X-Message-Number: 33243
Date: Sat, 15 Jan 2011 22:42:18 -0800 (PST)
Subject: a dehydrating moss gathers no ice

  Could dehydration finally put vitrification over the top? Full
vitrification without any ice formation is not currently available due to
cryoprotectant toxicity. Faced with the Scylla of ice formation on the
one hand and the Charybdis of cryoprotectant toxicity on the other, even
the best cryobiologist navigators have failed to avoid numerous
shipwrecks. However according to myth, Aeneas was able to bypass the
deadly strait altogether. Was Aeneas onto something? Below a moss avoids
freezing by dehydrating first. Quote: "an elegantly simple solution to
the problem of freezing; remove that which freezes". It is not widely
recognized that human skin has the ability to survive complete
desiccation, and in this regard rivals anhydrobiotic organisms. Not all
human cells can be expected to survive complete desiccation, though
partial dehydration is not a problem. Similarly human cells can survive
non-vitrifiable concentrations of some cryoprotectants. I would like to
propose a novel solution to the problem of cryoprotectant
toxicity. Perfuse first with safe non-vitrifiable concentrations of
cryoprotectants, then partially dehydrate tissue to fully prevent the
formation of any ice crystals.
  One possible method for dehydration might be a second perfusion with a
high molar PEG solution. PEG solution does not penetrate tissue, due to
its high molecular weight, but it is highly hygroscopic and can remove
moisture. The goal would be to achieve vitrifiable concentrations of
cryoprotectants in tissue, by gradual dehydration, but leave the absolute
level of cryoprotectants in the tissue below toxic levels. An example of
an extreme case would be human skin, where no cryoprotectants are needed,
since there is no water left.


Plant Cell Environ. 2010 Oct;33(10):1731-41. doi: 
Freeze avoidance: a dehydrating moss gathers no ice.

LennAC T, Bryant G, Hocart CH, Huang CX, Ball MC. Plant Science Division, 
Research School of Biology, The Centre for Advanced Microscopy, The Australian 
National University, Canberra, Australia.

  Using cryo-SEM with EDX fundamental structural and mechanical properties of 
  the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to 
  tolerance of freezing temperatures. In contrast to more complex plants, no ice
  accumulated within the moss during the freezing event. External ice induced 
  desiccation with the response being a function of cell type; water-filled 
  hydroid cells cavitated and were embolized at -4 A C while parenchyma cells of
  the inner cortex exhibited cytorrhysis, decreasing to a   20% of their 
  original volume at a nadir temperature of -20 A C. Chlorophyll fluorescence 
  showed that these winter acclimated mosses displayed no evidence of damage 
  after thawing from -20 A C while GCMS showed that sugar concentrations were 
  not sufficient to confer this level of freezing tolerance. In addition, 
  differential scanning calorimetry showed internal ice nucleation occurred in 
  hydrated moss at a  -12 A C while desiccated moss showed no evidence of 
  freezing with lowering of nadir temperature to -20 A C. Therefore the rapid 
  dehydration of the moss provides an elegantly simple solution to the problem 
  of freezing; remove that which freezes.
AC 2010 Blackwell Publishing Ltd.
PMID: 20525002

Transplantation. 2006 Jun 15;81(11):1583-8.

Human skin preserved long-term in anhydric pulverized sodium chloride retains 
cell molecular structure and resumes function after transplantation.

Olszewski WL, Moscicka M, Zolich D. Department of Surgical Research and 
Transplantology, Medical Research Center, Polish Academy of Sciences, Warsaw, 

  BACKGROUND: Human skin is needed to cover large areas of the body lost through
  burns, trauma, and extensive maxillofacial surgery. Contemporary methods of 
  skin storage are limited by the period of preservation to a few days. Our 
  previous findings showed that fixation and storage of human skin in anhydric 
  sodium chloride at room temperature for weeks or months preserves its 
  morphological and molecular structure. In this study, we examined whether skin
  grafts preserved in sodium chloride may be successfully transplanted.

METHODS: Skin was harvested from lower limbs of patients during elective 
surgery, placed in containers with anhydric salt powder, and kept at 22 degrees 
C for 3 to 12 weeks. Desalination and rehydration took place before 
transplantation. Desalinated fragments were transplanted onto the dorsum of scid

RESULTS: All grafts were accepted by recipients. Three weeks after 
transplantation, keratinocytes synthesized keratins 10, 16, and 17 and expressed
antigens specific for stem (p63) and transient (CD29) cells. Moreover, they 
proliferated vigorously, their basal layer cells incorporated bromdeoxyuridine 
and expressed proliferative cell nuclear antigen. Isolated from transplants and 
cultured in vitro, they remained viable and produced enzymes. Dermis retained 
its structure and expressed fibroblast-specific antigen. All graft cells 
remained human leukocyte antigen I.

