Sodium Hyaluronate Supplemented Culture Media as a New hMSC Chondrogenic Differentiation Media-Model for in vitro/ex vivo Screening of Potential Cartilage Repair Therapies

A major challenge of cell-based therapy for cartilage lesions is the preservation of the chondrogenic phenotype during ex vivo cell cultivation. In this in vitro study, the chondro-inductive capacity of two different hyaline cartilage-conditioned cell culture media on human chondrocytes in 3D spheroids was determined. Media were conditioned by incubation of 200 mg/mL vital or devitalized cartilage matrix in growth media over 35 days. The media were analyzed for the content of soluble procollagen type (Col) II and glycosaminoglycans (GAGs) as well as released TGF-β1, IGF-1 and IGFBP3. Unconditioned medium served as a negative control while the positive medium control was supplemented with TGF-β1 and IGF-1. Spheroid cultures prepared from human chondrocytes were cultivated at 37 °C, 5% CO2 and 21% O2 in the respective media and controls. After 14 and 35 days, the deposition of ECM components was evaluated by histological analysis. Vital cartilage-conditioned medium contained significantly higher levels of Col II and active TGF-β1 compared to medium conditioned with the devitalized cartilage matrix. Despite these differences, the incubation with vital as well as devitalized cartilage conditioned medium led to similar results in terms of deposition of proteoglycans and collagen type II, which was used as an indicator of re-differentiation of human chondrocytes in spheroid cultures. However, high density 3D cell cultivation showed a positive influence on re-differentiation.

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Vaginal Microbicide Gel for Delivery of IQP-0528, a Pyrimidinedione Analog with a Dual Mechanism of Action against HIV-1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01368-10 ◽

2011 ◽

Vol 55 (4) ◽

pp. 1650-1660 ◽

Cited By ~ 45

Author(s):

Alamelu Mahalingam ◽

Adam P. Simmons ◽

Shweta R. Ugaonkar ◽

Karen M. Watson ◽

Charlene S. Dezzutti ◽

...

Keyword(s):

Antiviral Activity ◽

Mechanical Stability ◽

Culture Media ◽

Ex Vivo ◽

Hydroxyethyl Cellulose ◽

Therapeutic Window ◽

Formulation Development ◽

Drug Release Profile ◽

Hiv 1

ABSTRACTPyrimidinediones, a novel class of compounds, have previously been shown to possess antiviral activity at nanomolar concentrations. One member of this class of compounds, IQP-0528, was selected as the lead molecule for formulation development owing to its stability at physiologically relevant conditions, wide therapeutic window, and antiviral activity in the nanomolar range. Here, we report the development of two vaginal gels—3.0% hydroxyethyl cellulose (HEC) formulation and a 0.65% Carbopol formulation—for the sustained delivery of IQP-0528. Stability studies under accelerated conditions confirmed the chemical stability of IQP-0528 and mechanical stability of the gel formulation for 3 months.In vitrorelease studies revealed that diffusion-controlled release of IQP-0528 occurred over 6 h, with an initial lag time of approximately 1 h. Based on the drug release profile, the 3.0% HEC gel was selected as the lead formulation for safety and activity evaluations. Thein vitroandex vivosafety evaluations showed no significant loss in cell viability or significant inflammatory response after treatment with a 3.0% HEC gel containing 0.25% IQP-0528. In anin vitroHIV-1 entry inhibition assay, the lead formulation showed an 50% effective concentration of 0.14 μg/ml for gel in culture media, which corresponds to ∼0.001 μM IQP-0528. The antiviral activity was further confirmed by using polarized cervical explants, in which the formulation showed complete protection against HIV infection. In summary, these results are encouraging and warrant further evaluation of IQP-0528 gel formulations inin vivomodels, as well as the development of alternative formulations for the delivery of IQP-0528 as a microbicide.

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Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing.

