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Successful long-term preservation of stem cells requires in-depth knowledge of physical, thermodynamical and chemical parameters and their mutual interactions.
The currently applied method for cryopreservation of “clumped” hES cells is not standardised and has a low efficiency: the survival rates after thawing are lower than 10% and the reproducibility of the results is very low. For adult stem cells, cryopreservation is based on the use of DMSO at high concentrations (e.g. 10%) as chemical cryoprotectant. The cryopreservation of cell cultures below minus 130°C in the presence of 5 to 15 % DMSO (v/v) and 5 to 20% (v/v) glycerol is the most widely used freezing procedure. However, a number of questions remain to be addressed regarding toxicity of DMSO to the cells, resulting in cell death during the freezing and after de-freezing of the cells. However, because of its toxicity DMSO adversely affects cell survival and glycerol can cause osmotic problems especially after thawing.
In addition, the cooling rate must be just slow enough to allow the cells to dehydrate, but quick enough to avoid excessive dehydration that results in cellular damage. Moreover, cells with less permeable membranes may require slower cooling rates.
Specific objectives of the CRYSTAL project in cryopreservation are:
- To develop optimal cryoprotectants or combinations of cryoprotectants with minimal cytotoxicity
- To establish surface-based freezing methods for stem cells on functionalised surfaces, on scaffolds or on feeder layers
- To define conditions for freezing cells in two- or multidimensional aggregates
CRYSTAL pursues a unique integrated approach using both somatic and embryonic stem cells to address the current shortcomings in stem cell banking (see also here).
CRYSTAL’s approach is threefold, recapitulating the workflow of a stem cell bank:
- First, the preparation of stem cells will be optimised. This will form the basis for the subsequent banking experiments by supplying haematopoietic and non-haematopoietic human stem cells together with protocols for their propagation. This aspect of CRYSTAL will systematically examine parameters determining stem cell vitality and define standardised parameters for their characterisation. By ensuring the quality and identity of human stem cell preparations, this work will provide a reference point for subsequent validation of stem cell vitality after freezing and allow the systematic evaluation of novel freezing protocols.
- The second part of CRYSTAL’s approach focuses on the actual cryopreservation step of stem cell banking. In many cases, the vitality of purified stem cell preparations after cryopreservation is low and precludes their immediate use in therapeutical approaches. CRYSTAL will put forward innovative protocols minimising the negative biochemical and biophysical impacts of freezing. Specifically, novel (organic and inorganic) cryoprotectants will be studied, optimised temperature gradients for freezing will be developed (slow vs. fast freezing) and the effect of the cell’s conformation during freezing will be studied to propose optimised cryopreservation conditions for all stem cell lines studied within CRYSTAL.
- Third, the protocols elaborated above will be validated using a set of standardised tests to judge the performance of a particular cryopreservation protocol by assessing cell identity and vitality. Viability alone is not a sufficient criterion to evaluate a thawed stem cell sample. Rather, aspects such as morphology, apoptotic status, differentiation and engraftment potential will be taken into consideration when developing standardised protocols for preparation, preservation and validation of stem cells for banking and therapeutic use. The validation carried out in the individual labs will be complemented by the development and use of an automated, self-controlling method of functional cell analysis on a single cell basis.
These three aspects of the CRYSTAL approach are interdependent and will be conducted in an iterative way so that results obtained in the validation section will be used as feedback for further rounds of the experimental approach.
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