EXTRACELLULAR VESICLES DERIVED FROM MESENCHYMAL STEM CELLS
20210113625 · 2021-04-22
Inventors
Cpc classification
C12N2501/165
CHEMISTRY; METALLURGY
A61K35/28
HUMAN NECESSITIES
International classification
Abstract
The present invention discloses a composition comprising extracellular vesicles (EVs) of placenta tissue derived CD106.sup.high CD151.sup.+Nestin.sup.+ mesenchymal stem cells (MSCs). In a first aspect, the invention relates to a particular method to prepare these EVs. In a second aspect, the invention relates to a therapeutic, a diagnostic, a veterinary or a cosmetic composition comprising the extracellular vesicles (EVs) obtained by said particular method. In a third aspect, the invention relates to a composition comprising these EVs, for use as a medicament for treating subjects suffering from an ischemic disease, a disorder of the circulatory system, an immune disease, an organ injury or an organ function failure.
Claims
1. A composition comprising extracellular vesicles (EV) obtained by: (i) culturing mesenchymal stem cells obtained from a biological tissue or fluid in a first culture medium deprived of growth factors, so as to generate a population of cultured undifferentiated mesenchymal stem cells, (ii) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing at least two pro-inflammatory growth factors, thereby generating CD106.sup.high CD151.sup.+Nestin.sup.+ mesenchymal stem cells, (iii) Culturing said MSCs in a EV-free culture medium, under conditions permitting their expansion; and iv) Purifying the EVs from the cells obtained in step (iii).
2. The composition of claim 1, wherein said second culture medium used in step (ii) contains at least two pro-inflammatory growth factors chosen among TNFα, IL1, IL4, IL12, IL18, and IFNγ, preferably chosen from IL1, IL4, IL12, and IL18.
3. The composition of claim 1, wherein said EV-free culture medium is supplemented with a mixture of IL1 and IL4, preferably of IL1β and IL4.
4. The composition of any one of claims 1 to 3, wherein said EVs are obtained from undifferentiated MSCs derived from a biological tissue selected from placenta, umbilical cord or placental membranes (chorion, amnion) or their components, or from a biological fluid such as umbilical cord blood, placenta blood or amniotic fluid.
5. The composition of any one of claims 1 to 4, wherein said EVs are obtained from undifferentiated MSCs derived from umbilical cord, preferably by cell isolation from an explant of umbilical cord fragment.
6. The composition of any one of claims 1 to 4, wherein said EVs are obtained from undifferentiated MSCs derived from placenta, preferably by cell isolation from a placenta tissue fragment.
7. The composition as defined in any one of claims 1 to 6, for use for treating subjects suffering from an ischemic disease, from a disorder of the circulatory system, from an immune disease, from an organ injury or disorder or from an organ function failure.
8. The composition for use according to claim 7, wherein said disease or disorder is chosen in the group consisting of: type-1 diabetes mellitus, type-II diabetes, GVHD, aplastic anemia, multiple sclerosis, Duchenne muscular dystrophy, rheumatoid arthritis, cerebral stroke, idiopathic pulmonary fibrosis, dilated cardiomyopathy, osteoarthritis, cirrhosis, liver failure, kidney failure, peripheral arterial occlusive disease, critical limb ischemia, peripheral vascular disease, heart failure, diabetic ulcer, fibrotic disorders, synechia and endometrial disorders.
9. The composition for use according to claim 7, wherein said disease or disorder is a skin or a mucous membrane disease or disorder, preferably a diabetic ulcer, an ulcer, a trauma, a burn, a scald, a wound or wound healing problem, a Decubitus ulcer, a wart, a synechia, an endometrial disorder or a fibrotic disorder of the gastro-intestinal tract such as anal fistula.
10. A pharmaceutical, veterinary, a diagnostic or a cosmetic composition containing the EVs as defined in any one of claims 1 to 6.
11. The pharmaceutical or veterinary composition of claim 10, further containing an hydrogel or other bio-compatible material.
12. A topical formulation containing the EVs as defined in any one of claims 1 to 6.
13. The pharmaceutical composition of claim 10 or 11 or the topical formulation of claim 12, for use for treating subjects suffering from an ischemic disease, from a disorder of the circulatory system, from an immune disease, from a fibrotic disorder, from an organ injury or from an organ function failure.
14. The pharmaceutical composition of claim 10 or 11 or the topical formulation of claim 12, for use for treating subjects suffering from a disease or disorder chosen in the group consisting of: type-1 diabetes mellitus, type-II diabetes, GVHD, aplastic anemia, multiple sclerosis, Duchenne muscular dystrophy, rheumatoid arthritis, cerebral stroke, idiopathic pulmonary fibrosis, dilated cardiomyopathy, osteoarthritis, cirrhosis, liver failure, kidney failure, peripheral arterial occlusive disease, critical limb ischemia, peripheral vascular disease, heart failure, diabetic ulcer, fibrotic disorders, synechia and endometrial disorders.
