Large-Scale Production of High-Capacity Adenoviral Vectors

Abstract

Source: Ehrke-Schulz, E., et al. Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5. J. Vis. Exp. (2016)

This video demonstrates the large-scale amplification of high-capacity adenoviral vectors in suspension-adapted human embryonic kidney cells expressing Cre recombinase for use in gene delivery applications requiring high-titer, helper-free vector preparations.

Protocol

1. Large Scale Amplification of High-capacity adenoviral vectors (HCAdV) in 116 cells Growing in Suspension

1. Add 900 ml of prewarmed (37 °C) fresh MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and hygromycin B (100 µg/ml) into a 3-L spinner culture flask (Figure 1B).
2. Remove medium from at least 10 individual 150-mm tissue culture dishes with 116 cells grown at a confluency of 90-100% and flush off cells with 10 ml of fresh pre-warmed (37 °C) MEM supplemented with FBS (10%) and hygromycin B (100 µg/ml) using a serological pipette. Pipette up and down several times to get a homogeneous ce....

Results

Figure 1. Schematic diagram for high-capacity adenoviral vector amplification and purification. (A) HCAdV DNA transfection and helper virus (HV) infection: Transfection of linearized HCAdV genome carrying the gene of inter.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hygromycin B	PAN Biotech	P02-015	Selection of CRE expressing 116 cells
DMEM	PAN Biotech	P04-03590	Hek293T cell culture medium
Minimal Essential Medium (MEM) Eagle	PAN Biotech	P04-08500	116 cell culture medium
Dulbecco’s phosphate buffer saline (DPBS)	PAN Biotech	P04-36500	Washing of cells, resuspension of cells
250-ml storage bottle	Sigma	CLS430281-24EA	Infection of 116 cells grown in suspension
500-ml PP CentrifugeTubes	Sigma	CLS431123-36EA	Sedimentation of cells from suspension culture
Spinner flask	Bellco	1965-61030	Growth of 116 cells in suspension