Kirigami-inspired stents for sustained local delivery of therapeutics.

Implantable drug depots have the capacity to locally meet therapeutic requirements by maximizing local drug efficacy and minimizing potential systemic side effects. Tubular organs including the gastrointestinal tract, respiratory tract and vasculature all manifest with endoluminal disease. The anatomic distribution of localized drug delivery for these organs using existing therapeutic modalities is limited. Application of local depots in a circumferential and extended longitudinal fashion could transform our capacity to offer effective treatment across a range of conditions. Here we report the development and application of a kirigami-based stent platform to achieve this. The stents comprise a stretchable snake-skin-inspired kirigami shell integrated with a fluidically driven linear soft actuator. They have the capacity to deposit drug depots circumferentially and longitudinally in the tubular mucosa of the gastrointestinal tract across millimetre to multi-centimetre length scales, as well as in the vasculature and large airways. We characterize the mechanics of kirigami stents for injection, and their capacity to engage tissue in a controlled manner and deposit degradable microparticles loaded with therapeutics by evaluating these systems ex vivo and in vivo in swine. We anticipate such systems could be applied for a range of endoluminal diseases by simplifying dosing regimens while maximizing drug on-target effects through the sustained release of therapeutics and minimizing systemic side effects.

Dissolved oxygen concentration regulates human hepatic organoid formation from pluripotent stem cells in a fully controlled bioreactor.

Developing technologies for scalable production of human organoids has gained increased attention for "organoid medicine" and drug discovery. We developed a scalable and integrated differentiation process for generation of hepatic organoid from human pluripotent stem cells (hPSCs) in a fully controlled stirred tank bioreactor with 150 ml working volume by application of physiological oxygen concentrations in different liver tissue zones. We found that the 20-40% dissolved oxygen concentration [DO] (corresponded to 30-60 mmHg pO2 within the liver tissue) significantly influences the process outcome via regulating the differentiation fate of hPSC aggregates by enhancing mesoderm induction. Regulation of the [DO] at 30% DO resulted in efficient generation of human fetal-like hepatic organoids that had a uniform size distribution and were comprised of red blood cells and functional hepatocytes, which exhibited improved liver-specific marker gene expressions, key liver metabolic functions, and, more important, higher inducible cytochrome P450 activity compared to the other trials. These hepatic organoids were successfully engrafted in an acute liver injury mouse model and produced albumin after implantation. These results demonstrated the significant impact of the dissolved oxygen concentration on hPSC hepatic differentiation fate and differentiation efficacy that should be considered ascritical translational aspect of established scalable liver organoid generation protocols for potential clinical and drug discovery applications.

Developing a Cost-Effective and Scalable Production of Human Hepatic Competent Endoderm from Size-Controlled Pluripotent Stem Cell Aggregates.

Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for potential research and therapeutic applications. However, current protocols for scalable and integrated differentiation of hPSCs limited due to high cost of growth factors and technical challenges. Here, hPSCs aggregates primed with 6 and 12 µM of CHIR99021 (CHIR), a Wnt agonist, in combination with different concentrations of high cost Activin A (10, 25, 50, 100 ng/mL). We sought to determine the appropriate treatment duration for efficient and cost-effective differentiation protocol for foregut definitive endoderm production in a dynamic suspension culture. Afterward, we evaluated the impact of the initial hPSC aggregate sizes (small: 86 ± 18 µm; medium: 142 ± 32 µm; large: 214 ± 34 µm) as critical bioprocess parameter on differentiation efficacy at the beginning of induction. The results indicated that 1-day priming of hPSCs as 3D aggregates (hPSpheres) with 6 µM CHIR followed by treatment with a low concentration of Activin (10 ng/mL) for 2 days resulted in efficient differentiation to definitive endoderm. This finding confirmed by the presence of ≥70% SOX17/FOXA2-double positive cells that highly expressed the anterior endodermal marker HEX. These endodermal cells differentiated efficiently into mature functional hepatocytes [60% albumin (ALB)-positive cells]. The results showed that the initial size of hPSC aggregates significantly impacted on the efficacy of differentiation. The medium sized-hPSpheres resulted in higher productivity and differentiation efficiency for scalable hepatocytes production, whereas small aggregates resulted in significant cell-loss after CHIR treatment and large aggregates had less efficacious endodermal differentiation. Differentiated cells exhibited multiple characteristics of primary hepatocytes as evidenced by expressions of liver-specific markers, indocyanine green and low-density lipoprotein uptake, and glycogen storage. Thus, this platform could be employed for scalable production of hPSC-derived hepatocytes for clinical and drug discovery applications.

Improving the biological function of decellularized heart valves through integration of protein tethering and three-dimensional cell seeding in a bioreactor.

