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.