Marys Medicine

Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate

Contents lists available at adipose-derived mesenchymal stem cells maintain highcapability to grow and differentiate q Minonzio , Mattia Corazza Luca Mariotta , Mauro Gola , Michele Zanzi , Eugenio Gandolfi Domenico De Fazio Gianni Soldati Swiss Stem Cell Foundation, In Pasquée, 6925 Gentilino, SwitzerlandMolecular Diagnostic Laboratory, In Pasquée, 6925 Gentilino, SwitzerlandCentre de Chirurgie Plastique, Lausanne, SwitzerlandPlastic Surgery, Academia Day Clinic, Chiasso, SwitzerlandVia Visconti di Modrone 8/10, Milano, Italy In recent years, there has been a shift toward tissue-engineering strategies using stem cells for plastic and Received 14 January 2014 reconstructive surgical procedures. Therefore, it is important to develop safe and reproducible protocols Accepted 9 July 2014 for the extraction of adipose-derived stromal cells (ASCs) to allow cells to be stored in liquid nitrogen for Available online 15 July future needs.
The aspirated liposuction obtained from healthy donors were immediately processed after the suction using a protocol developed in our laboratory. The resulting stromal vascular fraction (SVF) was then Adipose-derived stem cells characterized by the presence of adipose-derived stromal cells, at later stage frozen in liquid nitrogen.
Human cell therapy After that, cells were thawed and again characterized by adipose-derived stromal cells, cellular survival, Tissue engineeringTissue banking differentiation ability and Colony Forming Unit-Fibroblast like colonies (CFU-F).
Extraction and freezing of cells contained in the stromal vascular fraction demonstrate that thawed Adipose stem cells cells maintain the full capability to grow and differentiate in culture.
Mesenchymal stem cells The advent of adipose-derived stromal cells use in tissue engineering will assume a wide role in esthetic restoration in plastic surgery. It is thus important to develop clinically translatable protocolsfor the preparation and storage of adipose-derived stromal cells. Our results show that adipose-derivedstromal cells in serum free can easily be frozen and stored in liquid nitrogen with retention of 85% of cellviability and 180,890 cell/g yield plus normal proliferative capacity and differentiation potential com-pared with fresh controls. These observations set the basis for adipose-derived stromal cells banking.
Ó 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND stem cells (MSCs) were first isolated from bone marrow and then turned out to be able to regenerate rudiments The importance and the role of adipose tissue has been lately of bone and support hematopoiesis in vivo They also provided greatly re-evaluated after the discovery that adipose tissue is the an hemopoietic microenvironment in vitro and circulated in largest endocrine organ, which is able to interact with all major the blood between tissues . Plastic adherent populations organs via production of a wide range of hormones and cytokines isolated from bone marrow were proved to be functionally hetero- Furthermore, many groups working independently have geneous and fibroblast colony-forming unit-derived colonies were shown that adult stem cells derived from white adipose tissue made up of undifferentiated stem cells and progenitor cells. These can differentiate along multiple pathways raising great hope in cells were multipotent and they were able to differentiate into regenerative medicine, considering that adipose tissue can be an mesenchymal cells types, including osteoblasts, chondrocytes, abundant source of therapeutic cells and adipocytes. Because of the fact that MSCs are generated fromthe stromal component of bone marrow, they were later renamedas multipotent mesenchymal stromal cells (with the same Statement of funding: The authors would like to acknowledge the Swiss Stem acronym) to reflect their origin and biological properties MSCs Cell Foundation whose funding made this research possible.
are found in many tissues, including bone marrow, umbilical ⇑ Corresponding author. Fax: +41 919603707.
E-mail address: (G. Soldati).
cord, placental tissue and adipose tissue. However, adipose 0011-2240/Ó 2014 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license ).
