Cell culture

ASC from lipoaspirates from three donors were processed by the group of Prof. Karen Bieback (University of Heidelberg, Heidelberg, Germany) after informed consent. The Mannheim Ethics Commission II approved the study (vote 2011-215N-MA). The ASC were cultured using MEM- media, Gibco, ThermoFisher Scientific, 2,561,029) and 10% Fetal Bovine Serum (FBS, 10270-106, Gibco, MA, USA) at 37 with 5% CO2 and controlled humidity. These three ASC batches (referred to a N=3 biological replicates in the figure legends) were shipped as cryo-aliquots to the other two centers to be cultured under identical harmonized culture conditions from passage 46 as detailed previously [29]. Bioproducts were derived from the conditioned medium of either 3D cultured ASC, processed by size exclusion chromatography to yield (1) EV-SEC or the (2) protein-rich fraction, or 2D-cultured cells, processed by ultracentrifugation to yield (3) EV-UC or after concentration to yield (4) the conditioned medium (CM) or (5) the respective wash-off (CM-WO) (Fig.1).

At 80% confluence, ASC were passaged and seeded in a hollow-fibber bioreactor at a concentration of 14106 cells/cartridge (20kDa MWCO, 450cm2, C2025D, FiberCell System-KD Bio, France). Prior to injecting the cells, a pre-culture step was carried out to initiate and activate the bioreactor, first Dulbeccos phosphate-buffered saline (PBS) for 24h, followed by fibronectin coating over-night. After the pre-culture process, ASC were seeded in serum-free MEM- in the extra-capillary space, at 37 with 5% CO2 and controlled humidity for 7days without harvesting the supernatant, with continuous monitoring of glucose levels. Serum-containing medium was used as circulating medium, given that EVs and high molecular weight proteins cannot cross the 20 kD MWCO filter fiber and thus do not contaminate the cell-derived EVs harvested from the extra-capillary-space (according to the Hollow Fiber Bioreactor Protocol for Mesenchymal Stem Cells by fibercellsystems.com) ASC were cultured for 4weeks in the bioreactor and during this period, the supernatant was collected daily. Following centrifugation to remove cell debris (5min at 420g), the supernatant was stored at 80 until EV isolation by size exclusion chromatography (see below) was performed. Cells were harvested and counted to calculate the bioproduct per producer cell concentration.

The secretome obtained in vitro, also named conditioned media (CM), was generated from ASC at passage 4 to 6. Upon reaching 80% confluence, cells were washed with PBS and incubated for 24h in serum-free MEM- medium. The supernatant was collected and centrifuged for 5min at 400g to remove cell debris before being placed in centrifugal concentrator units of 3KDa molecular weight cut-off (UFC9003, Merck Millipore, USA). The CM was centrifuged for 90min at 3,000g, 4 using an Eppendorf 5810 R Centrifuge to achieve tenfold concentration. The flow-through resulted from the concentration step (thereafter named wash-off, CM-WO) was kept and used as a control. Concentrated conditioned media samples were stored at 80 until further use. Cells were harvested and counted to calculate the bioproduct per producer cell concentration.

When the cells reached 80% confluence, they were starved for 1624h in serum-free medium. The supernatant was collected and centrifuged for 20min at 3000g to remove cell debris and apoptotic cells. The supernatant was then ultracentrifuged for 2h at 100,000g, 4 using Beckman Coulter Optima L-100K Ultracentrifuge (Beckman Coulter, CA, USA) with the rotor type 70Ti. The EV pellet was resuspended in PBS supplemented with 1% DMSO. The suspension of EVs (EV-UC) was then stored at80 until further use. EVs were collected from ASC at 4-6th passage. Cells were harvested and counted to calculate the cell equivalents used for cell treatments.

After thawing at 4, samples were centrifuged for 10min at 300g and 20min at 4000g. After, the supernatant was filtered through a 0.2m syringe filter and concentrated with a 100kD MWCO concentration filter to a final volume of 10mL. The qEV10-IZON column 35mm was initially washed with sterile PBS, and then 10mL of the sample was added to concentrate it to the final volume of 1.5mL (Vivaspin 20, 100,000 MWCO PE, Sartorius). Each EV sample (EV-SEC) and the resultant supernatant containing the protein fraction (Protein-Rich Fraction) were collected, concentrated (Vivaspin 20, 100,000 MWCO PE, Sartorius) and stored at 80 until further use.

