Descriptive account of the first use of the LeVe CPAP System, a new frugal CPAP System, in adult patients with COVID-19 Pneumonitis in Uganda.

BACKGROUND: Continuous positive airway pressure (CPAP) has been a key treatment modality for Coronavirus Disease 2019 (COVID-19) worldwide. Globally, the demand for CPAP outstripped the supply during the pandemic. The LeVe CPAP System was developed to provide respiratory support for treatment of COVID-19 and tailored for use in low- and middle-income country (LMIC) settings. Prior to formal trial approval, received in November 2021, these devices were used in extremis to support critically unwell adult patients requiring non-invasive ventilatory support. METHODS: This is a retrospective descriptive review of adult patients with COVID-19 pneumonitis, who were treated with advanced respiratory support (CPAP and/or high-flow nasal oxygen, HFNO) at Mengo Hospital, Uganda. Patients were treated with the LeVe CPAP System, Elisa CPAP and/or AIRVO™ HFNO. Treatment was escalated per standard local protocols for respiratory failure, and CPAP was the maximum respiratory support available. Data were collected on patient characteristics, length of time of treatment, clinical outcome, and any adverse events. RESULTS: Overall 333 patients were identified as COVID-19 positive, 44 received CPAP ± HFNO of which 43 were included in the study. The median age was 58 years (range 28-91 years) and 58% were female. The median duration of advanced respiratory support was 7 days (range 1-18 days). Overall (all device) mortality was 49% and this was similar between those started on the LeVe CPAP System and those started non-LeVe CPAP System devices (50% vs 47%). CONCLUSIONS: The LeVe CPAP system was the most used CPAP device during the pandemic, bringing the hospital's number of available HFNO/CPAP devices from two to 14. They were a critical resource for providing respiratory support to the sickest group of patients when no alternative devices were available. The devices appear to be safe and well-tolerated with no serious adverse events recorded. This study is unable to assess the efficacy of the LeVe CPAP System; therefore, formal comparative studies are required to inform further use.

MiGut: A scalable in vitro platform for simulating the human gut microbiome-Development, validation and simulation of antibiotic-induced dysbiosis.

In vitro models of the human colon have been used extensively in understanding the human gut microbiome (GM) and evaluating how internal and external factors affect the residing bacterial populations. Such models have been shown to be highly predictive of in vivo outcomes and have a number of advantages over animal models. The complexity required by in vitro models to closely mimic the physiology of the colon poses practical limits on their scalability. The scalable Mini Gut (MiGut) platform presented in this paper allows considerable expansion of model replicates and enables complex study design, without compromising on in vivo reflectiveness as is often the case with other model systems. MiGut has been benchmarked against a validated gut model in a demanding 9-week study. MiGut showed excellent repeatability between model replicates and results were consistent with those of the benchmark system. The novel technology presented in this paper makes it conceivable that tens of models could be run simultaneously, allowing complex microbiome-xenobiotic interactions to be explored in far greater detail, with minimal added resources or complexity. This platform expands the capacity to generate clinically relevant data to support our understanding of the cause-effect relationships that govern the GM.

In vitro models to study Clostridioides difficile infection: current systems and future advances.

PURPOSE OF REVIEW: Clostridioides difficile infection (CDI) is the most common cause of healthcare-associated diarrhoea in western countries, being categorized as an urgent healthcare threat. Historically, researchers have relied on the use of in vivo animal models to study CDI pathogenesis; however, differences in physiology and disease prognosis compared with humans limit their suitability to model CDI. In vitro models are increasingly being used as an alternative as they offer excellent process control, and some are able to use human ex-vivo prokaryotic and/or eukaryotic cells. RECENT FINDINGS: Simulating the colonic environment in vitro is particularly challenging. Bacterial fermentation models have been used to evaluate novel therapeutics, explore the re-modelling of the gut microbiota, and simulate disease progression. However, they lack the scalability to become more widespread. Models that co-culture human and bacterial cells are of particular interest, but the different conditions required by each cell type make these models challenging to run. Recent advancements in model design have allowed for longer culture times with more representative bacterial populations. SUMMARY: As in vitro models continue to evolve, they become more physiologically relevant, offering improved simulations of CDI, and extending their applicability.

Bacterial biofilm colonization and succession in tropical marine waters are similar across different types of stone materials used in seawall construction.

