Conceived and designed the experiments: PL AA. Performed the experiments: PL KM GP YN AA. Analyzed the data: PL KM JFJ YN. Contributed reagents/materials/analysis tools: PL GP. Wrote the paper: PL MC JFJ AA.
Current address: National Institute of Livestock and Grassland Science, Osaka, Japan
The authors have declared that no competing interests exist.
The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies.
The possibility of storing blood cells and other cell lines in a dry state though the process of lyophilisation could be of immense benefit for clinical application. However, despite the optimism displayed in some articles
In addition to the long-term storage of blood cells, it has also been suggested that genetic banks, preferably in the form of cell lines, should be established from animal species threatened by extinction
Recently, our group has demonstrated that somatic sheep cells rendered non-viable by heat treatment retain the potential to generate normal offspring following nuclear transplantation
Sheep granulosa cells lyophilized according to the protocol described for mouse spermatozoa were used as nuclei donor in the first experiments
b, irregular pronuclei with fragmented DNA (10 hours after activation), aceto-orcein staining. ×400. c, d, comet analysis in pronuclear stage embryo reconstructed with a control (fresh) granulosa cell (c), and with a freeze–dried cell (d). ×40. e, f, SEM appearance of the lyophilized cells without (e) and with (f) trehalose. ×350
These results were not surprising. Spermatozoa, the only eukaryotic, nucleated cells successfully freeze dried to date, are structurally compatible with lyophilisation: they are small cells with a low degree of hydration, and their transcriptionally inactive DNA is tightly packed with protamines
Although a loss of cellular viability was expected, the frequency and extent of DNA damage was massive and could even be observed by cytological examination (
These results prompted us to seek means of preventing, or reducing the degree of DNA damage occurring during freeze drying.
The production of threalose by a number of plants, yeast, spores and a range of unicellular organisms confers them the ability to survive in almost total absence of water
Recently, using our device for extra cellular ice crystal propagation, we demonstrated that even extra-cellular threalose protects the cells during dehydration
The effect of trehalose inclusion in the freezing medium was immediately evident from the morphological appearance of the dry cells observed with Scanning Electron Microscope (SEM). The medium dried in the absence of this sugar showed an irregular splintered-like shape, while the presence of trehalose conferred a smooth and glassy appearance to the dry powder (
Staining with propidium iodine revealed that membrane damage occurred in 100% of the rehydrated lymphocytes and granulosa cells (
i, nuclear transfer of freeze-dried granulosa cell×60. j, blastocyst produced from nuclear transfer of freeze dried granulosa cells×40.
Freeze dried granulosa cells were prepared and dispatched from the Institute of Animal Science at the Volcani Centre in Israel to Teramo University in Italy by surface mail, and stored in the dark at room temperature for three years before use for nuclear transfer (
For nuclear transfer, cells were hydrated with Milli-Q grade water, washed twice with 199 medium plus antibiotics and BSA, and immediately injected into enucleated oocytes (
Enucleated oocytes injected with freeze dried granulosa cells initiated cleavage at similar rates to control embryos generated using fresh granulosa cells (
Source of cells | Cultured | 2-8 cells | Morula | Blastocyst |
Granulosa control | 129 | 43 (33.3%) |
31 (24.0%) |
27 (20.9%) |
Granulosa freeze-dried | 160 | 52 (32.5%) |
28 (17.5%) |
25 (15.6%) |
The culture were maintained for 7–8 day in medium SOF plus amino-acids and BSA, with FCS added a day 4. reconstructed embryos were checked every 24 hours for development.
Test χ2
P = 0.88;
P = 0.17;
P = 0.56
Total cells | ICM cells | Trophectoderm cells |
Control NT blastocysts | 28.4±7 |
50.2±12.0 |
78.6±14,8 |
||
Freeze dried NT blastocyst | 18.5±6 |
39.7±9.0 |
54.29±6.3 |
differ significantly for p<0.05
The embryos developed to blastocyst stage were effectively derived from the freeze dried cells and no from parthenogenetic activation of the oocytes. First, all oocytes were effectively enucleated, as assessed by Hoechst staining and UV examination. Second, microsatellite DNA analysis of the cloned blastocysts matched perfectly with the lyophilized donor cells.
We have demonstrated for the first time that lyophilized cells maintain the development potential when injected into enucleated oocytes. It is also worth noting that the cells used in this study were maintained in a dehydrated state at room temperature for three years, whereas lyophilised spermatozoa were stored for 4 months before Intra Cytoplasmatic Sperm Injection -ICSI
These findings suggest that our results may have important implications for biomedicine in the short term. For example, haematopoietic precursor cells are now widely used as a source of multipotent adult somatic cells for the treatment of degenerative disorders
In conclusion, we have demonstrated for the first time that somatic cells stored in a dehydrated state have the capacity to direct embryonic development of enucleated oocytes up to the blastocyst stage. These results represent a substantial breakthrough for the storage of a broad range of cell lines for biodiversity conservation efforts and possibly for biomedical applications.
All chemical were purchased by Sigma, unless otherwise stated.