ASC derived bioproducts were used at a ratio of 2:1 and 20:1 relative to recipient cells. To do so, we counted the number of ASC after harvesting and used it to relate the number of particles/volumes generated of EVs and CM respectively for each bioproduct.

After the isolation, the concentration of all the samples was measured (a) by Nanosight NS300 or (b) ZetaView.

After the isolation, the concentration of all the samples was measured (a) by Nanosight NS300 (Malvern Instruments Ltd., Malvern, UK) equipped with a 488nm laser module that utilizes Brownian motion and refraction index. The particle size scatters 10nm to 1000nm, although the optimized size range is 70300nm. It uses the scattered light to detect a particle and tracks its motion as a function of time. The particles scattered light was recorded with a light-sensitive camera under a 90 angle to the irradiation plane. This angle allows the Brownian motion of the EVs. Samples were diluted 1:100 in physiologic solution. For each sample, 3 videos of 60s at camera level 15 and threshold 5 were captured using a syringe pump 30. All the samples were characterized with NTA 3.2.16 Analytical software. The NTA settings were kept constant between samples.

After the isolation, the concentration of all the samples was measured b) by ZetaView (Particle Metrix GmbH, Germany). 1L of concentrated EVs was diluted in sterile-filtered PBS in a dilution 1:1,000 and visualized using the ZetaView (sensitivity 80%, shutter 100, 11 positions, 2 cycles; Particle Metrix, Germany).

Super-resolution microscopy pictures of EVs were obtained using a temperature-controlled Nanoimager S Mark II microscope from ONI (Oxford Nanoimaging, Oxford, UK) equipped with a 100 , 1.4NA oil immersion objective, an XYZ closed-loop piezo 736 stage, and 405nm/150mW, 473nm/1W, 560nm/1W, 640nm/1W lasers and triple emission channels split at 640/and 555nm. For sample preparation, we followed the manufacturers protocol using EV profiler Kit ONI (Alfatest, Rome, Italy). Before each imaging session, bead slide calibration was performed for aligning the channels, to achieve a channel mapping precision smaller than 12nm. Images were taken in dSTORM mode using 50% laser power for the 647nm channel, 30% laser power for the 488nm laser channel, and 30% for the 555 channel. Three-channels (2000 frames per channel) (647, 555 and 488) were acquired sequentially at 30Hz (Hertz) in total reflection fluorescence (TIRF) mode. Single-molecule data was filtered using NimOSsoftware (v.1.18.3, ONI) based on the point spread function shape, photon count and localization precision to minimize background noise and remove low-precision and non-specific colocalization. Data has been processed with the Collaborative Discovery (CODI) online analysis platform https://www.alto.codi.bio/ from ONI and the drift correction pipeline version 0.2.3 was used. Clustering analysis was performed on localizations and BD clustering-constrained parameters were defined (photon count 300-max, sigma 0200nm, p-value 01, localization precision 020nm). Colocalization was defined by a minimum number of localizations for each fluorophore/protein within a distance of 100nm or a distance used from the centroid position of a cluster.

MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) containing fluorescent labeled (FITC-PE) capture beads coupled to 37 exosomal surface epitopes and 2 isotope controls was used, following the manufacturers instructions (in detail: CD3, CD4, CD19, CD8, HLA-DR, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1, CD209, CD9, SSEA-4, HLA-ABC, CD63, CD40, CD62P, CD11c, CD81, MCSP, CD146, CD41b, CD42a, CD24, CD86, CD44, CD326, CD133-1, CD29, CD69, CD142, CD45, CD31, REA control, CD20, CD14, mIgG1 control). Briefly, 15L of beads were added to 120L of buffer or sample, including a total of 1109 EVs, and the complex was then incubated on a rotor overnight at 4. After the incubation and washing steps, a cocktail of APC fluorescent antibodies against tetraspanins (CD9, CD63 and CD81) was added (allowing the detection of beads bound EVs) and set on the rotor for 1h at room temperature. After washing, samples were detected using BD FACSCelestaTM Flow Cytometer (BD Bioscience, NJ, USA). Median background values of buffer control were subtracted, and samples were normalized to the median fluorescence intensity of tetraspanins.