Seawalls are important in protecting coastlines from currents, erosion, sea-level rise, and flooding. They are, however, associated with reduced biodiversity, due to their steep orientation, lack of microhabitats, and the materials used in their construction. Hence, there is considerable interest in modifying seawalls to enhance the settlement and diversity of marine organisms, as microbial biofilms play a critical role facilitating algal and invertebrate colonization. We assessed how different stone materials, ranging from aluminosilicates to limestone and concrete, affect biofilm formation. Metagenomic assessment of marine microbial communities indicated no significant impact of material on microbial diversity, irrespective of the diverse surface chemistry and topography. Based on KEGG pathway analysis, surface properties appeared to influence the community composition and function during the initial stages of biofilm development, but this effect disappeared by Day 31. We conclude that marine biofilms converged over time to a generic marine biofilm, rather than the underlying stone substrata type playing a significant role in driving community composition.

Evaluation of the novel vaginal contraceptive agent PPCM in preclinical studies using sperm hyaluronan binding and acrosome status assays.

BACKGROUND: Polyphenylene carboxymethylene (PPCM) sodium salt is a promising multipurpose technology for prevention of both sexually transmitted infections (STIs) and pregnancy. In preclinical studies, PPCM has demonstrated significant (1) antimicrobial activity against several important viral and bacterial pathogens and (2) contraceptive activity associated with premature acrosome loss. OBJECTIVE: To further evaluate a vaginal antimicrobial compound as a contraceptive agent in preclinical studies utilizing a repurposed hyaluronan binding assay (HBA). MATERIALS AND METHODS: Semen samples containing either neat semen or washed spermatozoa were treated with increasing concentrations of PPCM or calcium ionophore A23187 (positive control). Sperm inactivation was measured by two methods: (1) double acrosome staining (AS), and (2) a hyaluronan binding assay (HBA® ). Percentage of inactivated sperm was compared between untreated control sperm and those treated with PPCM or A23187. RESULTS: PPCM had a significant (p < 0.05) and dose-dependent effect on sperm inactivation in both assays, with HBA detecting a higher proportion of inactivated sperm than AS. PPCM did not affect sperm motility and exhibited equivalent responses in the neat and washed samples. DISCUSSION: Both HBA and AS confirmed that spermatozoa were rapidly inactivated at PPCM concentrations likely present in the vagina under actual use conditions and in a time-frame comparable to in vivo migration of spermatozoa out of seminal plasma into cervical mucus. CONCLUSION: PPCM vaginal gel may provide contraceptive protection as well as help with STI prevention. HBA may be a sensitive and much needed biomarker for sperm activity in future contraceptive development.

Sub-confluent culture of human mesenchymal stromal cells on biodegradable polycaprolactone microcarriers enhances bone healing of rat calvarial defect.

In the current emerging trend of using human mesenchymal stromal cell (MSCs) for cell therapy, large quantities of cells are needed for clinical testing. Current methods of culturing cells, using tissue culture flasks or cell multilayer vessels, are proving to be ineffective in terms of cost, space and manpower. Therefore, alternatives such as large-scale industrialized production of MSCs in stirred tank bioreactors using microcarriers (MCs) are needed. Moreover, the development of biodegradable MCs for MSC expansion can streamline the bioprocess by eliminating the need for enzymatic cell harvesting and scaffold seeding for bone-healing therapies. Our previous studies described a process of making regulated density (1.06 g/cm3) porous polycaprolactone biodegradable MCs Light Polycarprolactone (LPCL) (MCs), which were used for expanding MSCs from various sources in stirred suspension culture. Here, we use human early MSCs (heMSCs) expanded on LPCL MCs for evaluation of their osteogenic differentiation potential in vitro as well as their use in vivo calvarial defect treatment in a rat model. In summary, (i) in vitro data show that LPCL MCs can be used to efficiently expand heMSCs in stirred cultures while maintaining surface marker expression; (ii) LPCL MCs can be used as scaffolds for cell transfer for transplantation in vivo; (iii) 50% sub-confluency, mid-logarithmic phase, on LPCL MCs (50% confluent) exhibited higher secretion levels of six cytokines (interleukin [IL]-6, IL-8, Vascular endothelial growth factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), growth-regulated oncogene-α (GRO-α) and stromal cell-derived factor-1α (SDF-1α)) as compared with 100% confluent, stationary phase cultures (100% confluent); (iv) these 50% confluent cultures demonstrated better in vitro osteogenic differentiation capacity as compared with 100% confluent cultures (higher levels of calcium deposition and at earlier stage); the improved bone differentiation capacity of these 50% confluent cultures was also demonstrated at the molecular level by higher expression of early osteoblast genes Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), collagen type I, osterix and osteocalcin); and (v) in vivo implantation of biodegradable LPCL MCs covered with 50% heMSCs into rats with calvarial defect demonstrated significantly better bone formation as compared with heMSCs obtained from monolayer cultures (5.1 ± 1.6 mm3 versus 1.3 ± 0.7 mm3). Moreover, the LPCL MCs covered with 50% heMSCs supported better in vivo bone formation compared with 100% confluent culture (2.1 ± 1.3 mm3). Taken together, our study highlights the potential of implanting 50% confluent MSCs propagated on LPCL MCs as optimal for bone regeneration. This methodology allows for the production of large numbers of MSCs in a three-dimensional (3D) stirred reactor, while supporting improved bone healing and eliminating the need for a 3D matrix support scaffold, as traditionally used in bone-healing treatments.

