Patent ID: 12250944

EXAMPLES

The following examples are given for illustrative purposes, and should not be interpreted as limiting the scope of the invention in any way.

1. Introduction

3 different types of stem cells were used:Muscle stem cells (satellite cells) responsible for muscle growth and repair,Hematopoietic stem cells responsible for formation and renewal of the blood, andImmune system and mesenchymal stem cells, which are the most widely used stem cells in clinical applications.

For these 3 types of stem cells, exposure to natural neutrons and gamma-rays from radioactivity negatively impacts cell recovery after cryopreservation. It is hereafter shown that long-term storage using the method of the invention, i.e. in an almost neutron and natural radioactivity free environment, improves cell recovery after 6-month cryopreservation.

2. Material and Methods

Experimental Design

As a reference, the radiation dose received by a human over 1 year at the earth surface (2.4 mSv) is used.

The Modane Underground Laboratory (LSM) is located in the middle of the Fréjus tunnel. The laboratory is under 1700 m of rock. This is equivalent to 4800 meters of water as a shielding against cosmic rays. Inside the LSM cavity, the muon flux is 4 cosmic-rays/m2per day. As compared to the surface (1×107/m2/day), the cosmic ray flux is reduced by a 2.5 million factor. The neutrons flux is reduced by a 10 000 factor (10−6neutrons/cm2.s in LSM) as compared with the surface (10−2neutron/cm2·s)

The LSM also has a surface facility in Modane with a cosmic-ray flux of 2×107cosmic rays/m2per day.

The stem cells are stored in test tubes made of plastic. The stem cells were stored under 4 different exposure conditions with regard to ionizing radiations:

The “Altitude” storage conditions were designed to measure the effect of cosmic rays and natural radioactivity on different types of cell cryopreserved. Under “Altitude” conditions, the biological material was stored in cryotube and was exposed to standard cosmic ray and natural radioactivity flux at the LSM building in Modane city, corresponding to standard natural radioactivity at ground level with ambient neutron and gamma-ray conditions.

“Increased” conditions simulate a dose 32 times higher than natural exposure received by the stem cells. The “increased” conditions were obtained by adding ionizing radiation from natural source (2840 Bq of 232Th at secular equilibrium), specifically by exposing the cryotubes containing the biological material to a gamma-ray radioactive source. The doses were calculated and measured. The measurements of the induced doses were performed with a LiF dosimeter installed in a cryotube to have a phantom as representative as possible of the cells. Simulations performed with GEANT 3.1 simulation software are in good accordance with this measurement. The difference lies in the different geometry in the simulation, the tube is modelled by a flat bottom cylinder such as the cells pack, and for the measure the square is not fitting exactly the bottom of the tube. There is uncertainty of more than 10% on the real doses but it doesn't affect the results.

A two-month exposure under “Increased” conditions corresponds to 6 years exposure to natural ambient radioactivity.

“Standard” conditions correspond the usual storage conditions used at Institut Pasteur (Paris), wherein the biological material is stored in a common Dewar on the second basement. Taking into account the number of floors of the facility of Institut Pasteur above the surface, it is estimated that the protection against cosmic-rays is of the order of 100 cm of concrete. Which acts as a cosmic ray attenuating material. The neutron flux due to neutrons induced by muons only is estimated to 10−3neutron/cm2.s. Under “Standards” conditions, natural radioactivity is unchanged but the neutron flux from cosmic rays is reduced by a factor of 3 to 4 due to the subsurface storage.

“Deep underground” conditions correspond to storage conditions in a deep underground laboratory (−1700 m). At this depth, 99,999% of the cosmic ray flux is suppressed. The stem cells are installed in a cryostat shielded by 5 cm of archaeological lead and 10 cm standard lead corresponding to a reduction of 106of the natural gamma-ray flux. The storage is flushed with nitrogen to prevent radon to come in the vicinity of the cryotubes.

At 1700 m under Fréjus mountain, the laboratory is shielded against cosmic-rays, they are reduced by a factor 4 000 000 and then secondary induced particles (in particular induced fast neutrons) become negligible. The cells are installed in a cryostat protected against the natural radioactivity by an additional shielding made of 10 cm of low radioactivity lead and 5 cm of archaeological lead to shield from external radioactivity coming from rock. The gamma-ray flux is decreased by 6 orders of magnitude. The cells are put in a copper holder with 5 cm of copper as a shield for the hole in the shield induced by the cold finger. The cooling is monitored by pt100 thermometer. The cold is produced by the evaporation of nitrogen in a classical dewar and conducted to the cryotube by a 5 mm diameter copper rod. The evaporation of nitrogen is used to pressure a steel container preventing the radon to come in the vicinity of the cells (Scheme attached:FIG.10, configuration C without second shield). The materials used were tested for their radioactivity gamma and are all granted low radioactivity. The materials are below the detection limits of the detector so the doses induced to the cell is lower than the background of the germanium used for material selection. The shielding is not effective to protect the cells against neutron coming from rock produced by self-fission of heavy nuclei and (α,n) reaction on light nuclei. The spectrum of neutron in the lab is different from those encountered in the previous Condition increased because cosmic rays contain neutrons ranging up to energies of GeV instead of 9 MeV maximum energies in the underground lab. The experimental setups are not suited to extract data taking into account the spectra of neutrons so only integral counting is discussed.

