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Article: Bioprocessing Considerations towards the Manufacturing of Therapeutic Skeletal and Smooth Muscle Cells.

Franchi-Mendes, Teresa / Silva, Marília / Cartaxo, Ana Luísa / Fernandes-Platzgummer, Ana / Cabral, Joaquim M S / da Silva, Cláudia L

Bioengineering (Basel, Switzerland)

2023 Volume 10, Issue 9

Abstract: Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth ... ...

Abstract	Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth muscle, responsible for involuntary and voluntary contraction, respectively. In this review, several features and parameters involved in the bioprocessing of muscle cells are addressed. The cell isolation process is depicted, depending on the type of tissue (smooth or skeletal muscle), followed by the description of the challenges involving the use of adult donor tissue and the strategies to overcome the hurdles of reaching relevant cell numbers towards a clinical application. Specifically, the use of stem/progenitor cells is highlighted as a source for smooth and skeletal muscle cells towards the development of a cellular product able to maintain the target cell's identity and functionality. Moreover, taking into account the need for a robust and cost-effective bioprocess for cell manufacturing, the combination of muscle cells with biomaterials and the need for scale-up envisioning clinical applications are also approached.
Language	English
Publishing date	2023-09-09
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2746191-9
ISSN	2306-5354
ISSN	2306-5354
DOI	10.3390/bioengineering10091067
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Article ; Online: Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system.

Bandarra-Tavares, Hélder / Franchi-Mendes, Teresa / Ulpiano, Cristiana / Morini, Sara / Kaur, Navjot / Harris-Becker, Abigail / Vemuri, Mohan C / Cabral, Joaquim M S / Fernandes-Platzgummer, Ana / da Silva, Cláudia L

Cytotherapy

2024

Abstract: Background & aims: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable ... ...

Abstract	Background & aims: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. Methods: MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 10 Results: Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). Conclusions: Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.
Language	English
Publishing date	2024-03-11
Publishing country	England
Document type	Journal Article
ZDB-ID	2039821-9
ISSN	1477-2566 ; 1465-3249
ISSN (online)	1477-2566
ISSN	1465-3249
DOI	10.1016/j.jcyt.2024.03.001
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Article: From Promise to Reality: Bioengineering Strategies to Enhance the Therapeutic Potential of Extracellular Vesicles.

de Almeida Fuzeta, Miguel / Gonçalves, Pedro P / Fernandes-Platzgummer, Ana / Cabral, Joaquim M S / Bernardes, Nuno / da Silva, Cláudia L

Bioengineering (Basel, Switzerland)

2022 Volume 9, Issue 11

Abstract: Extracellular vesicles (EVs) have been the focus of great attention over the last decade, considering their promising application as next-generation therapeutics. EVs have emerged as relevant mediators of intercellular communication, being associated ... ...

Abstract	Extracellular vesicles (EVs) have been the focus of great attention over the last decade, considering their promising application as next-generation therapeutics. EVs have emerged as relevant mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. Given their natural ability to shuttle messages between cells, EVs have been explored both as inherent therapeutics in regenerative medicine and as drug delivery vehicles targeting multiple diseases. However, bioengineering strategies are required to harness the full potential of EVs for therapeutic use. For that purpose, a good understanding of EV biology, from their biogenesis to the way they are able to shuttle messages and establish interactions with recipient cells, is needed. Here, we review the current state-of-the-art on EV biology, complemented by representative examples of EVs roles in several pathophysiological processes, as well as the intrinsic therapeutic properties of EVs and paradigmatic strategies to produce and develop engineered EVs as next-generation drug delivery systems.
Language	English
Publishing date	2022-11-10
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2746191-9
ISSN	2306-5354
ISSN	2306-5354
DOI	10.3390/bioengineering9110675
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Article ; Online: Secretome of bone marrow mesenchymal stromal cells cultured in a dynamic system induces neuroprotection and modulates microglial responsiveness in an α-synuclein overexpression rat model.

