High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field

A new technique for seeding chondrocytes onto solvent-preserved human meniscus using the chemokinetic effect of recombinant human bone morphogenetic protein-2

Many investigators are currently studying the use of decellularized tissue allografts from human cadavers as scaffolds onto which patients’ cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. However, it is difficult to seed cells onto very dense regular connective tissue which has few interstitial spaces. Here, we discuss the development of a chemotactic cell seeding technique using solvent-preserved human meniscus. A chemokinetic response to recombinant human bone morphogenetic protein-2 (rhBMP-2) was observed in a monolayer culture of primary chondrocytes derived from femoral epiphyseal cartilage of 2-day-old rats. The rhBMP-2 significantly increased their migration upto 10 ng/ml in a dose-dependent manner. When tested with solvent-preserved human meniscus as a scaffold, which has few interstitial spaces, rhBMP-2 was able to induce chondrocytes to migrate into the meniscus. After a 3-week incubation, newly-formed cartilaginous extracellular matrix was synthesized by migrated chondrocytes throughout the meniscus, down to a depth of 3 mm. These findings demonstrate that rhBMP-2 may be a natural chemokinetic factor in vivo, which induces migration of proliferative chondrocytes into the narrow interfibrous spaces. Our results suggest a potential application of rhBMP-2 for the designed distribution of chondrocytes into a scaffold to be used for tissue engineering

New Insights into the Mechanisms of Embryonic Stem Cell Self-Renewal under Hypoxia: A Multifactorial Analysis Approach

Previous reports have shown that culturing mouse embryonic stem (mES) cells at different oxygen tensions originated different cell proliferation patterns and commitment stages depending on which signaling pathways are activated or inhibited to support the pluripotency state. Herein we provide new insights into the mechanisms by which oxygen is influencing mES cell self-renewal and pluripotency. A multifactorial approach was developed to rationally evaluate the singular and interactive control of MEK/ERK pathway, GSK-3 inhibition, and LIF/STAT3 signaling at physiological and non-physiological oxygen tensions. Collectively, our methodology revealed a significant role of GSK-3-mediated signaling towards maintenance of mES cell pluripotency at lower O2 tensions. Given the central role of this signaling pathway, future studies will need to focus on the downstream mechanisms involved in ES cell self-renewal under such conditions, and ultimately how these findings impact human models of pluripotency

Microfluidic approaches for producing lipid-based nanoparticles for drug delivery applications

The importance of drug delivery for disease treatment is supported by a vast literature and increasing ongoing clinical studies. Several categories of nano-based drug delivery systems have been considered in recent years, among which lipid-based nanomedicines, both artificial and cell-derived, remain the most approved. The best artificial systems in terms of biocompatibility and low toxicity are liposomes, as they are composed of phospholipids and cholesterol, the main components of cell membranes. Extracellular vesicles-biological nanoparticles released from cells-while resembling liposomes in size, shape, and structure, have a more complex composition with up to hundreds of different types of lipids, proteins, and carbohydrates in their membranes, as well as an internal cargo. Although nanoparticle technologies have revolutionized drug delivery by enabling passive and active targeting, increased stability, improved solubilization capacity, and reduced dose and adverse effects, the clinical translation remains challenging due to manufacturing limitations such as laborious and time-consuming procedures and high batch-to-batch variability. A sea change occurred when microfluidic strategies were employed, offering advantages in terms of precise particle handling, simplified workflows, higher sensitivity and specificity, and good reproducibility and stability over bulk methods. This review examines scientific advances in the microfluidics-mediated production of lipid-based nanoparticles for therapeutic applications. We will discuss the preparation of liposomes using both hydrodynamic focusing of microfluidic flow and mixing by herringbone and staggered baffle micromixers. Then, an overview on microfluidic approaches for producing extracellular vesicles and extracellular vesicles-mimetics for therapeutic applications will describe microfluidic extrusion, surface engineering, sonication, electroporation, nanoporation, and mixing. Finally, we will outline the challenges, opportunities, and future directions of microfluidic investigation of lipid-based nanoparticles in the clinic

