CENTER FOR REGENERATIVE MEDICINE of BOSTON UNIVERSITY and BOSTON MEDICAL CENTER
670 Albany Street
nd
2 Floor
Boston, MA 02118
Tel: 617-414-2969
http://www.bumc.bu.edu/stemcells/
iPSC CORE
Freezing hiPSCs growing onto feeders
Ideally, hiPSCs to be frozen should be between 60-80% confluent and most of the colonies in
the well should be large and healthy. Usually, 1-2 wells of a 6-well plate are frozen per vial.
You will need:






Collagenase Type IV (StemCell Technologies Cat# 07909)
1x PBS (Life Technologies Cat# 14190-144)
Cell scraper (Corning Cat# 3010)
Cold hESC medium
Cold 2x Freezing medium (60% FBS, 20% hESC medium, 20% DMSO)
Cryovials
1. Label the number of cryovials needed (usually 1 vial per well of a 6-well plate) and
place them in a -20C freezer.
2. Aspirate the medium from the hiPSC culture.
3. Wash once with 2 ml of 1x PBS.
4. Add 1 ml of Collagenase per well of a 6-well plate and incubate the cells for 3 – 5
minutes at RT (in the hood).
5. Observe the cells under the microscope. Some of the colonies will start to curl up on
the edges.
6. Aspirate the Collagenase and wash twice with 2 ml of 1x PBS.
7. Add 0.5 ml of cold hESC medium.
8. Scrape the cells from the bottom of the well until the colonies are all floating. Do not
pipette up and down when freezing hiPSCs!
9. Add 0.5 ml of cold 2x freezing medium dropwise while mixing the cells in the plate.
670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.
10. Place the total 1 ml in a cold cryovial.
11. Place the cryovial in a MrFrosty container or in a Styrofoam rack at -80C overnight and
transfer to the -150C the next day.
12. Log the vial on FreezerPro.
670 Albany Street, 2nd Floor. Boston, MA 02118. 617-414-2969.