Electrical control of calcium oscillations in mesenchymal stem cells using microsecond pulsed electric fields
© The Author(s). 2017
Received: 1 October 2016
Accepted: 14 March 2017
Published: 20 April 2017
Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions.
One or several electric pulses of 100 μs were used to induce Ca2+ spikes caused by the penetration of Ca2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked.
According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability.
An easy way to control Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2+ oscillations in these cells. Since microsecond electric pulse delivery constitutes a simple technology available in many laboratories, this new tool might be useful to further investigate the role of Ca2+ in human mesenchymal stem cells biological processes such as proliferation and differentiation.
KeywordsMesenchymal stem cells Calcium oscillations Calcium spikes Electroporation Electric pulses Electropermeabilization Electropulsation
Mesenchymal stem cells (MSCs) are multipotent stromal cells  originating from the embryonic mesoderm (mesenchyme) and present in many adult tissues such as bone marrow (bMSCs), adipose tissue (aMSCs), muscle, dermis, umbilical cord, etc. [2, 3]. These cells have gained a lot of momentum in the last decade due to their ability to differentiate into a wide variety of cells including osteoblasts, myoblasts, fibroblasts and chondrocytes. They also express key markers of cardiomyocytes, neuronal and endothelial cells . This ability makes them a very promising candidate for cell therapy and regenerative medicine in order to heal damaged tissues and organs. However, MSCs from different tissues are not the same. They have different differentiation capacities and transcriptomic signatures .
Human-adipose MSCs (haMSCs), derived from adipose tissue are amongst the most easily accessible MSCs, with high quantities, and without aggressive extraction procedures. They are more available than other MSCs as, for example, the human bMSCs (hbMSCs). In addition, they have a phenotype, surface markers , and gene expression profile similar to those of the hbMSCs, and they are easier to maintain and proliferate , which make them ideal MSCs to use .
These cells present spontaneous Ca2+ oscillations, implicating Ca2+ channels and pumps of the plasma membrane (PM) and the endoplasmic reticulum (ER) . These oscillations seem to start by an ATP autocrine/paracrine signaling  followed by inositol triphosphate (IP3)-induced Ca2+ release from the ER and further amplification from plasma membrane store-operated Ca2+ channels (SOCCs). Afterwards, the excess of Ca2+ is removed from the cytosol by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), the plasma membrane Ca2+ ATPase (PMCA), and the Na+/Ca2+ exchanger (NCX) . Ca2+ is one of the most important second messenger in the cell, and it regulates many important cellular processes such as ATP synthesis, apoptosis, cellular motility, growth, proliferation and gene expression. Hence, Ca2+ oscillations contain embedded information that has to be decoded by the cell, and Ca2+ signalling pathways play a key role in controlling cell behaviour and differentiation processes of MSCs.
It was shown that the Ca2+ oscillations frequency is different between undifferentiated MSCs and MSCs on route to differentiation and it differs between the various outcomes of the differentiation process (the level of differentiation and the differentiated cell type). While the MSCs exhibit regularly repeated Ca2+ oscillations, MSCs undergoing osteodifferentiation display a decrease in the frequency of their spontaneous Ca2+ oscillations while primary myoblasts present still another pattern of oscillations . This shows that each cell type possesses its own pattern of Ca2+ oscillation frequency and shape, but the exact correlation between Ca2+ oscillations and MSCs differentiation is still unclear.
Presently, pulsed electric fields (PEFs) are widely used in research as a non-invasive physical means to permeabilize cellular membranes. Using one or several pulses of ultrashort duration causes changes in the cell membrane structure that permits access to the cell cytosol to molecules that cannot cross the plasma membrane under normal conditions . Normally, Ca2+ is an ion that only crosses the plasma and ER membranes through channel proteins. Applying PEFs to cells in a medium containing Ca2+ (amongst other compounds) allows Ca2+ entry from the cell outside, and, if the electric field amplitude and pulse duration are high enough, Ca2+ is released from the cell inner stores . The electromagnetic fields (EMFs) have been already applied on hbMSCs to test their effect on the differentiation of the latter. Those EMFs were either direct currents of very low electric field amplitude (0.1 V/cm) and a long time exposition (30 minutes)  or biphasic electric currents (1.5 μA/cm2 for 250 μs) . These fields either activate voltage-operated Ca2+ (VOCCs) if their amplitude is large enough or do not alter the membrane potential if the electric field is too low. None of the electromagnetic fields used was permeabilizing the cell PM.