10.1101/2022.01.11.475795 ◽

2022 ◽

Author(s):

Rajeev Rai ◽

Winston Vetharoy ◽

Asma Naseem ◽

Zohar Steinberg ◽

Adrian James Thrasher ◽

...

Keyword(s):

Gene Editing ◽

Culture Media ◽

Ex Vivo ◽

Culture Conditions ◽

Fine Tuning ◽

Great Promise ◽

Hematopoietic Stem ◽

Monogenic Diseases

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

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FRI0389 TOWARDS ONE-STAGE ARTICULAR CARTILAGE REPAIR USING DEVICE BASED ARTHROSCOPIC MECHANICAL RELEASE OF SYNOVIAL MESENCHYMAL STEM CELLS WITH CHONDROGENESIS AUGMENTATION WITH AUTOLOGOUS PLATELET CONCENTRATE WITHOUT CELL CULTURE EXPANSION

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5717 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 792-793

Author(s):

A. Altaie ◽

E. Jones ◽

O. Wall ◽

D. Mcgonagle

Keyword(s):

Flow Cytometry ◽

Synovial Fluid ◽

Cartilage Repair ◽

Ex Vivo ◽

Synovial Cells ◽

The Novel ◽

Novel Device ◽

Chondrogenic Potential ◽

Before And After

Background:Synovial fluid contains resident mesenchymal stem cells (SF-MSCs) that are derived from the synovial membrane and may interact with superficial cartilage injury sites. We previously reported on a novel methodology for increasing the number of MSCs in the knee joints using synovial brushing combined with platelet lysate (PL) as a chondrogenic inducer [1, 2].Objectives:The purpose of this study was to evaluate autologous and allogenic PL as a chondrogenic inducer and the chondrogenic potential of the mobilised MSCs without further ex vivo expansion. The desired goal of the study was to provide in vitro proof of concept of direct chondrogensis without resort to MSC expansion protocols, since adequate MSCs towards repair could be mechanically procured in a minimally invasive fashion.Methods:SF-MSCs were derived from the joint cavity of patients undergoing arthroscopy procedures. For the mechanical release of MSCs ‘before’ and ‘after’ brushing the synovium with the novel device (Figure 1A), samples of irrigation fluid were collected and MSC numbers were evaluated by CFU-F assay and flow cytometry for stromal and immune populations. Standard chondrogenic assay was performed on uncultured and cultured expanded synovial MSCs. Pellet cultures were maintained in complete chondrogenic media (CCM), DMEM+50% autologous filtared platelet concentrate (fPC), 50% Stemulate (allogeneic human PL; Cook Regentec, Indianapolis, IN), or expansion media (control). Chondrogenesis was assessed by Glycosaminoglycan (GAG) andToluidine bluestaining. Autologous blood was processed through a gravity-based filtration system, HemaTrate®(HT; Cook Regentec, Indianapolis, IN), to produce a PC.Figure 1.Flow cytometry analysis of stromal and immune populations before’ and ‘after’ mechanically release of synovial with the novel device (CD90highCD45Lowin red circle) (A). Uncultured synovial cells after 21 days exposure to complete chondrogenic media Toulid Blue staining (B) Gags level (C) n=3.Results:Mechanically mobilized SF-MSC numbers increased as measured by CFU-F assay and flow cytometry for CD90HighCD45Lowcells (p<0.001), and CD14+HLA-DR+CD206+CD86+M2 macrophages also increased (p<0.05). The HT system significantly concentrated platelets and WBCs by 6- (p<0.0001) and 1.8-folds (p<0.001), respectively. Device-mobilized SF-MSC proliferation significantly increased after 6 days in DMEM + 10% PC (p<0.001) and correlated with PC platelet number (p<0.005). Autologous PC increased GAG levels compared to control (p<0.0001), and there was no significant difference compared to allogenic PL (p>0.5). Uncultured synovial cells produced significantly more GAG when cultured in CCM or DMEM + 50% autologous PC compared to control (p<0.0001). The GAG levels of uncultured synovial cells positively correlated with CFU-F (p<0.005). Chondrogenic potential of uncultured synovial cells that were mechanically mobilized with initial irrigation exhibited an increase (1.5-fold) in GAG levels (p<0.001) figure 1-B and also positively correlated with CFU-F (p<0.005).Conclusion:Synovial MSCs can be mechanically released in sufficient number to undergo in vitro chondrogenic induction with significant chondrogenic activity without the need for ex vivo culture expansion. In vitro, autologous PC can be used as chondrogenic inducer for uncultured SF-MSCs. The data presented here supports one stage arthroscopy procedures for cartilage repairReferences:[1]T.G. Baboolal, S.C. Mastbergen, E. Jones, S.J. Calder, F.P. Lafeber, D. McGonagle, Synovial fluid hyaluronan mediates MSC attachment to cartilage, a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction, Annals of the rheumatic diseases 75(5) (2016) 908-15.[2]A. Altaie, T.G. Baboolal, O. Wall, E. Jones, D. McGonagle, Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use, Cytotherapy 20(3) (2018) 375-384.Disclosure of Interests:Ala Altaie: None declared, Elena Jones: None declared, Owen Wall: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

