BrainPhys neuronal medium optimized for imaging and optogenetics in vitro.

The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology are advancing. In parallel, cellular reprogramming and organoid engineering are expanding the use of human neuronal models in vitro. This creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identify multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (i.e., suboptimal fluorescence signals, phototoxicity, and unphysiological neuronal activity). To overcome these issues, we develop a neuromedium called BrainPhys™ Imaging (BPI) in which we optimize the concentrations of fluorescent and phototoxic compounds. BPI is based on the formulation of the original BrainPhys medium. We benchmark available neuronal media and show that BPI enhances fluorescence signals, reduces phototoxicity and optimally supports the electrical and synaptic activity of neurons in culture. We also show the superior capacity of BPI for optogenetics and calcium imaging of human neurons. Altogether, our study shows that BPI improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting optimal neuronal viability and function.

Enumerating stem cell frequency: neural colony forming cell assay.

Recent reports have highlighted several parameters of the neurosphere culture or assay system which render it unreliable as a quantitative in vitro assay for measuring neural stem cell (NSC) frequency. The single-step semi-solid based assay, the Neural Colony Forming Cell (NCFC) assay is an assay which was developed to overcome some of the limitations of the neurospheres assay in terms of accurately measuring NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e. their proliferative potential). The NCFC assay and other improved tissue culture tools offer further advances in the promising application of NSCs for therapeutic use.

Methods to culture, differentiate, and characterize neural stem cells from the adult and embryonic mouse central nervous system.

Since the discovery of neural stem cells (NSC) in the embryonic and adult mammalian central nervous system (CNS), there have been a growing numbers of tissue culture media and protocols to study and functionally characterize NSCs and its progeny in vitro. One of these culture systems introduced in 1992 is referred to as the Neurosphere Assay, and it has been widely used to isolate, expand, differentiate and even quantify NSC populations. Several years later because its application as a quantitative in vitro assay for measuring NSC frequency was limited, a new single-step semisolid based assay, the Neural Colony Forming Cell (NCFC) assay was developed to accurately measure NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e., their proliferative potential). The evolution and continued improvements made to these tissue culture tools will facilitate further advances in the promising application of NSCs for therapeutic use.

RSD1235 blocks late INa and suppresses early afterdepolarizations and torsades de pointes induced by class III agents.

OBJECTIVE: RSD1235 is a novel antiarrhythmic drug with atria-selective electrophysiological actions on Na(+) and K(+) currents. The mechanism for its protection of ventricular repolarization was assessed by its action on Purkinje fibers, and by block of late sodium current active during repolarization. Further, RSD1235's ability to reverse the pro-arrhythmic actions of the class III agents dofetilide and clofilium was assessed in isolated Purkinje fibers and an in vivo model of torsades de pointes (TdP). METHODS: Action potential and early after-depolarization (EAD) recordings were made from in situ and isolated rabbit Purkinje fibers at 37 degrees C using floating sharp microelectrodes; late I(Na) was recorded using a whole-cell patch clamp technique of Nav1.5 expressed in HEK cells at 22 degrees C; In vivo, anesthetized methoxamine-sensitized rabbits were used to test the ability of RSD1235 to suppress clofilium-induced TdP. RESULTS: RSD1235 (0.5-30 microM) had minor dose-dependent effects on action potential duration (APD) at 50% and 90% repolarization in Purkinje fibers, but pre-treatment significantly attenuated the APD-prolonging effects of dofetilide (300 nM). EADs induced by 300 nM dofetilide were terminated by 30 microM RSD1235 in all experiments (n=7). RSD1235 blocked a late component of Na current (I(Na)), which can produce inward currents contributing to EAD formation. RSD1235 pre-treatment (1 micromol/kg/min) or acute infusions prevented/terminated TdP induced by clofilium in 8 of 9 rabbits, and reduced the duration of TdP episodes from 71 +/- 23 s in control to 17 +/- 7 and 14 +/- 14 s at infusion rates of 0.3 and 1.0 micromol/kg/min, respectively (n = 9, p < 0.001). CONCLUSION: RSD1235 itself has minor actions on repolarization in Purkinje fibers, but can reverse the AP-prolonging actions of class III agents and terminate arrhythmias in a model of TdP. We suggest that these protective actions of RSD1235 may result, at least in part, from its ability to inhibit late I(Na) during action potential repolarization.