The novelty of our study is the use of PEFs of a high field amplitude that are capable of permeabilizing the cell PM or both the PM and the ER. Our objectives were to test the effect of these pulses on the Ca2+ oscillations of the cells. Attached haMSCs presenting normal spontaneous Ca2+ oscillations were exposed to one single pulse with a duration of 100 μs and different electric field amplitudes. At low and moderate electric field amplitudes, the Ca2+ spike induced by such PEFs can be very similar to a spontaneous oscillation. At high electric field amplitudes, the electrically induced Ca2+ spikes inhibit the spontaneous oscillations occurrence for some time after which oscillations reappear. This inhibition allowed us to impose on the cells experimentally induced Ca2+ spikes at a desired frequency. Hence, we show here that it is possible to control haMSCs spontaneous Ca2+ oscillations by microsecond pulsed electric fields (μsPEFs) with a complete preservation of the cell viability. The delivery of μsPEFs constitutes an easy way to control Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes. Indeed, unlike chemical factors, which cause continuous effects, the direct link between the microsecond electric pulse(s) delivery and the ulterior occurrence/cancellation of the cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2+ oscillations in these cells. Since microsecond electric pulses delivery constitutes a simple technology available in many laboratories, the electrical control of Ca2+ oscillations could be in the future a promising tool to further investigate the role of Ca2+ in MSCs physiology and to better understand the correlation between the Ca2+ oscillations and MSCs biological processes such as proliferation and differentiation.
Cells and cell culture conditions
HaMSCs were isolated from surgical waste of individuals undergoing elective lipoaspiration. Samples were obtained after written informed consent from all the donors, in accordance with French and European legislations. The lipoaspirates were surgical waste and as such the French legislation (Art.L. 1245-2 du Code de la Santé Publique) establishes that the authorisation from an ethics committee is not required. Every experiment was done on cells before passage 8 from at least two donors (seven different donors in total). All haMSCs reacted similarly to the electric pulses, whoever the donor. The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with Glutamax and supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin and were cultured at 37 °C in a humidified incubator with 5% CO2. Cells were passed twice a week (every passage corresponds to one doubling time of the population). The multipotency capabilities of the cells were assessed by submitting them to differentiation conditions as previously reported in Liew et al. . Cells from all the donors have thus been differentiated in osteoblasts, adipocytes and chondrocytes. The cell culture chemicals were purchased from Fischer Scientific (Parc d’innovation Illkirch, France).
One day prior to the experiments, cells were seeded in 24-well plates at a density of 20 × 103 cells/cm2. After 1 day, the cells were incubated for 30 minutes with 5 μM of Fluo-4 AM (Fischer Scientific), a fluorescent Ca2+ marker, in a humidified 5% CO2 atmosphere at 37 °C in complete DMEM. The incubation buffer also contained 375 nM of the nuclear fluorescent dye Hoechst 33342. After incubation, the attached cells were rinsed three times with PBS (phosphate-buffered saline) and 500 μl of complete DMEM were added to the cells.
Microsecond pulse generator and electrodes
One single micropulse of 100 μs was delivered in all the experiments. A Cliniporator™ (Igea, Carpi, Italy) was used for the generation of the microsecond pulsed electric fields (μsPEFs). In order to treat the cells under the microscope, the pulse generator was connected to two parallel stainless steel rods of 1.2 mm diameter used as electrodes. The distance between them was 5 mm, and they were shaped to enter a 24-plate well and to reach the bottom of the dish. The whole system was set under a Zeiss Axiovert S100 epifluorescence inverted microscope.
