X-Message-Number: 28851
Date: Sat, 6 Jan 2007 20:09:51 -0800 (PST)
From: 
Subject: membrane coating as an alternative to cryoprotectants

[ I had been wondering about the ability of the cold-adapted arctic
woollybear caterpillar gynaephora groenlandica to survive -70 C. (see
below) This was quite a remarkable feat considering that only 0.1 M
glycerol was detected in woollybear hemolymph. As -70 C would appear to
be below its tissue glass transition temperature, then presumably this
insect would survive also at liquid nitrogen temperatures, provided
further cooling was slow enough to avoid fracture.
  With cryoprotectants present only at low concentrations, other
mechanisms must be operative to enable such a very high degree of cold
hardening. Now along comes a paper describing the superiority of wheat
protein extracts over DMSO in protecting a wide variety of cell types
against freezing injury. This protein extract apparently coats cell
membranes, and somehow protected cells against freezing injury in the
absence of colligative cryoprotectants. Possibly this coating protected
cells by preventing ice formation at the membrane interface.
  Perhaps something similar is happening to those woollybear
caterpillars. The current focus on preserving humans organs at low
temperatures is by using vitrification. Progress has been glacial, due to
the toxicity of high concentrations of conventional cryoprotectants
used. Vitrification is an ultralow temperature version of supercooling. In
nature supercooling is used by animals only at very high sub-zero
temperatures. Animals that survive low temperatures allow most of their
body water to freeze, yet somehow they are not harmed by this. Mother
nature has had billions of years to work out how to enable survival at
low temperatures. Humans have been trying to achieve same for only a few
decades. I suspect that efforts at supercooling/vitrification may be
backing the wrong horse. Perhaps it is high time to check out the
alternative.]

Biotechnol Bioeng. 2006 Nov 5;95(4):661-70.
Wheat extracts as an efficient cryoprotective agent for primary cultures
of rat hepatocytes.
Hamel F, Grondin M, Denizeau F, Averill-Bates DA, Sarhan F.
Departement des Sciences Biologiques, Universite du Quebec a Montreal,
C.P. 8888, Succursale Centre-ville, Montreal, Quebec H3C 3P8, Canada.
  Hepatocytes are an important physiological model for evaluation of
metabolic and biological effects of xenobiotics. They do not proliferate
in culture and are extremely sensitive to damage during freezing and
thawing, even after the addition of classical cryoprotectants. Thus
improved cryopreservation techniques are needed to reduce cell injury and
functional impairment. Here, we describe a new and efficient
cryopreservation method, which permits long-term storage and recovery of
large quantities of healthy cells that maintain high hepatospecific
functions. In culture, the morphology of hepatocytes cryopreserved with
wheat protein extracts (WPE) was similar to that of fresh
cells. Furthermore, hepatospecific functions such as albumin secretion
and biotransformation of ammonium to urea were well maintained during 4
days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes
cryopreserved with WPEs were similar to those in fresh hepatocytes. These
findings clearly show that WPEs are an excellent cryopreservant for
primary hepatocytes. The extract was also found to cryopreserve other
human and animal cell types such as lung carcinoma, colorectal
adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA,
cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T
cell leukemia. WPEs have potential as a universal cryopreservant agent of
mammalian cells. It is an economic, efficient and non-toxic agent.
PMID: 16927246

[Further quote from the above paper:
"After cryopreservation, it is plausible that the WPE proteins have
aggregated at the cell membrane of the hepatocytes, giving rise to their
apparent microscopic opacity."]

J Comp Physiol [B]. 1988;158(2):175-83.
Glycerol metabolism in a freeze-tolerant arctic insect: an in vivo 13C
NMR study.Kukal O, Serianni AS, Duman JG.
Department of Biological Sciences, University of Notre Dame, Indiana 46556.
  Freeze-tolerance in larvae of Gynaephora groenlandica is enhanced by
the accumulation of glycerol in the winter. Since summer larvae remain
freeze-tolerant despite the lack of glycerol, we investigated glycerol
metabolism as a function of acclimation and body temperature using
noninvasive 13C NMR spectroscopy. Major constituents of hemolymph isolated
from cold- and warm-acclimated larvae were identified with the aid of
standard NMR spectra and confirmed by TLC and GLC. Spectra obtained on
live, warm-acclimated larvae showed the presence of lipids, glycogen,
glucose, trehalose and amino acids. Similar spectra of cold-acclimated or
previously frozen larvae showed the additional presence of glycerol. In
vitro time-lapse 13C spectra of D-[1-13C]glucose added separately to
hemolymph or extracted fat body tissue showed that glycerol is
synthesized from glucose in the fat body tissue and distributed to the
peripheral tissue via hemolymph. In vivo time-lapse 13C spectra of cold-
and warm-acclimated larvae were obtained after injection with
D-[1-13C]glucose to monitor the production of labeled metabolic
intermediates and end-products. [13C]Glycerol was produced between -30
degrees C and 30 degrees C but accumulated only below 5 degrees C. Above 5
degrees C glycerol was degraded and the 13C label incorporated mainly
into glycogen. The mechanism underlying temperature control of glycerol
biosynthesis and degradation may provide a clue to the role of glycerol
in enhancing freeze-tolerance in these insects.
PMID: 3170824

[Further quote from the above paper:
"However, cold-acclimation (-15 C, -30 C) enhanced cold tolerance in the
larvae so they were able to tolerate temperatures lower than -70 C (the
lowest temperature tested, no mortality resulted;"]

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