitoring and Control of Soluble Signaling Factors Enables Enhanced Progenitor Cell Outputs from Human Cord Blood Stem Cell Cultures Elizabeth Csaszar,1,2 Kun Chen,1 Julia Caldwell,1 Warren Chan,1,2,3,4,5 Peter W. Zandstra1,2,3,6,7 1 Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S3E1, Canada; telephone: þ416-978-8888; fax: þ416-978-2666; e-mail: [email protected] 2 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S3E1, Canada 3 Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S3E1, Canada 4 Department of Chemistry, University of Toronto, Toronto, Ontario 5 Department of Materials Science and Engineering, University of Toronto, Toronto, Ontario 6 Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario M5G1L7, Canada 7 McEwen Centre for Regenerative Medicine, University Health Network, Toronto Medical Discovery Tower, Toronto, Ontario M5G1L7, Canada umbilical cord blood cell expansion system. By maintaining b ABSTRACT: Monitoring and control of primary cell cultures the concentration of TGF- 1 below an upper threshold is challenging as they are heterogenous and dynamically throughout the culture, we demonstrate enhanced ex vivo complex systems. Feedback signaling proteins produced from expansion of hematopoietic progenitor cells at higher input off-target cell populations can accumulate, inhibiting the cell densities and over longer culture periods. This study production of the desired cell populations. Although culture demonstrates the potential of a fully automated and strategies have been developed to reduce feedback inhibition, integrated real-time control strategy in stem cell culture they are typically optimized for a narrow range of process systems, and provides a powerful strategy to achieve highly fi parameters and do not allow for a dynamically regulated regulated and intensi ed in vitro cell manufacturing systems. response. Here we describe the development of a microbead- Biotechnol. Bioeng. 2014;111: 1258–1264. based process control system for the monitoring and control ß 2013 The Authors Biotechnology and Bioengineering of endogenously produced signaling factors. This system uses Published by Wiley Periodicals, Inc. quantum dot barcoded microbeads to assay endogenously KEYWORDS: produced signaling proteins in the culture media, allowing process control; hematopoietic stem cells; for the dynamic manipulation of protein concentrations. bioreactor; soluble signaling factors; protein detection This monitoring system was incorporated into a fed-batch bioreactor to regulate the accumulation of TGF-b1inan Primary stem cell cultures are complex and heterogeneous systems. In the in vitro culture of human hematopoietic This is an open access article under the terms of the Creative Commons Attribution- NonCommercial License, which permits use, distribution and reproduction in any progenitors, soluble factor-mediated feedback inhibition medium, provided the original work is properly cited and is not used for commercial limits the ex vivo expansion of hematopoietic stem and purposes. progenitor cells (HSPCs) (Kirouac and Zandstra, 2006). Elizabeth Csaszar and Kun Chen contributed equally to this work. Correspondence to: P.W. Zandstra Strategies to minimize the impact of emerging mature cell Received 30 July 2013; Revision received 20 October 2013; Accepted 18 November 2013 populations have provided some success towards controlling Accepted manuscript online 27 November 2013; endogenous factor accumulation (Csaszar et al., 2012; Article first published online 16 December 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25163/abstract). Madlambayan et al., 2005; Sandstrom et al., 1995; Yang DOI 10.1002/bit.25163 et al., 2010); however, these approaches are typically 1258 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 ß 2013 The Authors Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. optimized for a specific set of culture conditions and do not feeding schemes and found an exponential fed-batch feeding allow for the flexibility to easily accommodate changing scheme showed a trend of outperforming other strategies culture supplements or input cell compositions. One when normalized to total cumulative media use over 8 days approach to overcome this limitation would be a cell culture (Fig. 1C). The beneficial effects of the exponential feeding regulation system that allows for in-line or at-line monitoring regime were particularly evident on the primitive progenitor of soluble signaling factors in the culture media, thereby population, as measured by long-term culture-initiating cells providing dynamic response to system variations. A major (LTC-ICs) (Liu et al., 2013). challenge in