15. A dermatologic or a cosmetic composition containing the EVs as defined in any one of claims 1 to 6.
16. Use of the dermatologic or cosmetic composition as defined in claim 15 for regenerating the skin or mucosal cells, improving the skin or mucosal membrane aspect or for correcting a skin or mucosal membrane defect, such as dark patches, spots, acne, wrinkles, dryness.
17. Use of the dermatologic or cosmetic composition as defined in claim 15 for healing a skin injury or disorder, such as a burn, a scar, an angioma, a mole, or a wart.
18. A medical device containing the EV as defined in any one of claims 1 to 6.
19. The medical device of claim 18, wherein it is a bandage, a patch, a stent, an endoscope, or a syringe.
20. A delivery system containing the EV as defined in any one of claims 1-6.
21. The delivery system of claim 20, wherein it is micro or nano-vesicles of biopolymers, lipids or nanoparticles.
22. A method to prepare extracellular vesicles (EV), said method comprising the steps of: (i) culturing mesenchymal stem cells obtained from a biological tissue or fluid in a first culture medium deprived of growth factors, so as to generate a population of cultured undifferentiated mesenchymal stem cells, (ii) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing at least two pro-inflammatory growth factors, thereby generating CD106.sup.high CD151.sup.+Nestin.sup.+ mesenchymal stem cells, (iii) Culturing said MSCs in a EV-free culture medium, under conditions permitting their expansion; and iv) Purifying the EVs from the cells obtained in step (iii).
23. The method of claim 22, wherein said pro-inflammatory growth factors are chosen among TNFα, IL1, IL4, IL12, IL18, and IFNγ, preferably chosen from IL1, IL4, IL12, and IL18.
24. The method of claim 22 or 23, wherein said pro-inflammatory growth factor is a mixture of IL1 and IL4, preferably of IL1β and IL4.
25. The method of any one of claims 22 to 24, wherein said undifferentiated MSCs are derived from a biological tissue selected from placenta, umbilical cord or placental membranes (chorion, amnion) or their components, or from a biological fluid such as umbilical cord blood, placenta blood or amniotic fluid.
26. The method of any one of claims 22 to 25, wherein said undifferentiated MSCs are obtained by cell isolation from explant tissues.
27. The method of any one of claims 22 to 26, wherein said undifferentiated MSCs are derived from umbilical cord, preferably by cell isolation from an explant of umbilical cord fragment.
28. The method of any one of claims 22 to 26, wherein said undifferentiated MSCs are derived from placenta, preferably by cell isolation from a placenta tissue fragment.
29. The method of any one of claims 22 to 28, wherein said second culture medium contains between 1 and 100 ng/ml of Interleukine 1, preferably of Interleukine 1β.
30. The method of any one of claims 22 to 29, wherein said second culture medium contains between 1 and 100 ng/ml of Interleukine 4.
31. The method of any one of claims 22 to 30, wherein said EV-free culture medium is supplemented with a mixture of IL1 and IL4, preferably of IL1β and IL4.
32. The method of any one of claims 22 to 31, wherein the EVs are purified by ultrafiltration.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
[0167] For simplicity and illustrative purposes, the present invention is described by referring to exemplary embodiments thereof. It will be apparent, however, to one of ordinary skill in the art that the present invention may be practiced without limitation to these specific details. In other instances, well known methods have not been described in detail so as not to unnecessarily obscure the present invention.
[0168] All the steps 1-4 below have been performed as described in the example part of PCT/EP2017/082316 which is hereby incorporated by reference.
[0169] 1. Pro-Angiogenic Umbilical Cord-Derived Mesenchymal Stem Cells (MSC) Isolation
[0170] The umbilical cord has been removed from the transport solution and cut in 2-3 cm section long. Every segment containing a blood clot that cannot be removed was discarded, to avoid contamination by adherent blood cells. The sections were then disinfected by a bath of antibiotics and antifungal agents composed of αMEM+Vancomycin 1 g/L+Amoxicillin 1 g/L+Amikacin 500 mg/L+Amphotericin B 50 mg/L for 30 min at room temperature (RT). Antibiotics were extemporaneously dissolved in sterile water for injection.
[0171] The sections of umbilical cord were removed from the bath and quickly rinsed in 1×PBS at RT. The epithelial membrane was slightly sectioned without touching the vessels. The section was then detailed in slices of 0.5 cm thickness and disposed at the bottom of 150 cm.sup.2 plastic flask with lid. 6 to 10 slices per flask were disposed with at least a 1 cm radius circle of free space around each slice, and left to adhere for 15 min without medium at RT.
