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Abstract

Una clula madre posee la capacidad de replicarse y diferenciarse dando


lugar a diversos tipos de clulas especializadas. Las clulas madre se
pueden clasificar de tres maneras: a) Segn su potencial de diferenciacin
en

clulas

totipotenciales,

pluripotenciales,

multipotenciales

unipotenciales; b) Segn el tejido de origen en clulas madre embrionarias


o adultas, y c) Segn su capacidad de re-poblacin tisular in vivo en corto,
medio

largo

plazo

de

regeneracin.

Adems

de

las

diferentes

clasificaciones que son dadas a las clulas madre, tambin generan gran
inters los diferentes modelos de diferenciacin celular a los que pueden ser
conducidas, desde el modelo convencional clula madre-clula hija hasta
procesos

de

transdiferenciacin,

de-diferenciacin

re-diferenciacin

celular; es as como estos modelos son aplicados en la actualidad para


entender el fenmeno de la plasticidad que ha sido reconocido en este
tipo de clulas. La plasticidad de las clulas madre se reconoce como la
capacidad que poseen estas clulas para generar grupos celulares diferentes
a los de su tejido de origen; tal es el caso de la plasticidad identificada en
las clulas madre hematopoyticas que pueden formar hepatocitos y
miocitos en condiciones controladas. En la actualidad, existen controversias
debido a que la mayora de estudios sobre clulas madre son realizados a
partir de vulos donados en centros de fertilizacin humana lo que implica
un compromiso tico que no puede desconocerse; sin embargo, es posible
obtener clulas madre con caractersticas pluripotenciales de otras fuentes
diferentes a embriones humanos como la sangre de cordn umbilical. Las
investigaciones sobre la obtencin de progenitores celulares, especialmente

hematopoyticos, a partir de sangre de cordn umbilical, representa una


alternativa de investigacin en el estudio de la biologa de las clulas
madre, as como su aplicacin en alternativas de terapia de reemplazo
medular en individuos con patologas asociadas a la mdula sea como
leucemias y aplasias.

A Quick Guide for Stem Cell


Cryopreservation and Thawing
Karina Palomares December 23, 2014 A Quick Guide for Stem Cell Cryopreservation and
Thawing2014-12-23T09:08:58+00:00Cryopreservation, Stem cells No Comment

Efficient protocols for cryopreservation and thawing are critical to maintain human
embryonic stem cell colonies. Image credit: http://www.stemcell.ucsb.edu

Human embryonic stem cells have the potential for unlimited expansion
and differentiation into all the cell types of the human body, and are thus
attractive candidates for use in regenerative medicine. Since their
discovery in the late 1990s, our understanding and knowledge of hESCs
has advanced, but working with the cells can still be tricky. Back then,
researchers found that only 5% of hESCs were viable after a single
freeze-thaw cycle. Efficient protocols for stem cell cryopreservation and
thawing are critical for basic research, as well as for future clinical
applications. However, compared to other standard cell types,
cryopreservation of stem cells is quite difficult. Stem cells exhibit higher
rates of apoptosis and spontaneous differentiation after freezing and
thawing. If you are new to the stem cell field, The Scientist published a

useful guide for stem cell cryopreservation and thawing [1]. We have
summarized the key points below.
Dissociation Method: Conventional methods rely on non-enzymatic
dissociation of colonies to cryopreserve the hESCs in the form of cell
clumps. Following centrifugation, the cells are resuspended in a
cryopreservation solution consisting of culture medium and a
cryoprotective agent to minimize cellular damage during the freezing
process. Alternatively, some scientists have reported higher recovery
rates when colonies are dissociated with EDTA, which breaks the cells
into small, uniform-size clumps. Dissociation into near single-cell
suspensions allows an even distribution of the cyroprotectant.
Freezing Medium: A typical freezing medium consists of 90% FBS and
10% DMSO. However, due to potential toxicity and immune responses in
patients, researchers are switching to reagents that use lower
concentrations of DMSO and lack animal products. For example, the
commercially available freezing medium, CP-1, consists of hydroxyethyl
starch, a plant-based cryoprotectant, and DMSO in saline. One study
showed that addition of ethylene glycol further increases the
effectiveness of this media, achieving a post-thaw recovery of almost
50%.
Freezing Rate: Successful stem cell cryopreservation depends on a
gradual freezing rate. Typically, cells are stored in a -80C freezer for
several hours prior to long-term storage in liquid nitrogen. Controlledrate freezers are more effective in maintaining temperature control, but
are also quite expensive. One cost-effective alternative is a passive
freezing device, which cools cells at a rate of 1C per minute, down to
-80C.
Thawing: Unlike the freezing process, thawing should be quick. Vials can
be placed in a 37C water bath to facilitate the thawing process. Also,
the cells should be thoroughly washed to remove all traces of the
cryoprotectants.

Editor note: Its a shame the authors of this protocol did not know about
our new automated cell thawing system ThawSTAR which
dramatically standardizes the thawing of cryovials.
Recovery and Characterization: Up to 40-50% of the original stem cell
population can be recovered after a successful freezing and thawing.
However, some lines may exhibit a delayed onset of cell death, so it is
best to allow the cells to recover for a couple of days after the initial
plating. Once the cells have recovered, they should be examined for
pluripotency markers to ensure that theyre still stem cells.