Decellularized xenogeneic heart valves (DHVs) are promising products for valve replacement. However, the widespread clinical application of such products is limited due to the risk of immune reaction, progressive degeneration, inflammation, and calcification. Here, we have developed an optimized decellularization protocol for a xenogeneic heart valve. We improved the biological function of DHVs by protein tethering onto DHV and three-dimensional (3D) cell seeding in a bioreactor. Our results showed that heart valves treated with a Triton X-100 and sodium deoxycholate-based protocol were completely cell-free, with preserved biochemical and biomechanical properties. The immobilization of stromal derived factor-1α (SDF-1α) and basic fibroblast growth factor on DHV significantly improved recellularization with endothelial progenitor cells under the 3D culture condition in the bioreactor compared to static culture conditions. Cell phenotype analysis showed higher fibroblast-like cells and less myofibroblast-like cells in both protein-tethered DHVs. However, SDF-DHV significantly enhanced recellularization both in vitro and in vivo compared to basic fibroblast growth factor DHV and demonstrated less inflammatory cell infiltration. SDF-DHV had less calcification and platelet adhesion. Altogether, integration of SDF-1α immobilization and 3D cell seeding in a bioreactor might provide a novel, promising approach for production of functional heart valves.

Human embryonic stem cell-derived cardiovascular progenitor cells efficiently colonize in bFGF-tethered natural matrix to construct contracting humanized rat hearts.

Bioengineering of whole hearts using human embryonic stem cells (hESCs)-derived cardiovascular progenitor cells (CPCs) and natural matrices is a promising approach to overcome organ donor shortage threatening millions of patients awaiting for heart transplantation. Here, we developed a novel strategy for generation of heart constructs by repopulating engineered decellularized rat hearts using hESCs-derived CPCs. Careful expansion of CPCs in a scalable stirred-suspension bioreactor combined with step-wise seeding (60 million cells in 3 steps of 20 million per 1.5 h) onto decellularized hearts containing immobilized basic fibroblast growth factor (bFGF) resulted in improved retention of CPCs and differentiation to cardiomyocytes, smooth muscle cells and endothelial cells as evaluated by immunohistochemistry and qRT-PCR. We observed spontaneous and synchronous contractions of humanized hearts after 12 days of perfusion as well as advanced alignment of myofilaments. Our study provides a robust platform for generation of artificial human hearts and resolves major bottlenecks hindering further development of this technology.

Allogeneic cell therapy manufacturing: process development technologies and facility design options.

INTRODUCTION: 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.

Human Hair Reconstruction: Close, But Yet So Far.

Billions of dollars are annually invested in pharmaceutical industry and cosmetic sector with intent to develop new drugs and treatment strategies for alopecia. Because the hair looks an important characteristic of humans-an effective appendage in perception, expression of beauty, and preservation of self-esteem-the global market for hair loss treatment products is exponentially increasing. However, current methods to treat hair loss endure yet multiple challenges, such as unfavorable outcomes, nonpermanent and patient-dependent results, as well as unpredictable impacts, which limit their application. Over recent years, remarkable advances in the fields of regenerative medicine and hair tissue engineering have raised new hopes for introducing novel cell-based approaches to treat hair loss. Through cell-based approaches, it is possible to produce hair-like structures in the laboratory setting or manipulate cells in their native niche (in vivo lineage reprogramming) to reconstruct the hair follicle. However, challenging issues still exist with the functionality of cultured human hair cells, the proper selection of nonhair cell sources in cases of shortage of donor hair, and the development of defined culture conditions. Moreover, in the case of in vivo lineage reprogramming, selecting appropriate induction factors and their efficient delivery to guide resident cells into a hair fate-with the aim of reconstructing functional hair-still needs further explorations. In this study, we highlight recent advances and current challenges in hair loss treatment using cell-based approaches and provide novel insights for crucial steps, which must be taken into account to develop reproducible, safe, and efficient cell-based treatment.

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-ß, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Scalable Expansion of Human Pluripotent Stem Cell-Derived Neural Progenitors in Stirred Suspension Bioreactor Under Xeno-free Condition.

Recent advances in neural differentiation technology have paved the way to generate clinical grade neural progenitor populations from human pluripotent stem cells. These cells are an excellent source for the production of neural cell-based therapeutic products to treat incurable central nervous system disorders such as Parkinson's disease and spinal cord injuries. This progress can be complemented by the development of robust bioprocessing technologies for large scale expansion of clinical grade neural progenitors under GMP conditions for promising clinical use and drug discovery applications. Here, we describe a protocol for a robust, scalable expansion of human neural progenitor cells from pluripotent stem cells as 3D aggregates in a stirred suspension bioreactor. The use of this platform has resulted in easily expansion of neural progenitor cells for several passages with a fold increase of up to 4.2 over a period of 5 days compared to a maximum 1.5-2-fold increase in the adherent static culture over a 1 week period. In the bioreactor culture, these cells maintained self-renewal, karyotype stability, and cloning efficiency capabilities. This approach can be also used for human neural progenitor cells derived from other sources such as the human fetal brain.