Peripheral blood lymphocytes were isolated from Assaf breed ewes through a Ficoll-Paque density gradient; the purity of the cells (∼80%) was assessed by an automatic cell counter (Pentra 60, ABX, France). Cumulus Oocyte Complexes (COCs) collected from the ovaries of slaughtered Sarda and Assaf ewes were matured for 24 hours in medium TCM 199 plus 10 FCS, FSH, LH and estradiol in incubator at 38,5°C with 5% CO2. Expanded COCs were briefly incubated in hyaluronidase (300 USP units/ml, Sigma) and mechanically dissociated into a single cell population. In the first experiments, granulosa cells from Sarda breed ewes were freeze dried according the protocol described for sperm cells
Immediately before use for nuclear transfer, the ampoules were opened and 100 μl, or 2 mls of milliQ water added. After rehydration, cells were washed twice with medium 199 plus antibiotics and BSA before use for nuclear transfer. For each ampoule, viability was assessed on small aliquots of cells by propidium iodine staining; also the extend of DNA fragmentation was assessed with Comet essay in every replicate.
DNA integrity was evaluated using the single cell gel electrophoresis assay (aka comet assay, R&D Systems, USA) according to the manufacturer's instructions. Cells were diluted to a concentration of 105cells/ml in PBS. The cells were combined with molten LM agarose at a ratio of: 1∶10 (v/v), then 75 μl were placed on comet slides. The slides were put in the dark at 4°C for 10 minutes. Slides were then immersed in a pre cooled (4°C) lysis solution for 1 hour at 4°C. Afterward, the slides were immersed in a freshly prepared alkali solution, which consisted of 0.6g NaOH pellets, 250 μl of 250mM EDTA, pH 10.0 and 49.75ml deionized water, for 60 minutes in the dark at room temperature. Finally, the slides were washed in a TBE buffer (Tris base, Boric acid and EDTA) for 5 minutes and then the slides were submerged in TBE buffer in a horizontal electrophoresis apparatus. 1 volt per centimetre was applied for 10 minutes. In addition, each experiment was conducted with two controls; the first one was of cells that were previously treated with 100 μM of hydrogen peroxide for 10 minutes at 2°C–8°C as described in the manufacturer kit (this served as a positive control for the assay showing damaged DNA) and the second control was of fresh cells, indicating endogenous levels of damage within the cells. After the samples were dried they were stained with SYBR green. Scoring was done using a fluorescent microscope (Zeiss, Germany) connect to a digital camera (Sony, Japan) and analyzed using the Image J free software (NIH, USA).
Reconstructed embryos at pronuclear stage (12 hours after activation) were treated in the same way for the detection of DNA damage.
For evaluation of the morphology of dry samples Scanning Electron Microscopy was used. The samples were gold plated before being placed in the SEM. The voltage of the electron scatter was 25kv. We captured a 3D picture on a CRT screen.
Oocytes were matured in vitro in bicarbonate-buffered TCM-199 (Gibco) (275mOsm) containing 2 mM glutamine, 100 μM cysteamine, 0.3 mM sodium pyruvate, 10% fetal bovine serum (FBS) (Gibco), 5 μg/ml FSH (Ovagen), 5 μg/ml LH, 1 μg/ml estradiol in a humidified atmosphere of 5% CO2 in air at 39°C for 24 h
Oocytes were incubated in Hepes buffered 199 medium containing 4 mg/ml of BSA and 7.5 μg/ml of Cytochalasin B and 5 μg/ml of Hoechst 33342 for 15 minutes in incubator. Enucleation was carried out in Hepes buffered 199 medium plus BSA and Cytochalasin B with a Narishighe micromanipulator. Enucleate oocytes were allowed to recover from Cytochalasin B treatment then directly injected with freeze-dried cell. Reconstructed oocytes were activated in Hepes buffered medium 199 containing 5 μg/ml Ionomycin for 5 minutes, then incubated in SOF medium plus antibiotics and BSA containing 10 mM Dimethylaminopurine and 7.5 μg/ml Cytochalasin B for 3–5 hours.
Reconstructed embryos were transferred into 20-μl drops of SOF enriched with 1% (v:v) basal medium Eagle (BME)-essential amino acids, 1% (v∶v) minimum essential medium (MEM)-nonessential amino acids (Gibco), 1 mM glutamine, and 8 mg/ml fatty acid-free BSA (SOFaaBSA). Zygote cultures were maintained in an humidified atmosphere of 5% CO2, 7% O2, 88% N2 at 39°C, and the medium renewed on Day 3 and Day 5 of culture
Cloned blastocysts from both groups were incubated in 500 μl of solution 1 (PBS with 1% Triton X-100 and 100 μg/ml propidium iodide) for 20 s. Blastocyst were then directly transferred to 500 μl of solution 2 (100% ethanol with 25 μg/ml bisbenzimide Hoechst 33258) and stored at 4°C overnight. Blastocysts were then mounted onto a microscope slide in a drop of glycerol and flattened with a cover slide. Cell counting was performed directly on an inverted microscope fitted with an ultraviolet lamp and excitation filter (460 nm for blue and red fluorescence).
Cloned blastocysts were transferred in 200 μl of SOF medium and snap frozen in liquid nitrogen. DNA was extracted from the cloned blastocysts and from the donor cells using the kit Genomix, according with the manufacturer instruction. Genomic DNA from cloned embryos and donor cells have been amplified through a multiplex PCR for the following satellites: OarCP49, FCB11, OarAE129, FCB304, INRA063, MAF214, CSRD247 e HSC. These loci are part of a standard panel recognized by the International Society for Animal Genetics (ISAG).
All the experiments wee carried out in vitro, using ovaries collected from the slaughterhouse, without any experimental animals, therefore, we did not need to receive ethical approval from the local University Ethical Committee.