The main characteristics of these 4 sets of storage conditions are summarized in table 3 below.

TABLE 3DosesNeutron fluxDepth(mSv/(neutron/Condition(mwe)year)cm2.s)Standard102.43.10−3Increased076.6510−2Altitude02.410−2Deep4800<2.7 10−63.10−6Underground
Mice Injection Injury and Graft

All protocols were reviewed by the Institut Pasteur, the competent authority, for compliance with the French and European regulations on Animal Welfare and with Public Health Service recommendations. This project has been reviewed and approved (#CETEA 2015-0039) by the Institut Pasteur ethic committee (C2EA 89-CETEA). Unless specified 6 to 8 weeks old male mice were used in this study and housed on a 12:12 light/dark cycle in a pathogen free facility with controlled temperature and humidity. Food and drink were given ad libitum. For isolation of mesenchymal and hematopoietic stem cells bone marrow from either C57131/6 from Charles River or Tg:Actin-GFP mice were used. For isolation of muscle satellite cells Tg:Pax7-nGFP mice were used. Rag2−/−γC−/− immunocompromised mice were used for transplantation experiment as host. When grafting muscle stem cells mice were anesthetized with ketamine (Imalgene1000 100 mg/Kg Merial) and Xylazine (Rompun2% 20 mg/Kg Bayer) prior to surgery, injected 18 h before the transplantation with notexin 10 μl of 12.5 μg/ml (Latoxan) in the tibialis anterior. 10.000 muscle stem cells in 10 ∝l of 0.9% NaCl were grafted. For grafting hematopoietic stem cells Rag2−/−γC−/− immunocompromised mice were irradiated at 95cGy and transplanted 3 hours later intravenous (retro-orbital) with 20 ∝l of cells in suspension in 0.9% NaCl.

Muscle Cell Sorting, Count and Culture

Muscle dissection was done by removing all of the limb muscle from the mice, in cold DMEM. Muscles were then chopped with small scissors and put in a 50 ml Falcon® tube with collagenase 0.1% and trypsin 0.1% at 37° C. with gentle agitation. After 20 min, the supernatant was collected in 2% serum placed on ice, and the collagenase/trypsin solution was added to continue the digestion. Once muscle is completely digested, the solution was filtrated using 40 μm cell strainers. Satellite cells were cultured in 1:1 DMEM-Glutamax (Gibco #41965-039):MCDB201 (Sigma #M6770) containing 20% serum FBS (Biowest S1860). Cells were plated on Matrigel coating (BD Biosciences #354234) and kept in an incubator (37° C., 5% CO2) at an initial concentration of 2,000 cells per mm2. For satellite cell counting after grafting, only the Tibialis anterior muscle was dissected and digested as described earlier, and the totality of the tube was analysed to assess the number of satellite cells per muscle. FACS analyses were done using a FACSaria (Beckman). Analyses and quantitation were performed using Summit v4.3 software from DakoCytomation and FloJo software. Cells were labelled with propidium Iodide 10 μg/ml (Sigma-Aldrich #P4170) to exclude dead cells and displayed using the PE (Phycoerythrin, Red) channel on the FACS profile.

Isolation and Culture of Mesenchymal Stem Cells and Hematopoietic Stem Cells

MSC were harvested, cultured and characterized from C57BL/6J mice, as previously reported. Briefly, in anesthetized mice (injected intraperitoneally with 100 mg per kg body weight of ketamine and 5 mg per kg body weight of xylazine), femurs were flushed to recover bone marrow. For mesenchymal stem cells isolation, the cell suspension was filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-α, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45, and Ter119. Appropriate gates were constructed on a cell sorter to exclude dead cells and lineage (CD45(+)Ter-119(+))-positive cells. Cells were plated in tissue culture flasks, and cultured in 1 ml of complete medium at a density of 25×106 cells ml−1. Cells were incubated in plates at 37° C. with 5% CO2 in a humidified chamber. After 3 h, the supernatant was removed and non-adherent cells that accumulate on the surface of the dish were replaced by changing the medium. After an additional 8 h of culture, the medium was replaced with 1.5 ml of fresh complete medium. From this time cell were detached with light (0.01%) trypsin and washed to start the cryopreservation. For hematopoietic stem cells the same samples from flushed bone marrow were used but another set of antibodies used. To isolate HSC we used CD34low/−, SCA-1+, CD90/Thy1+/low, CD38+, c-Kit+, and Lin−. Appropriate gates were constructed on a cell sorter to exclude dead cells. Cells were washed and directly cryopreserved without any plating.