Marques, Cláudia Raquel / Campos, Jonas / Sampaio-Marques, Belém / Antunes, Filipa Ferreira / Dos Santos Cunha, Raquel Medina / Silva, Deolinda / Barata-Antunes, Sandra / Lima, Rui / Fernandes-Platzgummer, Ana / da Silva, Cláudia L / Sousa, Rui Amandi / Salgado, António José

Cytotherapy

2024

Abstract: Background aims: Parkinson's disease (PD) is the second most common neurodegenerative disorder. The etiology of the disease remains largely unknown, but evidence have suggested that the overexpression and aggregation of alpha-synuclein (α-syn) play key ... ...

Abstract	Background aims: Parkinson's disease (PD) is the second most common neurodegenerative disorder. The etiology of the disease remains largely unknown, but evidence have suggested that the overexpression and aggregation of alpha-synuclein (α-syn) play key roles in the pathogenesis and progression of PD. Mesenchymal stromal cells (MSCs) have been earning attention in this field, mainly due to their paracrine capacity. The bioactive molecules secreted by MSCs, i.e. their secretome, have been associated with enhanced neuronal survival as well as a strong modulatory capacity of the microenvironments where the disease develops. The selection of the appropriate animal model is crucial in studies of efficacy assessment. Given the involvement of α-syn in the pathogenesis of PD, the evidence generated from the use of animal models that develop a pathologic phenotype due to the action of this protein is extremely valuable. Therefore, in this work, we established an animal model based on the viral vector-mediated overexpression of A53T α-syn and studied the impact of the secretome of bone marrow mesenchymal stromal cells MSC(M) as a therapeutic strategy. Methods: Adult male rats were subjected to α-syn over expression in the nigrostriatal pathway to model dopaminergic neurodegeneration. The impact of locally administered secretome treatment from MSC(M) was studied. Motor impairments were assessed throughout the study coupled with whole-region (striatum and substantia nigra) confocal microscopy evaluation of histopathological changes associated with dopaminergic neurodegeneration and glial cell reactivity. Results: Ten weeks after lesion induction, the animals received secretome injections in the substantia nigra pars compacta (SNpc) and striatum (STR). The secretome used was produced from bone marrow mesenchymal stromal cells MSC(M) expanded in a spinner flask (SP) system. Nine weeks later, animals that received the viral vector containing the gene for A53T α-syn and treated with vehicle (Neurobasal-A medium) presented dopaminergic cell loss in the SNpc and denervation in the STR. The treatment with secretome significantly reduced the levels of α-syn in the SNpc and protected the dopaminergic neurons (DAn) within the SNpc and STR. Conclusions: Our results are aligned with previous studies in both α-syn Caenorhabditis elegans models, as well as 6-OHDA rodent model, revealing that secretome exerted a neuroprotective effect. Moreover, these effects were associated with a modulation of microglial reactivity supporting an immunomodulatory role for the factors contained within the secretome. This further supports the development of new studies exploring the effects and the mechanism of action of secretome from MSC(M) against α-syn-induced neurotoxicity.
Language	English
Publishing date	2024-02-18
Publishing country	England
Document type	Journal Article
ZDB-ID	2039821-9
ISSN	1477-2566 ; 1465-3249
ISSN (online)	1477-2566
ISSN	1465-3249
DOI	10.1016/j.jcyt.2024.02.008
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Article: Neurodifferentiation and Neuroprotection Potential of Mesenchymal Stromal Cell-Derived Secretome Produced in Different Dynamic Systems.

Marques, Cláudia Raquel / Fuzeta, Miguel de Almeida / Dos Santos Cunha, Raquel Medina / Pereira-Sousa, Joana / Silva, Deolinda / Campos, Jonas / Teixeira-Castro, Andreia / Sousa, Rui Amandi / Fernandes-Platzgummer, Ana / da Silva, Cláudia L / Salgado, António José

Biomedicines

2023 Volume 11, Issue 5

Abstract: Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of ... ...

Abstract	Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein (α-synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrow-derived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α-synuclein in one
Language	English
Publishing date	2023-04-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2720867-9
ISSN	2227-9059
ISSN	2227-9059
DOI	10.3390/biomedicines11051240
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Article ; Online: Scalable Manufacturing of Human Hematopoietic Stem/Progenitor Cells Exploiting a Co-culture Platform with Mesenchymal Stromal Cells.