In order to test the cell viability, another model of electrodes was designed. In this system, a thick cover of 10 cm2 containing two slots was designed to fit into a Petri dish of the same dimensions. Two plate electrodes of 2 mm thickness and 2 cm length were slipped into the slots until they touched the bottom of the Petri dish. This system does not permit observation of the cells under a microscope, it was used because it allows covering a larger surface (in order to have enough cells) than the system described above.
Images acquisition and analyses
Images of the cells were taken every 10 seconds for 10 to 20 minutes with a Zeiss AxioCam Hrc camera controlled by the Axio Vision 4.6 software (Carl Zeiss, Oberkochen, Germany). The electric pulse was delivered after at least 2 minutes of recording, and 2 seconds before the next image. The excitation and emission wavelengths used for Fluo-4 were 496 nm and 515 nm respectively. The nuclear dye Hoechst 33342 (λex = 350 nm, λem = 461 nm) was used to recognise the nuclei and to track the cells using the Cell Profiler (version 2.0) software (Broad Institute, Cambridge, MA, USA). The software allowed the automatic measurement of the fluorescence intensity signal of each cell on every image. Curves were plotted using a Matlab program (version 7.8.0, The MathWorks Inc., Natick, MA, USA). All the observations were done at room temperature. The minimum opening time of the shutter for the fluorescent light was about 500 ms. To decrease the light energy applied on the cells, a 90% density Filter NE110B (Thorlabs, Maisons-Lafitte, France) was used.
Cell viability assessment
One day prior to the experiments, cells were seeded at a density of 20 × 103 cells/cm2 in 10-cm2 Petri dishes containing a 12 × 32 mm cover slide. After 24 hours, medium was removed, cells were washed with PBS, and 1 ml of fresh medium was added. The electrodes were placed on the cover slide using the Petri dish cover containing two slots and a 100-μs pulse was delivered to the attached cells. After the pulse, the Petri dish was put in the incubator for 20 minutes. Then, the non-pulsed cells on the cover slides were scratched under a sterile hood. The area covered by the pulsed cells was recognised due to the loss of cells beneath the electrodes after the removal of the latter. The cover slide with the pulsed cells only was transferred to a Petri dish with fresh medium and placed for 2 hours in the incubator. Then medium was removed and the cells were trypsinised, centrifuged, and resuspended in 300 μl of a fresh medium. This volume was distributed in two wells of an opaque-walled 96-multiwell plate and put in the incubator for 24 hours. After 24 hours, cells were visualised on an epifluorescence microscope to compare their shape and level of confluence, and 150 μl of Cell-Titer Glo Reagent (Promega, Madison, WI, USA) were added to each well according to manufacturer’s protocol. The reagent caused cell lysis and the generation of a luminescent signal proportional to the amount of the ATP present in the medium. A GloMax luminometer (Promega) was used to read the luminescence. The ATP amount and therefore the luminescence intensity reflected the number of cells present in the culture .
Spontaneous Ca2+ oscillations in haMSCs in normal conditions and after one 100-μs electric pulse
Ca2+ spikes in response to different electric field amplitudes
Different outcomes on Ca2+ oscillations according to the electric field amplitude
Electrically mediated Ca2+ spikes manipulation after the 900 V/cm inhibition
Mesenchymal stem cells (MSCs) are multipotent stromal cells that have the ability to self-renew and to differentiate into a wide variety of cells of the mesodermal, ectodermal, and endodermal lineages . This is why they represent an important tool for cell therapy and regenerative medicine. However, the differentiation procedures may be long and not able to respond to the demand. The improvement and optimisation of these procedures is of a crucial importance to ensure the differentiated cells in a reasonable time. Many studies have tested the effects of physical stimuli on the differentiation outcomes. Altman et al. showed that the application of a cyclic mechanical stimulation (translational and rotational strain) to undifferentiated MSCs (embedded in a collagen gel) over a period of 21 days resulted in ligament cell lineage differentiation even without differentiation factors . Ward and colleagues studied the effect of the application of a 3–5% tensile strain to a collagen I substrate and concluded that it stimulated osteogenesis of attached hMSCs . Low-intensity ultrasounds, another form of mechanical stress, have been shown to enhance the chondrogenic differentiation . Sun et al. demonstrated that 0.1 V/cm electrical stimulation applied for 30 minutes per day for 10 days, on attached MSCs cultured with osteoinductive factors, accelerated their osteodifferentiation .