Continuous co-production of ethanol and xylitol from rice straw hydrolysate in a membrane bioreactor.

The present study was set to develop a robust and economic biorefinery process for continuous co-production of ethanol and xylitol from rice straw in a membrane bioreactor. Acid pretreatment, enzymatic hydrolysis, detoxification, yeast strains selection, single and co-culture batch fermentation, and finally continuous co-fermentation were optimized. The combination of diluted acid pretreatment (3.5 %) and enzymatic conversion (1:10 enzyme (63 floating-point unit (FPU)/mL)/biomass ratio) resulted in the maximum sugar yield (81 % conversion). By concentrating the hydrolysates, sugars level increased by threefold while that of furfural reduced by 50 % (0.56 to 0.28 g/L). Combined application of active carbon and resin led to complete removal of furfural, hydroxyl methyl furfural, and acetic acid. The strains Saccharomyces cerevisiae NCIM 3090 with 66.4 g/L ethanol production and Candida tropicalis NCIM 3119 with 9.9 g/L xylitol production were selected. The maximum concentrations of ethanol and xylitol in the single cultures were recorded at 31.5 g/L (0.42 g/g yield) and 26.5 g/L (0.58 g/g yield), respectively. In the batch co-culture system, the ethanol and xylitol productions were 33.4 g/L (0.44 g/g yield) and 25.1 g/L (0.55 g/g yield), respectively. The maximum ethanol and xylitol volumetric productivity values in the batch co-culture system were 65 and 58 % after 25 and 60 h, but were improved in the continuous co-culture mode and reached 80 % (55 g/L) and 68 % (31 g/L) at the dilution rate of 0.03 L per hour, respectively. Hence, the continuous co-production strategy developed in this study could be recommended for producing value-added products from this hugely generated lignocellulosic waste.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

UNLABELLED: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-ß and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. SIGNIFICANCE: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

Comparison of different ionic liquids pretreatment for corn stover enzymatic saccharification.

Recently, application of ionic liquids (ILs) has received much attention due to their special solvency properties as a promising method of pretreatment for lignocellulosic biomass. Easy recovery of ionic liquids, chemical stability, temperature stability, nonflammability, low vapor pressure, and wide liquidus range are among those unique properties. These solvents are also known as green solvents due to their low vapor pressure. The present study was set to compare the effect of five different ILs, namely, 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]), 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]), 1-ethyl-3-methyl imidazolium diethyl phosphate ([EMIM][DEP]), 1-allyl-3-methylimidazolium chloride ([AMIM][Cl]), and 1-ethyl-3-methylimidazolium-hydrogen sulfate ([EMIM][HSO4]), on corn stover in a bioethanol production process. The performance of ILs was evaluated based on the change observed in chemical structure, crystallinity index, cellulose digestibility, and glucose release. Overall, [EMIM][Ac]-pretreated corn stover led to significantly higher saccharification, with cellulose digestibility reaching 69% after 72 hr, whereas digestibility of untreated barley straw was measured at only 21%.

Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies.

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing.

Comparison of different ionic liquids pretreatment for barley straw enzymatic saccharification.

Recently, application of ionic liquids due to their special solvency properties as a promising method of pretreatment for lignocellulosic biomass has received much attention. Chemical stability, temperature stability, non-flammability, low vapor pressure, wide liquidus range, and non-toxicity are among those unique properties. These solvents are also known as green solvents due to non-toxicity and low vapor pressure. The present study was set to compare the effect of five different ionic liquids namely, 1-ethyl-3-methyl imidazolium acetate, 1-ethyl-3-methyl imidazolium diethyl phosphate, 1-butyl-3-methyl imidazolium chlorides, 1,3-dimethyl imidazolium dimethyl phosphate, and 1-butyl-3-methylimidazolium-trifluoromethane sulfonate on barley straw in bioethanol production process. The performance of ionic liquids was evaluated based on the change observed in chemical structure, crystallinity index, and cellulose digestibility. Overall, 1-ethyl-3-methyl imidazolium acetate was found most effective in pretreating barely straw for bioethanol production. To the best of our knowledge, the present study reports different ionic liquids; some for the first time, for barely straw pretreatment.

Draft genome sequence of Ureibacillus thermosphaericus strain thermo-BF, isolated from Ramsar hot springs in Iran.

Ureibacillus thermosphaericus strain Thermo-BF is an aerobic, thermophilic bacillus which has been characterized to biosynthesize gold nanoparticles. Here we present the draft genome sequence of Ureibacillus thermosphaericus strain Thermo-BF which consists of a 2,864,162-bp chromosome. This is the first report of a shotgun sequenced draft genome of a species in the Ureibacillus genus.

Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor.

Current protocols for the scalable suspension culture of human pluripotent stem cells (hPSCs) are limited by multiple biological and technical challenges that need to be addressed before their use in clinical trials. To overcome these challenges, we have developed a novel bioprocess platform for large-scale expansion of human embryonic and induced pluripotent stem cell lines as three-dimensional size-controlled aggregates. This novel bioprocess utilizes the stepwise optimization of both static and dynamic suspension culture conditions. After screening eight xeno-free media in static suspension culture and optimizing single-cell passaging in dynamic conditions, the scale-up from a static to a dynamic suspension culture in the stirred bioreactor resulted in a two- to threefold improvement in expansion rates, as measured by cell counts and metabolic activity. We successfully produced size-specific aggregates through optimization of bioreactor hydrodynamic conditions by using combinations of different agitation rates and shear protectant concentrations. The expansion rates were further improved by controlling oxygen concentration at normoxic conditions, and reached a maximum eightfold increase for both types of hPSCs. Subsequently, we demonstrated a simple and rapid scale-up strategy that produced clinically relevant numbers of hPSCs (∼2×10(9) cells) over a 1-month period by the direct transfer of "suspension-adapted frozen cells" to a stirred suspension bioreactor. We omitted the required preadaptation passages in the static suspension culture. The cells underwent proliferation over multiple passages in the demonstrated xeno-free dynamic suspension culture while maintaining their self-renewal capabilities, as determined by marker expressions and in vitro spontaneous differentiation. In conclusion, suspension culture protocols of hPSCs could be used to mass produce homogenous and pluripotent undifferentiated cells by identification and optimization of key bioprocess parameters that are complemented by a simple and rapid scale-up platform.

Evaluation of anti-phytoplasma properties of surfactin and tetracycline towards lime witches' broom disease using real-time PCR.

The anti-phytoplasma activities of surfactin (derived from Iranian native Bacillus subtilis isolates) and tetracycline towards Candidatus "Phytoplasma aurantifolia", the agent of lime Witches' broom disease, were investigated. HPLC was used to quantify the surfactin production in four previously characterized native surfactin-producing strains, and the one producing the highest amount of surfactin (about 1,500 mg/l) was selected and cultivated following optimized production and extraction protocols. Different combinations of purified surfactin and commercial tetracycline were injected into artificially phytoplasmainfected Mexican lime seedlings using a syringe injection system. An absolute quantitative real-time PCR system was developed to monitor the phytoplasma population shifts in the lime phloem during 3 months following the injections. The results revealed that the injections of surfactin or tetracycline had a significant inhibitory effect on Candidatus "P. aurantifolia". However, the combined treatment with both surfactin and tetracycline (1:1) resulted in the highest inhibition due to a synergic effect, which suppressed the phytoplasma population from about 2×10(5) to less than 10 phytoplasma units/g plant tissue.

Molecular and biochemical characterization of Iranian surfactin-producing Bacillus subtilis isolates and evaluation of their biocontrol potential against Aspergillus flavus and Colletotrichum gloeosporioides.

The characterization of surfactin-producing Bacillus subtilis isolates collected from different ecological zones of Iran is presented. Characterization was performed using blood agar, PCR, drop-collapse, and reverse-phase high-performance liquid chromatography (HPLC) analyses, and the isolates' biocontrol effects against the aflatoxin-producing agent Aspergillus flavus and the citrus antracnosis agent Colletotrichum gloeosporioides were studied. In total, 290 B. subtilis isolates were isolated from phylosphere and rhizosphere samples collected from fields and gardens of 5 provinces of Iran. Blood agar assays showed that 185 isolates produced different biosurfactants. Isolates containing the sfp gene, coding for surfactin, were detected using the PCR method. It was found that 14 different isolates contained the sfp gene. Drop-collapse assays, which detect isolates with high production of surfactin, showed that 7 isolates produced high levels of surfactin. It was found from HPLC analysis that the isolates containin the sfp gene produced between 55 and 1610 mg of surfactin per litre of broth medium. Four isolates, named BS119m, BS116l, N3dn, and BS113c, produced more than 1000 mg of surfactin per litre of broth. The highest surfactin production level was observed for isolate BS119m (1610 mg/L). The antagonistic potential of the sfp gene-containing isolates was determined using dual culture and chloroform vapour methods. Our bioassay results indicated that isolate BS119m showed high inhibitory effects against A. flavus (100%) and C. gloeosporioides (88%). Furthermore, the effect of purified surfactin on the growth of A. flavus was evaluated. Mycelia growth was considerably reduced with increasing concentration of surfactin, and 36%, 54%, 84%, and 100% inhibitions of mycelia growth were, respectively, observed at 20, 40, 80, and 160 mg/L after 7 days of incubation.