Technical feasibility

SECTION I: Testing



Has the R & D result been tested?






The following question is replied according to the reply in question 1


If yes



In what mode has the result been tested?

•             Prototype

•             Pilot Application

•             Alpha/BETA testing


1b. Please describe and discuss the testing results


If no:



Describe what type of testing does the R&D result need?
Objective of the project is the genotyping (genome sequencing) of sheep and cattle breeds that are primordial to be extinguished, and the construction of gene banks from them (cell lines). The research group has shown that for the first nuclei freeze-dried cells retain viability in response to nuclear transplantation (Loi P. et al., PLoS One. 2008, 20; 3 (8): e2978). The project will demonstrate that you can set up gene banks freeze-dried, then at very low operating costs in the long term.  The main application possibilities of the project are: 1) establishment of gene banks from freeze-dried animal species threatened with extinction 2) freeze-drying technology transfer to bio-medical prevalentedestinazione with banks, which preserve the samples in liquid nitrogen (-196 ° C). Regarding point 1, in Teramo there are species of domestic animals (sheep race pagliarola; goats Teramana goat) and wild (Apennine chamois, bear Marsicano, ect.) that are disappearing. The project would therefore have immediate application outcomes of races that characterize our territory. The European Community takes into account the large banks of biological material, for their strategic importance in the biomedical field (research, stem cells, freezing of gametes for assisted reproductive medicine).
1d. What is the time needed for testing?
2 years
1e. What is the cost needed for testing?

TOTAL for testing: 22.000 Euro



SECTION 2: Current Stage of Development



To what extent does the development team have technical resources for supporting the production of a new product? (Researchers, human resources, hardware, etc. )
Objective of the project is the genotyping (genome sequencing) of sheep and cattle breeds that are primordial to be extinguished, and the construction of gene banks from them (cell lines).


What are the technical issues that need to be tackled for full deployment, if needed?
For this project there is a collaboration between managers of companies and University so is possible work well. So there are not issues




What additional technical resources are needed for the production of this new product?
In order to optimize the search is useful presentation of the preliminary scientific results at national and international conferences.



Overall assessment of the current stage of technical development.
Actually, research is giving good results



