P. Schu et M. Reith, EVALUATION OF DIFFERENT PREPARATION PARAMETERS FOR THE PRODUCTION ANDCRYOPRESERVATION OF SEED CULTURES WITH RECOMBINANT SACCHAROMYCES-CEREVISIAE, Cryobiology, 32(4), 1995, pp. 379-388
In this article we describe the intensive investigation of defined phy
sicochemical parameters for their influence on the preparation, storag
e stability in liquid nitrogen, and the post-thaw productivity of a re
combinant strain of Saccharomyces cerevisiae expressing the human fact
or XIIIa protein, Preparation of industrially sized seed cultures and
their storage stability were monitored over more than a 1-year period.
Major parameters recorded before and after thawing were number of col
ony-forming units on agar slant media, plasmid retention, and expressi
on capacity of the recombinant protein. The viability of the cells aft
er reconstitution of the frozen batches was found to improve significa
ntly if a certain combination of prethaw growth conditions was applied
and amino acids were included in the cryoprotectant media. In additio
n, the influence of different concentrations of glycerol as a cryoprot
ectant was investigated and the specific freezing conditions which imp
roved the viability were identified. Even without complex freezing dev
ices for the controlled freezing of cultures, viability rates of 85% a
nd higher were obtained, enabling consistent production of the recombi
nant protein. (C) 1995 Academic Press, Inc.