Proteins extracted from Hela cells were used as cellular control, the pellet was resuspended in RIPA buffer (50mM TrisHCl, pH7.4, 150mM NaCl, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 0.1mM CaCl2, and 0.01mM MgCl2 supplemented with protease inhibitor cocktail (Thermo Fisher Scientific), incubate 30min in ice vortexing every 10min and centrifuge 20min at 20,000g. An equal volume of bioproducts (38L) was loaded and separated on 415% Mini-PROTEAN TGX Precast Gels (Bio-Rad, USA). Bioproducts and cell lysates were treated with protein loading dye (Laemmli sample buffer; Bio-Rad) with freshly added -mercaptoethanol 10%; v/v; Sigma, Germany) and boiled for 5min at 95 before SDS-PAGE. Proteins were subsequently blotted to a nitrocellulose blotting membrane (0.2m; 1,060,000; GE Healthcare, USA). Membranes were blocked in 5% BSA (Carl Roth, Germany) in 0.1% Tween in TBS (TBS-T). After blocking, blots were probed with the following primary antibodies diluted in 5% BSA/TBS-T: Calnexin (1:500 dilution, E-10, Santa Cruz Biotechnology). After overnight incubation at 4, membranes were washed 3 times with TBS-T and subsequently incubated with the secondary antibody dilution: Polyclonal Goat anti-mouse HRP (1:5000 dilution; P0447) for 1h at room temperature followed by washing. Blots were then developed using Western Bright ECL (541,004; Biozym Scientific, Germany) and protein bands were detected using the FusionCapt Advanced Solo 4 (Vilber, Germany).

The capacity of ASC or their bioproducts to inhibit induced proliferation of peripheral blood mononuclear cells (PBMCs) was analyzed as described before [27]. PBMCs were isolated from leukapheresis samples from healthy donors, provided by the German Red Cross Blood Donor Service in Mannheim (Mannheim Ethics Commission; vote number 2018-594N-MA). To assess their proliferation, PBMCs were labelled with proliferation dye Cytotell Green (ATT Bioquest, 22,253) (1:500 dilution) and seeded at a 1:10 ASC/bioproduction:PBMCs ratio in RPMI, supplemented with 10% FBS, 2% l-glutamine (PAN Biotech, P04-80100), 1% Penicillin/Streptomycin (PAN Biotech, P06-07100), and 200U/mL IL-2 (Promokine, C61240). PBMC proliferation was stimulated with phytohemagglutinin-L (PHA, 4.8g/mL (Biochrom, Merck Millipore, M5030)). PBMCs cultured alone without ASC in the absence and presence of PHA served as negative and positive controls, respectively. After 5days, PBMC proliferation was measured based on the dilution of Cytotell Green dye using a FACS Canto II (BD Biosciences) and the data were analyzed with FlowJo Software.

THP-1 monocyte-like cell line (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 growth medium with l-Glutamine (Sigma-Aldrich Ireland Ltd. Wicklow, Ireland) supplemented with 10% FBS (Sigma-Aldrich), 1% penicillin G (100U/mL) and streptomycin (100g/mL) solution (Sigma-Aldrich). In vitro assessment of phagocytic activity was done as described before [30]. THP-1 cells were seeded at a density of 5104 cells/well in dark 96 well-plates (Perkin Elmer Ireland Ltd. Dublin, Ireland) and exposed to 1g/mL of para-methoxyamphetamine (PMA, Sigma-Aldrich, St. Louis, MO, USA) for 48h to induce a macrophage-like phenotype. Cultures were washed with DPBS and fed with growth media for 24h. Afterwards, cells were activated with 100ng/mL of lipopolysaccharide (LPS, Sigma-Aldrich) for 24h. To measure the phagocytic capacity, Zymosan A FITC BioParticles (Thermo Fisher Ltd.) were used. Particles were opsonized with human serum (2mg/mL per 2107 particles, Sigma-Aldrich) for 1h and added to the cells in experimental media containing ASC bioproducts and growth media for 4h. Then, cells were washed twice with DPBS, fixed with 4% PFA for 15min and stained with Hoechst 33,342 (Invitrogen, Thermo Fisher Ltd). Images were taken on the Cytation 1 Imaging Reader at 20X (BioTek, with Gen5 Version 3.04 software, Swindon, UK). Six replicates were undertaken per condition and particle analysis was done by counting particle opsonization in a minimum of 200 cells per well.

