An In Vitro Model of Stretch Injury in Human Induced Pluripotent Stem Cell-Derived Neurons

Abstract

Source: Phillips, J. K. et al. Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format. J. Vis. Exp. (2018)

This video demonstrates the process of inducing stretch injury on human induced pluripotent stem cell-derived neurons (hiPSCNs). The cells are taken in a bottomless multi-well plate with a silicone membrane attached. Controlled mechanical stress is applied on the membrane to induce cytoskeletal disruption and cell membrane damage, inducing a stretch injury phenotype in the cells.

Protocol

1. Plate Fabrication

1. Plasma-treat a 96-well plate top with a 200 W plasma cleaner (see Table of Materials) for 60 s on high power, placing it bottom-side-up inside the plasma cleaner. Check for a purple glow visible through the window of the chamber, which indicates that an effective plasma has formed. This bonding technique is adapted from Sunkara et al.
2. Within 60 s, place the plate top in 200 mL of 1.5% (3-Aminopropyl) triethoxysilane (APTES) in deionized (DI) water for 20 min, bottom-side-down. This solution is unstable: prepare it no more than 60 s before introducing the plate.
   CAUTION:

Results

Figure 1: A labeled schematic of the injury device. (A) Top view, (B) isometric view, (C) front view, (D) right side view. The scale bar applies to the orthographic views (A, B and D).

....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
.010" Silicone Sheet	Specialty Manufacturing, Inc	#70P001200010	Polydimethylsiloxane (PDMS) sheet
Sparkleen	Fisher Scientific	#043204
Nunc 256665	Fisher Scientific	#12-565-600	Bottomless 96 Well Plate
Kim Wipes	ULINE	S-8115
Plasma Cleaner	Harrick Plasma	#PDC-001-HC
(3-Aminopropyl) triethoxysilane	Sigma-Aldrich	#440140	APTES
Parchment Paper	Reynolds	N/A
Dome Light	CCS inc	LFX2-100SW
Dome Light Power Supply	CCS inc	PSB-1024VB
Axial Diffuse Lighting Unit	Siemens	Nerlite DOAL-75-LED	Diffuse axial light
High Power LED Array	CREE	XLamp CXA2540	High Power LED Array
LED holder	Molex	1807200001	LED Holder
LED power supply	Mean Well	HLG-320H-36B	Constant Current Power Supply
FastCam Viewer software	Photron		camera softeware
Fastcam Mini UX50	Photron	N/A	High Speed Camera
Micro-NIKKOR 105mm f/2.8	Nikon	#1455	High Speed Camera Lens
0.1 mg/mL Poly-L-Ornithine	Sigma-Aldrich	#P4597
iCells	Cellular Dynamics International	#NRC-100-010-001
iCell media	Cellular Dynamics International	#NRM-100-121-001
iCell supplement	Cellular Dynamics International	#NRM-100-031-001
Laminin	Sigma-Aldrich	#L2020
voice coil actuator	BEI Kimco	LA43-67-000A
optical linear encoder	Renishaw	T1031-30A
servo drive	Copley Controls	Xenus XTL
Controller	National Instruments	cRIO 9024 Real Time PowerPC Controller
cRIO chassis	National Instruments	cRIO 9113
digital input module	National Instruments	NI 9411
data acquistion chassis	National Instruments	NI 9113
LabVIEW	National Instruments
hiPSCNs	Cellular Dynamics International