Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

Abstract

Source: Salick, M. R., et al. Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids. J. Vis. Exp. (2017).

This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

Protocol

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)

Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free stem cell maintenance media (SCMM) with a vitronectin or Geltrex substrate. If using an alternative, high-protein stem cell culturing media, it is advisable to transition SC cultures to SCMM for at least two passages before initiating organoid formation. The protocol has not been tested with feeder-based SC culture methods.

1. Using regular SC maintenance methods, bring cultures to 50%-70% confluence. This is typically 3-4 days after passaging, tho....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Xeno-Free Stem Cell Maintenance Media (SCMM), TeSR-E8	StemCell Technologies	5940	Use for stem cell maintenance and early stages of organoid formaiton. Store at 4 °C for up to two weeks after prepared from individual components
Enzyme-Free Detachment Reagent, ReLeSR	StemCell Technologies	5873
Enzymatic Detachment Reagent, Accutase	Gibco	A11105-01	Store aliquoted at -20 °C, do not keep thawed for longer than necessary
Y-27632	Selleck Chem	S1049	Reconstitute to 50mM in DMSO, and keep at -20 °C in 50µL aliquots.
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	D2650-100ML
Glutamate-supplemented DMEM/F12 (1X)	Gibco	10565-018	(+)2.438 g/L sodium bicarbonate, (+)sodium pyruvate
N-2 Supplement (100X)	Gibco	17502-048
MEM Non-Essential Amino Acids (MEM-NEAA)	Gibco	11140-050
Heparin	Sigma-Adrich	H4784-1G	Reconstitute to 2 mg/mL in sterile DI water and store at -20 °C in 250µL aliquots
Earle's Balanced Salt Solution (EBSS)	GE Healthcare Life Sciences	SH30029.02
Stericup Filter (500mL)	Millipore	220009-140
Costar Ultra-Low Attachment Plate, 96-Well, Round Bottom	Corning	7007
Low Residual Volume Reagent Reservoir (25mL)	Integra	4312
Pipet-Lite Multi Pipette L12-200XLS+	Rainin	17013810
Large Centrifuge, Culture Plate-Compatible	Thermo Scientific	75007210
Culture Plate Centrifuge Rotor	Thermo Scientific	75005706
Vero Cells	ATCC	CCL-81	For virus expansion, if necessary
Zika Virus (Uganda Strain)	ATCC	VR-1838	Other strains may be used
Zika Virus (Puerto Rico Strain)	ATCC	VR-1843	Other strains may be used