Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation
Published: July 31, 2024
Abstract
Source: Cvetkovic, C., et al. Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells. J. Vis. Exp. (2018).
This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacological interventions.
Protocol
1. Preparation and Maintenance of 3D Sphere Cocultures Dissociate hAstros from 3D aggregates in suspension. Gently dissociate human astrocyte (hAstro) aggregates with detachment solution when dark centers appear and remove spheres that spontaneously attach. Break spheres gently once a week to maintain sphere health and to avoid necrotic cores. NOTE: Do not exceed 5 min of treatment with detachment solution. …
Representative Results
Figure 1: Systematic and reproducible generation of bioengineered 3D neural spheres. (A) Microwell (µ-well) culture plates are used to form 3D neural spheres of desired density and neuron-to-astrocyte ratios in a systematic and user-specified manner, yielding consistent size and shape. Images are shown 1 day after sphere formation. Scale bars = 200 µm. (B</stron…
Divulgaciones
The authors have nothing to disclose.
Materials
| 6 well plate | Fisher Scientific | 08-772-1B | |
| 15 ml conical tubes | Olympus Plastics | 28-101 | |
| Accutase | Sigma | A6964-100ML | Detachment solution |
| AggreWell plate | Stemcell Technologies | 34850 | |
| Anti-Adherence Rinsing Solution | Stemcell Technologies | 7010 | Prevent cell adhesion to microwell plates |
| Anti/anti | Thermofisher | 15240062 | |
| B27 | Thermofisher | 17504044 | Media Supplement |
| BrainPhys neuronal medium | Stemcell Technologies | 5790 | Neurophysiological basal medium alternative |
| DMEM/F12 | Thermofisher | 10565-042 | With GlutaMAX supplement |
| DMH-1 | Stemcell Technologies | 73634 | HAZARD: Toxic if swallowed. Working concentration: 2 uM |
| Donkey serum | Lampire Biological Laboratories | 7332100 | Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer |
| Doxycycline Hydrochloride (Dox) | Sigma | D3072-1ml | HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL |
| Epidermal growth factor (EGF) | Peprotech | AF-100-15 | Working concentration: 10 ng/mL |
| Fibroblast growth factor-2 (FGF) | Peprotech | 100-18B | Working concentration: 10 ng/mL |
| Hemacytometer or automatic cell counter | Life Technologies | AMQAX1000 | |
| Matrigel membrane matrix | Corning | 354230 | ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled. |
| MEA 2100 System | Multichannel Systems | MEA2100 | |
| Rho-Kinase Inhibitor Y27632- (Y) | Tocris | 1254 | Working concentration: 10 uM |
| SB431542 | Stemcell Technologies | 72234 | Working concentration: 2 uM |
| Spinner flasks | Fisher Scientific | 4500-125 | |
| T25 Culture Flask | Olympus Plastics | 25-207 | Vented caps |
| Trypan blue | Invitrogen | T10282 |
Citar este artículo
Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation. J. Vis. Exp. (Pending Publication), e22392, doi: (2024).