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Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGROTM, AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGROTM-supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM-supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.

During tissue development, stem and progenitor cells are faced with fate decisions coordinated by microenvironmental cues. Although insights have been gained from in vitro and in vivo studies, the role of the microenvironment remains poorly understood due to the inability to systematically explore combinations of stimuli at a large scale. To overcome such restrictions, we implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high-throughput manner. Using embryonic stem cells as a model system, we derived definitive endoderm progenitors and applied them to the array platform to study the influence of ECM, including the interactions of ECM with growth factor signaling, on the specification of definitive endoderm cells towards the liver and pancreas fates. We identified ECM combinations that influence endoderm fate decisions towards these lineages, and demonstrated the utility of this platform for studying ECM-mediated modifications to signal activation during liver specification. In particular, defined combinations of fibronectin and laminin isoforms, as well as combinations of distinct collagen subtypes, were shown to influence SMAD pathway activation and the degree of hepatic differentiation. Overall, our systematic high-throughput approach suggests that ECM components of the microenvironment have modulatory effects on endoderm differentiation, including effects on lineage fate choice and cell adhesion and survival during the differentiation process. This platform represents a robust tool for analyzing effects of ECM composition towards the continued improvement of stem cell differentiation protocols and further elucidation of tissue development processes.Cellular microarrays can provide the capability to perform high-throughput investigations into the role of microenvironmental signals in a variety of cell functions. This study demonstrates the utility of a high-throughput cellular microarray approach for analyzing the effects of extracellular matrix (ECM) in liver and pancreas differentiation of endoderm progenitor cells. Despite an appreciation that ECM is likely involved in these processes, the influence of ECM, particularly combinations of matrix proteins, had not been systematically explored. In addition to the identification of relevant ECM compositions, this study illustrates the capability of the cellular microarray platform to be integrated with a diverse range of cell fate measurements, which could be broadly applied towards the investigation of cell fate regulation in other tissue development and disease contexts.

Strategies aiming at increasing the survival and paracrine activity of human mesenchymal stromal cells (MSCs) are of utmost importance to achieve the full therapeutic potential of these cells. Herein, we propose both physical and biochemical strategies to enhance the survival, homing, angiogenic, and immunomodulatory properties of MSCs in vitro. To that purpose, we compared the effect of exposing either 2D monolayer or 3D spheroids of MSCs to (i) hypoxia (2% O2 ) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O2 . 3D-cultured MSC spheroids evidenced higher survival upon exposure to oxidative stress and expressed higher levels of factors involved in tissue repair processes, namely tumor necrosis factor-stimulated gene-6, matrix metalloproteinase-2, and vascular endothelial growth factor. MSCs cultured as 3D spheroids and further exposed to hypoxia or hypoxic-mimetic conditions provided by DMOG synergistically favored the expression of the cell surface marker C-X-C chemokine receptor type-4, involved in homing processes to injured tissues, and adhesion to extracellular matrix components as fibronectin. These results highlight the role of ex vivo preconditioning approaches, presenting a novel strategy that combine biochemical stimuli with 3D spheroid organization of MSCs to maximize their tissue regeneration potential.

Replenishing neurons in patients with neurodegenerative diseases is one of the ultimate therapies for these progressive, debilitating and fatal diseases. Electrical stimulation can improve neuron stem cell differentiation but requires a reliable nanopatterned electroconductive substrate. Potential candidate substrates are polycaprolactone (PCL) – polyaniline:camphorsulfonic acid (PANI:CSA) nanofibers, but their nanobiophysical properties need to be finetuned. The present study investigates the use of the pseudo-doping effect on the optimization of the electroconductivity of these polyaniline-based electrospun nanofibers. This was performed by developing a new solvent system that comprises a mixture of hexafluoropropanol (HFP) and trifluoroethanol (TFE). For the first time, an electroconductivity so high as 0.2 S cm−1 was obtained for, obtained from a TFE:HFP 50/50 vol% solution, while maintaining fiber biocompatibility. The physicochemical mechanisms behind these changes were studied. The results suggest HFP promotes changes on PANI chains conformations through pseudo-doping, leading to the observed enhancement in electroconductivity. The consequences of such change in the nanofabrication of PCL-PANI fibers include an increase in fiber diameter (373 ± 172 nm), a decrease in contact angle (42 ± 3°) and a decrease in Young modulus (1.6 ± 0.5 MPa), making these fibers interesting candidates for neural tissue engineering. Electrical stimulation of differentiating neural stem cells was performed using AC electrical current. Positive effects on cell alignment and gene expression (DCX, MAP2) are observed. The novel optimized platform shows promising applications for (1) building in vitro platforms for drug screening, (2) interfaces for deep-brain electrodes; and (3) fully grown and functional neurons transplantation.