A bilayer swellable drug-eluting ureteric stent: Localized drug delivery to treat urothelial diseases.

A bilayer swellable drug-eluting ureteric stent (BSDEUS) is engineered and implemented, as a sustained drug delivery platform technology that enhances localized drug delivery to the highly impermeable urothelium, for the treatment of urothelial diseases such as strictures and carcinomas. On deployment, the device swells to co-apt with the ureteric wall and ensure drug availability to these tissues. BSDEUS consists of a stent spray-coated with a polymeric drug containing polylactic acid-co-caprolactone (PLC) layer which is overlaid by a swellable polyethylene glycol diacrylate (PEGDA) based hydrogel. In-vitro quantification of released drug demonstrated a tunable time-profile, indicating sustained delivery over 1-month. The PEGDA hydrogel overlayer enhanced drug release and transport into explanted porcine ureteric tissues ex-vivo, under a simulated dynamic fluid flow. A preliminary pilot in-vivo feasibility study, in a porcine model, demonstrated that the swollen hydrogel co-apts with the urothelium and thus enables localized drug delivery to the target tissue section. Kidney functions remained unaffected and device did not result in either hydronephrosis or systemic toxicity. This successful engineering of a bilayer coated stent prototype, demonstrates its feasibility, thus offering a unique solution for drug-based urological therapy.

Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

Fabrication of polycaprolactone-silanated ß-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications.

BACKGROUND CONTEXT: Interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site. PURPOSE: We recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device. STUDY DESIGN/SETTING: A combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds. MATERIALS AND METHODS: Freshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120-150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology. RESULTS: Progenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants. CONCLUSIONS: The combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.

Tunable Volumetric Density and Porous Structure of Spherical Poly-ε-caprolactone Microcarriers, as Applied in Human Mesenchymal Stem Cell Expansion.

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm3. Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm3, requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm3. The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 µm and densities ranging from 1.05-1.11 g/cm3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm3, achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.

Biodegradable poly-ε-caprolactone microcarriers for efficient production of human mesenchymal stromal cells and secreted cytokines in batch and fed-batch bioreactors.

Large numbers of human mesenchymal stromal cells (MSCs) used for a variety of applications in tissue engineering and cell therapy can be generated by scalable expansion in a bioreactor using microcarriers (MCs) systems. However, the enzymatic digestion process needed to detach cells from the growth surface can affect cell viability and potentially the potency and differentiation efficiency. Thus, the main aim of our study was to develop biocompatible and biodegradable MCs that can support high MSC yields while maintaining their differentiation capability and potency. After cell expansion, the cells that covered MCs can be directly implanted in vivo without the need for cell harvesting or use of scaffold. Poly-ε-caprolactone (PCL) is known as a biocompatible and biodegradable material. However, it cannot be used for generation of MCs because its high density (1.14 g/cm3) would exclude its applicability for suspension MCs in stirred reactors. In this article, we describe expansion and potency of MSCs propagated on low-density (1.06 g/cm3) porous PCL MCs coated with extracellular matrices (LPCLs) in suspended stirred reactors. Using these LPCLs, cell yields of about 4 × 104 cells/cm2 and 7- to 10-fold increases were obtained using four different MSC lines (bone marrow, cord blood, fetal and Wharton's jelly). These yields were comparable with those obtained using non-degradable MCs (Cytodex 3) and higher than two-dimensional monolayer (MNL) cultures. A fed-batch process, which demonstrated faster cell expansion (4.5 × 104 cells/cm2 in 5 days as compared with 7 days in batch culture) and about 70% reduction in growth media usage, was developed and scaled up from 100-mL spinner flask to 1-L controlled bioreactor. Surface marker expression, trilineage differentiation and clonogenic potential of the MSCs expanded on LPCL were not affected. Cytokine secretion kinetics, which occurred mostly during late logarithmic phase, was usually comparable with that obtained in Cytodex 3 cultures and higher than MNL cultures. In conclusion, biodegradable LPCL can be used to efficiently expand a variety of MSC lines in stirred scalable reactors in a cost-effective manner while maintaining surface markers expression, differentiation capability and high levels of cytokine secretion. This study is the first step in testing these cell-biodegradable porous MC aggregates for tissue engineering and cell therapy, such as bone and cartilage regeneration, or wound healing.