Live Video Microscopy

Cells isolated by FACS and cryopreserved were plated overnight on a 24-well glass bottom plate (P24G-0-10-F; MatTek) coated with matrigel (BD Biosciences #354234) and placed in an incubator in pre-equilibrated medium (1:1 DMEM Glutamax: MCDB [Sigma-Aldrich], 20% Fetal Calf Serum (FCS) (Biowest S1860)). The plate was then incubated at 37° C., 5% CO2 (Zeiss, Pecon). A Zeiss Observer.Z1 connected with a LCI PInN 10×/0.8 W phaseII objective and AxioCam camera piloted with AxioVision was used. Cells were filmed for up to 6 days, and images were taken every 30 min with brightfield and phase filters and MozaiX 3×3 (Zeiss). Raw data were transformed and presented as a video.

Immunostaining

Immunostaining was performed either on cryosections fixed with 4% paraformaldehyde (PFA EMS #15710) in cold PBS, permeabilized with 0.5% Triton X-100 20 min at room temperature, washed, and blocked with 10% BSA for 30 min; or on cells fixed with 4% paraformaldehyde (PFA EMS #15710) in cold PBS, permeabilized with 0.5% Triton X-100 with BSA 3% 20 min at room temperature. Sections or cells were incubated with primary antibodies overnight at 4° C. (Pax7 monoclonal DSHB; Myogenin clone F5D abcam ab1835; BrdU clone B44 BD Biosciences; γ-H2A.X clone JBw1 Merk05-636) and with Alexa-conjugated secondary antibodies 1/300 and Hoechst for 45 min. Sections were then analysed using an automated axioscan (Zeiss) or inverted Observer.Z1 Apotome (Zeiss). For BrdU immunostaining specifically cells were fixed with 4% paraformaldehyde, washed and unmasked with 2N HCl for 20 minutes at room temperature, neutralised with 0.1M borate and then processed as described for other primaries antibodies.

Image Analysis

For image analysis, the ImageJ 1.46r software was used. For counting the number of PLAP+ fibres and quantifying Pax7 and Myogenin expression the cells were counted in double blind. For videomicroscopy analysis “manual tracking was done” and single cells were manually followed to assess cell division and velocity.

Mitochondrial Membrane Potential and MitoTracker Deep Red and ROS Assays

Mitochondrial membrane potential was measured after cryopreservation. 200 nM Tetramethylrhodamine ethylamine (TMRE, Sigma-Aldrich) was 30 minutes at 37° C. Cells were also incubated for 30 min with the MitoTracker Deep Red staining (FM 8778S from Cell Signalling), a dye that stains mitochondria in live cells. ROS were measured by incubating CellRox (life technologies #C10422) 30 min at 37° C. Cells were analysed by cytometry.

RT-qPCR

Total RNA was isolated from cells using the RNAeasy Micro kit (Qiagen), and reverse-transcribed using Superscript II Reverse transcriptase (Invitrogen). Real-time quantitative PCR (RT-qPCR) was performed using Power Sybr Green PCR Master Mix (Applied Biosystems) and the rate of dye incorporation was monitored using the StepOne™ Plus RealTime PCR system (Applied Biosystems). Three biological replicates were used for each condition. Data were analyzed by StepOne Plus RT PCR software v2.1 and Microsoft excel. TBP transcript levels were used for normalisation of each target (=ΔCT). Real-time PCR CT values were analyzed using the 2-(DDCt) method to calculate the fold expression.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism software using appropriate tests (non-parametric Mann-Whitney or two-way anova unless specified) and a minimum of 95% confidence interval for significance; P values indicated on figures are <0.05 (*), <0.01 (**), and <0.001 (***). Figures display average values of all animals tested±s.d, or ±s.e.m. for RT-qPCR, or as specifically indicated for the other experiments.