G. Minonzio et al. / Cryobiology 69 (2014) 211–216 tissue-derived stems cells (even called adipose-derived stromal The syringe is hold vertically in the support stand for a few min- cells, ASCs) for autologous therapies are easier to obtain than MSCs utes to allow the separation of the phases, then the lower aqueous from other tissue sources, such as bone marrow, opening the door phase is discarded by pushing the piston. The sample is washed for potential Advanced Therapy Products twice. To free the cells in the aqueous phase the washed adipose human ASCs were successfully reprogrammed into tissue must be digested with the appropriate amount of Liberase embryonic stem cell-like colonies (induced pluripotent stem cell, MTF-S (Roche Applied Science, Basel, Switzerland) at a final con- iPS) faster and more efficiently than adult human fibroblasts centration of 0.28 Wünsch U/ml diluted in 10 ml DPBS (with Ca2+ , using the strategy developed by Yamanaka and co-workers.
and Mg2+). The sample is incubated for 45 min at 37 °C under con- cells are also increasingly appreciated in the plastic and stant but gentle agitation. Enzymatic reaction is stopped by aspira- reconstructive surgical procedures, where the shift toward tion of 30 ml of injectable 5% human albumin solution (CSL Behring tissue-engineering strategies using stem cells is now apparent AG, Bern, Switzerland) in the syringe. The syringe is then put back . Currently available reconstructive surgery using synthetic in vertical position to allow the separation of the phases. The lower materials or autologous fat transplants are often unsatisfactory, layer, which contains now the SVF cells, is carefully poured out into which is also due to the long-problems of volume maintenance.
a conical 50 ml centrifuge tube (TPP, Trasadingen, Switzerland).
Transplanted ASCs may overcome these problems via real stem The extracted adipose tissue is washed again with 40 ml 5% human cell-based regeneration of the tissues and thus introducing the albumin solution to increase cell yield. Finally, after filtration development of clinically translatable protocols for the preparation through 100 and a 40 lm sieve (Cell Strainer, BD Falcon, Basel, and storage of ASCs for tissue engineering.
Switzerland), SVF is centrifuged 400g, 5 min RT and the pellet this report we validate a safe and reproducible protocol to suspended another time in DPBS (without Ca2+ and Mg2+, PAA extract and freeze ASCs from lipo-aspirated and we demonstrate Laboratories, Pasching, Austria) or in tissue culture medium.
that ASCs can be frozen and thawed without damaging or compro- The SVF is then analyzed for cell count and number of nucleated mising their stem cell properties.
cells using an electronic cell counter (Hemocytometer – AxonLabABX Micros60).
Materials and methods characterization by FACS analysis Surgical techniques and adipose tissue sampling The cells of the SVF were characterized by cytofluorimetric analysis using a 10 channel Navios cytometer (Beckman Coulter, was performed during surgical esthetic procedures.
‘‘BC'', Nyon, Switzerland), as earlier . Briefly, roughly Women older than 18 years (range 18–53 years) in good health 500,000 cells from fresh SVF preparation were taken and centri- and HIV (Human Immunodeficiency Virus), HCV (Hepatitis C Virus) fuged 5 min at 400g. The pellet was re-suspended in 220 ll of and HBV (Hepatitis B Virus) negative were included in this study PBS without Ca2+/Mg2+ (Eurobio, CS1PBS01) with 1% human con- after obtaining their written informed consent.
verted AB serum (PAA, C11-021). 100 ll of cell suspensions were procedure started with a preemptive analgesia: put into 2 test tubes and stained with control antibodies IgG2a- Calecoxib 200 mg per os (400 mg for patients whose weight is over PE (BC, A12695), IgG1-KRO (BC, A96415), IgG1-APC-A750 (BC, 50 kg) about 1 h before surgery. Before going to the operating A71120) and Syto 40 (Invitrogen, S11351) for the control tube room, we administered an intravenous infusion with 100 ml of and CD146-PE (BC, A07483, CD146 is a cell adhesion antigen pres- NaCl 0.9%, Ranitidine 50 mg, Ondansetron 4 mg, Desametason ent mainly in endothelial cells), CD45-KRO (BC, A96416, CD45 is a 8 mg, Cefazolin 2 g and a sedation with Midazolam 1 mg bolus common leukocyte antigen found on all leukocytes), CD34-APC- I.V. Sedoanalgesia was performed with Sufentanil bolus I.V.