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% O2 , compared with atmospheric air) increased the in vitro angiogenic potential of the cultured cells. Envisaging the clinical manufacturing of an allogeneic, off-the-shelf MSC-based product, preconditioned UCM MSC maintain the angiogenic gene expression profile upon cryopreservation and delivery processes in the conditions of our study. These results are expected to contribute to the development of MSC-based therapies in the context of angiogenesis.

In recent years, human pluripotent stem (hPS) cells have started to emerge as a potential tool with application in fields such as regenerative medicine, disease modeling, and drug screening. In particular, the ability to differentiate human-induced pluripotent stem (hiPS) cells into different cell types and to mimic structures and functions of a specific target organ, resourcing to organoid technology, has introduced novel model systems for disease recapitulation while offering a powerful tool to provide a faster and reproducible approach in the process of drug discovery. All these technologies are expected to improve the overall quality of life of the humankind. Here, we highlight the main applications of hiPS cells and the main challenges associated with the translation of hPS cell derivatives into clinical settings and other biomedical applications, such as the costs of the process and the ability to mimic the complexity of the in vivo systems. Moreover, we focus on the bioprocessing approaches that can be applied towards the production of high numbers of cells as well as their efficient differentiation into the final product and further purification.

The development of cellular therapies to treat hematological malignancies has motivated researchers to investigate ex vivo culture systems capable of expanding the number of hematopoietic stem/progenitor cells (HSPC) before transplantation. The strategies exploited to achieve relevant cell numbers have relied on culture systems that lack biomimetic niche cues thought to be essential to promote HSPC maintenance and proliferation. Although stromal cells adhered to 2-D surfaces can be used to support the expansion of HSPC ex vivo, culture systems aiming to incorporate cell-cell interactions in a more intricate 3-D environment can better contribute to recapitulate the bone marrow (BM) hematopoietic niche in vitro.Herein, we describe the development of a 3-D co-culture system of human umbilical cord blood (UCB)-derived CD34

Chimeric antigen receptor (CAR) T cell therapy for the treatment of acute myeloid leukemia (AML) has the risk of toxicity to normal myeloid cells. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients but is absent on normal myeloid and erythroid cells. Since CD7 expression by malignant blasts is also linked with chemoresistance and poor outcomes, targeting this antigen may be beneficial for this subset of AML patients. Here, we show that expression of a CD7-directed CAR in CD7 gene-edited (CD7(KO)) T cells effectively eliminates CD7(+) AML cell lines, primary CD7(+) AML, and colony-forming cells but spares myeloid and erythroid progenitor cells and their progeny. In a xenograft model, CD7 CAR T cells protect mice against systemic leukemia, prolonging survival. Our results support the feasibility of using CD7(KO) CD7 CAR T cells for the non-myeloablative treatment of CD7(+) AML.

Bone regeneration, following fracture, relies on autologous and allogenic bone grafts. However, majority of fracture population consists of older individuals with poor quality bone associated with loss and/or modification of matrix proteins critical for bone formation and mineralization. Allografts suffer from same limitations and carry the risk of delayed healing, infection, immune rejection and eventual fracture. In this work, we apply a synergistic biomimetic strategy to develop matrices that rapidly form bone tissue - a critical aspect of fracture healing of weight bearing bones. Collagen matrices, enhanced with two selected key matrix proteins, osteocalcin (OC) and/or osteopontin (OPN), increased the rate and quantity of synthesized bone matrix by increasing mesenchymal stem/stromal cell (MSC) proliferation, accelerating osteogenic differentiation, enhancing angiogenesis and showing a sustained bone formation response from MSC obtained from a variety of human tissue sources (marrow, fat and umbilical cord). In vivo assessment of OC/OPN mineralized scaffolds in a critical sized-defect rabbit long-bone model did not reveal any foreign body reaction while bone tissue was being formed. We demonstrate a new biomimetic strategy to rapidly form mineralized bone tissue and secure a sustained bone formation response by MSC from multiple sources, thus facilitating faster patient recovery and treatment of non-union fractures in aging and diseased population. Acellular biomimetic matrices elicit bone regeneration response from MSC, obtained from multiple tissue sources, and can be used in variety of scaffolds and made widely available.