3. Results

In order to assess the long term effect of cell exposure to natural ionising radiations (cosmic rays and natural radioactivity) when cryopreserved, muscle stem satellite cells (SCs) were isolated from Tg:Pax7nGFP transgenic mouse1,2. SCs were stored in the 4 conditions described above. The cells were harvested at 2, 5, 7 months post cryopreservation. We assessed the number of DNA double strand break (DSB) as they are considered to be the most relevant lesion for the deleterious effects of radiations3,4(FIG.1a). An increase of the average number of DSB measured with γ-H2A.X immunostaining5was observed in the cryopreserved cells stored under “Standard” conditions as well as under “Surface” conditions, in all time points investigated (FIG.1a-d). Interestingly the SCs kept under “Deep underground” conditions displayed no statistically significant change in the number of foci observed after 7 months of cryopreservation (FIG.1b-d). The average increase in the number of foci was due to an increase in the number of cells damaged by ionising radiation (FIG.1e). To investigate the DSB disappearance kinetics we plated the cells after 7 months cryopreservation. We observed a persistence of DSB in with an efficient DSB repair taking place only 24 hours post-plating in the Condition Standard (FIG.1f) confirming previous data showing a decreased capacity to repair DSB upon very low irradiation exposure.5The use of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are very promising and there is an extensive research on those two cell types. The previous observations on SCs was confirmed by applying the same techniques and it was shown that HSCs and MSCs display fewer foci when cryopreserved under “Deep Underground” conditions (FIG.5a-d). A better preservation of the DNA integrity was observed upon preservation under “Deep underground” conditions when cells were preserved for extended periods of time.

Repair of radiation-induced DSBs in quiescent cells appears to occur primarily by Non-Homologous End Joining (NHEJ).6-8NHEJ-type repair is an error prone mechanism, which often leads to misrepaired DSBs that may result in chromosomal deletions, insertions or translocations, and subsequent genomic instability.9Thus, the genes responsible for NHEJ repair were identified in the three investigated lineages, and it was also investigated whether their expression is activated upon cryopreservation in the three storage conditions. Key regulators of NHEJ following irradiation include Ku70, Ku80, XRCC4, DNA-PKcs, DNA Ligase4, Cernunnos or XRCC4-like factor (XLF) and Artemis (Atc-ip)12. It is known that SCs transcribed NHEJ genes.7RT-qPCR of the DNA damage repair genes showed that after 7 months of cryopreservation, the genes involved in NHEJ DNA repair were much more expressed than when SCs were kept in a cosmic ray free environment (FIG.2a). Homologous recombination (HR) increased after cryopreservation in irradiated group but to a much lesser extent (FIG.2a). Those results were confirmed with a westernblot. An analysis of the anti-apoptotic (Bcl2, Bcl-xL) and pro-apoptotic (Bax, Bak) genes11showed marked differences in their expression under “Deep underground” conditions, “Standard” conditions and “Increased” conditions. Under the “Deep underground” cryopreservation conditions, it was observed an up regulation of anti-apoptotic genes whereas in the irradiation exposed conditions we observed an up-regulation of pro apoptotic genes (FIG.2a). These results were already detected as soon as 2 and 5 months post cryopreservation (corresponding to 5.25 and 10.5 years of preservation) (FIG.6a;FIG.7a) and further confirmed in other cell types HSCs and MSCs at all time points investigated (FIG.6b,c;FIG.7b,c;FIG.7b,cFIG.8ba,b). When looking at apoptosis we observed a 25% decrease of the number of cleaved caspase 3 positive cells 7 months post cryopreservation when the SCs are preserved under «Deep Underground» conditions (FIG.2c-e). These results were confirmed with Annexin V staining (FIG.8c). Cell cryopreservation under «Deep Underground» conditions also resulted in less reactive oxygen species (ROS) upon thawing the cells (FIG.2f). This is key as ROS can affect signalling pathways by directly reacting with various proteins to alter processes that regulate cell cycle progression, apoptosis, quiescence or differentiation; key features of stem cells12-14. Interestingly, TMRE measuring the mitochondrial membrane potential showed reverse staining 7 months post cryopreservation, also indicating the better state of SCs when kept away from radiations (FIG.8d) and no change in the mitochondrial mass were observed as measured by mitotracker staining (FIG.8e). The cells are less prone to enter apoptosis and have less ROS when kept under «Deep Underground» conditions for extended periods of time.

The “Increased” conditions were designed to test the influence of neutron (Altitude condition) versus gamma-rays. The same number of foci as when cells were kept with radioactivity was observed (FIG.3a) and the gene expression showed the same level of repair DNA NHEJ expression (FIG.3b). However, in absence of additional radioactivity, no significant elevation of ROS nor TMRE level was detected (FIG.3c,d). It was also observed a decreased level of apoptotic cells compared with the cryopreservation with enhanced radioactivity (FIG.3d).