[0172] After adhesion, complete medium (αMEM+5% Clinical Grade Platelet Lysate+2 U/mL heparin) was added carefully, to keep the explants adherent to the bottom of the flask. The flasks were then incubated at 37° C., 90% humidity and 5% CO2.
[0173] The culture medium was changed after 5 to 7 days.
[0174] At day 10 after isolation, the migration of the cells out of the explants was controlled by inverted microscopy. If a circle of adherent cells was visible around most of the explants, they were carefully removed, by picking them out of the flask, through the lid, with a sterile, disposable, single-use pair of tweezers.
[0175] From this step, the confluency of the cells was visually checked every other day and, if needed, a medium change was performed at day 17.
[0176] When the cells reached 70-90% confluency or at D20, the medium was removed, cells were washed with 30 mL of 1×PBS per flask. Cells were then removed with trypsin and collected with the old medium and centrifuged 10 min at 300 g. Supernatant was discarded and cells were then suspended in a cryopreservation solution consisting in αMEM+100 mg/mL HSA (Human Serum Albumin)+10% DMSO (DiMethyl SulfOxide) and cryopreserved.
[0177] 2. MSC Cells Thawing and Culture
[0178] Cells were thawed following a classical protocol. Briefly, cryotubes were removed for liquid nitrogen and quickly plunged into a 37° C. water bath. As soon as there was no ice left in the tube, cells were diluted in preheated (37° C.) complete medium (αMEM+0.5% (v/v) ciprofloxacin+2 U/mL heparin+5% (v/v) Platelet Lysate (PL) and quickly centrifuged (300 g, RT, 5 min).
[0179] After centrifugation, cells were suspended in preheated complete medium, and assessed for number and viability (blue trypan/Malassez hemocytometer).
[0180] Cells were seeded at 4000 cells/cm.sup.2 in plastic culture flask in complete medium, and incubated (90% humidity, 5% CO2, 37° C.).
[0181] 3. MSC Cells Stimulation
[0182] After a few days of expansion, cells were checked for confluency. When confluency reached 30 to 50%, the old medium was discarded and replaced either by fresh complete medium for unstimulated condition, or by fresh medium completed with 10 ng/mL IL-1β and 10 ng/mL IL-4.
[0183] Cells were then incubated at least 2 days before flow cytometry experiment.
[0184] This stimulation step was instrumental in conferring a pro-angiogenic phenotype to the MSC, as described in PCT/EP2017/082316.
[0185] Several cells batches have been tested in a Boyden chamber assay for their angiogenic activity (see
[0186] Briefly, ECFC (Endothelial Colony Forming Cells) migration in response to a pro-angiogenic gradient was assessed in a 24-well modified Boyden chamber, on polycarbonate membrane filter with 8 μm pore diameter (BD Biosciences) coated with 20 μg/mL fibronectin from bovine plasma (Sigma-Aldrich—F1141). Before the experiment, the bottom of a 24-well plate wells was seeded with the MSC cells at a density of 6000 cells/cm.sup.2 in sextuplicate and cultivated 4 days in αMEM+1% SVF. A medium control row was realized, filled with αMEM+1% FBS.
[0187] After an overnight starvation in EBM-2 basal medium (Lonza—CC-3156) supplemented with 0.2% FBS, ECFC were loaded in starving medium into the upper part of the microchamber at 200,000 cells/well.
[0188] For each condition VEGF (Miltenyi—130-109-383) was added in three of the six wells at a concentration of 50 ng/mL as a positive control.
[0189] After 5 h of incubation, cells on the upper surface of the membrane filter were removed by wiping with a cotton swab. Then, all membranes were MGG-stained, mounted and photographed.
TABLE-US-00001 Insert 1 Insert 2 Insert 3 (n = 3) (n = 3) (n = 3) MEM-α 1% 95.00 196.67 259.67 SVF − VEGF (Medium CTRL−) MEM-α 1% 509.33 390.00 494.33 SVF avec VEGF (Medium CTRL+) 101 − VEGF 234.00 177.00 258.00 101 + VEGF 439.00 433.67 573.00 201 − VEGF 330.00 257.00 224.33 201 + VEGF 648.33 537.33 635.33 301 − VEGF 333.67 257.33 320.67 301 + VEGF 564.33 648.00 599.67
[0190] In
[0191] As Boyden chambers prevent cell to cell contact, this effect is necessarily mediated through soluble extracellular mediators, either soluble proteins or extracellular vesicles.
[0192] 4. MSC Cells Harvesting and Cryopreservation
[0193] After 2 to 3 days of expansion/stimulation, cells were checked for confluency. If confluency was up to 80%, the cells were harvested. Briefly, the old medium was discarded and cells were washed with 1×DPBS. Trypsin EDTA was added and cells were incubated 5 min at 37° C. Trypsin was neutralized with at least 2× the volume of medium, and cell suspension was harvested and assessed for number and viability.
