Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

Abstract

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.

Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids

“…Human organoids from two different iPSC lines were cultured in spinner flasks containing medium mixture of DMEM/F12 and Neural Basal Medium (in 1:1 ratio), supplemented with 1:200 N2, 1:100 B27 w/o vitamin A, 1:100 L-glutamine, 0.05 mM MEM non-essential amino acids (NAA), 100 U/ml penicillin, 100 mg/ml streptomycin, 1.6 g/L insulin (Sigma-Aldrich) and 0.05 mM b-mercaptoethanol (all from Life Technologies if not stated) and supplemented with 0.5 mmol dorsomorphin (SigmaAldrich, USA) and 5 mM SB431542 (Selleckchem, USA). Differentiated cortical neurons from two different iPSC lines were maintained in a medium consisting of BrainPhys basal medium (Bardy et al, 2015) supplemented with 1x B27 (without vitamin A, Thermo scientific, USA), 1x N2 (Thermo scientific, USA), 20ng/ml BDNF (Peprotech, USA), 20ng/ml GDNF (Peprotech, USA), 20ng/ml NT3, 1 mM cAMP (Sigma, USA) and 0.2 mM ascorbic acid (Sigma, USA). Fresh medium was added every 2-3 days.…”

“…In detail, coverslips were coated with poly-L-ornithine (PLO)-/laminin and the NPCs were seeded on the coverslips as a monolayer. 48 hr later, NPCs were switched to cortical neuronal differentiation medium consisting of BrainPhys basal medium (Bardy et al, 2015) supplemented with 1x B27 (without vitamin A, Thermo scientific, USA), 1x N2 (Thermo scientific, USA), 20ng/ml BDNF (Peprotech, USA), 20ng/ml GDNF (Peprotech, USA), 20ng/ml NT3, 1 mM cAMP (Sigma, USA) and 0.2 mM ascorbic acid (Sigma, USA). Fresh medium was added every 2-3 days.…”

Programming and Reprogramming Cellular Age in the Era of Induced Pluripotency

“…Strategies to trigger neuronal maturation has been even more challenging. Improved cell culture media formulations(Bardy et al, 2015) and the use of astrocyte co-culture can accelerate electrophysiological maturation of hPSC-derived neurons. However, there is no evidence that those conditions accelerate other aspects of neuronal maturation such as the acquisition of late stage neuronal markers including expression of PV in cortical interneuron cultures.…”

Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons?

“…It should be noted, though, that primary rat cortical cultures exhibit higher and stable levels of bursting and spiking following more prolonged culture (DIV10-14; Robinette et al, 2011;de Groot et al, 2016;Dingemans et al, submitted) and that over time the degree of neuronal activity in DOPA.4U and HIP cultures may still develop to levels that equal or exceed those of iCell neurons at DIV4-6. While this should be taken into account in future studies, such studies may also need to focus on exploring the influence of different culture media on the speed and degree of neuronal development (see, e.g., Bardy et al, 2015). Notably, our data on spontaneous network activity is derived from iCell and DOPA.4U neurons in the absence of (added) astrocytes.…”