Peripheral artery disease (PAD) is a debilitating and prevalent condition characterized by blockage of the arteries, leading to limb amputation in more severe cases. Mesenchymal stem/stromal cells (MSC) are known to have intrinsic regenerative properties that can be potentiated by the introduction of pro-angiogenic genes such as the vascular endothelial growth factor (VEGF). Herein, the use of human bone marrow MSC transiently transfected with minicircles encoding for VEGF is proposed as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The VEGF gene was cloned in minicircle and conventional plasmid vectors and used to transfect bone marrow-derived MSC ex vivo. VEGF expression was evaluated both by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The number of VEGF transcripts following MSC transfection with minicircles increased 130-fold relative to the expression in non-transfected MSC, whereas for the plasmid (pVAX1)-based transfection, the increase was 50-fold. Compared to the VEGF basal levels secreted by MSC (11.1 ± 3.4 pg/1,000 cells/day), significantly higher values were detected by enzyme-linked immunosorbent assay after both minicircle and pVAX1 transfection (644.8 ± 82.5 and 508.3 ± 164.0 pg/1,000 cells/day, respectively). The VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays, without affecting the expansion potential ex vivo, as well as multilineage differentiation capacity or immunophenotype of MSC. Although preclinical in vivo studies are required, these results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach for an improved angiogenesis in the context of PAD.

The diverse bacterial populations that comprise the commensal microbiome of the human intestine play a central role in health and disease. A method that sustains complex microbial communities in direct contact with living human intestinal cells and their overlying mucus layer in vitro would thus enable the investigation of host-microbiome interactions. Here, we show the extended coculture of living human intestinal epithelium with stable communities of aerobic and anaerobic human gut microbiota, using a microfluidic intestine-on-a-chip that permits the control and real-time assessment of physiologically relevant oxygen gradients. When compared to aerobic coculture conditions, the establishment of a transluminal hypoxia gradient in the chip increased intestinal barrier function and sustained a physiologically relevant level of microbial diversity, consisting of over 200 unique operational taxonomic units from 11 different genera and an abundance of obligate anaerobic bacteria, with ratios of Firmicutes and Bacteroidetes similar to those observed in human faeces. The intestine-on-a-chip may serve as a discovery tool for the development of microbiome-related therapeutics, probiotics and nutraceuticals.

Cell-based microarrays are valuable platforms for the study of cytotoxicity and cellular microenvironment because they enable high-throughput screening of large sets of conditions at reduced reagent consumption. However, most of the described microarray technologies have been applied to two-dimensional cultures, which do not accurately emulate the in vivo three-dimensional (3D) cell-cell and cell-extracellular matrix interactions.Herein, we describe the methodology for production of alginate- and Matrigel-based 3-D cell microarrays for the study of mouse and human pluripotent stem cells on two different chip-based platforms. We further provide protocols for on-chip proliferation/viability analysis and the assessment of protein expression by immunofluorescence.

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up.

Cell-based microarrays are valuable platforms for the study of cytotoxicity and cellular microenvironment because they enable high-throughput screening of large sets of conditions at reduced reagent consumption. However, most of the described microarray technologies have been applied to two-dimensional cultures, which do not accurately emulate the in vivo three-dimensional (3D) cell-cell and cell-extracellular matrix interactions.Herein, we describe the methodology for production of alginate- and Matrigel-based 3-D cell microarrays for the study of mouse and human pluripotent stem cells on two different chip-based platforms. We further provide protocols for on-chip proliferation/viability analysis and the assessment of protein expression by immunofluorescence.

The development of cellular therapies to treat hematological malignancies has motivated researchers to investigate ex vivo culture systems capable of expanding the number of hematopoietic stem/progenitor cells (HSPC) before transplantation. The strategies exploited to achieve relevant cell numbers have relied on culture systems that lack biomimetic niche cues thought to be essential to promote HSPC maintenance and proliferation. Although stromal cells adhered to 2-D surfaces can be used to support the expansion of HSPC ex vivo, culture systems aiming to incorporate cell-cell interactions in a more intricate 3-D environment can better contribute to recapitulate the bone marrow (BM) hematopoietic niche in vitro.Herein, we describe the development of a 3-D co-culture system of human umbilical cord blood (UCB)-derived CD34+ cells and BM mesenchymal stem/stromal cell (MSC) spheroids in a microwell-based platform that allows to attain large numbers of spheroids with uniform sizes. Further comparison with a traditional 2-D co-culture system exploiting the supportive features of feeder layers of MSC is provided, while functional in vitro assays to assess the features of HSPC expanded in the 2-D vs. 3-D MSC co-culture systems are suggested.

Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.

