scholarly journals Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2158
Author(s):  
Pauline François ◽  
Laurent Giraudo ◽  
Julie Veran ◽  
Baptiste Bertrand ◽  
Chloé Dumoulin ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Subset ◽  
Stromal Vascular Fraction ◽  
Cost Effective ◽  
Advanced Therapy Medicinal Product ◽  
Automated Method ◽  
Advanced Therapy ◽  
Development And Validation

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

scholarly journals Fibronectin Adsorption on Electrospun Synthetic Vascular Grafts Attracts Endothelial Progenitor Cells and Promotes Endothelialization in Dynamic In Vitro Culture

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 778 ◽  
Author(s):  
Ruben Daum ◽  
Dmitri Visser ◽  
Constanze Wild ◽  
Larysa Kutuzova ◽  
Maria Schneider ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Cell Activation ◽  
Vascular Endothelial Cells ◽  
Endothelial Progenitor ◽  
Advanced Therapy Medicinal Product ◽  
Endothelial Colony Forming Cells ◽  
Advanced Therapy ◽  
Custom Made

Appropriate mechanical properties and fast endothelialization of synthetic grafts are key to ensure long-term functionality of implants. We used a newly developed biostable polyurethane elastomer (TPCU) to engineer electrospun vascular scaffolds with promising mechanical properties (E-modulus: 4.8 ± 0.6 MPa, burst pressure: 3326 ± 78 mmHg), which were biofunctionalized with fibronectin (FN) and decorin (DCN). Neither uncoated nor biofunctionalized TPCU scaffolds induced major adverse immune responses except for minor signs of polymorph nuclear cell activation. The in vivo endothelial progenitor cell homing potential of the biofunctionalized scaffolds was simulated in vitro by attracting endothelial colony-forming cells (ECFCs). Although DCN coating did attract ECFCs in combination with FN (FN + DCN), DCN-coated TPCU scaffolds showed a cell-repellent effect in the absence of FN. In a tissue-engineering approach, the electrospun and biofunctionalized tubular grafts were cultured with primary-isolated vascular endothelial cells in a custom-made bioreactor under dynamic conditions with the aim to engineer an advanced therapy medicinal product. Both FN and FN + DCN functionalization supported the formation of a confluent and functional endothelial layer.

scholarly journals Development of Autologous Platelet-Rich Plasma Mixed-Microfat as an Advanced Therapy Medicinal Product for Intra-Articular Injection of Radio-Carpal Osteoarthritis: From Validation Data to Preliminary Clinical Results

2019 ◽  
Vol 20 (5) ◽  
pp. 1111 ◽  
Author(s):  
Alice Mayoly ◽  
Aurélie Iniesta ◽  
Caroline Curvale ◽  
Najib Kachouh ◽  
Charlotte Jaloux ◽  
...  
Keyword(s):  
Medicinal Product ◽  
Platelet Rich Plasma ◽  
Stromal Vascular Fraction ◽  
Clinical Results ◽  
Advanced Therapy Medicinal Product ◽  
Validation Data ◽  
Painful Condition ◽  
Advanced Therapy ◽  
Functional Scores

Wrist osteoarthritis (OA) is one of the most common conditions encountered by hand surgeons with limited efficacy of non-surgical treatments. The purpose of this study is to describe the Platelet-Rich Plasma (PRP) mixed-microfat biological characteristics of an experimental Advanced Therapy Medicinal Product (ATMP) needed for clinical trial authorization and describe the clinical results obtained from our first three patients 12 months after treatment (NCT03164122). Biological characterization of microfat, PRP and mixture were analysed in vitro according to validated methods. Patients with stage four OA according to the Kellgren Lawrence classification, with failure to conservative treatment and a persistent daily painful condition >40 mm according to the visual analog scale (VAS) were treated. Microfat-PRP ATMP is a product with high platelet purity, conserved viability of stromal vascular fraction cells, chondrogenic differentiation capacity in vitro and high secretion of IL-1Ra anti-inflammatory cytokine. For patients, the only side effect was pain at the adipose tissue harvesting sites. Potential efficacy was observed with a pain decrease of over 50% (per VAS score) and the achievement of minimal clinically important differences for DASH and PRWE functional scores at one year in all three patients. Microfat-PRP ATMP presented a good safety profile after an injection in wrist OA. Efficacy trials are necessary to assess whether this innovative strategy could delay the necessity to perform non-conservative surgery.

