Harvesting Adeno-Associated Virus from Transfected Mammalian Cells

Abstract

Source: Rghei, A. D., et al. Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models. J. Vis. Exp. (2021)

This video demonstrates the recovery of crude adeno-associated virus from transfected mammalian cells using chemical lysis and centrifugation, enabling high-yield AAV production for downstream purification and preclinical gene therapy applications.

Protocol

1. Harvesting AAV (adeno-associated virus) and chemical lysis of the transfected HEK293 (human embryonic kidney 293) cells

1. Shake the cell culture chamber vigorously to dislodge the cells until the media appears cloudy from dislodged cells and pour into four 500 mL centrifuge tubes.
2. Centrifuge the tubes at 18,000 x g for 30 min at 4 °C to pellet the cells. Pour the clarified supernatant into 1 L polyethylene terephthalate copolyester (PETG) bottle.NOTE: If one does not have access to a high-speed centrifuge, centrifuge at 12,000 x g for 40 min. The pelleted cells may not be solid at this speed and will slide as pouring out supernatant.
3. Resus....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 chamber cellstack	Corning	3271
1L PETG bottle	Thermo Fisher Scientific	2019-1000
Dulbecco's Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose, L-glutamine	Cytiva Life Sciences	SH30022.01
Fetal Bovine Serum (FBS)	Thermo Fisher Scientific	SH30396.03
HEK293 cells	American Tissue Culture Collection	CRL-1573	Upon receipt, thaw the cells and culture as described in manufacturer’s protocol. Once cells have been minimally passaged and are growing well, freeze a subfraction for future in aliquots and store in liquid nitrogen. Always use cells below passage number 30. Once cultured cells have been passaged more than 30 times, it is recommended to restart a culture from the stored aliquots
Large Volume Centrifuge Tube Support Cushion	Corning	CLS431124	Support cushion must be used with large volume centrifuge tubes uless the centrifuge rotor has the approriate V-bottom cushions
Large Volume Centrifuge Tubes	Corning	CLS431123-6EA	500 mL centrifuge tubes