Advanced cell-based therapies are promising approaches for stimulating full regeneration of cartilage lesions. In addition to a few commercially available medicinal products, several clinical and preclinical studies are ongoing worldwide. In preclinical settings, high-quality cartilage tissue has been produced using combination strategies involving stem or progenitor cells, biomaterials, and biomolecules to generate a construct for implantation at the lesion site. Cell numbers and mechanical stimulation of the constructs are not commonly considered, but are important parameters to be evaluated in forthcoming clinical studies. We review current clinical and preclinical studies for advanced therapy cartilage regeneration and evaluate the progress of the field.

Stem cell’s unique properties confer them a multitude of potential applications in the fields of cellular therapy, disease modelling and drug screening fields. In particular, the ability to differentiate neural progenitors (NP) from human induced pluripotent stem cells (hiPSCs) using chemically-defined conditions provides an opportunity to create a simple and straightforward culture platform for application in these fields. Here, we demonstrated that hiPSCs are capable of undergoing neural commitment inside microwells, forming characteristic neural structures resembling neural rosettes and further give rise to glial and neuronal cells. Furthermore, this platform can be applied towards the study of the effect of neurotoxic molecules that impair normal embryonic development. As a proof of concept, the neural teratogenic potential of the antiepileptic drug valproic acid (VPA) was analyzed. It was verified that exposure to VPA, close to typical dosage values (0.3 to 0.75 mM), led to a prevalence of NP structures over neuronal differentiation, as confirmed by analysis of the expression of neural cell adhesion molecule, as well as neural rosette number and morphology assessment. The methodology proposed herein for the generation and neural differentiation of hiPSC aggregates can potentially complement current toxicity tests such as the humanized embryonic stem cell test for the detection of teratogenic compounds that can interfere with normal embryonic development.

Currently, promising outcomes from clinical trials of allogeneic cells, especially allogeneic mesenchymal stromal cells, fibroblasts, keratinocytes, and human cardiac stem cells, have encouraged research institutions, small and medium enterprises (SMEs), and big pharmaceutical companies to invest and focus on developing allogeneic cell therapy products. Commercial and large-scale production of allogeneic cell therapy products requires unique capabilities to develop technologies that generate safe and effective allogeneic cells/cell lines and their fully characterized master/working banks. In addition, it is necessary to design robust upstream and downstream manufacturing processes, and establish integrated, well-designed manufacturing facilities to produce high quality affordable products in accordance with current GMP regulations for the production of cell therapy products. Areas covered: The authors highlight: the recent advances in the development of allogeneic products, the available options to develop robust manufacturing processes, and facility design considerations. Expert opinion: Currently, there are multiple challenges in development of allogeneic cell therapy products. Indeed, the field is still in its infancy; with technologies and regulations still under development, as is our understanding of the mechanisms of action in the body and their interaction with the host immune system. Their characterization and testing is also an emerging and very complex area.

This study proposes to use alginate encapsulation as a strategy to assess the paracrine activity of 3D- and 2D-cultured human bone marrow mesenchymal stem/stromal cells (BM MSC) in the setting of wound repair and regeneration processes. A side-by-side comparison of MSC culture in three different 3D configurations (spheroids, encapsulated spheroids and encapsulated single cells) versus 2D monolayer cell culture is presented. The results reveal enhanced resistance to oxidative stress and paracrine potential of 3D spheroid-organized BM MSC. MSC spheroids (148 ± 2 μm diameter) encapsulated in alginate microbeads evidence increased angiogenic and chemotactic potential relatively to encapsulated single cells, as supported by higher secreted levels of angiogenic factors and by functional assays showing the capability of encapsulated MSC to promote formation of tubelike structures and migration of fibroblasts into a wounded area. In addition, a higher expression of the anti-inflammatory factor tumor necrosis factor alpha-induced protein 6 (TSG-6) was demonstrated by RT-PCR for encapsulated and non-encapsulated spheroids. Culture of spheroids within an alginate matrix maintains low aggregation levels below 5% and favors resistance to oxidative stress. These are important factors towards the establishment of more standardized and controlled systems, crucial to explore the paracrine effects of 3D-cultured MSC in therapeutic settings.