Fernandes-Platzgummer, Ana / Andrade, Pedro Z / Cabral, Joaquim M S / da Silva, Cláudia Lobato

Methods in molecular biology (Clifton, N.J.)

2020 Volume 2286, Page(s) 107–120

Abstract: In the context of hematopoietic cell transplantation, hematopoietic stem/progenitor cells (HSPC) from the umbilical cord blood (UCB) present several advantages compared to adult sources including higher proliferative capacity, abundant availability and ... ...

Abstract	In the context of hematopoietic cell transplantation, hematopoietic stem/progenitor cells (HSPC) from the umbilical cord blood (UCB) present several advantages compared to adult sources including higher proliferative capacity, abundant availability and ease of collection, non-risk and painless harvesting procedure, and lower risk of graft-versus-host disease. However, the therapeutic utility of UCB HSPC has been limited to pediatric patients due to the low cell frequency per unit of UCB. The development of efficient and cost-effective strategies to generate large numbers of functional UCB HSPC ex vivo would boost all current and future medical uses of these cells. Herein, we describe a scalable serum-free co-culture system for the expansion of UCB-derived CD34
MeSH term(s)	Biomedical Technology/methods ; Biomedical Technology/standards ; Cells, Cultured ; Coculture Techniques/methods ; Coculture Techniques/standards ; Hematopoietic Stem Cells/cytology ; Humans ; Mesenchymal Stem Cells/cytology ; Practice Guidelines as Topic ; Primary Cell Culture/methods ; Primary Cell Culture/standards ; Regenerative Medicine/methods ; Regenerative Medicine/standards
Language	English
Publishing date	2020-06-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/7651_2020_289
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Article ; Online: Optimized operation of a controlled stirred tank reactor system for the production of mesenchymal stromal cells and their extracellular vesicles.

Fernandes-Platzgummer, Ana / Cunha, Raquel / Morini, Sara / Carvalho, Marta / Moreno-Cid, Juan / García, Carmen / Cabral, Joaquim M S / da Silva, Cláudia L

Biotechnology and bioengineering

2023 Volume 120, Issue 9, Page(s) 2742–2755

Abstract: The therapeutic effects of human mesenchymal stromal cells (MSC) have been attributed mostly to their paracrine activity, exerted through small-secreted extracellular vesicles (EVs) rather than their engraftment into injured tissues. Currently, the ... ...

Abstract	The therapeutic effects of human mesenchymal stromal cells (MSC) have been attributed mostly to their paracrine activity, exerted through small-secreted extracellular vesicles (EVs) rather than their engraftment into injured tissues. Currently, the production of MSC-derived EVs (MSC-EVs) is performed in laborious static culture systems with limited manufacturing capacity using serum-containing media. In this work, a serum-/xenogeneic-free microcarrier-based culture system was successfully established for bone marrow-derived MSC cultivation and MSC-EV production using a 2 l-scale controlled stirred tank reactor (STR) operated under fed-batch (FB) or fed-batch combined with continuous perfusion (FB/CP). Overall, maximal cell numbers of (3.0 ± 0.12) × 10
MeSH term(s)	Humans ; Mesenchymal Stem Cells ; Batch Cell Culture Techniques ; Extracellular Vesicles/metabolism ; Regenerative Medicine ; Cell Proliferation
Language	English
Publishing date	2023-06-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	280318-5
ISSN	1097-0290 ; 0006-3592
ISSN (online)	1097-0290
ISSN	0006-3592
DOI	10.1002/bit.28449
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Article ; Online: Developing a Cell-Microcarrier Tissue-Engineered Product for Muscle Repair Using a Bioreactor System.

Cartaxo, Ana Luísa / Fernandes-Platzgummer, Ana / Rodrigues, Carlos A V / Melo, Ana M / Tecklenburg, Katja / Margreiter, Eva / Day, Richard M / da Silva, Cláudia L / Cabral, Joaquim M S

Tissue engineering. Part C, Methods

2023 Volume 29, Issue 12, Page(s) 583–595

Abstract: Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, ... ...