SECTION 3: Deployment


Define the demands for large scale production in terms of
  • Materials
Production of somatic cell lines by freeze-drying; optimization procedures of lyophilization for the cells;  nuclear transplantation of somatic cells lyophilized in enucleated oocytes sheep; verification of the vitality of the cells to rehydration
  • technologies, tools, machineries
i) Production of somatic cell lines from freeze-dried.
The cells to be freeze-dried will fibroblasts and lymphocytes of sheep. Fibroblasts are taken from adult subjects by skin biopsy, mechanical and enzymatic dissociation of tissue (collagenase, EDTA) and primary cultures grown
in standard culture conditions (38.5 ° C) in 5% CO2 in air in complex medium (DMEM) with added fetal serum and antibiotics.The lymphocytes are isolated from peripheral blood by centrifugation in gradient of Ficoll ® and processed
immediately for the lyophilization.
ii) optimization of the procedures of lyophilization for the cells. The technology platform will be represented by the departure of our previous work in collaboration with Core Dynamics (Loi P et., PLoS ONE, 2009, Nathan et al. PLoS
ONE, 2009). The freeze will be preceded by the freezing of cells in medium containing 0.5-1.5 M trehalose. The freeze will be implemented in close collaboration with Dr. Amir Arav of Core Dynamics. The amendments will
tested dall’assegnista research in order to improve the efficiency are:
a) changes in the ionic composition of freezing medium
b) the freezing of cells at different stages of the cell cycle.
With regard to the point a), will be used based solutions of KCl in place of NaCl, commonly used for the formulation of medium. The rational and that the presence of K ions in the extracellular medium would be better tolerated in
case of damage of the membrane. This assumption is supported by preliminary results The other variation that will be tested is the increase in Ca + + ions in the medium of freezing, H2O of re-hydration. Studies of sea urchin oocytes have shown
that the Ca + + promotes repair of extensive lesions of the cell membrane (Togo T, Alderton JM, Steinhardt RA. The mechanism of cell membranes repair. Zygote. 2000; 8
Suppl 1: S3, McNeil PL, Kirchhausen T. An emergency response team for membrane repair. Nat Rev Mol Cell Biol. 2005, 6: 499-505). It is therefore expected an improvement in cell viability after rehydration to those changes.
b) Freezing of cells at different stages of the cell cycle. Generally, the cells are detached from the flasks or Petri dishes and the culture were frozen during the proliferation phase, where cells are found in all phases of the cell cycle (M, G1, S, G2).
No particular attention was paid when the cryo tolerance of various phases of cell cycle, since the current freezing protocols are unsatisfactory in terms of efficiency (Maryman et al., 2006, cryopreservation of living
cells: Principles and Practice. Transfusion 47, 935-945). However, the lyophilization comprises an additional stress to the cells, it is important to freeze the cells in anticipation of the further physical stress that must be met during the sublimation
water. Protocols to synchronize the cells in these two stages of the cell cycle does not present any difficulty and are easily applied (serum starvation for quiescence and
treatment with colchicine for synchronization in M ​​phase).
v) lyophilized somatic cell nuclear transplantation into enucleated oocytes. This is the most challenging part of the project. Immature oocytes aspirated from ovaries taken at slaughtering will be matured in vitro according to the protocols made
developed by our laboratory (G. Ptak, P. Loi, M. Dattena, Tischner M, Cappai P. (1999) Offspring from one month old lambs: Studies on the developmental capability of prepubertal oocytes. Biology of Reproduction 61, 1568 - 1574; G. Ptak, M. Dattena,
Loi P., Tischner M., Cappai P. (1999) Ovum pick up in sheep.Efficiency of in vitro embryo production, vitrification and birth of offspring. Theriogenology 52, 1105-1114).).
The reconstructed embryos will be cultured in vitro for 7 days as described (Dattena M., G. Ptak, Loi P., Cappai P. (2000) Survival of vitrified in vitro and in vivo produced sheep blastocysts. Theriogenology, 53: 1511-1519 ; G. Ptak, P. Loi, M. Dattena, Tischner M, Cappai P. (2000) Follow-up of Lambing after transfer of in vitro-produced embryos. Theriogenology 53, 316; G. Ptak, P. Loi,Dattena M., Tischner M, Cappai P. (1999) Offspring from one month old lambs: Studies on the developmental capability of
prepubertal oocytes. Biology of Reproduction 61, 1568-1574; G. Ptak, M. Dattena, Loi P, Tischner M, Cappai P. (1999) Ovum pick up in sheep. Efficiency of in vitro embryo production, vitrification and birth of offspring. Theriogenology 52, 1105-111). In this way, we will evaluate the functionality of the nuclear freeze-dried cells. The embryos thus produced will be analyzed with RT-PCR for the expression of genes responsible for the
totipotency (OCT4, SOX3, Nanog, CDX2).
vi) verification of the vitality of the cells to rehydration.
This is the most technically simple project and one that presents less risk. The cells will be re-hydrated with milly-Q water and analyzed for: functional integrity of the cell membrane (Live / Dead kit, Sigma) proliferative capacity (serial passages in culture medium DMEM with more serum and antibiotics); integrity of chromosomes (comet assay , karyotype).



  • Staff effort
This research may be supported by major farmers






SECTION 4: Overall Assessment



What is you overall assessment of the technical feasibility of the research result?

Research is underway









Please put X as appropriate. 1 2 3 4 5
Adequacy of testing activity undertaken so far         x
Adequacy and availability of technical resources of the development team         x
Current development stage       x  
Overall technical feasibility         x




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