Genomic instability is recognized as one of the most important hurdles in the expanding field of stem cell-based therapies. In the recent years, an accumulating body of evidence has shown that human stem cells undergo a diverse program of biological changes upon ex vivo cultivation that include numerical and structural chromosomal abnormalities, point mutations, variation of telomere length, and epigenetic instability. As the field moves forward, the growing awareness of the risk factors associated with human genome plasticity strongly advocates for the use of extensive genetic screening as part of a quality control platform to attest to the safety of stem cell-based products. Here we present a timely and comprehensive review that addresses the current status and emerging trends of the field, ultimately underscoring the need to implement new regulatory standards able to streamline the route to therapeutic applications. Stem Cells 2014;32:2824–2832

The present work aims to achieve additional insight on a mechanism describing the fundamental steps involved in the esterification reactions catalyzed by cutinase. The synthesis of ethyl caproate has been used as a model system to obtain a suitable kinetic model to estimate the activation energies involved in the various steps of the reaction pathway. Kinetic measurements have been made for the enzymatic esterification of caproic acid with ethyl alcohol catalyzed by recombinant Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. Different temperature conditions, from 25 to 50 °C, were tested for two different alcohol/acid molar ratios ( R = 1 and R = 2). The third ordered Ping Pong Bi Bi mechanism with alcohol inhibition was shown to be able to describe the experimental results. The model shows that the productivity decreases as the reaction temperature increases.

Neurodegenerative diseases compromise the quality of life of increasing numbers of the world’s aging population. While diagnosis is possible no effective treatments are available. Strong efforts are needed to develop new therapeutic approaches, namely in the areas of tissue engineering and deep brain stimulation (DBS). Conductive polymers are the ideal material for these applications due to the positive effect of conducting electricity on neural cell’s differentiation profile. This novel study assessed the biocompatibility of polybenzimidazole (PBI), as electrospun fibers and after being doped with different acids. Firstly, doped films of PBI were used to characterize the materials’ contact angle and electroconductivity. After this, fibers were electrospun and characterized by SEM, FTIR and TGA. Neural Stem Cell’s (NSC) proliferation was assessed and their growth rate and morphology on different samples was determined. Differentiation of NSCs on PBI - CSA fibers was also investigated and gene expression (SOX2, NES, GFAP, Tuj1) was assessed through Immunochemistry and qPCR. All the samples tested were able to support neural stem cell (NSC) proliferation without significant changes on the cell’s typical morphology. Successfully differentiation of NSCs towards neural cells on PBI – CSA fibers was also achieved. This promising PBI fibrous scaffold material is envisioned to be used in neural cell engineering applications, including scaffolds, in vitro models for drug screening and electrodes.

Novel bioengineering strategies for the ex vivo fabrication of native-like tissue-engineered cartilage are crucial for the translation of these approaches to clinically manage highly prevalent and debilitating joint diseases. Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, expensive, and not very versatile. In the current study, a novel, cost-effective, and customizable perfusion bioreactor totally fabricated by additive manufacturing (AM) is proposed for the study of the effect of fluid flow on the chondrogenic differentiation of human bone-marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly(ɛ-caprolactone) (PCL) scaffolds. hBMSCs are first seeded and grown on PCL scaffolds and hBMSC-PCL constructs are then transferred to 3D-extruded bioreactors for continuous perfusion culture under chondrogenic inductive conditions. Perfused constructs show similar cell metabolic activity and significantly higher sulfated glycosaminoglycan production (≈1.8-fold) in comparison to their non-perfused counterparts. Importantly, perfusion bioreactor culture significantly promoted the expression of chondrogenic marker genes while downregulating hypertrophy. This work highlights the potential of customizable AM platforms for the development of novel personalized repair strategies and more reliable in vitro models with a wide range of applications.

Differentiation of pluripotent stem cells (PSCs) into β cells could provide insulin independence for type 1 diabetes (T1D) patients. This approach would reduce the clinical complications that most patients managed on intensive insulin therapy (IIT) face. However, bottlenecks of PSC manufacturing and limited engraftment of encapsulated cells hinder the long-term effectiveness of these therapies. A bioprocess decision-support tool is combined with a disease state-transition model to evaluate the cost-effectiveness of the stem cell-based therapy against IIT. Clinical effectiveness is assessed in quality-adjusted life years (QALYs). Manufacturing costs per patient reduce from $430 000 to $160 000 with optimization of batch size and annual demand. For 96% of the patients, cell therapy improves the quality of life compared to IIT. Cost savings are achieved for 2% of the population through prevention of renal disease. The therapy is cost-effective for 3.4% of patients when a willingness to pay (WTP) of up to $150 000 per QALY is considered. A 75% cost reduction in the cell therapy price increases cost-effectiveness likelihood to 51% at $100 000 per QALY. This study highlights the need for scalable manufacturing platforms for stem cell therapies, as well as to prioritizing access to the therapy to patients with an increased likelihood of costly complications.

In the version of this Article originally published, the authors mistakenly cited Fig. 5d in the sentence beginning ‘Importantly, the microbiome cultured in these primary Intestine Chips...’; the correct citation is Supplementary Table 2. This has now been amended.