scholarly journals Influence of Hypothermic Storage Fluids on Mesenchymal Stem Cell Stability: A Comprehensive Review and Personal Experience

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1043
Author(s):  
Aneta Ścieżyńska ◽  
Marta Soszyńska ◽  
Patrycja Szpak ◽  
Natalia Krześniak ◽  
Jacek Malejczyk ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Viability ◽  
Advanced Therapy Medicinal Product ◽  
In Vivo Models ◽  
Therapeutic Value ◽  
Advanced Therapy ◽  
Key Factor ◽  
Cell Stability ◽  
Preservation Solutions

Mesenchymal stem cells have generated a great deal of interest due to their potential use in regenerative medicine and tissue engineering. Examples illustrating their therapeutic value across various in vivo models are demonstrated in the literature. However, some clinical trials have not proved their therapeutic efficacy, showing that translation into clinical practice is considerably more difficult and discrepancies in clinical protocols can be a source of failure. Among the critical factors which play an important role in MSCs’ therapeutic efficiency are the method of preservation of the stem cell viability and various characteristics during their storage and transportation from the GMP production facility to the patient’s bedside. The cell storage medium should be considered a key factor stabilizing the environment and greatly influencing cell viability and potency and therefore the effectiveness of advanced therapy medicinal product (ATMP) based on MSCs. In this review, we summarize data from 826 publications concerning the effect of the most frequently used cell preservation solutions on MSC potential as cell-based therapeutic medicinal products.

Polyamines and Related Nitrogen Compounds in the Chemotherapy of Neglected Diseases Caused by Kinetoplastids

2018 ◽  
Vol 18 (5) ◽  
pp. 321-368 ◽  
Author(s):  
Juan A. Bisceglia ◽  
Maria C. Mollo ◽  
Nadia Gruber ◽  
Liliana R. Orelli
Keyword(s):  
Drug Targets ◽  
Redox Homeostasis ◽  
Cost Effective ◽  
Structural Features ◽  
Mammalian Host ◽  
Neglected Diseases ◽  
Nitrogen Compounds ◽  
Chemical Structures

Neglected diseases due to the parasitic protozoa Leishmania and Trypanosoma (kinetoplastids) affect millions of people worldwide, and the lack of suitable treatments has promoted an ongoing drug discovery effort to identify novel nontoxic and cost-effective chemotherapies. Polyamines are ubiquitous small organic molecules that play key roles in kinetoplastid parasites metabolism, redox homeostasis and in the normal progression of cell cycles, which differ from those found in the mammalian host. These features make polyamines attractive in terms of antiparasitic drug development. The present work provides a comprehensive insight on the use of polyamine derivatives and related nitrogen compounds in the chemotherapy of kinetoplastid diseases. The amount of literature on this subject is considerable, and a classification considering drug targets and chemical structures were made. Polyamines, aminoalcohols and basic heterocycles designed to target the relevant parasitic enzyme trypanothione reductase are discussed in the first section, followed by compounds directed to less common targets, like parasite SOD and the aminopurine P2 transporter. Finally, the third section comprises nitrogen compounds structurally derived from antimalaric agents. References on the chemical synthesis of the selected compounds are reported together with their in vivo and/or in vitro IC50 values, and structureactivity relationships within each group are analyzed. Some favourable structural features were identified from the SAR analyses comprising protonable sites, hydrophobic groups and optimum distances between them. The importance of certain pharmacophoric groups or amino acid residues in the bioactivity of polyamine derived compounds is also discussed.