A750 (BC, A89309, CD34 is an hematopoietic progenitor cell anti- (0.05 lg/kg) and Propofol continuous infusion.
gen found on hematopoietic stem cells), 7-AAD and Syto 40 for access points of the cannula were infiltrated with a physio- the positive tube, respectively. All antibodies were used accord- logic solution containing 0.1% lidocaine and 1:100,000 adrenalin.
ingly to the manufacturer's instructions. After 20 min of incuba- The composition and the quantity of the infiltrated solution tion, erythrocytes were lysed with 1 mL of VersaLyse (BC, depended on the volume of the adipose tissue to be removed and A09777). Before acquisition, 100 ll Flow-Count Fluorospheres it corresponded to a 1:1 proportion with the aspirated amount. A (BC, 7547053) were added to the test tube. Post-acquisition, the negative pressure of 400 mm Hg was applied to the cannula data were analyzed with the Kaluza software (BC). Briefly, the connected to a 60 ml syringe for aspiration.
DNA marker Syto 40 was used to exclude cellular debris (i.e. neg-ative) and 7-amino-actinomycin D (7-AAD) was used for dead Isolation of stromal vascular fraction (SVF) and live cell discrimination and therefore for assessing the cellularviability . ASCs were identified in the CD45 and CD146 neg- isolation of the SVF was performed by means of a protocol ative and CD34 positive fraction . Finally, Flow-Count Fluor- we developed in our laboratories . This isolation protocol is ospheres were used to directly determinate the absolute number of based on the use of a 100 ml syringe (Omnifix 100 ml with Luer ASCs by applying the formula: Absolute Count (cells/ll) = (Total Adaptor, B. Braun AG, Melsungen, Germany) as a separation funnel Number of Cells Counted/Total Number of Fluorospheres Coun- (Patent pending). The protocol is based on the fact that adipose ted)  Flow-Count Fluorospheres Assayed Concentration.
tissue and hydrophilic fluids spontaneously separate in two phaseswith no need of centrifugation. The piston of the syringe is used to ing Unit (CFU-F) assay take in or to expel the solutions used to wash the sample, to disso-ciate the suctioned fat, or to extract the cells from the dissociated The CFU-F assay was performed as already described elsewhere adipose tissue. The syringe is hold in a vertical position using a and used to evaluate the frequency of mesenchymal progenitors in laboratory apparatus stand with support rings. Therefore, all the the SVF fraction. Therefore, freshly extracted nucleated cells were necessary manipulations for the extraction of ASCs are performed plated at two cell concentrations (5000 and 10,000 cells) in stan- inside the syringe and last about 70 min. The first step is to wash dard 100  20 mm tissue culture dishes (growth area 58.95 cm2, the sample with 40 ml Dulbecco's PBS (DPBD, with Ca2+ and BD Falcon, Basel, Switzerland) and cultured in MEM/5% converted Mg2+, PAA Laboratories, Pasching, Austria) by gentle agitation.
human serum/1% antibiotics for 14 days. The plates were then G. Minonzio et al. / Cryobiology 69 (2014) 211–216 washed with DPBS, fixed in 2% formaldehyde (Sigma–Aldrich, Buchs, Switzerland)/0.2% Glutaraldehyde (AppliChem, Darmstadt,Germany) for 5 min and stained with crystal violet solution analyzed the adipose-derived stromal vascular fraction of (Sigma–Aldrich, Buchs, Switzerland) for 10 min. After washing more than 130 liposuction procedures. We show here the obtained the plates with water, the number of colonies were counted. A col- data from N = 44 adipose tissue samples before cell culture. On ony consisting of more than 50 cells was defined as a CFU-F.
average, we obtained 75.3 g of fat tissue per sample and 180,890total nucleated cells/g. The procedure developed in our laboratoryallows the extraction of nucleated cells in a safe and the reproduc- tion and thawing of SVF ible way by showing an average cell viability of 85.05% as mea-sured by 7-AAD stain (and , left panel).
Fresh SVF cells were centrifuged 5 min at 400g, re-suspended in cells were characterized by FACS analysis and considered 25 ml ice-cold solution of injectable 5% human albumin solution to be CD45 and CD146 negative and CD34 positive. On the 44 sam- with 5% ME2SO (Dimethylsulfoxide, WAK-Chemie Medical GmbH, ples considered we found an average of 26.44% of ASCs, following Steinbach, Germany) and transferred into a freezing 25 ml cryobag the characterization by FACS method ().