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering applications and cell-based therapies. Large cell doses (>1 × 106 cells kg-1 ) and Good Manufacturing Practices (GMP)-compliant processes are however required for clinical purposes. Here, a serum- and xenogeneic-free (S/XF) microcarrier-based culture system is established for the expansion of human umbilical cord matrix (UCM)- and adipose tissue (AT)-derived MSC using the Vertical-Wheel system (PBS-0.1 MAG; PBS Biotech). UCM and AT MSC are expanded to maximum cell densities of 5.3 ± 0.4 × 105 cell mL-1 (n = 3) and 3.6 ± 0.7 × 105 cell mL-1 (n = 3), respectively, after 7 days of culture, while maintaining their identity, according to standard criteria. An economic evaluation of the process transfer from T-flasks to PBS-0.1 MAG shows a reduction in the costs associated with the production of a dose for an average 70 kg adult patient (i.e., 70 million cells). Costs decrease from $17.0 K to $11.1 K for UCM MSC and from $21.5 K to $11.1 K for AT MSC, proving that the transition to Vertical-Wheel reactors provides a cost-effective alternative for MSC expansion. The present work reports the establishment of a scalable and cost-effective culture platform for the manufacturing of UCM and AT MSC in a S/XF microcarrier-based system.

The large cell doses (>1 × 10(6) cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system.

Pulsed field gradient spin echo high resolution magic angle spinning nuclear magnetic resonance spectroscopy is a powerful technique to characterize confined biosystems. We used this approach to assess the diffusion of solvent and reaction species within sol–gel matrices differing in enzyme loading.

This communication presents quantitative studies of the dynamic adhesion behavior of mesenchymal stem cells (MSCs) enabled by the combination of cell-surface receptor–ligand interactions and three-dimensional hydrodynamic control by microtopography., National Institutes of Health (U.S.) (Grant HL-095722), National Institutes of Health (U.S.) (Grant HL-097172), National Science Foundation (U.S.) (CAREER Award 0952493), Korea (South). Ministry of Science, ICT and Future Planning (National Research Foundation of Korea. Pioneer Research Center Program 2013M3C1A3064777), National Research Foundation of Korea (Framework of International Cooperation Program 2013K2A1A2053078)

One of the key challenges in stem cell bioprocessing is the large-scale cultivation of stem cells in order to meet the demanding meaningful cell numbers needed for biomedical applications, especially for clinical settings. Mouse embryonic stem cells [1] , used as a model system herein, were cultivated on microcarriers in a fully controlled stirred tank reactor (STR) [2] . The impact of varying the concentration of dissolved oxygen (at 5%, 10%, 20% and 30% DO) and operating under a continuous perfusion mode on cell growth and pluripotency maintenance was investigated. In addition, in order to further optimize the feeding strategy of the STR operating under continuous perfusion toward maximal cell production, the influence of different medium residences times (12 h, 24 h, 32 h, 48 h and 96 h) was evaluated. Overall, the maximal cell concentration of 7.9–9.2 × 106 cells/mL were attained after 11 days, with no passaging required, under a DO of 10–20% in the continuous perfused bioreactor with cell retention and medium residences times of 32–48 h. Importantly, mESC expanded under these conditions, retained the expression of pluripotency markers (Oct4, Nanog and Ssea-1), as well as their differentiation potential into cells of the three embryonic germ layers. The STR-based cultivation platform optimized herein represents a major contribution toward the development of large-volume production systems of differentiated cell derivatives for a wide range of biomedical applications.

The world's energy supply is mainly composed of fossil fuels, which are a non-renewable source of energy that is rapidly running out. To overcome this concern, industry has been focusing on the production of biofuels such as biodiesel. A range of approaches has been considered to transform oils into applicable biodiesel: dilutions, microemulsifications, pyrolysis and transesterification. The latter method consists of the conversion of triglycerides to a mixture of alkyl esters and glycerol, in the presence of an acyl acceptor and a catalyst. Due to high selectivity when using enzymes as catalysts, and mild operating conditions, biocatalytic transesterification has proven to be an efficient method. Cutinase, from the superfamily α/β hydrolases, is an enzyme with lipolytic activity that effectively catalyses transesterification reactions. This article highlights the use of cutinase microencapsulated in bis(2ethylhexyl) sodium sulfosuccinate (AOT)-reversed micelles to perform the biocatalytic transes...

