Estudio de la cinética de la crioconservación de tejidos vegetale

González Benito, Elena

Molina García, Antonio Diego

Schneider Teixeira, Aline

Servicio de Coordinación de Bibliotecas de la Universidad Politécnica de Madrid

[4] Fahy GM et al.. Cryopreservation of organs by vitriﬁcation:perspectives and recent
advances. Cryobiology 2004;48(2):157–78.
doi:10.1016/j.cryobiol.2010.10.030
Biophysics of cryobiology
27. Progress of vitriﬁcation: From single cell to multiple objects. L.L. Kuleshova * 1,
S.S. Gouk 1, R. Magalhaes 1, F. Wen 1, Y.N. Wu2, G.S. Dawe 3,4, H. Yu 5,6, 1 Low
Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin
School of Medicine, National University of Singapore, Singapore, 2Department of
Orthopaedic Surgery, NUS Tissue Engineering Program, Yong Loo Lin School of
Medicine, National University of Singapore, Singapore, 3Department of
Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore, 4Neurobiology and Ageing, Centre for Life Sciences, National University of
Singapore, Singapore, 5Department of Physiology, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore, 6 Institute of Bioengineering and
Nanotechnology, Agency for Science, Technology and Research, Singapore
Vitriﬁcation achieves cryopreservation without the formation of ice crystals, and
therefore beneﬁts cells and tissues known to be vulnerable to ice damage during con-
ventional freezing. It is widely reported that oocytes, stem cells and other sensitive
cells are adversely affected by freezing. Vitriﬁcation offers an improvement in both
survival and non-lethal cell injury. Several areas of life sciences can signiﬁcantly ben-
eﬁt from development of vitriﬁcation strategy. Vitriﬁcation of single object is now
very well understood. My success on development of vitriﬁcation strategy leading
to the world-ﬁrst live birth from a vitriﬁed oocyte is the last step in the line. This
was accomplished 100 years after vitriﬁcation as a concept was proposed in physics,
namely in 1898. Advancement in the area leads to development of vitriﬁcation strat-
egies for multiple objects.
We developed vitriﬁcation of multiple objects such as neuronal stem cells in their
spherical form and more complex tissue engineered constructs involving hepatocytes
for urgent metabolic support during liver failure. The formulation of improved vitri-
ﬁcation solutions is an integral part in developing this vitriﬁcation strategy. To
achieve this goal, microencapsulated hepatocytes and hepatocyte spheroids were
used as the model systems. In regards to implementation of this new cryopreserva-
tion strategy in clinical practice, we were the ﬁrst to originate the idea that serum/
proteins are not essential during vitriﬁcation, and published successful results which
eliminating their usage; this is signiﬁcant because addition of serum and proteins
may induce differentiation during passaging or compromise the sterility of stem cells
if they are used for therapeutic transplantation. We also proved efﬁcacy of this strat-
egy for a platform technology involving human adult stem cells. As we have achieved
vitriﬁcation of large number of neurospheres sufﬁcient for human treatment, we
envision feasibility of vitriﬁcation of mesenchymal stem cells in large quantities.
We reported a way to achieve effective preservation of large constructs involving pri-
mary cells used as a part of bioartiﬁcial liver device. Cryobiology of tissue-engineered
complexes has now been developed in my laboratory, and this research will provide a
vital therapeutic opportunity.
We attempted to conﬁrm that vitriﬁcation has an advantage of causing less
mechanical stress to the biological material over freezing on cellular level. In a recent
study we observed the actin underlining of the cellular membrane and also the actin
distribution through the cytoskeleton, two of the sub-cellular arrangements that
would be greatly affected by mechanical damage. Our study on the effect of different
cryopreservation strategies on the cell actin distribution and membrane thinning (or
most speciﬁcally the thinning of the actin band underlying the cellular membrane)
clearly showed that the detrimental effects of cryopreservation by freezing are much
more prominent than those incurred by the hepatocytes preserved using the vitriﬁca-
tion approach. Therefore, we may conclude that vitriﬁcation as the concept of cryo-
preservation has advanced from single non-sensitive cells to the sensitive cell (e.g.
human egg) and currently handling of multiple biological objects, even in up-scaled
conditions. There is no doubt in universality of vitriﬁcation strategy.
Conﬂict of interest: None declared.
Source of funding: None declared.
doi:10.1016/j.cryobiol.2010.10.031
28. Fast measurement of cooling and heating rates in standard cryopreservation protocols
based on vitriﬁcation. Aline Schneider Teixeira * 1, Marha Elena Gonzalez-Benito 2,
Antonio Diego Molina-Garcha 1, 1 Instituto del Frı´o-ICTAN (CSIC), José Antonio Novais
10, 28040 Madrid, Spain, 2Dept. Biologı´a Vegetal, Universidad Politécnica de Madrid,
Ciudad Universitaria, 28040 Madrid, Spain
Some cryopreservation protocols rely on fast transfer from liquid to glassy state,
avoiding the formation of intracellular ice crystals. Ice can be equally formed during
cooling and warming processes. Protocols include additives and steps designed to
reduce freezing equilibrium (Tf) and increase glass transition (Tg) temperatures, as
well as to confer speciﬁc protection to cells against several associated stresses gener-
ated during the cryopreservation process (cold pre-treatment, dehydration and oxida-
tive stress). These additives and pre-treatments also contribute to reduce the