implementing control strategies for endoge- Given the apparent benefits of the exponential feeding nously secreted proteins is their non-linear and dynamic regimen, we predicted that adjusting the media dilution secretion profiles (Csaszar et al., 2012) and the low scheme based on the actual measurements of soluble factors concentration at which they are bioactive. As a result, as opposed to a pre-determined algorithm would further dynamic regulation of signaling proteins has not yet been enhance the regulation that could be achieved by providing a integrated into stem cell culture systems. sample-specific dilution scheme. It was further predicted that Herein, we describe the development and demonstration monitoring the concentration of a sentinel endogenously of a real-time control system for monitoring and controlling produced factor would provide a good initial indication of the signaling proteins in a human HSPC expansion bioprocess culture dynamics of the global secreted factor milieu and using a microbead detection assay. Microbead-based bio- TGF-b1 was selected as an initial candidate for the sensing platforms provide a compelling option for integrat- development of the system. TGF-b1 is a potent inhibitor ing antibody based protein detection into culture platforms of HSPC expansion (Csaszar et al., 2012; Fortunel et al., 2000) as they have faster reaction kinetics and higher throughput which rapidly accumulates at high levels (>1,000 pg/mL) in capacity of biomolecule conjugation compared to planar this culture system in a manner that mimics the behavior of a detection techniques (Wilson et al., 2006). We developed a number of secreted factors (Fig. 1B). Importantly, measured microbead sandwich antibody detection system for the rapid levels of TGF-b1 are negatively correlated to total cell measurement of TGF-b1, a critical inhibitory factor of expansion (Fig. 1D), suggesting that maintaining a low human hematopoiesis (Fortunel et al., 2000). By integrating concentration of TGF-b1 should be beneficial to cell this detection assay into a HSPC culture system (Csaszar expansion. Because TGF-b1 accumulates in culture in a et al., 2012), we demonstrate the ability of the strategy to latent form and is difficult to measure with antibody expand hematopoietic progenitors at higher cell seeding detection without an activation step (Lyons et al., 1988), densities and for longer culture times than we can achieve we developed the assay to monitor the latency-associated with alternative culture methods. peptide (LAP) as a TGF-b1 surrogate. LAP in a propeptide As a first step, it was investigated whether the human that binds strongly to TGF-b1 to form the Small Latent hematopoietic culture system would likely benefit from the Complex (Rifkin, 2005; Young and Murphy-Ullrich, 2004), incorporation of a dynamic process control system. The and its concentration correlates strongly to that of TGF-b1 robust ex vivo expansion of umbilical cord blood (UCB) protein (Fig. 1E). derived HSPCs is highly desired for hematopoietic stem cell In order for the measurement of LAP to be used to control transplantation, however, cell variability can limit the cell culture, it is critical for the detection assay reaction to be reproducibility of this expansion. To assess the variability rapid, sensitive, and have high precision and accuracy. The þ in this system, we measured the purity of CD34 progenitors reaction procedure should be simple and easy to automate for following a lineage depletion process (commonly performed device production. We have previously developed a quantum prior to seeding cells for culture) and found the frequency to dot (QD)-barcoded microbead platform for biosensing be highly variable from sample to sample. This input cell (Fournier-Bidoz et al., 2008; Klostranec et al., 2007). This variability manifests itself into differences in the kinetics platform is optimized for detection of genetic targets using and types of mature cell production, ultimately impacting 4-micron size beads (Gao et al., 2011; Giri et al., 2011). the total cell expansion achieved in a non-linear manner Microbeads doped with QDs can detect many molecules (Fig. 1A). In a previous study, we demonstrated that simultaneously in a single reaction chamber in less than 1 h. experimental differences in hematopoietic cell culture out- Here we show that a QD-magnetic nanoparticle microbead puts could be recapitulated by varying the secretion rates of system can meet the requirements for LAP detection. The endogenous regulators in silico (Kirouac et al., 2009). A large detection assay is based on a
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