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A Method for the Evaluation of Early Osseointegration of Implant Materials Ex Vivo: Human Bone Organ Model

Materials ◽

10.3390/ma14113001 ◽

2021 ◽

Vol 14 (11) ◽

pp. 3001

Author(s):

Sergej Zankovic ◽

Michael Seidenstuecker ◽

Wolf C. Prall ◽

Johannes Loos ◽

Franziska Maderer ◽

...

Keyword(s):

Culture Media ◽

Ex Vivo ◽

Human Bone ◽

Cell Counting ◽

In Vitro Tests ◽

Implant Materials ◽

Replacement Surgery ◽

Organ Model ◽

Total Knee

In the present work, an ex vivo organ model using human bone (explant) was developed for the evaluation of the initial osseointegration behavior of implant materials. The model was tested with additive manufactured Ti6Al4V test substrates with different 3D geometries. Explants were obtained from patients who underwent total knee replacement surgery. The tibial plateaus were used within 24 h after surgery to harvest bone cylinders (BC) from the anterior side using hollow burrs. The BCs were brought into contact with the test substrate and inserted into an agarose mold, then covered with cell culture media and subjected to the external load of 500 g. Incubation was performed for 28 days. After 28d the test substrate was removed for further analysis. Cells grown out BC onto substrate were immunostained with DAPI and with an antibody against Collagen-I and alkaline phosphatase (ALP) for visualization and cell counting. We show that cells stayed alive for up to 28d in our organ model. The geometry of test substrates influences the number of cells grown onto substrate from BCs. The model presented here can be used for testing implant materials as an alternative for in vitro tests and animal models.

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New ex vivo calcification model for intact murine aortic valves

European Heart Journal ◽

10.1093/ehjci/ehaa946.3718 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

B Kruithof ◽

V Van De Pol ◽

T Los ◽

K Lodder ◽

M.C De Ruiter ◽

...

Keyword(s):

