Differentiating Human Neuronal Stem Cells into 3D Culture Using a Porous Scaffold

Abstract

Source: Stevanato, L. et al., Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes. J. Vis. Exp. (2015)

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.

1. 3D Culture Using 12-well Plate
   1. Use an aseptic technique throughout the procedure. In a biological safety cabinet, re-hydrate scaffolds by adding 2 ml/well of 70% ethanol prepared using water for irrigation (WFIr). Avoid touching the scaffolds by dispensing the 70% ethanol down the wall of each well.
   2. Carefully aspirate the 70% ethanol and immediately wash the scaffolds with 2 ml/well Dulbecco's Modified Eagle Medium F12 (DMEM: F12) for 1 min. Repeat the wash procedure twice. Carefully aspirate the DMEM: F12 after the final wash.
   3. Coat scaffolds by adding 2 ml/well of laminin (10 µg/ml) in DMEM: F12. Incubate plate at 37 °C in a 5% CO₂ humidified incubator for a minimum of 2 hr. Wash the plate with warm DMEM: F12.
   4. Seed each scaffold with approximately 500,000 hNSCs in a volume of 150 µl of RMM+growth factors (GFs).
   5. Incubate the plate for 3 hr at 37 °C in a 5% CO₂ humidified incubator to allow cells to settle into the scaffolds.
   6. Add 3.5 ml of RMM to each well, taking care not to dislodge cells from scaffolds.
   7. Incubate the plate for 7 days; replace RMM medium (3.5 ml/well) after 1 day (first feeding) and again after 2-3 days from the first feeding.

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
Laminin	AMS Biotech	3400-010-10
Water for irrigation	Baxter Healthcare	UKF7114
Alvetex 12 well plate	Reinnervate Ltd	AVP002
Ethanol	Merck	1.00986.1000