Ca2+ oscillations play an important role in the transduction of many physical stimuli in the cells. For example, tissue strain and shear stress on mouse tibia are transduced by repetitive Ca2+ spikes in the osteocytes. The spikes frequency and amplitude depend on the mechanical magnitude . Electromagnetic fields (EMFs) can also promote MSCs differentiation to osteoblasts through Ca2+-related mechanisms. The exposition of MSCs to pulsed EMFs for 10 minutes every day results in the enhancement of osteogenesis early stages .
Ca2+ oscillations are a universal mode of Ca2+ signalling in both excitable [24, 25], and non-excitable cells . In non-excitable cells such as hMSCs, Ca2+ oscillations are typically initiated by a receptor-triggered production of IP3 (after the binding of an agonist to the receptor)  and the subsequent Ca2+ release from the ER , which stimulates the store-operated calcium entry (SOCE) via the SOCCs . It was shown that the Ca2+ oscillation frequency is different between undifferentiated MSCs and MSCs on route to differentiation and it differs between the various differentiated cell types . According to Titushkin et al., the MSCs Ca2+ oscillations are suppressed after the addition of neuroinductive factors, but reappear in less than 7 days after neurodifferentiation . Sun and colleagues observed that multipotent MSCs present 8.06 ± 2.64 Ca2+ spikes per 30 minutes of observation, and that this number decreases to 3.66 ± 2.42 after 28 days of incubation with osteoinductive factors .
In our study, we decided to analyse the effect of a type of short electric pulses termed microsecond pulsed electric fields (μsPEFs) on the spontaneous Ca2+ oscillations of haMSCs because many of the physical stimuli are transduced by cytosolic Ca2+ concentration rapid changes (oscillations) in the cells. The ultrashort pulses (one thousandth to one ten thousandth shorter than the pulses of 100 μs used in our study) called nanopulses or nanosecond pulsed electric fields (nsPEFs) have been used already to induce Ca2+ bursts in the cell, which resulted from the electropermeabilization of the ER, the plasma membrane or both [13, 30]. However, contrary to the 100-μs electric pulses, the technology to deliver nanopulses is not yet spread in the laboratories and is not simple to use.
The haMSCs loaded with the fluorescent marker Fluo-4 asynchronously displayed periodic changes in their cytosolic Ca2+ concentration detected by the periodic increase of their fluorescence. The videos of the haMSCs showed that not all the cells presented Ca2+ oscillations at a specific time. Some of the cells never displayed oscillations during the recording time (20 minutes). This observation can be related to the fact that MSCs display Ca2+ oscillations only during the phases G1 and S of the cell cycle, and not all the cells in the visualisation area of an experiment were in those phases of the cell cycle. Ca2+ oscillations increase the levels of cell cycle regulators such as cyclins A and E and probably control cell cycle progression and cell proliferation, via the regulation of cyclin levels (amongst other mechanisms) . In addition, the haMSCs presenting Ca2+ oscillations displayed them at different rhythms and frequencies: the oscillations were asynchronous, and at a given time, each cell was in a different phase of a Ca2+ oscillation and the oscillations frequencies displayed wide variations between cells. The average duration of an oscillation was around 2 minutes.
