3D Spheroid Culture Workflow using iPSC-induced Human Neurons
Poster May 31, 2019
Kaiping Xu1, Zhong-Wei Du1, Anju Dang2, Ben Dungar1, Kurt Laha1
Human induced pluripotent stem cells (iPSC) derived neurons are now considered a more relevant in vitro model system for psychiatric and neurological diseases. They can be used for the development of physiological cell models, human disease models, and drug library screening. Three dimensional (3D) cultures are also being pursued as a more physiologically relevant system because they provide a microenvironment, cell-to-cell interactions, and biological processes that better represent in vivo conditions. The implementation of 3D spheroid culture plays an important role as an alternative approach for drug development and therapeutic applications in central neural system (CNS) disorders. To develop a neuronal 3D spheroid culture system, we tested iPSC-derived human motor neurons and cortical glutamatergic neurons using the S-BIO PrimeSurface Ultra Low Attachment Micoplates. After 2 hours, plated neurons started to settle down at the bottom of the well and form large clusters. On day 3, 3D spheroids could be seen clearly under phase contrast. On day 7, the spheroids were more condensed. The size of the 3D spheroids was proportional to the number of neurons seeded per well. In addition to the morphological assessments, a cytotoxicity assay and MEA assay were performed to demonstrate the suitable of 3D spheres as a platform for various applications. In both assays, the spheroids yielded expected results. The results presented here demonstrate the feasibility of generating uniform and reproducible spheroids using human neurons and the potential application for neurotoxicity studies.
Spinal muscular atrophy (SMA) is an inheritable cause of infant mortality that is characterized by the loss of lower motor neurons and skeletal muscle atrophy. The degeneration of motor neurons is caused by insufficient levels of survival motor neuron (SMN) protein, which is encoded by two nearly identical genes SMN1 and SMN2. Most cases of SMA harbour homozygous deletions of the SMN1 gene and retain at least one copy of SMN2.READ MORE
In order to generate a robust protocol for MEA recording on hiPSC- derived neurons, we evaluated several conditions, which could affect culture performance (1.neuron seeding density; 2.seeding medium; 3.astrocyt eco-culture). These conditions were evaluated with BrainXell’s hiPSC-derived spinal motor neurons, cortical glutamatergic neurons and mixed cortical neurons.READ MORE