(Pall Europe Ltd., Portsmouth, England). Cells were frozen by were then checked for the ability to form CFU-F colonies.
means of a programmable freezer (Consartic GmbH, Schoellkrip- The average value for colony formation in fresh samples was pen, Germany) under the following ‘‘controlled-rate'' conditions: 5.8  10 3 colonies, where a colony was defined to have more than from 4 °C to 0 °C in 6 min, then hold for 15 min at 0 °C. From 0 °C 50 clonal cells We also checked the CFU-F after thawing 2 °C in 9 min and then hold for 2 min at by seeding cells at three different concentrations for three different 35 °C in 25.5 min and finally from 100 °C in 13 min.
For what regards thawing, the cryobag was immersed in a 37 °C water bath for 2–3 min. Immediately after being thawed, the cellswere carefully aspirated, mixed with an equal volume of injectable 5% human albumin solution in a 50 ml TPP conical tube and centri- Characterization of SVF in N = 44 adipose tissue samples.
fuged at 400g for 5 min.
culture and differentiation Adipose-derived mesenchymal stem cells were cultured until passage 2 in basal Ham's F12/IMDM (1:1) medium (Cell Culture Technologies, Gravesano, CH) supplemented with various growth factors and referred as to serum free medium (Patent pending).
Cells were then plated at a density of 3  103/cm2 onto multi wells plates (PureCoat ECM Mimetic Cultureware, BD Biosciences, Bedford, USA) for induction. Half of the wells cells were cultured in the conditions specified here above, i.e. serum free medium (basal Ham's F12/IMDM (1:1) medium supplemented with growth factors) and referred as non-induced cells, whereas in the remaining wells cells were induced to osteoblasts, adipocytes and chondrocytes by means of different induction media. For osteoinduction we used the serum free medium supplemented with 3 mM Sr2+ and 10– 200 nM Vitamin D. Cell differentiation was confirmed at day 21 by Alizarin Red staining. Briefly, the cells were fixed in 10% forma- lin for 30 min RT and incubated 30 min RT in Alizarin Red staining.
The formation of red calcium deposits is a marker of osteogenic dif- ferentiation. For adipogenic induction serum free medium was supplemented with Epidermal Growth Factor (EGF, cyt-217, ProS- pec-Tany Technogene Ltd., East Brunswick, USA) and Rosiglitazone (Sigma–Aldrich, Buchs, Switzerland). Adipogenesis was assessed by Oil Red staining. Briefly, cells fixed in 10% formalin for 30 min RT were incubated in fresh Oil O Red water solution for 5 min RT. Induced cells were visible as cells containing consistent red deposits in vacuoles. Chondrogenic differentiation was assessed by induction of ASCs using the micro mass method. Briefly, ASCs were gently centrifuged in a 15 ml conical tube to form small pel- lets and then cultured for 21 days in the serum free medium sup- plemented with sodium pyruvate, Bone Morphogenic Protein 6 (BMP6), Transforming Growth Factor Beta 3 (TGF-beta3), Fibro- blast Growth Factor beta (beta-FGF) and Prostaglandin E2 (PGE2).
Chondrogenic pellets were fixed in 10% formalin for 30 min RT.
Samples were then embedded in paraffin and sections stained with Alcian Blue. Control cells did not retain a spheroid shape and Fresh adipose tissue samples were processed for ASCs extraction as described.
showed no specific staining while induced cells showed a strong Volume, cells/g, viability and percentage of ASCs are reported in Table. (SVF, stromal blue signal.
vascular fraction; ASCs, adipose-derived stem cells.)

G. Minonzio et al. / Cryobiology 69 (2014) 211–216 Fig. 1. Viability of stromal vascular fraction cells before and after a freeze/thaw cycle. FACS analysis for 7-AAD stain in fresh extracted SVF cells (left) and the same sample ofSVF cells thawed after a freeze cycle (right). Dead cells are gated at the right of both panels. (7-AAD, 7-amino-actinomycin; SS, side scatter.) The results are showed in . Three different samples were pla-ted at 3000 cells/cm2 and cultured for 20 days. Cells showed a clas-sical growth pattern with an early lag-phase in the first 7 days anda subsequent exponential growth. After 20 days in culture, cellsreached a concentration of 42,550 cells/cm2 i.e. a 13 expansionof the initial seeded number.
The same cells were induced to differentiate into adipocytes, osteocytes and chondrocytes and representative results are shownin Cells were clearly inducible to the specified phenotypes.