[0194] Cells were centrifuged 10 min at 300 g. Supernatant was discarded and cells were then suspended in a cryopreservation solution consisting in αMEM+100 mg/mL HSA+10% DMSO and cryopreserved.
[0195] 5. MSC Cells Thawing and Conditioned Medium Production
[0196] Cells were thawed following a classical protocol. Briefly, cryotubes or bags were removed for liquid nitrogen and quickly plunged into a 37° C. water bath. As soon as there was no ice left in the tube, cells were diluted in preheated (37° C.) αMEM and quickly centrifuged (300 g, RT, 5 min).
[0197] After centrifugation, cells were suspended in preheated complete medium, and assessed for number and viability (blue trypan/Mallassez hemocytometer).
[0198] Cells were seeded at a density of 2000 cells/cm.sup.2 in the necessary number of 300 cm.sup.2 plastic culture flask to produce the requested amount of conditioned medium (1T300=30 mL of conditioned medium). Cells were incubated at 90% humidity, 5% CO2, 37° C., with αMEM complemented with 0.5% (v/v) ciprofloxacin, 2 U/mL heparin and 8% (v/v) of centrifuged PL. Centrifuged PL was prepared by centrifuging PL 1 h at 6000 g and 10° C., and recovering the supernatant.
[0199] After 5 days the medium was changed. When the cells reached 80% confluency (at day 6), the medium was removed and cells were washed 3 times with PBS. 50 ml/T300 of uncomplemented αMEM was added for 24H. After 24H the medium was changed to 30 mL of either uncomplemented αMEM, or αMEM complemented with 8% of vesicles deprived PL (platelet lysate).
[0200] Cells were allowed to secrete for 36H and the medium was retrieved, centrifuged 5 min at 400 g and RT in a Heraeus Multifuge 3 S-R. The supernatant was retrieved and frozen at −80° C.
[0201] Cells were trypsinized and assayed for their number and viability.
[0202] 6. Extra-Cellular Vesicles Isolation and Characterization
[0203] Extra-cellular vesicles derived from such MSC can be isolated by any methods known in the art, such as, but not limited to, ultracentrifugation, ultrafiltration, density gradient, size-exclusion chromatography, kit-based precipitation, immune-affinity capture, microfluidic devices.
[0204] In this experiment, the fraction enriched in extra-cellular vesicles was separated by ultrafiltration of the conditioned medium followed by Size Exclusion Liquid Chromatography (SEC).
[0205] The analysis of number and size distribution of extra-cellular vesicles was performed using Nanoparticle Tracking Analysis (Nanosight).
[0206] Also, the content of the EVs of the invention has been assessed by Western Blot, using the following conditions.
[0207] Material and Reagents:
[0208] Lysis buffer (RIPA):
TABLE-US-00002 Sodium deoxycholate 1% SDS 0.1%.sup. Tris. Cl pH 7.4 20 mM EDTA 1 mM NaCl 150 mM Triton x100 1% Protease Inhibitor Cocktail (CIP 100X), Sigma ref. P8340
[0209] Cells Lysis:
[0210] 100 μl of cold RIPA containing CIP at 1× final concentration has been added to 1×10.sup.6 MSC cells.
[0211] The resulting mixture has been incubated for 10 mn on ice, centrifuged for 15 mn at 4° C. for 20 min and then the supernatant has been recovered.
[0212] The dosage of proteins has been done by using the micro BCA Protein Assay Kit Pierce Ref 23235.
[0213] The electrophoresis was done on Novex Nosex 4-12% Bis-Tris Protein Gels 1.5 mm, 10 wells (Life Technologies, NP0335PBOX) or NuPAGE® MOPS SDS Running Buffer (20×) (Life Technologies, NP0001). The samples have been denatured for 10 mn at 70° C. with ¼ volume of LDS buffer (Invitrogen NP0007, 4×) with or without DTT (500 mM). The transfer was done with Mini Trans-Blot Electrophoretic cell membrane transfer, ref: 170-3930 on a membrane of PVDF Amersham, ref: RPN303LFP activated with absolute ethanol and rinsed.
[0214] The following Materials were used for revealing the proteins:
[0215] TBS10X, BioRad ref. 1706435
[0216] Blocking buffer (TBS milk): TBS 1X_Tween 0.1% skimmed milk 5%
[0217] Washing Buffer (TBS): TBS 1X_Tween 0.1%
[0218] AC Buffer (TBS_AC): TBS 1X_Tween 0.1% _Milk 0.3%
[0219] The presence of membrane proteins CD9 (Biolegend—312102) and CD81 (Biolegend—349501) was analyzed in Western Blot (
[0220] Fluorimetry has been performed by using Alexa Fluor 680 antibody, Thermofisher GAM (ref: A21058) or GAR (ref: A21076) at 1/10000.sup.th.
