X-Message-Number: 33081
Date: Fri, 26 Nov 2010 23:32:35 -0800 (PST)
From:
Subject: Successful restoration of function of frozen and thawed isola...
Elami et al used a directional freezing device from Core Dynamics (see below).
The full research report stated concern over cryoprotectant toxicity. Perhaps
somebody might wish to have a talk with them about donating some vitrification
solution for their use.
J Thorac Cardiovasc Surg. 2008 Mar;135(3):666-72, 672.e1.
Successful restoration of function of frozen and thawed isolated rat hearts.
Elami A, Gavish Z, Korach A, Houminer E, Schneider A, Schwalb H, Arav A. The
Department of Cardiothoracic Surgery and the Joseph Lunenfeld Cardiac Surgery
Research Center, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Abstract
OBJECTIVE: Long-term organ preservation for transplantation may allow
optimal donor-recipient matching with potential reduction in the incidence
and severity of rejection. Complete cessation of metabolism may be obtained
by freezing. Previous attempts to freeze intact mammalian hearts were
limited to -3.6 degrees C, restricting tissue ice content to 34%. We
hypothesized that our method will allow recovery of function of the intact
rat heart after freezing to -8 degrees C, a temperature at which most of the
tissue water is frozen.
METHODS: Isolated rat hearts were attached to a Langendorff apparatus. After
normothermic perfusion, cold cardioplegia was induced followed by perfusion with
a cryoprotecting agent. Hearts were than frozen to -8 degrees C (45 +/- 8
minutes), thawed, and reperfused (60 minutes).
RESULTS: All frozen and thawed hearts regained normal electric activity. At -8
degrees C, ice content was 64.36% +/- 13%. The use of 10% ethylene glycol for
cryoprotection (n = 13) resulted in recovery (mean +/- standard deviation) of
49.7% +/- 21.8% of +dP/dt, 48.0% +/- 23.5% of -dP/dt, 65.2% +/- 30.8% of
coronary flow, and 50.4% +/- 23.9% of left ventricular developed pressure.
Hearts in this group (n = 4) maintained 81.3% +/- 10% viability compared with
69.3% +/- 14% (not significant) in control hearts kept at 0 degrees C for the
same duration. Energy stores, represented by adenosine triphosphate and
phosphocreatine, were depleted to 12.2 +/- 6.1 micromol/g dry weight and 22.5
+/- 6.4 micromol/g dry weight, respectively, compared with 19.0 +/- 2.5
micromol/g dry weight and 36.6 +/- 3.0 micromol/g dry weight, respectively (P <
.05) in the control hearts. The integrity of muscle fibers and intracellular
organelles after thawing and reperfusion was demonstrated by electron
microscopy.
CONCLUSION: We demonstrate for the first time the feasibility of functional
recovery after freezing and thawing of the isolated rat heart while maintaining
structural integrity and viability.
PMID: 18329491
____________________________________________
Directional freezing does not use a varying magnetic field to suppress the
formation of large ice crystals. I wonder whether there might not be a synergism
between these two technologies.
http://www.coredynamics.com/
Cell Preservation
using Core Dynamics' Cryopreservation Technologies
Freeze dried and reconstituted leukocytes
Core Dynamics is developing new methods for freezing, thawing and freeze-drying
cells. The company's unique freezing and thawing technology allows precise
control, and in a repeatable process, with high recovery, survival and function
of cells after thawing.
Cells, preserved with the Company's technologies and specially developed
protocols can be stored for long time periods at room temperature and
subsequently rehydrated with resultant high rates of cell viability and
functionality. The company's preservation technologies are already providing
means for cell storage of cell types that are being studied for therapeutic
purposes.
This breakthrough is based in part on the innovative freezing technology called
Multi Thermal Gradient (MTGT) freezing developed by the company. This freezing
method, also known as directional freezing, achieves low temperature freezing
without the use of glycerol or DMSO, the additives commonly used today to freeze
cells.
Core Dynamics has expanded the scope of its basic directional freezing
technology to include a number of different freezing methods adapted to
different materials. The directional freezing, coupled with the Company's
freeze-drying technology, are making it possible to freeze-dry a variety of cell
types.
Core Dynamics provides companies and institutions the opportunity to collaborate
on the development of cell preservation methods and protocols to suit specific
cell types.
Company Profile
>From science fiction to science that reads like fiction, the field of
cryopreservation has captured the imagination of many. We are fascinated by
natural phenomena such as the wood frog (Rana sylvatica), which can freeze
during its winter hibernation when 35-45% of the frog's body may turn to ice.
Or, for example certain, Antarctic fish that thrive in ice water and
tardigrades, tiny 1mm invertebrates that can freeze in winter and completely dry
out during the summer.