Biodegradable ECM-coated PCL microcarriers support scalable human early MSC expansion and in vivo bone formation.

BACKGROUND AIMS: Human mesenchymal stromal cells or marrow stromal cells (MSCs) are of great interest for bone healing due to their multi-potency and trophic effects. However, traditional MSC expansion methods using 2-dimensional monolayer (MNL) flasks or cell stacks are limited by labor-intensive handling, lack of scalability, the need for enzymatic cell harvesting and the need for attachment to a scaffold before in vivo delivery. Here, we present a biodegradable microcarrier and MSC bioprocessing system that may overcome the abovementioned challenges. METHODS: We cultured human early MSCs (heMSCs) on biodegradable polycaprolactone microcarriers (PCL MCs) coated with extracellular matrix (ECM) and evaluated the in vitro osteogenic differentiation and in vivo bone formation capacity of ECM-coated PCL MC-bound heMSCs compared with conventional MNL-cultured cells. RESULTS: We found that heMSCs proliferate well on PCL MCs coated with a fibronectin, poly-l-lysine, and fibronectin (FN+PLL+FN) coating (cPCL MCs). During in vitro osteogenic induction, heMSCs cultured on cPCL MCs displayed a 68% increase in specific calcium deposition compared with cultures on MNL. In a mouse ectopic mineralization model, bone mass was equivalent for MNL-expanded and cPCL MC-bound heMSC implants but higher in both cases when compared with cell-free cPCL MC implants at 16 weeks post-implantation. In summary, compared with MNL cultures, biodegradable MC MSC cultures provide the benefits of large-scale expansion of cells and can be delivered in vivo, thereby eliminating the need for cell harvesting and use of scaffolds for cell delivery. These results highlight the promise of delivering heMSCs cultured on cPCL MCs for bone applications.

Bioabsorbable radiopaque water-responsive shape memory embolization plug for temporary vascular occlusion.

We describe the preparation, characterization and evaluation of a biodegradable radiopaque water-triggered shape memory embolization plug for temporary vascular occlusion. The shape memory occluding device consists of a composite of a radio-opaque filler and a poly (dl-lactide-co-glycolide) (PLGA) blend, which was coated with a crosslinked poly (ethylene glycol) diacrylate (PEGDA) hydrogel. The mechanical properties, the degradation timeframe, the effect of programming conditions on the shape memory behaviour and the extent of radio-opacity for imaging were evaluated. Based on the tests, the mechanism responsible for the water-induced shape memory effect in such an embolization plug was elucidated. Suitable materials were optimized to fabricate an embolic plug prototype and its in vitro performance was evaluated as an occlusion rate (using a custom-built set up) and its biocompatibility. Finally, a feasibility study was conducted in vivo in a rabbit model to investigate the ease of device deployment, device migration and extent of vessel occlusion. The in vivo results demonstrated that the prototypes were visible under fluoroscopy and complete vascular occlusion occurred within 2 min of deployment of the prototypes in vivo. In conclusion, the developed embolization plug enables controlled and temporary vascular embolization, and is ready for safety studies.

Fabrication of uniform-sized poly-ɛ-caprolactone microspheres and their applications in human embryonic stem cell culture.

The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres, which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution, as well as the resulting PCL microsphere size, are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets, within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c, the microfluidic device is operated in dripping mode, which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity, resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins, the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells.

Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures.

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously, we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article, we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%), which leads to generation of cells/MC aggregates (400 µm in size) and high cell yields (2.4-3.5×10(6) cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (>90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover, tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10(8) cells.

Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture.