Stem cell-derived products have the potential to represent promising therapeutic approaches for the treatment of a wide range of conditions. Neurodegenerative diseases, like Parkinson’s disease or Huntington’s disease, neurological disorders, cardiac failure, and blood disorders, among others, may one day be treated using cellular therapies and regenerative medicine approaches based on stem cells. Furthermore, owing to the potential positive impact in healthcare systems, translation of stem cell technologies into clinical applications will bring a broad social and economic advantage worldwide. However, to fully realize this potential, advanced bioprocessing systems are needed to deliver sufficient numbers of cells in compliance with stringent regulatory landscapes and that can be used in a safe and effective manner. This review presents and summarizes recent advancements in the field of stem cell engineering, in particular novel technologies for the interrogation of stem cell fate and systems for the robust manufacturing of cells under standardized, reproducible and strictly controlled conditions. c � 2013 Society of Chemical Industry

Physiologically low O2 tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0–5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O2 tensions (3–10%). We hypothesized that O2 concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34+-enriched cells was studied in co-culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O2. 2% O2 resulted in a significantly lower CD34+ cell expansion (25-fold vs 60-, 64- and 92-fold at day 10 for 5%, 21%, 10% O2, respectively). In turn, 10% O2 promoted the highest CD34+CD90+ cell expansion, reaching 22 ± 5.4- vs 5.6 ± 2.4- and 5.7 ± 2.0-fold for 2%, 5% and 21% O2, respectively, after 14 days. Similar differentiation patterns were observed under different O2 tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O2. Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O2 when compared to higher O2 concentrations (5–21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O2, can maximize the ex vivo expansion of UCB stem/progenitor cells in co-culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.

Umbilical cord blood (UCB) transplantation has observed a significant increase in recent years, due to the unique features of UCB haematopoietic stem/progenitor cells (HSCs) for the treatment of blood-related disorders. However, the low cell numbers available per UCB unit significantly impairs the widespread use of this source for transplantation of adult patients, resulting in graft failure, delayed engraftment and delayed immune reconstitution. In order to overcome this issue, distinct approaches are now being considered in clinical trials, such as double-UCB transplantation, intrabone injection or ex vivo expansion. In this article the authors review the current state of the art, future trends and challenges on the ex vivo expansion of UCB HSCs, focusing on culture parameters affecting the yield and quality of the expanded HSC grafts: novel HSC selection schemes prior to cell culture, cytokine/growth factor cocktails, the impact of biochemical factors (e.g. O2) or the addition of supportive cells, e.g. mesenchymal stem/stromal cell (MSC)-based feeder layers) were addressed. Importantly, a critical challenge in cellular therapy is still the scalability, reproducibility and control of the expansion process, in order to meet the clinical requirements for therapeutic applications. Efficient design of bioreactor systems and operation modes are now the focus of many bioengineers, integrating the increasing 'know-how' on HSC biology and physiology, while complying with the GMP standards for the production of cellular products, i.e. through the use of commercially available, highly controlled, disposable technologies. Copyright © 2013 John Wiley & Sons, Ltd.

Patients with stress urinary incontinence mainly suffer from malfunction of the urethra closure mechanism. We established the decellularization of porcine urethras to produce acellular urethra bioscaffolds for future tissue engineering applications, using bioscaffolds or bioscaffold-derived soluble products. Cellular removal was evaluated by H&E, DAPI and DNA quantification. The presence of specific ECM proteins was assessed through immunofluorescence staining and colorimetric assay kits. Human skeletal muscle myoblasts, muscle progenitor cells and adipose-derived stromal vascular fractions were used to evaluate the recellularization of the acellular urethra bioscaffolds. The mechanochemical decellularization system removed ~93% of tissue’s DNA, generally preserving ECM’s components and microarchitecture. Recellularization was achieved, though methodological advances are required regarding cell seeding strategies and functional assessment. Through microdissection and partial digestion, different urethra ECM-derived coating substrates were formulated (i.e. containing smooth or skeletal muscle ECM) and used to culture MPCs in vitro. The skeletal muscle ECM substrates enhanced fiber formation leading to the expression of the main skeletal muscle-related proteins and genes, as confirmed by immunofluorescence and RT-qPCR. The described methodology produced a urethra bioscaffold that retained vital ECM proteins and was liable to cell repopulation, a crucial first step towards the generation of urethra bioscaffold-based Tissue Engineering products.

