McLaughlin-McEwen Pluripotent Stem Cell Core Training Facility

PROTOCOLS AND STANDARD OPERATING PROCEDURES

Summary of Contents:

hESC feeding and passaging/splitting
1 day after thawing of hESCs, and everyday after that, the media should be changed to ensure good, healthy colonies. To feed cells, simply aspirate the old hESC media and replace with 3ml of fresh hESC media. 7-10 days later split cells (1:1-1:3).

Freezing hESCs
Freezing hESCs will allow you to create a stock for future use. In addition to this, it will allow for the collection of stocks of early passage cells.

MEF thawing and feeding
hESCs may be grown on feeders. The feeder cells provide the ES cells with different soluble factors required for successful maintenance of the cell line.

Irradiating, and plating MEFs
Before MEFs can be used with ES cultures, they must be irradiated to stop proliferation. The idea is to stop the cells from proliferating without killing them.

Freezing irradiated MEFs
It will always be necessary to maintain feeder cells frozen and ready for future use. Freezing can be done with unirradiated cells, but is possible with irradiated cells. In this fashion, it is possible to have a stock of irradiated MEFs, ready to plate and use as feeders. Freeze 3x106 cells/vial (ml).

MEF conditioned medium (MEF CM) preparation
hESCs are normally cultured in the presence of feeder cells. The purpose of these cells is to provide the embryonic stem cells with essential nutrients. At times hESCs are not grown in the presence of MEFs, but they still require the nutrients these cells provide. A way around this is to condition the standard media in the presence of MEFs. In this manner, the media used for the hESCs contains the nutrients produced by the MEFs, but the hESCs themselves are not grown on MEFs.

MEF CM PREPARATION

Irradiated MEFs in a T75 flask are used to condition standard hESC medium. CM is collected daily for 7 days, and further supplemented with bFGF. The CM is then filtered and aliquoted (modified from Geron Corportation, 2002).