Abstract	Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provides a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restoring continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. In this study, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. First, patient-isolated SkMDCs were expanded using planar static culture systems. Second, by using a single-use PBS-MINI Vertical-Wheel
MeSH term(s)	Humans ; Cell Culture Techniques/methods ; Fecal Incontinence ; Quality of Life ; Bioreactors ; Muscles
Language	English
Publishing date	2023-11-21
Publishing country	United States
Document type	Journal Article
ZDB-ID	2420585-0
ISSN	1937-3392 ; 1937-3384
ISSN (online)	1937-3392
ISSN	1937-3384
DOI	10.1089/ten.TEC.2023.0122
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Zs.A 5500/C: Show issues

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Article: Hypothermic Preservation of Adipose-Derived Mesenchymal Stromal Cells as a Viable Solution for the Storage and Distribution of Cell Therapy Products.

Branco, André / Tiago, Ana L / Laranjeira, Paula / Carreira, Maria C / Milhano, João C / Dos Santos, Francisco / Cabral, Joaquim M S / Paiva, Artur / da Silva, Cláudia L / Fernandes-Platzgummer, Ana

Bioengineering (Basel, Switzerland)

2022 Volume 9, Issue 12

Abstract: Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily ... ...

Abstract	Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g., 2-8 °C) could be a feasible alternative. Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity was verified by flow cytometry, and a set of assays was performed to evaluate functionality. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor.
Language	English
Publishing date	2022-12-14
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2746191-9
ISSN	2306-5354
ISSN	2306-5354
DOI	10.3390/bioengineering9120805
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Article ; Online: Modulation of the in vitro angiogenic potential of human mesenchymal stromal cells from different tissue sources.

Pinto, Diogo S / Ahsan, Tabassum / Serra, Joana / Fernandes-Platzgummer, Ana / Cabral, Joaquim M S / da Silva, Cláudia L

Journal of cellular physiology

2020 Volume 235, Issue 10, Page(s) 7224–7238

Abstract: Mesenchymal stromal cells (MSCs) have been widely exploited for the treatment of several conditions due to their intrinsic regenerative and immunomodulatory properties. MSC have demonstrated to be particularly relevant for the treatment of ischemic ... ...

Abstract	Mesenchymal stromal cells (MSCs) have been widely exploited for the treatment of several conditions due to their intrinsic regenerative and immunomodulatory properties. MSC have demonstrated to be particularly relevant for the treatment of ischemic diseases, where MSC-based therapies can stimulate angiogenesis and induce tissue regeneration. Regardless of the condition targeted, recent analyses of MSC-based clinical trials have demonstrated limited benefits indicating a need to improve the efficacy of this cell product. Preconditioning MSC ex vivo through microenvironment modulation was found to improve MSC survival rate and thus prolong their therapeutic effect. This workstudy aims at enhancing the in vitro angiogenic capacity of a potential MSC-based medicinal product by comparing different sources of MSC and culture conditions. MSC from three different sources (bone marrow [BM], adipose tissue [AT], and umbilical cord matrix [UCM]) were cultured with xenogeneic-/serum-free culture medium under static conditions and their angiogenic potential was studied. Results indicated a higher in vitro angiogenic capacity of UCM MSC, compared with cells derived from BM and AT. Physicochemical preconditioning of UCM MSC through a microcarrier-based culture platform and low oxygen concentration (2% O
MeSH term(s)	Adipose Tissue/cytology ; Bone Marrow Cells/cytology ; Cell Differentiation ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Cellular Microenvironment/physiology ; Cryopreservation ; Culture Media, Serum-Free ; Humans ; Immunophenotyping ; In Vitro Techniques ; Mesenchymal Stem Cells/cytology ; Mesenchymal Stem Cells/physiology ; Neovascularization, Physiologic/genetics ; Oxygen ; Transcriptome ; Umbilical Cord/cytology
Chemical Substances	Culture Media, Serum-Free ; Oxygen (S88TT14065)
Language	English
Publishing date	2020-02-09
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3116-1
ISSN	1097-4652 ; 0021-9541
ISSN (online)	1097-4652
ISSN	0021-9541
DOI	10.1002/jcp.29622
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