This video showcases the development and differentiation of human neural stem cells within a three-dimensional scaffold. A laminin coating on the scaffold promotes cell attachment, while a growth factor-free neural medium induces cell differentiation into neural progenitor cells, which extend their processes within a porous architecture in multiple directions, forming a three-dimensional culture
Protocol
1. HNSC differentiation on Three-Dimensional (3D) Culture NOTE: Isolate and derive human neural stem cells (hNSCs) from an ethically approved tissue described in a previous publication. Please follow the ethical and institutional guidelines while following this procedure. 3D Culture Using 12-well Plate Use an aseptic technique throughout the procedure. In a biological safety cabinet, re-hydrate scaffolds by adding 2 ml/well of 70% …
Offenlegungen
The authors have nothing to disclose.
Materials
DMEM:F12
Invitrogen
21331-020
For RMM medium preparation
Human Albumin Solution
Baxter health care limited
1501052
For RMM medium used at a final concentration of 0.03%
Transferrin, Human recombinant
Sigma
T8158
For RMM medium used at a final concentration of 5 µg/ml
Putrescine DiHCl
Sigma
P5780
For RMM medium used at a final concentration of 16.2 µg/ml
Insulin, Human recombinant
Sigma
I9278
For RMM medium used at a final concentration of 5 µg/ml
Progesterone
Sigma
P6149
For RMM medium used at a final concentration of 60 ng/ml
L-Glutamine
Invitrogen
25030-24
For RMM medium used at a final concentration of 2 mM
Sodium Selenite
Sigma
S9133
For RMM medium used at a final concentration of 40 ng/ml
bFGF
Peprotech
AF-100-15
To be added to RMM medium, used at a final concentration of 10 ng/ml
EGF
Peprotech
AF-100-188
To be added to RMM medium, used at a final concentration of 20 ng/ml
4-OHT
Sigma
H7904
To be added to RMM medium, used at a final concentration of 100 nM