Core Dynamics is a biotechnology company that is stretching the horizons of the
science of cryopreservation. The company is active in the research and
development of unique freezing, thawing and freeze drying technologies that are
being applied in work with cell preservation, blood transfusion, and tissue and
organ transplantation. The company has developed unique protocols, equipment and
solutions for cryopreservation of cells, of tissues such as osteo-articular
cartilage and of whole organs.
Core Dynamics hopes to develop additional applications through research
partnerships involving its advanced cryopreservation technologies.
Core Dynamics' Research and Development center is located in Ness Ziona, Israel,
with a staff of over 30 research scientists and support personnel. Company
management has wide experience in the fields of medicine, cryopreservation and
cell biology. The company's commercial offices are located in Rockville,
Maryland.
Research in Freezing, Thawing and Freeze-Drying
Core Dynamics' research has led to the successful freezing and lyophilization of
white blood cells and stem cells. The ability to preserve and store cells at
room temperature, thereby negating the logistical problems involved in
transportation and storage in freezers and liquid nitrogen will open a world of
possibilities for research and work with many varieties of cells.
Applying the company's MTG freezing technology and freezing protocols to
cartilage has led to the successful long term preservation of osteo-articular
allograft plugs. In a series of clinical trials over the past year preserved
plugs have been transplanted in patients suffering from cartilage damage in the
knee.
In the area of whole organs Core Dynamics has successfully transplanted frozen
thawed ovaries in large animals. In the near future this technology will be
applied to human ovaries for the purpose of fertility preservation in young
female patients undergoing chemo and radiotherapy.
Core Dynamics is confident that supplies of lyophilized (freeze dried) cells and
preserved tissues and organs screened for contamination will revolutionize the
field of cell therapy, the blood transfusion and transplantation industries and
other research involving a wide variety of cells.
__________________________________
Reprod Biomed Online. 2010 Jan;20(1):48-52. Epub 2009 Oct 31.
Ovarian function 6 years after cryopreservation and transplantation of whole
sheep ovaries.
Arav A, Gavish Z, Elami A, Natan Y, Revel A, Silber S, Gosden RG, Patrizio P.
Institute of Animal Science, Agricultural Research Organization, Volcani Center,
Bet Dagan, Israel.
Abstract
Whole ovary cryopreservation and transplantation has been proposed as a
method for preserving long-term ovarian function. This work reports ovarian
function 6years post transplantation of frozen-thawed whole sheep ovaries.
Three 9-month-old Assaf sheep underwent unilateral oophorectomy to provide
organs for the experiments. After perfusing with cold University of
Wisconsin solution supplemented with 10% dimethyl sulphoxide, ovaries were
cryopreserved using unidirectional solidification freezing technology. After
thawing, ovaries were re-perfused and re-transplanted orthotopically by
microvascular re-anastomosis, to the contralateral ovarian pedicle after
removing the remaining ovary. Six years following transplantation and after
inducing superovulation, the sheep were killed and the ovaries analysed. Two
ovaries had normal size and shape showing some recent corpora lutea, while
the third showed atrophic changes. A total of 36 antral follicles were
counted by transillumination and four germinal vesicle oocytes were
aspirated and matured in vitro to metaphase II. Serum progesterone
concentrations were indicative of ovulatory activity in one of the three
sheep. Histological evaluations revealed normal tissue architecture, intact
blood vessels and follicles at various stages. Currently, this is the
longest recorded ovarian function after cryopreservation and
re-transplantation. Cryopreservation of whole ovaries, using directional
freezing combined with microvascular anastomosis, is a promising method for
preserving long-term reproductive capacity and endocrine function.
Copyright (c) 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All
rights reserved.
PMID: 20158987
Rejuvenation Res. 2008 Aug;11(4):765-72.
Cryopreservation of whole murine and porcine livers.
Gavish Z, Ben-Haim M, Arav A. Core Dynamics Ltd., Nes Ziona, Israel.
Abstract
Preservation of vascularized organs, such as the liver, is limited to 24 h
before destructive processes disqualify them for transplantation. This
narrow window of opportunity prevents the performance of optimal pathogen
screening and matching tests and possibly results in the need for
retransplantation. Numerous problems are associated with freezing and
thawing a whole liver while preserving its viability upon thawing, including
complicated geometry, poor heat transfer, release of latent heat, and the
difficulty of generating a uniform cooling rate. On the basis of our past
success with sheep ovaries, we have now applied our novel freezing technique
to a larger solid organ, the liver. Whole rat and pig livers were frozen
and thawed using directional solidification apparatus, and viability of
these livers was tested by means of integrity and functionality in vitro and
in auxiliary liver transplantation. The thawed rat and porcine livers were
intact and demonstrated >80% viability. Histology revealed normal
architecture. Bile production and blood flow following auxiliary
transplantation were normal as well. Our encouraging results in applying
this novel cryopreservation technique in rat and pig livers suggest that
this method may enable better human organ donor-recipient matching in the
future.