[0221] Chemiluminescence has been performed by using HRP Antibody, Bio Rad GAM (ref: 170-6516) or GAR (ref: 170-6515) at 1/5000th.
[0222] 7. Comparison of EV Derived from UCMSCs Stimulated by LPS and EV Derived from UCMSCs Stimulated by the Combination of IL1β and IL4 (EVs of the Invention).
[0223] Dongdong Ti & al (Journal of translational medicine, 2015) describes extra-cellular vesicles purified from umbilical cord MSCs that are pre-conditioned with the pro-inflammatory factor LPS.
[0224] Yunbing Wu & al (BioMed Research International, 2017) describes anti-inflammatory properties of extra-cellular vesicles derived from unstimulated mesenchymal stem cells obtained from human umbilical cord.
[0225] None of these publications proposes to add IL1β and/or IL4 for stimulating the MSCs from which the EVs of the invention are derived.
[0226] The purpose of the present example is to demonstrate that EV derived from the CD106.sup.high CD151.sup.+Nestin.sup.+ MSCs of the invention are distinct from those described in the prior art, because they are secreted by cells that have been cultivated in a particular conditioning medium containing pro-inflammatory growth factors such as IL1β and IL4.
[0227] A comparison of the EV of the invention with the EV derived from unstimulated MSCs, or from MSCs stimulated by LPS demonstrated that the EVs of the invention exhibit molecular characteristics that distinguish they indeed from the EVs of the prior art, e.g., in terms of protein content or surface markers.
[0228] 7.1. Production of Conditioned Media (Culture Supernatants) of Umbilical Cord-Derived MSCs Cultured in 3 Different Conditioning Media:
[0229] 7.1.1. Preparation of the 3 Conditioning Media
[0230] Three conditioning media were prepared as follows:
[0231] NS=Control medium (without conditioning pro-inflammatory factor):
[0232] For a 675 mL αMEM bag (final concentration: 10 ng/mL) [0233] 1—Put an injection site in one of the outlet port. [0234] 2— Inject with a needle 290 μL of heparin 5000 U/mL (final concentration 2 U/mL). Ensure that no heparin is left in the injection site by suction/discharge. [0235] 3—Put an injection site in the outlet port of a bag of Clinical Platelet Lysate (CPL) and take 56.8 mL of centrifuged CPL with a 50 mL LL syringe.
[0236] S1=IL1β—IL4 conditioning medium (“second medium” of the method as described in WO 2018/108859):
[0237] For a 675 mL αMEM bag (final total interleukine concentration: 10 ng/mL) [0238] 4—Put an injection site in one of the outlet port. [0239] 5— Inject with a needle 290 μL of heparin 5000 U/mL (final concentration 2 U/mL). Ensure that no heparin is left in the injection site by suction/discharge. [0240] 6—Put an injection site in the outlet port of a bag of CPL and take 56.8 mL of centrifuged CPL with a 50 mL LL syringe. [0241] 7—Inject the CPL in the αMEM bag and homogenise with the syringe. [0242] 8—Cytokine addition: [0243] 1. Thaw a 73.2 μL aliquot of each interleukine (IL) (C=100 μg/mL). [0244] 2. Use a 1 mL needled syringe to sample 400 μL of complete medium, and mix it with the ILs. [0245] 3. Take the medium containing the ILs and add it to the bag of complete medium.
[0246] LPS Conditioning Medium:
[0247] For a 675 mL αMEM bag (final LPS concentration: 100 ng/mL) [0248] 1—Put an injection site in one of the outlet port. [0249] 2—Inject with a needle 290 μL of heparin 5000 U/mL (final concentration 2 U/mL). Ensure that no heparin is left in the injection site by suction/discharge. [0250] 3—Put an injection site in the outlet port of a bag of CPL and take 56.8 mL of centrifuged CPL with a 50 mL LL syringe. [0251] 4— Inject the CPL in the αMEM bag and homogenise with the syringe. [0252] 5—LPS addition: [0253] 1. Thaw a 73.2 μL aliquot of LPS (C=1 mg/mL). [0254] 2. Use a 1 mL needled syringe to sample 400 μL of complete medium, and mix it with the LPS. [0255] 3. Take the medium containing the LPS and add it to the bag of complete medium.
[0256] Centrifuged Clinical Platelet Lysate (CPL) Preparation:
[0257] Centrifuged CPL was used for the stimulation step, in order to start “cleaning” the cells from exogenous EV derived from the CPL.
[0258] 1—Put an injection site in the outlet port.
[0259] 2—Transfer the CPL in 50 mL plastic tubes.
[0260] 3—Centrifuged at 6000 g and 4° C. for 1 hour.
[0261] 4—Transfer the supernatant in a new container.