scholarly journals Systematic Review: Allogenic Use of Stromal Vascular Fraction (SVF) and Decellularized Extracellular Matrices (ECM) as Advanced Therapy Medicinal Products (ATMP) in Tissue Regeneration

2020 ◽  
Vol 21 (14) ◽  
pp. 4982 ◽  
Author(s):  
Pietro Gentile ◽  
Aris Sterodimas ◽  
Jacopo Pizzicannella ◽  
Laura Dionisi ◽  
Domenico De Fazio ◽  
...  
Keyword(s):  
Systematic Review ◽  
Meta Analysis ◽  
Stromal Vascular Fraction ◽  
Lateral Epicondylitis ◽  
Medicinal Products ◽  
Advanced Therapy Medicinal Products ◽  
Advanced Therapy ◽  
Meta Analyses ◽  
Clinical Experiments

Stromal vascular fraction (SVF) containing adipose stem cells (ASCs) has been used for many years in regenerative plastic surgery for autologous applications, without any focus on their potential allogenic role. Allogenic SVF transplants could be based on the possibility to use decellularized extracellular matrix (ECM) as a scaffold from a donor then re-cellularized by ASCs of the recipient, in order to develop the advanced therapy medicinal products (ATMP) in fully personalized clinical approaches. A systematic review of this field has been realized in accordance with the Preferred Reporting for Items for Systematic Reviews and Meta-Analyses-Protocols (PRISMA-P) guidelines. Multistep research of the PubMed, Embase, MEDLINE, Pre-MEDLINE, PsycINFO, CINAHL, Clinicaltrials.gov, Scopus database, and Cochrane databases has been conducted to identify articles and investigations on human allogenic ASCs transplant for clinical use. Of the 341 articles identified, 313 were initially assessed for eligibility on the basis of the abstract. Of these, only 29 met all the predetermined criteria for inclusion according to the PICOS (patients, intervention, comparator, outcomes, and study design) approach, and 19 have been included in quantitative synthesis (meta-analysis). Ninety-one percent of the studies previously screened (284 papers) were focused on the in vitro results and pre-clinical experiments. The allogenic use regarded the treatment of perianal fistulas, diabetic foot ulcers, knee osteoarthritis, acute respiratory distress syndrome, refractory rheumatoid arthritis, pediatrics disease, fecal incontinence, ischemic heart disease, autoimmune encephalomyelitis, lateral epicondylitis, and soft tissue defects. The information analyzed suggested the safety and efficacy of allogenic ASCs and ECM transplants without major side effects.

scholarly journals Facile sonochemical-assisted synthesis of orthorhombic phase black phosphorus/rGO hybrids for effective photothermal therapy

Nanophotonics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 3023-3034
Author(s):  
Weiyuan Liang ◽  
Dou Wang ◽  
Xiaohui Ren ◽  
Chenchen Ge ◽  
Hanyue Wang ◽  
...  
Keyword(s):  
Electrode Materials ◽  
Orthorhombic Phase ◽  
Cost Effective ◽  
Black Phosphorus ◽  
Antitumor Effects ◽  
Covalent Bonds ◽  
Preparation Conditions ◽  
Reduced Graphene

AbstractTwo-dimensional black phosphorus (BP) has been demonstrated to be promising in photoelectronic devices, electrode materials, and biomedicine owing to its outstanding properties. However, the application of BP has been hindered by harsh preparation conditions, high costs, and easy degradation in ambient condition. Herein, we report a facile and cost-effective strategy for synthesis of orthorhombic phase BP and a kind of BP-reduced graphene oxide (BP/rGO) hybrids in which BP remains stable for more than 4 weeks ascribed to the formation of phosphorus-carbon covalent bonds between BP and rGO as well as the protection effect of the unique wrinkle morphology of rGO nanosheets. Surface modification BP/rGO hybrids (PEGylated BP/rGO) exhibit excellent photothermal performance with photothermal conversion efficiency as high as 57.79% at 808 nm. The BP/rGO hybrids exhibit enhanced antitumor effects both in vitro and in vivo, showing promising perspectives in biomedicine.