Mini-review: advances in 3D bioprinting of vascularized constructs

3D in vitro constructs have gained more and more relevance in tissue engineering and in cancer-modeling. In recent years, with the development of thicker and more physiologically relevant tissue patches, the integration of a vascular network has become pivotal, both for sustaining the construct in vitro and to help the integration with the host tissue once implanted. Since 3D bioprinting is rising to be one of the most versatile methods to create vascularized constructs, we here briefly review the most promising advances in bioprinting techniques

Performance of a Defect-Mapping Microperimetry Approach for Characterizing Progressive Changes in Deep Scotomas

Purpose: To examine whether a microperimetry testing strategy based on quantifying the spatial extent of functional abnormalities (termed "defect-mapping" strategy) could improve the detection of progressive changes in deep scotomas compared to the conventional thresholding strategy. Methods: A total of 30 healthy participants underwent two microperimetry examinations, each using the defect-mapping and thresholding strategies at the first visit to examine the test-retest variability of each method. Testing was performed using an isotropic stimulus pattern centered on the optic nerve head (ONH), which acted as a model of a deep scotoma. These tests were repeated at a second visit, except using a smaller stimulus pattern and thereby increasing the proportion of test locations falling within the ONH (to simulate the progressive enlargement of a deep scotoma). The extent of change detected between visits relative to measurement variability was compared between the two strategies. Results: Relative to their effective dynamic ranges, the test-retest variability of the defect-mapping strategy (1.8%) was significantly lower compared to the thresholding strategy (3.3%; P < 0.001). The defect-mapping strategy also captured a significantly greater extent of change between visits relative to variability (-4.70 t-1) compared to the thresholding strategy (2.74 t-1; P < 0.001). Conclusions: A defect-mapping microperimetry testing strategy shows promise for capturing the progressive enlargement of deep scotomas more effectively than the conventional thresholding strategy. Translational Relevance: Microperimetry testing with the defect-mapping strategy could provide a more accurate clinical trial outcome measure for capturing progressive changes in deep scotomas in eyes with atrophic retinal diseases, warranting further investigations

Microtechnology for stem cell culture

Advances in stem cell research in recent decades have been aided by progress in the development of novel technologies aimed at biological systems. At the same time mimicking stem cell niches in vitro has become crucial for both basic stem cell research and the development of innovative therapies based on stem cells. Innovative microscale technologies can contribute to our quantitative understanding of how phenomena at the microscale can determine stem cell behavior based on our increasing ability to control culture conditions and the throughput of data while reducing times and costs. In particular, microtechnologies must be designed and developed to capture the complexity of cell\u2013substrate, cell\u2013cell, and cell\u2013soluble environment interactions considering the characteristic time and length scales of biological phenomena. While acknowledging the advantages of applying these technologies to stem cell culture, this chapter focuses on issues related to the control and mimicking of microenvironmental cues of the stem cell niche, such as substrate properties, cell topology, the soluble environment, and the electrophysiology

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study

none8noAdvanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the eciency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfuidic platform used to accurately tune the virus delivery from a diusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors (AdVs) carrying the EGFP transgene were used at dierent multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfuidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efciency of infection. Finally, we introduced the concept of eective MOI, a more appropriate variable for microfuidic infections that considers the number of adenoviruses in solution per cell at a certain time.noneE Cimetta; M Franzoso; M Trevisan; E Serena; A Zambon; S Giulitti; L Barzon; N ElvassoreCimetta, Elisa; Franzoso, Mauro; Trevisan, Marta; Serena, Elena; Zambon, Alessandro; Giulitti, Stefano; Barzon, Luisa; Elvassore, Nicol

Micropatterning topology on soft substrates affects myoblast proliferation and differentiation

Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 μm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 μm wide lanes compared to (39 ± 7)% in 500 μm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 μm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models