Oil Red staining evidenced adipoinduction by red deposits in vac-uoles (panel A: induced and D: control), whereas Alizarin-S staining was used for osteoinduction and showed the formationof red calcium deposits as a marker of osteogenic differentiation, panel B: induced and E: control). Sections of chondro-induced samples stained with Alcian Blue showed a strong bluesignal , panel C, induced and F, control). We found all testedsamples to be inducible for the differentiation of adipocytes, osteo-cytes and chondrocytes.
Fig. 2. FACS analysis of fresh adipose-derived SVF cells. FACS analysis for thecharacterization of ASCs in fresh extracted SVF. ASCs cells are CD45 , CD34+ and and are gated in the right lower panel. (FACS, fluorescent activated cell sorter; ASCs, adipose-derived stem cells; SVF, stromal vascular fraction; CD, clusterof differentiation.) Tissue engineering keeps promise for the restoration of the soft tissue esthetic function and for the treatment of known diseasesthat have currently no therapy option . In this regard, the stor- samples and the results showed that the freezing protocol do not age of ASCs is still for long time the initial step for future cell ther- affect the clonogenic ability of ASCs. Conversely, the CFU-F number apies using ASCs for regenerative purposes. There are actually was shown to increase after thawing (data not shown).
more than 60 clinical trials worldwide involving ASCs in the treat- fresh SVF cells were successively challenged by a freezing ment of human disease (clinicaltrials.gov Current standard and thawing cycle. N = 15 samples were used in the freezing/thaw- clinical strategies for soft tissue augmentation primarily include ing procedure as described above. SVF samples ranging from the use of synthetic implants and fillers. However, various compli- 6.66  105 to 3.94  106 total cells were taken in consideration.
cations derived from the foreign body, such as capsular contracture shows the results of these experiments. Cell samples were or displacement, lead to implant removal or replacement at a kept frozen for periods ranging from 14 to 193 days. Viability of relatively high rate.
SVF cells was measured by FACS analysis and gave an average Free fat transfer gives unpredictable results, where graft reab- value of 89.6% ranging from 81% to 98%. The total ASCs content sorption can vary between patients, although it seems to work well of each fresh sample ranged from 237,938 to 1,092,925 with an for small defects correction average value of 587,753 cells. After thawing, cells were counted Mixing autologous ASCs with a portion of suctioned fat and for ASCs number and viability. We could demonstrate the viability injecting subcutaneously back into the target site is another results over 15 samples, ranging from 71.7% to 98.3% and average strategy which is recently used to overcome these problems and recovery rates of 79.82% of living ASCs after the freeze/thaw to provide a ‘‘living scaffold'' for stem cells It has become crucial to develop safe and reproducible protocols cells after a freezing/thawing cycle are important because for the extraction and storage of ASCs that can adhere to the strict the freezing process prolongs cells' life and makes them available European regulation concerning the Advanced Therapy Medicinal for future therapies based on expanded ASCs. To check whether Products (ATMPs). Storage of the SVF could be seen as an interme- the thawed cells can grow and differentiate again after the freez- diary GMP product to be needed in the future for many differenti- ing/thawing cycle, we cultivated and differentiated 3 samples of ation protocols to be developed. One step in this direction is the thawed SVF-cells in 0.1% human serum supplemented medium.
possibility to store frozen cells for long periods of time in liquid

G. Minonzio et al. / Cryobiology 69 (2014) 211–216 2Post-thaw characterization of SVF in N = 15 adipose tissue samples.
Total ASCs recovered ASCs recovery (%) SVF samples were characterized for total cell content, total ASC content and viability and frozen in 10% DMSO in liquid nitrogen following the protocol described in Materialsand Methods. Post-thawing results are shown for ASCs recovery and percentage of viability recovered after the freeze cycle.
nitrogen and to be able to use them after thawing, i.e. for cellamplification and/or differentiation.