Electric pulses of 100 μs duration are already applied in many biotechnological and medical applications, notably in anticancer electrochemotherapy , tumour ablation , and cell transfection : by permeabilizing the plasma membrane temporally they allow the internalisation of non-permeant molecules of interest like drugs or nucleic acids. Classically, eight successive pulses of 100 μs are used in biomedical sciences. The technology is spread in many laboratories and is very simple to use. In our study, we applied a single electric pulse because, when several pulses are delivered, pulses repetition frequency may impact the efficacy of cell membrane permeabilization as we have recently shown in our group . When an electric pulse of 100 μs was applied to the cells, an electrically induced Ca2+ spike was observed synchronously in a given percentage of the cells (as a function of the electric field amplitude). Moreover, the electric field amplitude should be enough to cause the transmembrane voltage to reach a permeabilization threshold. This threshold depends on the size of the molecule to be internalised. This is why, at 100 V/cm, which is a very low electric field amplitude, no Ca2+ spike was observed because there was no permeabilization and hence no Ca2+ entry. 120 V/cm was the lowest electric field amplitude at which some cells presented Ca2+ spikes. This threshold is very low compared to other ones previously reported, because the Ca2+ has a small size compared to other classical markers such as yo-pro-1 iodide, propidium iodide or bleomycin that are those frequently used (Hanna et al., submitted). At 450 V/cm, 100% of the cells responded to the electric pulse. The origin of the Ca2+ causing the Ca2+ spike was not always the same: it could be the result of Ca2+ entry from the external medium only, or the combination of Ca2+ entry from cells outside and Ca2+ release from the ER. Our group demonstrated recently that the μsPEFs could permeabilize not only the cell membrane (as usually observed and stated) but also the internal organelles membranes (Hanna et al., submitted). The origin of the Ca2+ spike depends on the electric field amplitude. If the latter is below 500 V/cm, the pulse permeabilizes only the plasma membrane (Hanna et al., submitted), and the Ca2+ spike is primarily the result of Ca2+ entry through the plasma membrane followed by an amplification due to Ca2+ entry from the voltage-operated Ca2+ channels (VOCCs), activated by the membrane depolarisation . However, above 500 V/cm, the electric pulses will also affect the ER membranes, permeabilize them and cause Ca2+ release from the ER (Hanna et al., submitted). Hence, the Ca2+ spike at high electric field amplitudes, resulted primarily of a massive Ca2+ entry through the plasma membrane (due to the generation of larger pores at the higher field amplitudes) also followed by the external Ca2+ entry through the VOCCs, the Ca2+ release from the ER and the subsequent stimulation of the store-operated Ca2+ channels (SOCCs) . This explains why the Ca2+ spike induced by electric fields below 500 V/cm had a very similar shape to the spontaneous Ca2+ oscillations with a gradual increase and same amplitude, whereas the Ca2+ spike induced by electric fields above 500 V/cm had higher amplitude than the spontaneous oscillations, and presented a sharper rise (due to the abrupt penetration of Ca2+ through the permeabilized membranes). At 600 V/cm, three different outcomes were observed, mainly due to four factors: first the distribution of the electric field which results in field amplitude small differences in the area between the two electrodes; second, the cells are heterogeneous in size and according to Schwan equation , the electric field impact will be different between the smallest and the largest cells; third, the orientation of the cell with respect to the electric field lines, which has an impact on the induced transmembrane potential that will lead to the cell membrane permeabilization ; fourth, the position of the cells are in the cell cycle that could lead to different responses to the pulses. However, at 750 V/cm, it seems that the electric field has exceeded a certain threshold above which no more heterogeneity was observed and nearly all the cells had their Ca2+ oscillations inhibited.
