Porous microspheres support mesenchymal progenitor cell ingrowth and stimulate angiogenesis

Porous microspheres have the potential for use as injectable bone fillers to obviate the need for open surgery. Successful bone fillers must be able to support vascularisation since tissue engineering scaffolds often cease functioning soon after implantation due to a failure to vascularise rapidly. Here, we test the angiogenic potential of a tissue engineered bone filler based on a photocurable acrylate-based high internal phase emulsion (HIPE). Highly porous microspheres were fabricated via two processes, which were compared. One was taken forward and investigated for its ability to support human mesenchymal progenitor cells and angiogenesis in a chorioallantoic membrane (CAM) assay. Porous microspheres with either a narrow or broad size distribution were prepared via a T-junction microfluidic device or by a controlled stirred-tank reactor of the HIPE water in oil in water (w/o/w), respectively. Culture of human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells showed proliferation over 11 days and formation of cell-microsphere aggregates. In-vitro, hES-MP cells were found to migrate into microspheres through their surface pores over time. The presence of osteoblasts, differentiated from the hES-MP cells, was evidenced through the presence of collagen and calcium after 30 days. Microspheres pre-cultured with cells were implanted into CAM for 7 days and compared with control microspheres without pre-cultured cells. The hES-MP seeded microspheres supported greater angiogenesis, as measured by the number of blood vessels and bifurcations, while the empty scaffolds attracted host chick cell ingrowth. This investigation shows that controlled fabrication of porous microspheres has the potential to create an angiogenic, bone filling material for use as a cell delivery vehicle

DESIGN AND DEVELOPMENT OF A MICROFLUIDIC DEVICE FOR THE ASSESSMENT OF FIRST-PASS METABOLISM

The aim of the thesis is to develop a microfluidic platform in order to mimic the first pass metabolism of oral ingested compounds. In the first part of the thesis, there is an introduction about the in vivo mechanism involved in the in process of first pass metabolism. First pass metabolism is strictly correlated to oral bioavailability of new developed drugs. The prediction of the dose of drug that reaches the blood flow and the target is fundamental. The organ involved in the first pass metabolism are principally the intestine, where a first metabolic process takes place, and the liver where the quote of drugs is metabolized again. New bioengineered in vitro model to assess first pass metabolism are explained, with a particular attention on 3D intestine and liver model. Furthermore, the first chapter is focused on the recent studies on organ-on-chip device that can recapitulate the in vivo physiology and microenvironment, with the relative steps of fabrications. To achieve the reproduction of the first pass metabolism on chip, we first focussed on the production of an innovative hepatic three dimensional tissues and then on the development of a organotypic intestinal tissues. In the chapter 2 it is presented the comparison of two kind of hepatic 3D model: spheroids and microtissues. The 3D-hepatic model chosen, was cultured into the new developed liver-on-chip device in order to have a perfusion culture. The chapter 3 is focused on the fabrication of an organotypic intestinal 3D tissues cultured in both in static and dynamic conditions. In particular a gut-on-chip microfluidic device was fabricated in order to obtain an air-liquid interface culture. The combination of the two hepatic and intestine model on chip, is addressed in chapter 4. In this last chapter a microfluidic biochip, can accommodate both hepatic microtissues and 3D human intestinal equivalent. By the selective communication of the two tissues recreated into the biochip, it is possible to simulate in vitro the mechanism of orally ingested drugs

Advanced 3D cell culture techniques in micro-bioreactors, Part II: Systems and applications

In this second part of our systematic review on the research area of 3D cell culture in micro-bioreactors we give a detailed description of the published work with regard to the existing micro-bioreactor types and their applications, and highlight important results gathered with the respective systems. As an interesting detail, we found that micro-bioreactors have already been used in SARS-CoV research prior to the SARS-CoV2 pandemic. As our literature research revealed a variety of 3D cell culture configurations in the examined bioreactor systems, we defined in review part one “complexity levels” by means of the corresponding 3D cell culture techniques applied in the systems. The definition of the complexity is thereby based on the knowledge that the spatial distribution of cell-extracellular matrix interactions and the spatial distribution of homologous and heterologous cell–cell contacts play an important role in modulating cell functions. Because at least one of these parameters can be assigned to the 3D cell culture techniques discussed in the present review, we structured the studies according to the complexity levels applied in the MBR systems

Bio-inks for 3D bioprinting : recent advances and future prospects

In the last decade, interest in the field of three-dimensional (3D) bioprinting has increased enormously. 3D bioprinting combines the fields of developmental biology, stem cells, and computer and materials science to create complex bio-hybrid structures for various applications. It is able to precisely place different cell types, biomaterials and biomolecules together in a predefined position to generate printed composite architectures. In the field of tissue engineering, 3D bioprinting has allowed the study of tissues and organs on a new level. In clinical applications, new models have been generated to study disease pathogenesis. One of the most important components of 3D bio-printing is the bio-ink, which is a mixture of cells, biomaterials and bioactive molecules that creates the printed article. This review describes all the currently used bio-printing inks, including polymeric hydrogels, polymer bead microcarriers, cell aggregates and extracellular matrix proteins. Amongst the polymeric components in bio-inks are: natural polymers including gelatin, hyaluronic acid, silk proteins and elastin; and synthetic polymers including amphiphilic block copolymers, PEG, poly(PNIPAAM) and polyphosphazenes. Furthermore, photocrosslinkable and thermoresponsive materials are described. To provide readers with an understanding of the context, the review also contains an overview of current bio-printing techniques and finishes with a summary of bio-printing applications