show here that SVF extracted cells can be frozen and thawed without losing their ability to grow and differentiate in a few studies examined the role of frozen storage of adi- pose tissue. One of them has recently described the storage of entire adipose tissue at various temperatures for periods longer than 1 year to see whether the tissue was still capable of adipo-genic differentiation. Cells isolated from the tissue proved to be a reliable source of human ASCs and adipocytes Early research studies described a domestic 18 °C storage of adipose tissue for 2 weeks. Injection of fat tissue in nude mice demonstrated the sur- Fig. 3. Growth curve of frozen/thawed ASCs. N = 3 samples (gray lines) of adipose- vival of this tissue as compared to a control group of non-frozen derived mesenchymal stem cells (ASCs) submitted to a freeze/thaw cycle were tissue A simple freezing technique was recently used by stor- cultured for 21 days in a medium supplemented with 1% human serum. (ASCs,adipose-derived stem cells; red line = mean ± SEM). (For interpretation of the ing fat tissues at 196 °C in liquid nitrogen for up to 8 days dem- references to color in this figure legend, the reader is referred to the web version of onstrating a good maintenance of mitochondrial metabolic activity in the frozen grafts . Remarkably, in both experimentsfat tissue samples were frozen without the addition of a cryopro- Fig. 4. Differentiation assay of thawed ASCs. Cells were grown confluent to 70% and induced for 9 days to differentiate to osteo-, chondro- or adipo-lineages. Staining wasperformed as described in Materials and methods, where, induced and non-induced samples were treated the same way. Oil-O-Red staining for adipocytes, Alizarin-S stainingfor osteocytes and Alcian Blue staining for chondrocytes were used. Induced cells incubated in fresh Oil-O-Red were visible as cells containing consistent red deposits invacuoles (Panel A: induced cells; panel D: control); osteocytes induction, evaluated with Alizarin-S staining, is shown by the formation of red calcium deposits (Panel B:inducted cells, Panel E: control) and chondrocyte induction, as evaluated by Alcian Blue staining, show in Panel C (induced cells) robust fiber tracts of collagen matrix (blackarrows) and several chondrocyte-like cells (dashed circles at arrowhead points) as compared to Panel F (control non-induced) where only marginally developed collagen-likefibers can be evidenced (black arrows), chondrocyte-like cells cannot be clearly identified and Alcian Blue stained areas are rather sparse (arrowheads). (ASCs, adipose-derived stem cells.) G. Minonzio et al. / Cryobiology 69 (2014) 211–216 tective agent. Another study reported the use of a cryoprotective agent to better save and keep viable tissues after thawing .
we have to consider that adipose tissue is the source of ASCs responsible for the biological effect observed in regenerative medicine. Thus, for long conservation purposes we should only consider the stromal vascular fraction (SVF) by isolat- ing it from the carrier tissue. Indeed, the vast majority of studies report the separation, growth and differentiation of the SVF and all clinical trials to date using ASCs have been designed on this par- ticular fraction of cells, where a large number of stem cells have studies about the cryoconservation of human SVF- cells extracted from adipose tissues are rare (for a review see ). Recently, it has been described a method for liquid nitrogen storage of SVF-cells , where thawed SVF-cells has been shown to differentiate into adipocytes and endothelial cells. Unfortunately, this study used a freezing medium containing fetal bovine serum thus avoiding the possibility to use cells as an Advanced Cell Ther- apy Product.
presence of serum in the freezing medium was also chal- lenged in another study and reported to be not necessary by the authors. They suggested indeed that post-thaw ASCs viability, adi- pogenic and osteogenic differentiation can be maintained even when ASCs cells are frozen in the absence of serum but with a min- imal concentration of 2% ME2SO in DMEM which represents a step forward to the use of these cells as therapeutic agents. Other reagents like sericin, a protein hydrolysate very rich in serine, has been used in the freezing medium and found to be effective on the survival of ASCs and in their differentiation potential .
are pluri-potential cells and can thus give rise to many target tissues, like bone, tendons, cartilages, heart and nerves, opening the door to the real world of Advanced Therapy Products that, in a first time, will be autologous-based but could in the near future be engineered to everyone's need.
designed and validated a protocol to extract and freeze SVF stem cells from adipose tissues that allows thawed cells to main- tain their growth and differentiation potential. Overall, our data show that the SVF can be easily frozen following defined standard conditions for cell freezing. The yield after the procedure, in terms of cell survival number and percentage of viable cells, is high enough to be safely used for banking purposes.
results need further confirmation and we are actively working on the GMP-validation of the whole process to be able to store SVF-cells as a real medicinal drug, allowing thus the patient to dispose of his own cells for cell therapies in the near

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