X-Message-Number: 32719
Date: Sun, 18 Jul 2010 10:07:27 -0700 (PDT)
From: 
Subject: EGCG enables freeze-drying of human cells


The addition of EGCG to help induce freezing and drying tolerance has increased 
human cell viability to about 90%. I think cryonics should take a good look at 
how this was achieved below. The advantages of this optimized freeze-drying 
process are as follows:

1. superior (90%) cell viability
2. no recrystalation damage upon thawing
3. tolerance for high storage temperatures


As a consequence of (3) optimized freeze-drying offers the potential for very 
long term storage. Currently due to economic constraints, cryonics suffers from 
the disadvantage of negligible chances for successful storage of human remains 
in a viable condition for hundreds of years. In principle, optimized 
freeze-drying should not be vulnerable to such a limitation.


I realize that nobody is going to switch from freezing to freeze-drying over 
night on the basis of one experiment. However it is apparent from this as well 
as other research that further investigations into ECGC, or flavonoids in 
general is long overdue. Indeed, it may well turn out that successful 
vitrification of human organs will in future depend on flavonoid additions to 
vitrifcation solutions.

PLoS One. 2009;4(4):e5240. Epub 2009 Apr 21.

Freeze-drying of mononuclear cells derived from umbilical cord blood followed by
colony formation.
Natan D, Nagler A, Arav A. Core Dynamics Ltd, Ness-Ziona, Israel.
Abstract

    BACKGROUND: We recently showed that freeze-dried cells stored for 3 years at
    room temperature can direct embryonic development following cloning. 
    However, viability, as evaluated by membrane integrity of the cells after 
    freeze-drying, was very low; and it was mainly the DNA integrity that was 
    preserved. In the present study, we improved the cells' viability and 
    functionality after freeze-drying. METHODOLOGY/PRINCIPAL FINDINGS: We 
    optimized the conditions of directional freezing, i.e. interface velocity 
    and cell concentration, and we added the antioxidant EGCG to the freezing 
    solution. The study was performed on mononuclear cells (MNCs) derived from 
    human umbilical cord blood. After freeze-drying, we tested the viability, 
    number of CD34(+)-presenting cells and ability of the rehydrated 
    hematopoietic stem cells to differentiate into different blood cells in 
    culture. The viability of the MNCs after freeze-drying and rehydration with 
    pure water was 88%-91%. The total number of CD34(+)-presenting cells and the
    number of colonies did not change significantly when evaluated before 
    freezing, after freeze-thawing, and after freeze-drying (5.4 x 10(4)+/-4.7, 
    3.49 x 10(4)+/-6 and 6.31 x 10(4)+/-12.27 cells, respectively, and 
    31+/-25.15, 47+/-45.8 and 23.44+/-13.3 colonies, respectively). CONCLUSIONS:
    This is the first report of nucleated cells which have been dried and then 
    rehydrated with double-distilled water remaining viable, and of 
    hematopoietic stem cells retaining their ability to differentiate into 
    different blood cells.
PMID: 19381290
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667668/pdf/pone.0005240.pdf


[Unlike freeze-dried cells, frozen cells are very vulnerable to increases in 
storage temperature above liquid nitrogen temperatures.]

Exp Clin Transplant. 2009 Mar;7(1):50-5.

The effect of freezing on the recovery and expansion of umbilical cord blood 
hematopoietic stem cells.

Beshlawy AE, Metwally HG, Khalek KA, Hammoud RF, Mousa SM. Pediatric Department,
Kasr Al-Aini School of Medicine, Cairo University, Cairo, Egypt.
Abstract

    OBJECTIVES: Cell populations residing in waste tissues (cord blood, 
    umbilical cord, and placenta) may be collected without any medical or 
    ethical contraindications concerning the mother or newborn baby. Cord blood 
    hematopoietic stem cells are routinely used for clinical transplants; 
    however, the low cell dose of the graft limits their therapeutic efficacy as
    it is associated with increased delayed or failed engraftment. The cell 
    dose can be increased, and the efficacy of cord blood transplant potentially
    improved, by ex vivo expansion before transplant. MATERIALS AND METHODS: 
    Twelve umbilical cord blood samples were included. The effect of cord blood 
    storage at -80 degrees C on CD34+ cell count (mean -/+ standard deviation 
    [SD]), cell viability (mean -/+ SD percent), and cell cycle status (percent 
    quiescent versus dividing) was estimated. Ex vivo culture of cord blood 
    mononuclear cells was done before storage, and after 1 week of freezing, and
    after 2 weeks of freezing. Ex vivo liquid culture was performed with media 
    supplemented with stem cell factor, interleukin-3 (IL-3), and both. RESULTS:
    The count of CD34+ cells in pre-expansion aliquots decreased from 15.00 +/-
    9.96 x 106 cells before freezing to 7.70 -/+ 3.20 x 106 cells after 2 weeks
    of freezing (P = .024). Cell viability in pre-expansion aliquots decreased 
    from 99.5% -/+ 1.0% before freezing, to 52.5% -/+ 27.5% after 1 week of 
    freezing (P = .013) and to 32.5% -/+ 9.5% after 2 weeks of freezing (P = 
    .001). Mean fold of cell expansion and proportion of quiescent versus 
    dividing cells did not change significantly from before to after freezing, 
    and was not significantly different for culture with stem cell factor, IL-3,
    or both. CONCLUSION: Although freezing decreased cell count and viability, 
    it did not impair the expansion potential of cord blood hematopoietic cells.
    Whether IL-3 or stem cell factor should be considered as essential 
    components of expansion media is uncertain.
PMID: 19364313
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