PMID: 18729808
Semin Reprod Med. 2009 Nov;27(6):438-42. Epub 2009 Oct 5.
Directional freezing: a solution to the methodological challenges to preserve
large organs.
Arav A, Natan Y. Animal Science, Beit Dagan, Israel.
Abstract
Although 60 years have passed since the first successful freezing of cells,
whole organ freezing is in its initial phase. Heat and mass transfer has
limited the success of large tissue and organ cryopreservation either by
slow freezing or vitrification. In this article we discuss the limiting
factors for the successful freezing of whole organs, such as homogeneous
cooling rate, supercooling, latent heat, and recrystallization. We show how
the use of directional freezing technology provides solutions to these
problems. Whole ovary cryopreservation and transplantation has been proposed
as a new method for preserving long-term ovarian function as opposed to
ovarian cortical slices. Fertility preservation will benefit from the
success of whole ovary freezing and transplantation.
Thieme Medical Publishers.
PMID: 19806511
J Endod. 2010 Aug;36(8):1336-40. Epub 2010 Jun 23.
Effects of cryopreservation of intact teeth on the isolated dental pulp stem
cells.
Lee SY, Chiang PC, Tsai YH, Tsai SY, Jeng JH, Kawata T, Huang HM. School of
Dentistry, Taipei Medical University, Taipei, Taiwan.
Abstract
INTRODUCTION: Human dental pulp stem cells (DPSCs) have been reported to be
useful material for future regenerative medicine. Clinically,
cryopreservation of intact teeth can successfully preserve the periodontal
ligament for future autotransplantation; however, the effects of
cryopreservation procedure on the properties of DPSCs are still unclear. The
aim of this study was to test whether DPSCs isolated from cryopreserved
teeth can express stem cell-specific markers.
METHODS: In this study, a novel programmable freezer coupled to a magnetic field
was used to perform the cryopreservation experiments. The tested DPSCs were
isolated from magnetically cryopreserved and non-cryopreserved fresh teeth with
an enzyme digestion procedure. The success rate of isolation, growth curves,
morphology, stem cell-specific markers, and the differentiation capacity of the
isolated cells were evaluated and compared.
RESULTS: The isolation rate of dental pulp cells from magnetically cryopreserved
teeth was 73%. After culture for 5 generations, there was no significant
difference in cell viability between cells isolated from magnetically
cryopreserved teeth and those isolated from fresh teeth. There were also no
visible differences between the 2 groups of dental pulp cells in morphology,
expression of stem cell markers, or osteogenic and adipogenic differentiations.
CONCLUSIONS: The results suggest that cryopreserved whole teeth can be used for
autotransplantation and provide a viable source of DPSCs.
Copyright 2010 American Association of Endodontists. Published by Elsevier Inc.
All rights reserved.
PMID: 20647092
Cryobiology. 2010 Aug;61(1):73-8. Epub 2010 May 15.
Cryopreservation of periodontal ligament cells with magnetic field for tooth
banking.
Kaku M, Kamada H, Kawata T, Koseki H, Abedini S, Kojima S, Motokawa M, Fujita T,
Ohtani J, Tsuka N, Matsuda Y, Sunagawa H, Hernandes RA, Ohwada N, Tanne K.
Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima
University Graduate School of Biomedical Science, 1-2-3 Kasumi, Minami-ku,
Hiroshima 734-8553, Japan.
Abstract
The purpose of this study was to establish a long-term tooth
cryopreservation method that can be used for tooth autotransplantation.
Human periodontal ligament (PDL) cells were frozen in 10% dimethyl sulfoxide
(Me(2)SO) using a programmed freezer with a magnetic field. Cells were
cryopreserved for 7 days at -150 degrees C. Immediately after thawing, the
number of surviving cells was counted and the cells were cultured; cultured
cells were examined after 48 h. Results indicated that a 0.01 mT of a
magnetic field, a 15-min hold-time, and a plunging temperature of -30
degrees C led to the greatest survival rate of PDL cells. Based on these
findings, whole teeth were cryopreserved under the same conditions for 1
year. The organ culture revealed that the PDL cells of cryopreserved tooth
with a magnetic field could proliferate as much as a fresh tooth, although
the cells did not appear in the cryopreserved tooth without a magnetic
field. Histological examination and the transmission electron microscopic
image of cryopreserved tooth with a magnetic field did not show any
destruction of cryopreserved cells. In contrast, severe cell damage was seen
in cells frozen without a magnetic field. These results indicated that a
magnetic field programmed freezer is available for tooth cryopreservation.
(c) 2010 Elsevier Inc. All rights reserved.
PMID: 20478291
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