Mass Generation, Neuron Labeling, and 3D Imaging of Minibrains

Abstract

Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains.

Neuro-toxicogenomic mapping of TMT induced neurotoxicity using human minibrain reveals associated adverse outcome events

“…Neural stem cells derived from induced pluripotent stem cells (NSC hIPSC ) (ThermoFisher, #A3890101) were seeded with 2E5 cells per 6 well-plates and processed for minibrain generation according to the protocol previously described (Govindan et al, 2021). Trimethyltin chloride (TMT) (Merck-Sigma 146498) compound at a concentration of 0,1, 2.5, 5 μM was added to 4-month-old mature minibrains and was treated for 1 day (acute), 3 days (intermediate) and 7 days (chronic).…”

“…In this study we use minibrains developed in our lab as an in vitro model to map molecular events that lead to TMT induced neurotoxicity. Previously we described a pipeline to mass generate human minibrains, perform large scale immunolabeling, viral mediated neuron labeling and 3D imaging that would facilitate reconstruction of neuron morphology and detection of molecular markers in 3D for screening studies (Govindan et al, 2021). Here, we utilize BRB-seq, an ultra-low-cost transcriptomics pipeline that allows time and cost-efficient multiplexing of large numbers of samples to setup our neuro-toxicogenomic assay in exposure to TMT.…”

SpikeOnChip : A Custom Embedded Platform for Neuronal Activity Recording and Analysis

“…Engineered human neural tissues are small human-derived iPS cell neuropsheres/minibrains [6]. These tissues, even though the cellular brain organization is not reproduced, incorporate all the key types of cells and present a behaviour similar to human brain tissue, making it a good biological model for the study of brain-related diseases and toxicology screening [6], [14]- [16].…”

“…Although the use of engineered human neural tissue has many advantages over animal models, there are constraints on the tissue culture method. The long time of maturation (typically eight weeks of culture [6], up to several months) makes growing tissue cultures directly within MEA biochips complex to handle and expensive. The best culturing and experimental setup for such 3D tissues is to grow them at air-liquid interface, where the tissues are not immersed into nutritive medium, but remain in direct contact with air to provide sufficient oxygenation while being fed through a porous membrane [6]- [8].…”

“…The long time of maturation (typically eight weeks of culture [6], up to several months) makes growing tissue cultures directly within MEA biochips complex to handle and expensive. The best culturing and experimental setup for such 3D tissues is to grow them at air-liquid interface, where the tissues are not immersed into nutritive medium, but remain in direct contact with air to provide sufficient oxygenation while being fed through a porous membrane [6]- [8]. Mature tissues grown on a air-liquid interface can then be placed into adapted porous substratebased MEA biochips connected to external micro-fluidics for medium supply during experimental recordings/measurements.…”