Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

Summary

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Abstract

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage¹. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration². In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor³. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

Protocol

Procedure

Coating slides with fibronectin

Prior to the assembling of the microfluidic device, sterilize glass slides with fibronectin. Coat the slides in a biohood to maintain sterility. Align a sterile PDMS spacer along the edges of the slide and mark the side that is facing up with a permanent marker. Pipette 1 ml of a 5 μg/ml solution of fibronectin (Sigma) inside the PDMS spacer over the entirety of the glass slide. Leave the slides undisturbed for an hour, then dry completely using an N₂ gun. The slides can be stored at 4^oC for later experiments.

Assembling the devic....

Discussion

Fig. 1 shows trajectories of three NSC strains, the parental cells, the wtEGFR and the ΔEGFR (control). Each set of trajectories were obtained from a 5 hours long movie. For parental cells, cell motility peaked when EGF concentration was ~ 10ng/ml; for wtEGFR cells, cell motility peak shifted to 100ng/ml or above; for ΔEGFR cells, the cell motility was independent of EGF concentration as expected.

The wtEGF cells with 100ng/ml were the most motile cells, they moved about 1........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco’s Modified Eagle Medium (DMEM) (1X)	Reagent	Invitrogen	11971-025
CO2-independent media	Reagent	Invitrogen	18045-088	A non-HEPES proprietary medium suitable for supporting cell growth for a variety of epithelial, fibroblast, and lymphoid cell lines in atmospheric conditions, contains no L-glutamine
Trypsin-EDTA	Reagent	Invitrogen	25200-072
PBS	Reagent	Invitrogen	14190-144	Phosphate-buffered saline, without calcium or magnesium
Pen/Strep	Reagent	Invitrogen	15140-122	Contains 10,000 units of penicillin (base) and 10,000 g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline
FBS	Reagent	Invitrogen	10437-028	Fetal Bovine Serum, Qualified
Horse Serum	Reagent	Invitrogen	16050-130	Horse Serum, EIA tested (negative) serum
Fibronectin	Reagent	Sigma-Aldrich	F0895	Fibronectin from human plasma, also called cold insoluble globulin
EGF	Reagent	Sigma-Aldrich	E4127	Epidermal Growth Factor from murine submaxillary gland, lyophilized
Agarose	Reagent	Fisher Scientific	BP1356-500