Marine bacteria have evolved to survive in the marine environment by using unique physiological, biochemical and metabolic features and the ability to produce enzymes and compounds which may have commercial value. The Azores archipelago presents several ecosystems with strong volcanic activity where bacteria thrive under e.g. high temperatures. In this study, samples collected in the island of Sao Miguel were screened for biocatalysts possessing e.g. lipase, esterase, amylase, and inulinase activities. After isolation of several hundred bacterial strains, high throughput screening methods allowed the fast identification of biocatalysts. The first cultivation tests were performed on 24-wells microtiter plates with online oxygen monitoring and bacteria able to grow within 24 h were selected for further process development. Bacteria able to produce the desired enzymes were selected for the first round of tests. Four Bacillus strains presented high inulinase activity. The next step in process development was the determination of key parameters for enzyme activity such as temperature, pH, salinity and substrate concentration. The highest inulinase activity, 2.2 gsugars /gprotein h, was attained when the supernatant of a culture of a Bacillus subtilis strain was used in a magnetically stirred bioreactor. This study demonstrates how bacterial strains from marine environments may be used successfully in biotechnological processes.

Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources-bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP(+) cells 48 hr post-transfection, 58 ± 7.1% and 54 ± 3.8%, respectively, versus 33 ± 4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP(+) cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) has triggered the need for high cell doses in a vast number of clinical applications. This demand requires the development of good manufacturing practices (GMP)-compliant ex vivo expansion protocols that should be effective to deliver a robust and reproducible supply of clinical-grade cells in a safe and cost-effective manner. Controlled stirred-tank bioreactor systems under xenogeneic (xeno)-free culture conditions offer ideal settings to develop and optimize cell manufacturing to meet the standards and needs of human MSC for cellular therapies. Herein we describe two microcarrier-based stirred culture systems using spinner flasks and controlled stirred-tank bioreactors under xeno-free conditions for the efficient ex vivo expansion of human bone marrow and adipose tissue-derived MSC.

Due to their unique features, mesenchymal stem/stromal cells (MSC) have been exploited in clinical settings as therapeutic candidates for the treatment of a variety of diseases. However, the success in obtaining clinically-relevant MSC numbers for cell-based therapies is dependent on efficient isolation and ex vivo expansion protocols, able to comply with good manufacturing practices (GMP). In this context, the 2-dimensional static culture systems typically used for the expansion of these cells present several limitations that may lead to reduced cell numbers and compromise cell functions. Furthermore, many studies in the literature report the expansion of MSC using fetal bovine serum (FBS)-supplemented medium, which has been critically rated by regulatory agencies. Alternative platforms for the scalable manufacturing of MSC have been developed, namely using microcarriers in bioreactors, with also a considerable number of studies now reporting the production of MSC using xenogeneic/serum-free medium formulations. In this review we provide a comprehensive overview on the scalable manufacturing of human mesenchymal stem/stromal cells, depicting the various steps involved in the process from cell isolation to ex vivo expansion, using different cell tissue sources and culture medium formulations and exploiting bioprocess engineering tools namely microcarrier technology and bioreactors.

A new support for enzyme immobilisation, an alloy of niobium ore and graphite, was tested with: invertase from baker's yeast and inulinase from Aspergillus niger. The efficiency of immobilisation was about 30% for invertase and 72% for inulinase. The maximum activities values were observed for both enzymes at: pH 4.5, 50°C and 500 g/L of sucrose. The hydrolysis of inulin (5% w/v) by the free inulinase and invertase presented specific productivity of reducing sugars lower after immobilisation, about 15 and 1.5 times, respectively; with the hydrolysis of sucrose (50% w/v) the reductions observed were of 14 and 3.5 times, respectively. © 2012 Canadian Society for Chemical Engineering

The use of microchannel reactor based technologies within the scope of bioprocesses as process intensification and production platforms is gaining momentum. Such trend can be ascribed a particular set of characteristics of microchannel reactors, namely the enhanced mass and heat transfer, combined with easier handling and smaller volumes required, as compared to traditional reactors. In the present work, a continuous production process of 4-cholesten-3-one by the enzymatic oxidation of cholesterol without the formation of any by-product was assessed. The production was carried out within Y-shaped microchannel reactors in an aqueous–organic two-phase system. Substrate was delivered from the organic phase to aqueous phase containing cholesterol oxidase and the product formed partitions back to the organic phase. The aqueous phase was then forced through a plug-flow reactor, containing immobilized catalase. This step aimed at the reduction of hydrogen peroxide formed as a by-product during cholesterol oxidation, to avoid cholesterol oxidase deactivation due to said by-product. This setup was compared with traditional reactors and modes of operation. The results showed that microchannel reactor geometry outperformed traditional stirred tank and plug-flow reactors reaching similar conversion yields at reduced residence time. Coupling the plug-flow reactor containing catalase enabled aqueous phase reuse with maintenance of 30% catalytic activity of cholesterol oxidase while eliminating hydrogen peroxide. A final production of 36 m of cholestenone was reached after 300 hours of operation.