In order to investigate whether the cryopreservation conditions have a functional impact the previous experiment was repeated. A serial passage and FACS analysis of each cell types and conditions was performed in vitro and in vivo. SCs were isolated, cryopreserved under “Standard”, “Increased” and “Deep underground” conditions, and at 7 months post-cryopreservation the cells were thawed and live video microscopy was performed to assess their behaviour. After preservation under «Deep Underground» conditions the cells took shorter time to perform the first division (FIG.4a). This data was further confirmed by cultivating the SCs with BrdU 24 h. It was showed that fewer cells were in S-phase after cryopreservation in an irradiated environment (FIG.9a-c). The second division was also faster when cells were cryopreserved under «Deep Underground» conditions. Notably, no differences in cell-cycle time were observed for subsequent divisions in any of the groups studied (FIG.4b). The same observation was made for velocity of the cells, with faster SCs when kept under «Deep Underground» conditions but only for the first and second divisions (FIG.4c). When investigating in vitro the differentiation potential of the SCs kept in all different conditions we observed by immunostaining a faster decrease of the nuclear protein Pax7 (marker of quiescent SCs15, (FIG.4d)) and faster expression of Myogenin (marker of differentiation of SCs16, (FIG.4e-g)).

The differentiation and self-renewal potential of these subpopulations was then examined in vivo in transplantation and regeneration assays. Triple transgenic mice were used for quantifications: Tg:CAG-hPLAP carrying the human placental alkaline phosphatase gene that is expressed ubiquitously19, Tg:MLC3F-nlacZ-2E that marks differentiated myonuclei20and Tg:Pax7-nGFP to isolate the SCs by cytometry.1,2SCs were isolated by FACS, and 10,000 GFP+ SCs were transplanted in cryodamaged tibialis anterior (TA) muscle of immunocompromised Rag2−/−:gC−/−mice. SCs from irradiated conditions were injected in the left TA, SCs from cosmic ray free environment in the right TA. Twenty-eight days later, analysis by immunofluorescence showed a large area of newly generated PLAP+ myofibers and no differences could be detected (FIG.4h-j). To assess the long-term regenerative capacity of the SCs cryopreserved in different conditions in vivo, an initial transplantation was performed with 10,000 SCs isolated by FACS (FIG.4k). Four weeks later, several thousand GFP+ SCs were collected, pooled (respecting the condition of storage), and used for transplant into the pre-injured TA muscle of secondary recipient mice. Subsequent serial transplantations were performed in a similar manner (four rounds maximum;FIG.4k). A drop in cell numbers was often observed between transplanted and harvested cells, likely due to cell death immediately after transplantation19. After the first round of grafting an average of 64% more cells were collected under «Deep Underground» conditions with respect to “Standard” conditions. The second round led to a 25% increase in SCs recovery and the difference was lost after the third round. However, it is noteworthy that the quantity of cells recovered was always higher in the condition of storage cosmic ray free. HSCs isolated from Tg:Actin-GFP and injected intravenously into 95cGy irradiated Rag2−/−:gC−/−mice showed more efficiency to make white blood cells (FIG.9d). However, after 8 weeks the same quantity of GFP+ cells was found in the blood and the bone marrow in both condition of storage (FIG.9e). After cryopreservation in an irradiated environment, the SCs take longer time to exit quiescence but differentiate faster. They sustain fewer rounds of serial transplantations. For HSCs the participation of the cells to the blood tissue is also faster. However, upon completion of the regeneration, no differences were detected in those paradigms.

4. Conclusions

In sum, it was shown that the method of the invention prevents DSB, which is otherwise inevitable upon long-term storage. It was also shown that neutrons exposure is the main source of energy responsible for DSB, and that gamma-rays and neutrons induce higher ROS concentrations in cells. Low repair of DSB in vitro was observed, which highlights the importance of completely protecting stem cells upon cryopreservation: otherwise, cells would keep the DSB over time, and even could end up damaging the surrounding cells by “bystander” effect. In addition, quiescent stem cells rely on the NHEJ error prone mechanism, which renders them vulnerable to mutagenesis following DNA damage. Thus, to preserve the genetic stability of stem cells for extended periods of time (for instance for up to 80-90 years) and be able to use them afterwards, it seems necessary to keep them protected from cosmic ray and natural radioactivity aggressions (which are cumulative), using the method of the invention.

The method of the invention further enables to overcome the burden of non- or less-functional stem cells, when patients will need to recover the material after up to 100-year cryopreservation.

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