Aortic Valve ◽

Culture Media ◽

Ex Vivo ◽

Funding Source ◽

Aortic Valves ◽

Aortic Sinus ◽

Intact Mouse ◽

Jude Medical ◽

Endochondral Calcification

Abstract Introduction Calcific aortic valve disease (CAVD) is a common, progressive disease of the aortic valves for which no medical treatment exists and surgery is the only therapeutic solution. The discovery of novel pharmacological treatment for CAVD has been hampered due to the lack of suitable test-systems. Purpose We aimed to establish an ex vivo calcification model for intact mouse aortic valves. Methods We used the ex vivo flow model for mouse hearts (Miniature Tissue Culture System) to induce calcification with culture media supplemented with 1) β-glycerophosphate, ascorbic acid, and dexamethasone (OSM) or 2) inorganic phosphates (PI) and compared this with in vitro calcification of mouse valvular interstitial cells (mVICs). Results Ex vivo cultured aortic valve leaflets calcified in the presence of PI, but not OSM. RUNX was upregulated in both OSM and PI conditions, whereas alkaline phosphatase (ALP) and cyclooxygenase-2 (COX2) were differentially expressed. Apoptosis was not observed, together indicating that osteogenic calcification took place. Interestingly, both OSM and PI were able to induce calcification in vitro, revealing in vitro-ex vivo differences. In addition to the calcification in the aortic valves, endochondral calcification was present in the sinus near the hinge of the aortic valve in both PI and OSM conditions as evidenced by the expression of collagen II, aggrecan and ALP. Conclusions Together these data show that we have established an ex vivo calcification model for mouse aortic valves. Osteogenic calcification is induced in the aortic leaflets, whereas endochondral calcification is induced in the aortic sinus, reflecting the calcification found in human CAVD. Using this model, we can now study the initiation and progression of aortic valve calcification. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Dutch Heart Foundation, grant number 2013T093. Grants received from GE Healthcare, Lantheus medical imaging, St Jude Medical, Medtronic, Boston Scientific, Biotronik, and Edwards Lifesciences

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Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment

International Journal of Molecular Sciences ◽

10.3390/ijms19092635 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2635 ◽

Cited By ~ 7

Author(s):

Jagadeesh Venkatesan ◽

Oliver Gardner ◽

Ana Rey-Rico ◽

David Eglin ◽

Mauro Alini ◽

...

Keyword(s):

Gene Transfer ◽

Cartilage Repair ◽

Chondrogenic Differentiation ◽

Three Dimensional ◽

Cartilage Defects ◽

Human Mscs ◽

Lacz Gene ◽

Potential Benefits ◽

Hydrodynamic Environment

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair.

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Treatment With Cladribine Selects IFNγ+IL17+ T Cells in RRMS Patients – An In Vitro Study

Frontiers in Immunology ◽

10.3389/fimmu.2021.743010 ◽

2021 ◽

Vol 12 ◽

Author(s):

Minodora Dobreanu ◽

Doina Ramona Manu ◽

Ion Bogdan Mănescu ◽

Manuela Rozalia Gabor ◽

Adina Huţanu ◽

...

Keyword(s):

T Cell ◽

Experimental Model ◽

Culture Media ◽

Ex Vivo ◽

Interleukin 2 ◽

In Vitro Study ◽

Cell Receptor ◽

T Cell Subsets ◽

Multiparametric Flow Cytometry

BackgroundMultiple sclerosis (MS) is an incurable autoimmune disease mediated by a heterogeneous T cell population (CD3+CD161+CXCR3−CCR6+IFNγ−IL17+, CD3+CXCR3+CCR6+IFNγ+IL17+, and CD3+CXCR3+IFNγ+IL17− phenotypes) that infiltrates the central nervous system, eliciting local inflammation, demyelination and neurodegeneration. Cladribine is a lymphocyte-depleting deoxyadenosine analogue recently introduced for MS therapy as a Disease Modifying Drug (DMD). Our aim was to establish a method for the early identification and prediction of cladribine responsiveness among MS patients.MethodsAn experimental model was designed to study the cytotoxic and immunomodulatory effect of cladribine. T cell subsets of naïve relapsing-remitting MS (RRMS) patients were analyzed ex vivo and in vitro comparatively to healthy controls (HC). Surviving cells were stimulated with rh-interleukin-2 for up to 14days. Cell proliferation and immunophenotype changes were analyzed after maximal (phorbol myristate acetate/ionomycin/monensin) and physiological T-cell receptor (CD3/CD28) activation, using multiparametric flow cytometry and xMAP technology.ResultsEx vivo CD161+Th17 cells were increased in RRMS patients. Ex vivo to in vitro phenotype shifts included: decreased CD3+CCR6+ and CD3+CD161+ in all subjects and increased CD3+CXCR3+ in RRMS patients only; Th17.1 showed increased proliferation vs Th17 in all subjects; CD3+IL17+ and CD3+IFNγ+IL17+ continued to proliferate till day 14, CD3+IFNγ+ only till day 7. Regarding cladribine exposure: RRMS CD3+ cells were more resistant compared to HC; treated CD3+ cells proliferated continuously for up to 14 days, while untreated cells only up to 7 days; both HC/RRMS CD3+CXCR3+ populations increased from baseline till day 14; in RRMS patients vs HC, IL17 secretion from cladribine-treated cells increased significantly, in line with the observed proliferation of CD3+IL17+ and CD3+IFNγ+IL17+ cells; in both HC/RRMS, cladribine led to a significant increase in CD3+IFNγ+ cells at day 7 only, having no further effect at day14. IFNγ and IL17 secreted in culture media decreased significantly from ex vivo to in vitro.ConclusionsCD3+ subtypes showed different responsiveness due to selectivity of cladribine action, in most patients leading to in vitro survival/proliferation of lymphocyte subsets known as pathogenic in MS. This in vitro experimental model is a promising tool for the prediction of individual responsiveness of MS patients to cladribine and other DMDs.