[0262] 7.1.2. Thawing and Amplification:
[0263] Umbilical cord-derived mesenchymal stem cells, stored in gaseous nitrogen after isolation and passage 1, have been thawed following a classical protocol. Briefly, the bag was removed from storage and quickly plunged into a 37° C. water bath. As soon as there was no ice left in the bag, cells were diluted in preheated (37° C.) complete medium (αMEM+0.5% (v/v) ciprofloxacin+2 U/mL heparin+5% (v/v) PL) and quickly centrifuged (300 g, RT, 5 min).
[0264] After centrifugation, the cells were suspended in preheated complete medium, and assayed for cell count and viability (blue trypan/Malassez hemocytometer).
[0265] The cells were seeded at 2000 cells/cm.sup.2 in two cell stacks 1 (CS1) in complete medium, and incubated (90% humidity, 5% CO2, 37° C.) for seven days.
[0266] After seven days, the CS1 were rinsed with 100 mL of PBS, and 25 mL of Trypzean per CS1 were added to harvest the cells. After 10 min in the incubator the Trypzean was neutralized with a total of 100 mL of complete medium (50 mL per CS1). Cells were pooled and assessed for number and viability.
[0267] 7.1.3. Stimulation:
[0268] T300 were seeded at 6000 cells/cm.sup.2.
[0269] After one day, the medium was changed for the appropriate conditioning medium. The cells were stimulated for 2 days without medium change.
[0270] 7.1.4. Starving and EV Secretion:
[0271] After the stimulation the medium was discarded and the cells were rinsed three times with PBS. Cells were then starved 24H with αMEM+/−a pro-inflammatory factor (combination of 10 ng/mL of IL1β and of IL4 or 100 ng/mL of LPS).
[0272] After the starving period, the cells were again rinsed three times with PBS then each flask was loaded with 30 mL of αMEM+/−a pro-inflammatory factor (IL or LPS) for 72 hours.
[0273] The supernatant was collected in 50 mL plastic tubes and centrifuged for 5 min at 400 g at room temperature. The supernatant of each condition was pooled in a 500 mL bottle and a small aliquot of 1 ml was separately frozen at −80° C. along the bottles.
[0274] For each condition, 3 T300 flasks were trypsinized to evaluate the number of cells, and cells were cryopreserved in cryotubes.
[0275] Cell Culture and Conditioning Reagents:
TABLE-US-00003 Manufacturer Reagent Reference Batch number Macopharma αMEM BC0110020 112611484DM Macopharma Clinical Platelet Multi PL100i 11217335DM Lysate (virally inactivated) Sigma Trypzean T3449-500ML RNBG4130 and Sanofi Heparin CIP 70579 CHOAY 5000 3400955243861 UI/mL Macopharma PBS BC0120030 11233414DM CellGenix IL-1β 201-GMP-01M 1011QB11a CellGenix IL-4 204-GMP-01M 1003PC22b Macopharma CS1 Cellstack 1 29815014 Dutscher 300 cm.sup.2 culture 190301 20180414 flasks Pharma Ciprofloxacine Cipro 16102089 Sigma LPS L6529-1MG 028M4138V
[0276] 7.2. Phenotypic Characterisation of the Cells Contained in the 3 Conditioned Media
[0277] A phenotypic characterization of the cells cultured using the 3 conditioning media was performed by cytometry analysis to assess the efficiency of the stimulation.
[0278] As expected, the cells conditioned in the media S1 and LPS exhibited a different phenotype, the pro-angiogenic marker CD106 being expressed at higher levels in the conditioned media S1 (stimulation of the cells by ILs as described in WO 2018/108859).