scholarly journals Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues

2021 ◽  
Vol 22 (15) ◽  
pp. 7920
Author(s):  
Myroslava Mytsyk ◽  
Giulia Cerino ◽  
Gregory Reid ◽  
Laia Gili Sole ◽  
Friedrich S. Eckstein ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Therapeutic Potential ◽  
Stromal Vascular Fraction ◽  
Human Adipose Tissue ◽  
Bioreactor Culture ◽  
Proliferating Cells ◽  
Angiogenic Potential ◽  
Engineered Tissues

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

scholarly journals Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Zeping Qiu ◽  
Jingwen Zhao ◽  
Fanyi Huang ◽  
Luhan Bao ◽  
Yanjia Chen ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Myocardial Fibrosis ◽  
Ventricular Remodeling ◽  
Cardiac Fibroblasts ◽  
Cost Effective ◽  
Intramyocardial Injection ◽  
Undesirable Consequence ◽  
Adverse Remodeling

AbstractMyocardial fibrosis and ventricular remodeling were the key pathology factors causing undesirable consequence after myocardial infarction. However, an efficient therapeutic method remains unclear, partly due to difficulty in continuously preventing neurohormonal overactivation and potential disadvantages of cell therapy for clinical practice. In this study, a rhACE2-electrospun fibrous patch with sustained releasing of rhACE2 to shape an induction transformation niche in situ was introduced, through micro-sol electrospinning technologies. A durable releasing pattern of rhACE2 encapsulated in hyaluronic acid (HA)—poly(L-lactic acid) (PLLA) core-shell structure was observed. By multiple in vitro studies, the rhACE2 patch demonstrated effectiveness in reducing cardiomyocytes apoptosis under hypoxia stress and inhibiting cardiac fibroblasts proliferation, which gave evidence for its in vivo efficacy. For striking mice myocardial infarction experiments, a successful prevention of adverse ventricular remodeling has been demonstrated, reflecting by improved ejection fraction, normal ventricle structure and less fibrosis. The rhACE2 patch niche showed clear superiority in long term function and structure preservation after ischemia compared with intramyocardial injection. Thus, the micro-sol electrospun rhACE2 fibrous patch niche was proved to be efficient, cost-effective and easy-to-use in preventing ventricular adverse remodeling.

scholarly journals EXTH-46. ARTIFICIAL INTELLIGENCE-BASED IDENTIFICATION OF COMBINED VANDETANIB AND EVEROLIMUS IN THE TREATMENT OF ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii97-ii97
Author(s):  
Diana Carvalho ◽  
Peter Richardson ◽  
Nagore Gene Olaciregui ◽  
Reda Stankunaite ◽  
Cinzia Emilia Lavarino ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Cell Viability ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Serine Threonine Kinase ◽  
Orthotopic Xenografts ◽  
Extended Survival

Abstract Somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2 receptor, are found in a quarter of children with the currently incurable brain tumour diffuse intrinsic pontine glioma (DIPG). Treatment of ACVR1-mutant DIPG patient-derived models with multiple inhibitor chemotypes leads to a reduction in cell viability in vitro and extended survival in orthotopic xenografts in vivo, though there are currently no specific ACVR1 inhibitors licensed for DIPG. Using an Artificial Intelligence-based platform to search for approved compounds which could be used to treat ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an approved inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has been trialed in DIPG patients with limited success, in part due to an inability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits both ABCG2 (BCRP) and ABCB1 (P-gp) transporter, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumour burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Based on these preclinical data, three patients with ACVR1-mutant DIPG were treated with vandetanib and everolimus. These cases may inform on the dosing and the toxicity profile of this combination for future clinical studies. This bench-to-bedside approach represents a rapidly translatable therapeutic strategy in children with ACVR1 mutant DIPG.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

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