Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies.

Airway cholinergic nerves play a key role in airway physiology and disease. In asthma and other diseases of the respiratory tract, airway cholinergic neurons undergo plasticity and contribute to airway hyperresponsiveness and mucus secretion. We currently lack human in vitro models for airway cholinergic neurons. Here, we aimed to develop a human in vitro model for peripheral cholinergic neurons using human pluripotent stem cell (hPSC) technology. hPSCs were differentiated towards vagal neural crest precursors and subsequently directed towards functional airway cholinergic neurons using the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic neurons were characterized by ChAT and VAChT expression, and responded to chemical stimulation with changes in Ca2+ mobilization. To culture these cells, allowing axonal separation from the neuronal cell bodies, a two-compartment PDMS microfluidic chip was subsequently fabricated. The two compartments were connected via microchannels to enable axonal outgrowth. On-chip cell culture did not compromise phenotypical characteristics of the cells compared to standard culture plates. When the hPSC-derived peripheral cholinergic neurons were cultured in the chip, axonal outgrowth was visible, while the somal bodies of the neurons were confined to their compartment. Neurons formed contacts with airway smooth muscle cells cultured in the axonal compartment. The microfluidic chip developed in this study represents a human in vitro platform to model neuro-effector interactions in the airways that may be used for mechanistic studies into neuroplasticity in asthma and other lung diseases.

Tumor cell capture from blood by flowing across antibody-coated surfaces.

The load of circulating tumor cells (CTC) is related to poor outcomes in cancer patients. A sufficient number of these cells would enable a full characterization of the cancer. An approach to probe larger blood volumes, allowing for the detection of more of these very rare CTC, is the use of leukapheresis. Currently available techniques allow only the analysis of a small portion of leukapheresis products. Here, we present a method that uses flow rather than static conditions which allows processing of larger volumes. We evaluated the conditions needed to isolate tumor cells from blood while passing antibody coated surfaces. Results show that our set-up efficiently captures cancer cells from whole blood. Results show that the optimal velocity at which cells are captured from blood is 0.6 mm s-1. Also, it can be concluded that the VU1D9 antibody targeting the EpCAM antigen has very high capture efficiency. When using an antibody that does not capture 100% of all cells, combining multiple antibodies on the capture surface is very beneficial leading to an increase in cell capture and is therefore worthwhile considering in any cancer cell capture methodology.