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A comparison of BMP2 delivery by coacervate and gene therapy for promoting human muscle-derived stem cell-mediated articular cartilage repair

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1434-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Xueqin Gao ◽

Haizi Cheng ◽

Hassan Awada ◽

Ying Tang ◽

Sarah Amra ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Raman Spectroscopy ◽

Articular Cartilage ◽

Cartilage Repair ◽

Chondrogenic Differentiation ◽

Human Muscle ◽

Articular Cartilage Repair ◽

Sustain Release

Abstract Background Osteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study. Method In this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair. Results We observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining. Conclusions We conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

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Sodium Hyaluronate Nanocomposite Respirable Microparticles to Tackle Antibiotic Resistance with Potential Application in Treatment of Mycobacterial Pulmonary Infections

Pharmaceutics ◽

10.3390/pharmaceutics11050203 ◽

2019 ◽

Vol 11 (5) ◽

pp. 203 ◽

Cited By ~ 7

Author(s):

Irene Rossi ◽

Francesca Buttini ◽

Fabio Sonvico ◽

Filippo Affaticati ◽

Francesco Martinelli ◽

...

Keyword(s):

Drug Resistance ◽

Efflux Pump ◽

Respiratory Infections ◽

Ex Vivo ◽

Sodium Hyaluronate ◽

Drug Level ◽

Aqueous Environment ◽

Macrophage Infection ◽

Efflux Pump Inhibitor

Tuberculosis resistant cases have been estimated to grow every year. Besides Mycobacterium tuberculosis, other mycobacterial species are responsible for an increasing number of difficult-to-treat infections. To increase efficacy of pulmonary treatment of mycobacterial infections an inhalable antibiotic powder targeting infected alveolar macrophages (AMs) and including an efflux pump inhibitor was developed. Low molecular weight sodium hyaluronate sub-micron particles were efficiently loaded with rifampicin, isoniazid and verapamil, and transformed in highly respirable microparticles (mean volume diameter: 1 μm) by spray drying. These particles were able to regenerate their original size upon contact with aqueous environment with mechanical stirring or sonication. The in vitro drugs release profile from the powder was characterized by a slow release rate, favorable to maintain a high drug level inside AMs. In vitro antimicrobial activity and ex vivo macrophage infection assays employing susceptible and drug resistant strains were carried out. No significant differences were observed when the powder, which did not compromise the AMs viability after a five-day exposure, was compared to the same formulation without verapamil. However, both preparations achieved more than 80% reduction in bacterial viability irrespective of the drug resistance profile. This approach can be considered appropriate to treat mycobacterial respiratory infections, regardless the level of drug resistance.

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