TABLE-US-00004 S1 = IL-Stimulated as described in WO Unstimulated 2018/108859 LPS-Stimulated Cell number 808 000 cells 1 693 000 cells 1 666 000 cells Viability 91% 95% 98% CD90+ 99.96% 99.38% 99.99% CD106+ 12.21% 89.93% 23.14% HLA-DR+ 0.87% 1.88% 0.33% CD31+ 7.28% 1.11% 0.25% CD34+ 7.56% 0.62% 0.56% CD73+ 99.94% 99.94% 99.99% CD45+ 4.66% 1.34% 0.82% CD151+ 97.39% 99.25% 100.00% CD105+ 99.59% 99.70% 99.94%
[0279] Cytometry Reagents Used for Cytometry Analysis:
TABLE-US-00005 Manufacturer Reagent Reference Batch number Dutscher 1X DPBS P04-36500 9110816 R&D System Human polyvalent IgG 1-001-A (2 mg/mL in PBS) LFB HSA 20% Vialebex 20% Sigma 37% filtered Formalin F1635 Miltenyi MACS Comp Bead 130-097-900 5180522233 Kit anti-Mouse Igk Dutscher 96-wells conic bottom 651101 plate BD Falcon Cytometry tubes 352053 Miltenyi CD34-PE, human 130-081-002 5180511513 Beckman CD31-FITC, human IM143U 33 Coulter Beckman CD90-FITC, human IM1839 35 Coulter Miltenyi Anti-HLA-DR-FITC, 130-111-788 5171121099 human Miltenyi CD105-PE, human 130-094-941 5170105175 Miltenyi CD45-FITC, human 130-110-631 5180209339 Miltenyi Mouse IgG2a-PE 130-091-835 5160804636 Miltenyi Mouse IgG1-FITC 130-092-213 5160804642 Miltenyi REA Control (S)-FITC 130-104-610 5161209090 Miltenyi MACS Comp Bead 130-104-693 5160901338 Kit anti-Mouse REA BD PE Mouse IgG1, □ 555749 6070641 Biosciences Isotype Control BD PE Mouse 555647 8025927 Biosciences Anti-Human CD106 51-10C9 RUO BD PE Mouse 550257 6237614 Biosciences Anti-Human CD73 AD2 RUO BD PE Mouse 556057 5134578 Biosciences Anti-Human CD151 14A2.H1 RUO
[0280] 7.3. EV Purification
[0281] In this experiment, the fractions enriched in extra-cellular vesicles were separated by ultrafiltration of the 3 conditioned media.
[0282] The analysis of the number and size distribution of extra-cellular vesicles was performed using Nanoparticle Tracking Analysis (Nanosight 300 from Malvern-Panalytical).
[0283] The results of EV purification are presented below:
[0284] Yield:
TABLE-US-00006 NS S1 LPS Concentration (vesicles/mL) 2.90E+09 8.65E+08 2.81E+09 Initial Volume in 525 538 539 conditioned medium or MC 0.45μ Conc° EV after tangential 5.96E+10 1.43E+11 8.54E+10 ultrafiltration (particules/mL) Number of frozen tubes (/0.5 mL) 12 12 11 Number of EV/tube 2.98E+10 7.15E+10 4.27E+10 Total number of EV 3.58E+11 8.58E+11 4.70E+11 Total Protein Concentration (mg/mL) 2.0 2.3 2.4
[0285] Size:
TABLE-US-00007 Conditioned Medium (MC) 0.45μ EV NS SD S1 SD LPS SD NS SD S1 SD LPS SD mode 145.1 6.3 136.7 7.2 138.1 7.2 134.6 3.1 140.5 5.5 131.4 4.2 D10 127.2 5.4 111.7 2 113 1.2 121.1 2.8 123.1 1.5 119.4 1.1 D50 222.6 6.9 147.3 4.1 146.8 2.2 159.7 5.3 159.8 1.6 153.3 4.3 D90 439 16.4 209.5 11.7 242 4.1 277.5 10.8 269.4 6.1 278.3 7
[0286] Yield/Cells:
TABLE-US-00008 Yield EV in the EV/cells/hour Cells conditioned EV after Yield of secretion Condition harvested Viability medium purification EV/cells (72 h) Unstimulated 1.10E+08 95.81% 1.52E+12 3.58E+11 3251 45 S1-Stimulated 1.02E+08 90.68% 4.65E+11 8.58E+11 8412 117 LPS-stimulated 1.04E+08 91.75% 1.51E+12 4.70E+11 4526 63
[0287] 7.3. Characterisation of the EVs Obtained from NS/S1/LPS-Conditioned MSCs
[0288] 7.3.1. EV and MSC Protein Content Analysis by Western Blot
[0289] The content of the EVs and the MSCs obtained in the 3 conditions (NS/S1/LPS) has been assessed by Western Blot using the following conditions.
[0290] Material and Reagents:
[0291] Lysis buffer (RIPA): [0292] Sodium deoxycholate 1% [0293] SDS 0.1% [0294] Tris.HCl pH 7.4 20 mM [0295] EDTA 1 mM [0296] NaCl 150 mM [0297] NP40: 1%
[0298] Protease Inhibitor Cocktail (CIP 100×), Sigma ref. P8340
[0299] Cell Lysis (for the MSCs):
[0300] Add 100 μl/1×10.sup.6 MSC cells of cold RIPA containing CIP at 1× final concentration.
[0301] Incubate for 10 mn in ice, centrifuge at 4° C. for 20 min at 10000 g and then recover the supernatant.
[0302] The EV containing fraction was solubilized following addition of an equal volume of ice-cold 2×RIPA lysis buffer.
[0303] The dosage of proteins has been done by using the micro BCA Protein Assay Kit Pierce Ref 23235.
