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.
- 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% ethanol prepared using water for irrigation (WFIr). Avoid touching the scaffolds by dispensing the 70% ethanol down the wall of each well.
- 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.
- 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.
- Seed each scaffold with approximately 500,000 hNSCs in a volume of 150 µl of RMM+growth factors (GFs).
- Incubate the plate for 3 hr at 37 °C in a 5% CO2 humidified incubator to allow cells to settle into the scaffolds.
- Add 3.5 ml of RMM to each well, taking care not to dislodge cells from scaffolds.
- 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.
Divulgazioni
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 |
| Laminin | AMS Biotech | 3400-010-10 | |
| Water for irrigation | Baxter Healthcare | UKF7114 | |
| Alvetex 12 well plate | Reinnervate Ltd | AVP002 | |
| Ethanol | Merck | 1.00986.1000 |
