Brainphys hpsc neuron kit

After local anesthesia, 5-mm diameter biopsy specimens were obtained from 10 cm above the lateral malleolus of the study participants. The skin tissues were cultured at 37°C in a 5% CO₂ atmosphere with Dulbecco's modified Eagle medium (DMEM; GIBCO, Cat. #C11995500BT, USA) containing 10% fetal bovine serum (GIBCO, Cat. #10099141C, USA) and 1% penicillin and streptomycin (Solarbio, Cat. #P7630, China); the culture medium was changed every 3 days. Fibroblasts were obtained after the skin tissues had been cultured for 15 days; the fibroblasts were cultured using the same method as was used for the skin tissues. Then, we induced the fibroblasts into iPSCs with a CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher, Cat. #A16517, USA). Next, neural progenitor cells (NPCs) were induced from the iPSCs with a STEMdiff™ SMADi Neural Induction Kit (STEM CELL, Cat. #08581, Canada). Finally, we obtained neurons from the NPCs with a BrainPhys™ hPSC Neuron Kit (STEM CELL, Cat. #05795, Canada) following the instructions provided in the product manual. The neurons were cultured at 37°C with a 5% CO₂ atmosphere using a BrainPhys™ hPSC Neuron Kit (STEM CELL, Catalog #08581, Canada).

Two neural precursor cell lines (NPCs XCL1 [#70901], XCL4[#70902]) were purchased from StemCell Technology, Inc. (Vancouver, Canada) and differentiated into neurons according to the manufacturer’s protocol (catalog numbers 08500 and 08510). Briefly, NPCs were plated onto Poly-l-Ornithine (PLO) and Laminin (MilliporeSigma, Burlington, MA) coated plates and cultured in STEMdiff™ Neuron Differentiation Media for 2 to 4 passages. Neuron maturation was achieved by changing from Neuron Differentiation to STEMdiff™ Neuron Maturation Media or Brainphys media (BrainPhys™ hPSC Neuron Kit, StemCell Technology) for 3 to 4 weeks. Neuronal maturation was confirmed in each culture by immunohistochemical staining of neuronal markers such as Tuj1 and Map2a. Cells were maintained at 5% oxygen and 5% carbon dioxide, and media changes were done every other day. All experiments were done in triplicates with 6 to 8 replicates in each experiment.