reprogrammed cells were called induced pluripotent stem (iPS)
cells and were similar to mouse ESCs. Subsequently, different cell
types including human somatic cells were reprogrammed to pluripotency by forced expression of the same or slightly different
transcription factors [7, 8]. Unlike ESCs, iPSCs can be generated
from small biopsies taken from patients, making them patientspecific. Therefore, properly generated and handled iPSCs are
expected not to induce any immune response when transplanted
for tissue repair and other treatments. Moreover, by reprogramming patients’ own cells, it is possible to generate disease-specific
iPSC models that can be used for drug testing and development of
personalized treatment plans for patients [9]. Considering their
self-renewal and differentiation capacity into any cell type of all
three germ layers, hiPSCs hold great potential for regenerative
medicine, tissue engineering, and the treatment of degenerative
diseases. While the benefits of using hiPSCs for clinical applications
are evident, there is a lack of viable clinical-grade cryopreservation
methods needed to preserve these cells and, thus, to realize their
potential clinical applications. One challenge with banking human
pluripotent stem cells (hPSCs) is that they are highly sensitive to
cryopreservation and dissociation to single cells [10]. Although
high serum concentrations and adding a selective Rho-associated
kinase (ROCK) inhibitor such as Y-27632 to freeze-thaw and
subsequent culture media somewhat improved the outcome of
hPSC cryopreservation, the overall post-thaw recovery still remains
unsatisfactory [11–14]. To add to this, clinical-grade cryopreservation protocols prohibit the use of serums and animal products due
to the biosafety and reproducibility issues; therefore, development
of a xeno-free and chemically defined method is essential for cryobanking stem cells [15]. Since serum contains numerous factors
that are beneficial to cell attachment and growth, overcoming
oxidative stresses, buffering pH, binding and neutralization of
toxic molecules, and reduction of shear stresses, it might be critical
to implement such beneficial properties of serum when developing
chemically defined, xeno-free culture and cryopreservation methods. Indeed, mimicking some of the beneficial effects of serum by
including antioxidants, amino acids, vitamins, and polymers in the
freezing medium while combining low concentrations of two
penetrating cryoprotectants to reduce their toxicity [16] resulted
in development of a successful xeno-free and chemically defined
cryopreservation method for adipose-derived stem cells
[15]. Using the same concept, we formulated a defined medium
for hiPSCs with some modifications and tested it against the conventional undefined freezing medium (i.e., 10% DMSO, 20% KSR,
and 10 μM ROCK inhibitor Y-27632 in DMEM/F-12) and
unfrozen controls. Our experiments showed that the chemically
defined, xeno-free freezing medium formulated for hiPSCs yields
satisfactory results although not completely comparable to the
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Juliette Seremak and Ali Eroglu
cells and were similar to mouse ESCs. Subsequently, different cell
types including human somatic cells were reprogrammed to pluripotency by forced expression of the same or slightly different
transcription factors [7, 8]. Unlike ESCs, iPSCs can be generated
from small biopsies taken from patients, making them patientspecific. Therefore, properly generated and handled iPSCs are
expected not to induce any immune response when transplanted
for tissue repair and other treatments. Moreover, by reprogramming patients’ own cells, it is possible to generate disease-specific
iPSC models that can be used for drug testing and development of
personalized treatment plans for patients [9]. Considering their
self-renewal and differentiation capacity into any cell type of all
three germ layers, hiPSCs hold great potential for regenerative
medicine, tissue engineering, and the treatment of degenerative
diseases. While the benefits of using hiPSCs for clinical applications
are evident, there is a lack of viable clinical-grade cryopreservation
methods needed to preserve these cells and, thus, to realize their
potential clinical applications. One challenge with banking human
pluripotent stem cells (hPSCs) is that they are highly sensitive to
cryopreservation and dissociation to single cells [10]. Although
high serum concentrations and adding a selective Rho-associated
kinase (ROCK) inhibitor such as Y-27632 to freeze-thaw and
subsequent culture media somewhat improved the outcome of
hPSC cryopreservation, the overall post-thaw recovery still remains
unsatisfactory [11–14]. To add to this, clinical-grade cryopreservation protocols prohibit the use of serums and animal products due
to the biosafety and reproducibility issues; therefore, development
of a xeno-free and chemically defined method is essential for cryobanking stem cells [15]. Since serum contains numerous factors
that are beneficial to cell attachment and growth, overcoming
oxidative stresses, buffering pH, binding and neutralization of
toxic molecules, and reduction of shear stresses, it might be critical
to implement such beneficial properties of serum when developing
chemically defined, xeno-free culture and cryopreservation methods. Indeed, mimicking some of the beneficial effects of serum by
including antioxidants, amino acids, vitamins, and polymers in the
freezing medium while combining low concentrations of two
penetrating cryoprotectants to reduce their toxicity [16] resulted
in development of a successful xeno-free and chemically defined
cryopreservation method for adipose-derived stem cells
[15]. Using the same concept, we formulated a defined medium
for hiPSCs with some modifications and tested it against the conventional undefined freezing medium (i.e., 10% DMSO, 20% KSR,
and 10 μM ROCK inhibitor Y-27632 in DMEM/F-12) and
unfrozen controls. Our experiments showed that the chemically
defined, xeno-free freezing medium formulated for hiPSCs yields
satisfactory results although not completely comparable to the
570
Juliette Seremak and Ali Eroglu