CONCLUSION: Human skin preserved in anhydric sodium chloride at room temperature
for months can be successfully transplanted to scid mice. We propose the 
concept of "spore-like" keratinocyte stem cells to explain the long-term ex vivo
survival of keratinocytes. The mechanism of survival of fibroblasts remains to 
be determined.
PMID: 16770248

Ann Transplant. 2004;9(4):37-9.

Human skin preserved in anhydric sodium chloride for months can be successfully 

Olszewski WL, Moscicka M, Zolich D. Department of Surgical Research and 
Transplantology, Medical Research Center, Polish Academy of Sciences, Warsaw, 

  Human skin can be preserved in pulverized sodium chloride dehydrated at 240C 
  for 2 hours at room temperature for periods of weeks or months and 
  successfully transplanted to scid mouse, retaining its normal morphological 
  structure. Fragment of skin of a size of 10 x 10 x 6 mm were harvested during 
  elective vascular and orthopaedic surgery of lower limbs, dried of blood and 
  placed in anhydric sodium chloride powder in tight sealed containers. Prior to
  transplantation to scid mice, the specimens were desalinated and rehydrated. 
  Specimes preserved for 1 to 6 months and harvested 3-4 weeks after 
  transplantation revealed intensive incorporation of bromdeoxyuridine (BrdU) 
  into basal keratinocytes. They expressed p63 and CD29 (stem cells, and 
  transient cells antigens), PCNA (proliferating cell nuclear antigen) and 
  cytokeratin 16 specific for proliferating keratinocytes. Dermal fibroblasts 
  and few large HLA II cells showed a normal structure. Bacterial flora of skin 
  did not change after grafting. We conclude that human skin can survive in a 
  dehydrated state in sodium chloride for months and after transplantation the 
  epidermal basal layer cells give rise to keratinocyte progenies. Skin 
  fibroblasts and some resident immune cells can also survive.
PMID: 15884435

Cryobiology. 2001 Sep;43(2):182-7.
Recovery of human mesenchymal stem cells following dehydration and rehydration.

Gordon SL, Oppenheimer SR, Mackay AM, Brunnabend J, Puhlev I, Levine F. Osiris 
Therapeutics, Inc., 2001 Aliceanna Street, Baltimore, MD 21231-3043, U.S.A.

  As cell therapies advance from research laboratories to clinical application, 
  there is the need to transport cells and tissues across long distances while 
  maintaining cell viability and function. Currently cells are successfully 
  stored and shipped under liquid nitrogen vapor. The ability to store these 
  cells in the desiccated state at ambient temperature would provide tremendous 
  economic and practical advantage. Human mesenchymal stem cells (hMSCs) have 
  broad potential uses in tissue engineering and regeneration since they can 
  differentiate along multiple lineages and support hematopoeisis. The current 
  research applied recent technological advances in the dehydration and storage 
  of human fibroblasts to hMSCs. Three conditions were tested: air-dried, 
  air-dried and stored under vacuum (vacuum only), and incubated with 50 mM 
  trehalose + 3% glycerol and then air-dried and stored under vacuum (vacuum + 
  trehalose). Plates containing dehydrated hMSCs were shipped from San Diego to 
  Baltimore overnight in separate FedEx cardboard boxes. The hMSCs were 
  rehydrated with 3 ml of hMSC medium and were able to regain their 
  spindle-shaped morphology and adhesive capability. In addition, they 
  maintained high viability and proliferation capacity. Rehydrated and passaged 
  cells continued to express the characteristic hMSC surface antigen panel. 
  Additionally, cells showed constitutive levels of mRNA for a stromal factor 
  and, when exposed to reagents known to induce differentiation, demonstrated 
  upregulation of two tissue-specific messages indicative of differentiation 
  potential for fat and bone. While our preliminary findings are encouraging, we
  still need to address consistency and duration of storage by considering 
  factors such as cell water content, oxygen concentration, and the presence of 
  free radicals.
PMID: 11846472

As with anhydrobiotic organisms, humans cells require gradual desiccation in 
order to maintain viability.

J Physiol. 2004 Jul 1;558(Pt 1):181-91. Epub 2004 May 14.

Response of human cells to desiccation: comparison with hyperosmotic stress 

Huang Z, Tunnacliffe A. Institute of Biotechnology University of Cambridge, 
Tennis Court Road, Cambridge CB2 1QT, UK.
Comment in:  J Physiol. 2004 Jul 1;558(Pt 1):3.

