Introduction

This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed iPS cells, the initial idea came via word of mouth reports of its effectiveness to increase the efficiency of viral transduction. It has most commonly been used on retrovirus and lentivirus factors. Initial evaluation of this method showed 5–10× increase in transduction efficiency.

Flow Chart

Protocol Steps

Materials

Fibroblast Media

WiCell hES Media

Notes

^*Retroviral integration is dependent on actively dividing cells. Reprogramming efficiencies also have been shown to increase with increased proliferation rate of the starting cell population. For these reasons it is important to start reprogramming experiments with robust and actively dividing/growing cells. For human fibroblast we typically start with 50,000–100,000 cells per well (of 6 well). We typically transduce the fibroblast a day or two after passaging when the cells are sub-confluent, but are touching or nearly touching each other, and that ample open areas between the cells are still present. An extra well can be plated and counted at the time of infection if desired.

^**We have had success using WiCell hES Media or mTeSR1. WiCell hES media (made in-house) is significantly less expensive than commercially purchased mTeSR1. We have had success plating transduced fibroblast onto matrigel coated wells and feeding with WiCell media (no MEFs, no conditioned media). Then, switching to mTeSR1 once colonies start to emerge and picking colonies directly onto matrigel/mTeSR1 conditions for expansion and characterization.

Alternatively (traditional approach) the transduced cells are passaged onto mitotically inactivated MEF feeder cells, and fed every day with WiCell (or similar) hES cell media. Make fresh media, store at 4 deg C and use within 2 weeks. Make fresh media as needed.

*

Last revised August 28, 2012. Published December 10, 2012. This chapter should be cited as: Berggren, W.T., Lutz, M., and Modesto, V., General Spinfection Protocol (December 10, 2012), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.85.1, http://www​.stembook.org.