Poster AA15 - Development of an Aptamer-Based Biosensor for Real-Time Monitoring of Cell Attachment

Introduction: A major goal of our group is to transform artisanal tissue engineering (TE) into tissue manufacturing. To this end, as many steps as possible should be converted to feedback-controlled automated processes. Cell seeding onto scaffolds is a critical step in many TE applications, but it is usually done “open loop” with no immediate feedback on cell coverage of the scaffolds. Destructive tests like histology waste the construct, and are, thus, not useful in this context. Aptamers (single-stranded DNA or RNA oligonucleotides generated by a process termed SELEX (systematic evolution of ligand by exponential enrichment)) are designed to bind to specific target molecules or whole cells with high affinity. Aptamers change conformation after binding to their target, which can be used for real-time non-invasive, imaging-based measurements. In this study we show that appropriate aptamers can be used to provide a cell-seeding feedback signal.

Materials and

Methods: We designed an aptamer-based molecular beacon specific to MDA-MB231 cancer cells. The molecular beacon contains a 3’ fluorescent FAM tag with a BHQ-1 black-hole quencher in close proximity (in the absence of target), and a biotinTEG for anchorage via streptavidin. Upon cell binding the aptamer converts to its open conformation, where the fluorophore and the quencher do not interact. The recovered fluorescent signal is used for detection of cell binding to the scaffold. Scaffolds were functionalized with the aptamer, 24h later cells were seeded and fluorescence was quantified every 15 minutes for 24 hrs. Cell counts from live/dead assays were compared to the fluorescent signal from the aptamer for validation. A streptavidin coated plate was used as a proof of principle to test the ability of the molecular beacon to produce a signal upon cell binding.

Results, Conclusions, and Discussions: The molecular beacon aptamer attached to streptavidin coated plates produced fluorescence signal after cells were seeded at different densities. The signal increased with cell number following a power law. Next, we tested the molecular beacon using collagen scaffolds. The cell-specific aptamer provided a quantifiable fluorescent signal upon the addition of cells. Their presence otherwise had no measurable impact on cell viability by live-dead assays.
The novel aptamer-based cell seeding biosensors described here will provide the afferent arm of a real-time feedback loop to be used to automate cell-seeding inside bioreactors. Preliminary results suggest that the aptamers have no impact on cell viability, but longer-term comparative validation studies will be performed. Additionally, the development of more compact data acquisition hardware that will interface with our bioreactor systems will be required and is ongoing.

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