The Ca2+ spikes obtained looked like the Ca2+ spikes induced by even shorter electric pulses (the so-called nanopulses, of a duration of e.g. 10 ns, that can directly affect the cell internal membranes such as those of the endoplasmic reticulum ). In our study, an electrically induced Ca2+ spike with properties very near to the oscillations did not inhibit the latter, could be applied at any time of an oscillation without interfering with the latter, and after the pulse, the oscillations continued normally at their own rhythm, and with their normal shape. Nevertheless, a high-amplitude Ca2+ spike inhibited the Ca2+ oscillations. The percentage of cells that were unable to display further Ca2+ oscillations increased proportionally with the electric field amplitude of the inhibitory electric pulse. The Ca2+ oscillations inhibition could last some minutes or be longer, to reach tens of minutes, also depending on the electric field amplitude of the inhibitory electric pulse.
The inhibition of the Ca2+ oscillations for some time allowed us to study the state of the cell during this period, to determine whether the cell was in a “refractory state” or if it was still able to respond to an electric pulse. The application of several pulses after a 900 V/cm pulse inhibition showed that the cells could still react to the pulse and present Ca2+ spikes with different amplitudes depending on the applied electric field amplitude.
Seven days after exposure, MSCs showed similar stem cell surface antigens expression compared with non-treated MSCs.
Four days after exposure to the eight electric pulses, MSCs were cultured in osteogenic and adipogenic differentiation media and successfully differentiated into osteoblasts and adipocytes.
Eleven days after exposure to the electric pulses, MSCs cultures yielded similar cell growth rates compared with non-treated MSCs cultures.
The electrically induced Ca2+ spikes can have wide effects on the Ca2+ oscillations frequency, amplitude and duration. Whereas the nanosecond pulses allowed only to add a Ca2+ spike to the oscillations , the microsecond pulses display more complex effects. The electric field amplitude allowed us either to induce a Ca2+ spike without affecting the oscillations, or to inhibit the Ca2+ oscillations and impose on the cell Ca2+ oscillations at a desired frequency and amplitude. It is worth noting that the microsecond technology is cheaper than the nanosecond one, it is more available in the laboratories, and simple generators can deliver it. These considerations add further value to the observations reported here. Taking into account that each type of differentiated cell from MSCs has its own Ca2+ oscillation characteristics, and that these oscillations may play an important role in the decision of MSCs fate, the μsPEFs constitute a novel useful tool to control the oscillations and the differentiation of MSCs.
hMSCs show great promise for regenerative medicine. As a function of the pulse(s) electric field amplitude, we report here that it is possible to generate in hMSCs a supplementary calcium spike with properties close to those of their spontaneous calcium oscillations, or, on the contrary, to inhibit their spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. The delivery of short PEFs of 100 μs, a robust technology easy to apply, could be therefore a promising way to investigate the role of the Ca2+ in MSCs physiology and biological processes such as proliferation and differentiation.
Adipose tissue mesenchymal stem cells
Bone marrow mesenchymal stem cells
Dulbecco’s Modified Eagle Medium
Mesenchymal stem cells
Pulsed electric fields
Plasma membrane Ca2+ ATPase
Sarco/endoplasmic reticulum Ca2+ ATPase
Store-operated Ca2+ channels
Voltage-operated Ca2+ channels
Microsecond pulsed electric fields
The authors would like to thank Dr. Bassim Al Sakere for the lipoaspirates.
This work was supported by CNRS, Gustave Roussy, Université Paris-Sud, the ITMO Cancer in the frame of the Plan Cancer 2015-2019 (project PC201517) and the Fondation EDF. The research was also conducted in the scope of the EBAM European Associated Laboratory (LEA) and of the COST action BM 1309 EMF-MED.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request
HH, FMA and LMM designed the study, contributed to data interpretation, analysed the data and drafted the manuscript. HH performed the experiments. All authors edited and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
All the authors have read and approved the manuscript for publication.
Ethics approval and consent to participate
HaMSCs were isolated from surgical waste of individuals undergoing elective lipoaspiration. Samples were obtained after written informed consent from all the donors, in accordance with French and European legislations. The lipoaspirates were surgical waste and as such the French legislation (Art.L. 1245-2 du Code de la Santé Publique) establishes that the authorization from an ethics committee is not required.
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