Effects of Type IV Collagen and Laminin on the Cryopreservation of Human Embryonic Stem Cells

Sun Jong Kim, Jong Hyuk Park, Jeoung Eun Lee, Jin Mee Kim, Jung Bok Lee,
Shin Yong Moon, Sung Il Roh, Chul Geun Kim, Hyun Soo Yoona

Division of Stem Cell Biology, Medical Research Center, MizMedi Hospital;
Department of Life Science,
College of Natural Sciences, Hanyang University;
Stem Cell Research Center, Seoul, Korea

Cryopreservation of hESCs

The undifferentiated hESC clumps were harvested for routine
passaging as described above and then transferred to the
freezing medium (90% SR with 10% dimethylsulfoxide
[DMSO]; frozen control group) with serial increments of
DMSO (0%, 2%, 4%, 6%, 8%, and 10%). Human type IV
collagen (Sigma, St. Louis) or human laminin (1, 2, and 5
µg/ml; Sigma) were added to the freezing medium in each
experimental group.

Thirty to 40 clumps of hESCs were
loaded into a straw (IMV Technologies, L’Aigle Cedex,
France) with slow freezing (at the rate of –1°C per minute
until –80°C, seeding at –7°C) using a cell freezer (CryoMagic,
Miraebiotech, Seoul, Korea) and then stored in liquid
nitrogen.

At least 48 hours after cryopreservation, the
hESC clumps were rapidly thawed in a water bath at 37°C,
and DMSO in the thawing medium was gradually diluted
(10%, 8%, 6%, 4%, 2%, and 0%). Thawed hESC clumps
were then washed twice in the hESC culture medium and
plated on a fresh feeder layer.