Production and Utility of Extracellular Vesicles with 3D Culture Methods

In recent years, extracellular vesicles (EVs) have emerged as promising biomarkers, cell-free therapeutic agents, and drug delivery carriers. Despite their great clinical potential, poor yield and unscalable production of EVs remain significant challenges. When using 3D culture methods, such as scaffolds and bioreactors, large numbers of cells can be expanded and the cell environment can be manipulated to control the cell phenotype. This has been employed to successfully increase the production of EVs as well as to enhance their therapeutic effects. The physiological relevance of 3D cultures, such as spheroids, has also provided a strategy for understanding the role of EVs in the pathogenesis of several diseases and to evaluate their role as tools to deliver drugs. Additionally, 3D culture methods can encapsulate EVs to achieve more sustained therapeutic effects as well as prevent premature clearance of EVs to enable more localised delivery and concentrated exosome dosage. This review highlights the opportunities and drawbacks of different 3D culture methods and their use in EV research

Three-Dimensional (3D) Printed Microneedles for Microencapsulated Cell Extrusion

Cell-hydrogel based therapies offer great promise for wound healing. The specific aim of this study was to assess the viability of human hepatocellular carcinoma (HepG2) cells immobilized in atomized alginate capsules (3.5% (w/v) alginate, d = 225 µm ± 24.5 µm) post-extrusion through a three-dimensional (3D) printed methacrylate-based custom hollow microneedle assembly (circular array of 13 conical frusta) fabricated using stereolithography. With a jetting reliability of 80%, the solvent-sterilized device with a root mean square roughness of 158 nm at the extrusion nozzle tip (d = 325 μm) was operated at a flowrate of 12 mL/min. There was no significant difference between the viability of the sheared and control samples for extrusion times of 2 h (p = 0.14, α = 0.05) and 24 h (p = 0.5, α = 0.05) post-atomization. Factoring the increase in extrusion yield from 21.2% to 56.4% attributed to hydrogel bioerosion quantifiable by a loss in resilience from 5470 (J/m3) to 3250 (J/m3), there was no significant difference in percentage relative payload (p = 0.2628, α = 0.05) when extrusion occurred 24 h (12.2 ± 4.9%) when compared to 2 h (9.9 ± 2.8%) post-atomization. Results from this paper highlight the feasibility of encapsulated cell extrusion, specifically protection from shear, through a hollow microneedle assembly reported for the first time in literature

Development of microspheres for biomedical applications: a review

An overview of microspheres manufactured for use in biomedical applications based on recent literature is presented in this review. Different types of glasses (i.e. silicate, borate, and phosphates), ceramics and polymer-based microspheres (both natural and synthetic) in the form of porous , non-porous and hollow structures that are either already in use or are currently being investigated within the biomedical area are discussed. The advantages of using microspheres in applications such as drug delivery, bone tissue engineering and regeneration, absorption and desorption of substances, kinetic release of the loaded drug components are also presented. This review also reports on the preparation and characterisation methodologies used for the manufacture of these microspheres. Finally, a brief summary of the existing challenges associated with processing these microspheres which requires further research and development are presented

Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare

Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications

Silk fibroin/gelatin microcarriers as scaffolds for bone tissue engineering

Microcarrier cell scaffolds have potential as injectable cell delivery vehicles or as building blocks for tissue engineering. The use of small cell carriers allows for a ‘bottom up’ approach to tissue assembly when moulding microparticles into larger structures, which can facilitate the introduction of hierarchy by layering different matrices and cell types, while evenly distributing cells through the structure. In this work, silk fibroin (SF), purified from Bombyx mori cocoons, was blended with gelatin (G) to produce materials composed of varying ratios of the two components (SF: G 25:75, 50:50, and 75:25). Cell compatibility to these materials was first confirmed in two-dimensional culture and found to be equivalent to standard tissue culture plastic, and better than SF or G alone. The mechanical properties of the blends were investigated and the blended materials were found to have increased Young's moduli over SF alone. Microcarriers of SF/G blends with defined diameters were generated in a reproducible manner through the use of an axisymmetric flow focussing device, constructed from off-the-shelf parts and fittings. These SF/G microcarriers supported adhesion of rat mesenchymal stem cells with high degrees of efficiency under dynamic culture conditions and, after culturing in osteogenic differentiation medium, cells were shown to have characteristics typical of osteoblasts. This work illustrates that microcarriers composed of SF/G blends are promising building blocks for osteogenic tissue engineering

Hydrogel microparticles for biosensing

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field. Keywords: Hydrogel; Biosensor; Microparticle; Multiplex assayNovartis Institutes of Biomedical Research (Presidential Fellowship)Novartis Institutes of Biomedical Research (Education Office)National Cancer Institute (U.S.) (Grant 5R21CA177393-02)National Science Foundation (U.S.) (Grant CMMI-1120724)Institute for Collaborative Biotechnologies (Grant W911NF-09-0001)United States. Army Research Offic