[0304] The electrophoresis was done on Novex Nosex 4-12% Bis-Tris Protein Gels 1.5 mm, 10 wells (Life Technologies, NP0335PBOX) or NuPAGE® MOPS SDS Running Buffer (20×) (Life Technologies, NP0001). The samples have been denatured for 10 mn at 70° C. with ¼ volume of LDS sample buffer (Invitrogen NP0007, 4×) with or without DTT (500 mM). The transfer was done with Mini Trans-Blot Electrophoretic cell membrane transfer, ref: 170-3930 on a membrane of PVDF Amersham, ref: RPN303LFP activated with absolute ethanol and rinsed.
[0305] The following Materials were used for revealing the proteins:
[0306] TBS10X, BioRad ref. 1706435
[0307] Blocking buffer (TBS milk): TBS 1X_Tween 0.1% skimmed milk 5%
[0308] Washing Buffer (TBS): TBS 1X_Tween 0.1%
[0309] AC Buffer (TBS_AC): TBS 1X_Tween 0.1% _Milk 0.3%
[0310] Fluorimetry has been performed by using Alexa Fluor 680 antibody, Thermofisher GAM (ref: A21058) or GAR (ref: A21076) at 1/10000.sup.th.
[0311] Chemiluminescence has been performed by using HRP Antibody, Bio Rad GAM (ref: 170-6516) or GAR (ref: 170-6515) at 1/5000th.
[0312] Antibodies anti-CD9: Biolegend—312102
[0313] Antibodies anti-CD81: Biolegend—349501
[0314] Antibodies anti-Calnexin: Elabscience—E-AB-30723
[0315] Antibodies anti-VCAM: Bio-Rad VMA00461
[0316] Antibodies anti-CD200: Bio-Techne 2AF2724
[0317] Results:
[0318] As expected, the pro-angiogenic marker CD106/VCAM was detectable in column B (MSCs of the invention) and completely absent in column C (MSCs in LPS condition), and in column A (negative control), as expected. The membrane glycoprotein CD200, a second pro-angiogenic marker, was present in the MSCs of the S1 fraction and not in the MSCs in the LPS fraction, nor in the column A (negative control) (see
[0319] In the purified EVs (
[0320] The reticulum marker Calnexin was not detected, showing that the EV fraction was correctly purified.
[0321] The pro-angiogenic marker CD106/VCAM was detectable in column B (EVs of the invention) and completely absent in column C (EVs in LPS condition), and in column A (negative control), showing that the membrane markers of the MSCs of the invention are transferred to the EVs (
[0322] The membrane glycoprotein CD200, a second pro-angiogenic marker, was present in the EVs of the S1 fraction and not in the EVs of the LPS fraction, nor in the column A (negative control)(
[0323] 7.3.2 Angiogenesis Protein Array
[0324] The EVs derived from the MSCs cultivated in NS, S1 and LPS conditioned media were compared with an angiogenesis proteome array (R&D Systems—ARY007).
[0325] The lysis Buffer was prepared as follows: [0326] 1. For 50 mL: Dissolve 157.6 mg of Tris-HCL in 10 mL of water, 400.3 mg of NaCl in 10 mL of water and 37.2 mg of EDTA in 10 mL of water. [0327] 2. Adjust to pH=8. [0328] 3. Add 0.5 mL of triton X-100, 5 mL of Glycerol, 50 μL of Aprotinin 10 mg/mL, 50 μL of Leupeptin 10 mg/mL and 500 μL of Pepstatin 1 mg/mL. [0329] 4. QSP 50 mL of sterile water.
[0330] The EVs derived from the MSCs obtained in the NS, S1 and LPS media were solubilized in the lysis buffer: [0331] NS: 150 μL of EVs were suspended in 1 mL of Lysis Buffer [0332] S1: 130.4 μL of EV were suspended in 1 mL of Lysis Buffer [0333] LPS: 125 μL of EV were suspended in 1 mL of Lysis Buffer
[0334] A total amount of proteins in the lysates of 300 μg was quantified with a BCA assay, thereby confirming the efficiency of the lysis.
[0335] The EVs lysates were then pipeted up and down for resuspension and gently rocked at 2-8° C. for 30 minutes, then micro-centrifuged at 14,000×g for 5 minutes. The supernatant was transferred into a clean test tube and assayed following the manufacturer's instructions.
[0336] Angiogenesis Proteome Array Reagents:
TABLE-US-00009 Manufacturer Reagent Reference Batch number R&D Angiogenesis proteome ARY007 Systems array Merk Tris-HCL 1547-0100 Sigma NaCl S3014 Biosolve EDTA 05142357 Prolabo Triton X-100 81338 Invitrogen Glycerol 15514-011 Tocris Aprotinin 4139 3 Tocris Leupeptin 1167 20 Tocris Pepstatin A 1190 20 Versylene Eau Sterile B230521 13MFP291 Fresnuis Thermo Micro BCA ™ 23252 MC154396 Scientific Protein Assay Kit
[0337] The graph on
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