HUVEC were seeded in 48-well plates at 84,000 cells/cm2 and cultured overnight. Subsequently, a p200 tip was used to create a scratch in each monolayer. Cultures were washed with DPBS before adding EVs/CM as described before. Complete EndoGRO-LS medium was used as a positive control, while EndoGRO-LS without FBS and VEGF served as a negative control. Scratches were imaged immediately after the addition of CM (0h) and after 8- and 24-h incubation using the automated Cytation 1 Imaging Reader at 4X. Six replicates were undertaken, and the total area of each scratch was measured using Image J. The percentage of closure was calculated relative to time 0h.

20,000 ASC were seeded in a 96-well Essen ImageLock plate and cultured overnight. Then, a 96-pin WoundMaker was used to create precise and reproducible wounds in all the wells. After the wound, the cells were washed 2 times with DPBS and ASC bioproducts added in different concentrations. Plates were then cultured in an IncuCyte ZOOM incubator and every 3h were taken a picture with the software. The results were analyzed after 24h. Relative Wound Density algorithm was used to report data.

Human umbilical vascular endothelial cells (HUVEC) either from Lonza or prepared as described before [31] and cultured until the 6th passage in EndoGRO-LS Complete Culture Media Kit (SCME001, Sigma-Aldrich, St. Louis, MO, USA). In vitro formation of capillary-like structures was performed on growth factorreduced Matrigel (356,231, Corning, NY, USA, center 1 and 3) or geltrex (Geltrex LDEV-free reduced growth factor matrix; Thermo Fisher Scientific, United States, center 2) HUVEC cells were treated with EVs or CM as described before, seeded at a density of 10103cells/well on a 48-well plate. Positive control was full EndoGro-LS medium, negative control medium without VEGF and FBS (as used for all the conditions). Cells were periodically observed with a Nikon TE2000E inverted microscope (Nikon, Tokyo, Japan), and experimental results were recorded after 16h; 3 images were taken per well. Image analysis was performed with the ImageJ software v.1.53c, using the Angiogenesis Analyzer (center 1,3). The data from three independent experiments were expressed as the meanSD of tube length in arbitrary units per field. Center 2 used live cell imaging (Incucyte Zoom) to assess network formation as described before [32].

Presence of vascular endothelial growth factor (VEGF) on ASC bioproducts was determined by solid phase sandwich ELISA using the human VEGF DuoSet ELISA (R&D Systems, USA) according to manufacturers instructions. The samples were read immediately at 450nm with a wavelength correction at 570nm using a VICTOR X4 multilabel plate reader (Perkin Elmer, Waltham, Massachusetts, USA). Levels of cytokines were quantified against an eight-point standard curve using twofold serial dilutions in reagent diluent.

The Pierce BCA Protein Assay Kit (ThermoFisher Scientific, UK) was used to determine protein concentration. In order to quantify the total amount of protein, samples were first lysed with RIPA buffer 4:1 (ThermoFisher Scientific, UK) for 30min on ice. The assay was carried out as per manufacturers instructions. The absorbance values were read in a VICTOR X4 plate reader (Perkin Elmer) at a 550nm wavelength, and the protein concentrations of the samples were quantified against the standard curve.

Statistical analysis was performed using GraphPad prism v9.4.2 (GraphPad software, USA). Data are expressed as meanstandard deviation (SD). N indicates biological replicates; n indicates technical replicates. Statistical differences among groups were calculated using ordinary two-way analysis of variance (ANOVA) and Tukeys post-hoc test when group distributions were normal (ShapiroWilks test) and variances of populations were equal (Bartletts test). When either or both assumptions were violated, non-parametric analysis was conducted; KruskalWallis test used to perform multiple comparison analysis and Dunns multiple comparison test for pairwise comparison. Results were considered statistically significant when p>0.05.

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Adipose stromal cells bioproducts as cell-free therapies ... - Journal of Translational Medicine