probability of ice nucleation at all temperatures, mainly through micro-viscosity
increase. Still, there is a risk of ice formation in the temperature region comprised
between Tfand Tg. Consequently, fast cooling and warming, especially in this region,
is a must to avoid ice-derived damage.
In spite of the importance of temperature change rates, they have been scarcely
experimentally measured. Temperature evolution of samples simulating real cryo-
preservation protocols has been measured in this work and the results are compara-
tively presented.
A very fast temperature data acquisition system was employed (50 measure-
ments per second), producing enough data points in the interval of interest (consid-
ered here from 0 C to 150 C), even in the fastest cases (1–2 s). The precision of the
experimental measurement system employed was evaluated and a good reproducibil-
ity was found among repeats, in spite of the experimental variation inherent to cus-
tom cryopreservation practice. Several techniques based in fast cooling published in
the literature (vitriﬁcation, encapsulation-dehydration, droplet) were studied,
together with the described warming procedures. A large difference in temperature
change rates can be found among methods, ascribed to differences in mass, heat
capacity and thermal conductivity of samples and barriers (containers). Differences
between cooling and warming rates in the same experimental set were smaller
and, for most cases, suggested no additional ice formation risk during the warming
process. In some cases in which protocols included a deﬁnite warming time period
in a thermostatic bath, it was observed that this time elapsed before the sample tem-
perature had risen from 0 C.
The probability of ice formation under Tfand over Tg always exits. Although pre-
treatments can largely limit this probability, both by molecular mobility and freez-
able water content reduction, they may also cause decreases in viability, due to dehy-
dration or cytotoxic derived damages, and produce alterations in the recovered
material, also derived from too drastic stress conditions. A reduction in these stresses
can be made compatible with successful cryopreservation by increasing cooling and
warming rates.
Conﬂict of interest: None declared.
Source of funding: This work was funded from project ‘‘CRIOFIS” (AGL2007-
65938-C02-02/AGR) from the Spanish ‘‘Plan Nacional de Investigaciyn CientHﬁca,
Desarrollo e Innovaciyn Tecnolygica 2004-2007”, M.E.C..
doi:10.1016/j.cryobiol.2010.10.032
29. Identiﬁcation of supercooling-facilitating (anti-ice nucleating) hydrolyzable tannins
from xylem parenchyma cells in katsura tree (Cercidiphyllum japonicum). Donghui Wang
* 1, Jun Kasuga 2, Chikako Kuwabara 1, Yukiharu Fukushi 1, Keita Arakawa 1, Seizo
Fujikawa 1, 1 Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589,
Japan, 2 Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University,
Morioka 020-8550, Japan
Xylem parenchyma cells of katsura tree exhibit deep supercooling capability to
40 C. Our previous studies showed that diverse kinds of supercooling-facilitating
(anti-ice nucleating) substancesmight exist in these xylemparenchyma cells, although
we have currently identiﬁed only four kinds of supercooling-facilitating ﬂavonol glyco-
sides in the xylem parenchyma cells of katsura tree (Kasuga et al., Plant Cell Environ. 31
(2008) 1335–1348). In this study, therefore, we tried to identify other types of superco-
oling-facilitating substances from xylem parenchyma cells in katsura tree.
Four to six-year-old twigs from an adult katsura tree (C. japonicum) grown in an
experimental ﬁeld of Hokkaido University were used for isolation of new compounds
with supercooling-facilitating capability. Crude xylem extracts were extracted with
methanol from xylem and they were separated by liquid–liquid extraction using ethyl
acetate (EtOAc). The resultant EtOAc fraction was further separated to about 20 frac-
tions by silica gel column chromatography. All these silica gel fractions exhibited
supercooling-facilitating capability, and our previous study showed the existence of
ﬂavonol glycosides in the silica gel fraction with the highest supercooling capability
(Kasuga et al., ibid). In this study, we used different silica gel fractions that also exhib-
ited high supercooling capability in order to identify different types of supercooling-
facilitating substances. One silica gel fraction, which exhibited high supercooling
capability in second order among all silica gel fractions, was separated by Sephadex
LH-20 column chromatography and then subjected to HPLC analysis. Finally, chemical
structures of compounds in three peaks of HPLC, which exhibited high supercooling
capability, were analyzed by using a MS and NMR.
From deep supercooling xylem parenchyma cells in katsura tree, we identiﬁed
three kinds of supercooling-facilitating hydrolyzable tannins. These were 2,3,6-tri-
O-galloyl-a,b-D-hamamelose (Kurigalin), 1,2,3,6-tetra-O-galloyl-b-D-glucopyranose,
and 1,2,6-tri-O-galloyl-b-D-glucopyranose. Our results suggested that the presence
of diverse kinds of supercooling-facilitating substances, including previously identi-
ﬁed ﬂavonol glycosides and hydrolyzable tannins, in xylem parenchyma cells pro-
duced high supercooling capability in xylem parenchyma cells of katsura tree.
370 Abstracts / Cryobiology 61 (2010) 362–408


Fast measurement of cooling and heating rates in standard cryopreservation protocols based on vitrification

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Fast measurement of cooling and heating rates in standard cryopreservation protocols based on vitrification

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