INTRODUCTION: Myocardial infarction is accompanied by a significant loss of cardiomyocytes (CMs). Functional CMs, differentiated from human embryonic stem cells (hESCs), offer a potentially unlimited cell source for cardiac disease therapies and regenerative cardiovascular medicine. However, conventional production methods on monolayer culture surfaces cannot adequately supply the large numbers of cells required for such treatments. To this end, an integrated microcarrier (MC) bioprocessing system for hESC propagation and subsequent CM differentiation was developed. METHODS: Production of hESC-derived CMs was initially established in monolayer cultures. This control condition was compared against hESC expansion on laminin-coated MC with cationic surface charge, in a stirred serum-free defined culture. Following expansion, the hESC/MC aggregates were placed in a CM differentiation medium, using Wnt signalling modulators in four different culture conditions. This process eliminated the need for manual colony cutting. The final optimized protocol was tested in stirred spinner flasks, combining expansion and differentiation on the same MC, with only media changes during the culture process. RESULTS: In the propagation phase, a 15-fold expansion of viable pluripotent HES-3 was achieved, with homogeneous sized aggregates of 316 ± 11 µm. Of the four differentiation conditions, stirred spinner flask cultures (MC-Sp) provided the best controlled aggregate sizes and yielded 1.9 × 106 CM/ml, as compared to 0.5 × 106 CM/ml using the monolayer cultures method: a four-fold increase in CM/ml. Similar results (1.3 × 106 CM/ml) were obtained with an alternative hESC H7 line. The hESC/MC-derived CM expressed cardiac-specific transcription factors, structural, ion channel genes, and exhibited cross-striations of sarcomeric proteins, thus confirming their cardiac ontogeny. Moreover, E-4031 (0.3 µM) prolonged the QT-interval duration by 40% and verapamil (3 µM) reduced it by 45%, illustrating the suitability of these CM for pharmacological assays. CONCLUSIONS: We have demonstrated a robust and scalable microcarrier system for generating hESC-derived CM. This platform is enabled by defined microcarrier matrices and it integrates cell propagation and differentiation within a continuous process, in serum-free culture media. It can generate significant numbers of CM, which are potentially suitable for future clinical therapies.

Cationic surface charge combined with either vitronectin or laminin dictates the evolution of human embryonic stem cells/microcarrier aggregates and cell growth in agitated cultures.

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 µm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 µm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment.

A novel geometry for a laboratory-based larval settlement assay.

A novel configuration, consisting of two apposing surfaces bounding a vertical water column, is presented and evaluated for settlement assays using cyprids of Balanus amphitrite. Assays were conducted on planar surfaces, ranging from hydrophobic polystyrene to hydrophilic glass and including CH(3)- and NH(3) (+)-terminated self-assembled monolayers (SAMs). Identical apposing surfaces generated settlement rates comparable to those obtained in prior studies, while a choice assay yielded consistent results, with individual replicates each indicating the preferred surface for settlement. As gravity favours contact with the lower apposing surface, cyprids trapped at the air/water interface settled on or around the perimeter where the water column meets the lower substratum. These cyprids are capable of selecting a settlement location and are thus not lost to the assay. The assay geometry lends itself to assessing cyprid exploration and settlement on planar surfaces with chemical patterning, including relief microstructures, without using a confining material or requiring the coating of a three-dimensional well.

Temporal application of topography to increase the rate of neural differentiation from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine, especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells, but these predominantly rely on biochemical cues. Recently, differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However, the means by which these topographical cues improve neuronal yield remains unknown. In this study, we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 µm gratings increase the rate of neural differentiation, and that an additional culture period of 2 µm gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added, thus generating hPSC-derived neural cells in a more cost effective and efficient manner.

Understanding the nano-topography changes and cellular influences resulting from the surface adsorption of human hair keratins.

Recent interest in the use of human hair keratins as a biomaterial has grown, fuelled by improvements in keratin extraction methods and better understanding of keratin bioactivity. The use of keratins as a bioactive coating for in vitro cell culture studies is an attractive proposition. In this light, the surface adsorption of human hair keratins onto tissue culture polystyrene surfaces has been investigated. Keratin density, nano-topography and hydrophobicity of keratin coated surfaces were characterized. To understand the cellular influence of these coated surfaces, murine L929 fibroblasts were cultured on them and evaluated for cytotoxicity, proliferation, metabolic activity and detachment behaviors compared to collagen type 1 coated surfaces. Keratins were deposited up to a density of 650 ng/cm(2) when a coating concentration of 80 µg/ml or higher was used. The surface features formed by adsorbed keratins also changed in a coating concentration dependent manner. These surfaces improved L929 mouse fibroblast adhesion and proliferation in comparison to uncoated and collagen type 1 coated tissue culture polystyrene. Furthermore, the expression of fibronectin was accelerated on surfaces coated with solutions of higher keratin concentrations. These results suggest that human hair keratins can be used as a viable surface coating material to enhance substrate compliance for culturing cells.