  Increasing interest in anhydrobiosis ('life without water') has prompted the 
  use of mammalian cells as a model in which candidate adaptations suspected of 
  conferring desiccation tolerance can be tested. Despite this, there is no 
  information on whether mammalian cells are able to sense and respond to 
  desiccation. We have therefore examined the effect of desiccation on stress 
  signalling pathways and on genes which are proposed to be expressed in 
  response to water loss through osmotic stress. Depending on the severity of 
  the drying regime, human cells survived for at least 24 h. Both SAPK/JNK and 
  p38 mitogen-activated protein kinases (MAPKs) were activated within 30 min by 
  desiccation as well as by all osmotica tested, and therefore MAPK pathways 
  probably play an important role in both responses. Gene induction profiles 
  differed under the two stress conditions, however: quantitative polymerase 
  chain reaction (PCR) experiments showed that AR, BGT-1 and SMIT, which encode 
  proteins governing organic osmolyte accumulation, were induced by 
  hypersalinity but not by desiccation. This was surprising, since these genes 
  have been proposed to be regulated by ionic strength and cell volume, both of 
  which should be significantly affected in drying cells. Further investigation 
  demonstrated that AR, BGT-1 and SMIT expression was dependent on the nature of
  the osmolyte. This suggests that their regulation involves factors other than
  intracellular ionic strength and cell volume changes, consistent with the 
  lack of induction by desiccation. Our results show for the first time that 
  human cells react rapidly to desiccation by MAPK activation, and that the 
  response partially overlaps with that to hyperosmotic stress.
PMID: 15146043
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Nat Rev Gastroenterol Hepatol. 2010 Oct;7(10):557-64. Epub 2010 Aug 24.
The safety of osmotically acting cathartics in colonic cleansing.

Nyberg C, Hendel J, Nielsen OH. Department of Gastroenterology, Medical Section,
Herlev Hospital, University of Copenhagen, 75 Herlev Ringvej, DK-2730 Herlev, 

  Efficient cleansing of the colon before a colonoscopy or a radiological 
  examination is essential. The osmotically acting cathartics (those given the 
  Anatomical Therapeutic Chemical code A06AD) currently used for this purpose 
  comprise products based on three main substances: sodium phosphate, 
  combinations of polyethylene glycol and electrolyte lavage solutions 
  (PEG-ELS), and magnesium citrate. All these preparations give adequate 
  cleansing results and have similar profiles in terms of the frequency and type
  of mild to moderate adverse effects. However, serious adverse events, such as
  severe hyperphosphatemia and irreversible kidney damage owing to acute 
  phosphate nephropathy, have been reported after use of sodium-phosphate-based 
  products. The aim of this Review is to provide an update on the potential 
  safety issues related to the use of osmotically acting cathartics, especially 
  disturbances of renal function and water and electrolyte balance. The 
  available evidence indicates that PEG-ELS-based products are the safest 
  option. Magnesium-citrate-based, hypertonic products should be administered 
  with caution to elderly individuals and patients who are prone to develop 
  disturbances in water and electrolyte balance. Sodium-phosphate-based products
  can occasionally cause irreversible kidney damage and should not be routinely
  used in bowel-cleansing procedures.
PMID: 20736921

J Biochem. 2008 Jun;143(6):841-7.

CH2-units on (poly-)ethylene glycol radially dehydrate cytoplasm of resting 
skinned skeletal muscle.

Kimura M, Takemori S. Department of Molecular Physiology, Jikei University 
School of Medicine, Minato-ku, Tokyo 105-8461, Japan
Corrected and republished from:  J Biochem. 2008 Jan;143(1):123-9.

  Observing the optical cross-section and electron micrographs of mechanically 
  skinned fibres of frog skeletal muscle, we found that ethylene glycols (EGs) 
  of small (mono-, di-, tri- and tetra-EGs; M(r) 62-194) and medium (poly-EGs; 
  M(r) 900 and 3350) molecular weights efficiently dehydrate the fibres to 
  shrink them radially without microscopic inhomogeneity. The medium-sized 
  poly-EGs at 30% weight/weight concentration absorbed almost all the evaporable
  water from the fibre. Passive tension measurement at near slack sarcomere 
  spacing indicated that this dehydration by EGs did not accompany longitudinal 
  fibre shrinkage. Chemically relevant fully hydric alcohols (glycerol, 
  threitol, ribitol and mannitol; M(r) 92-182) showed no appreciable dehydrating
  ability on fibres. An intimate correlation was found between fibre 
  dehydration and CH(2)-concentration of the solutions. Viscosity measurements 
  indicated that the hydrodynamic radii of the alcohols were comparable to those
  of the small EGs. Therefore, hydrodynamic radii are not a primary determinant
  of the dehydrating ability. Additionally, CH(2)-concentration of EGs but not 
  alcohols was found to correlate intimately with the measured viscosity of the 
  bulk solution of EGs. These results suggested that the interaction between 
  water molecules and CH(2)-units in crowded cytoplasm of skeletal muscle 
  affects cytoplasm as a whole to realize anisotropic fibre shrinkage.
PMID: 18583358

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