Liquid Marble as Bioreactor for Engineering Three-Dimensional Toroid Tissues.

Liquid marble is a liquid droplet coated with hydrophobic powder that can be used as a bioreactor. This paper reports the three-dimensional self-assembly and culture of a cell toroid in a slow-releasing, non-adhesive and evaporation-reducing bioreactor platform based on a liquid marble. The bioreactor is constructed by embedding a hydrogel sphere containing growth factor into a liquid marble filled with a suspension of dissociated cells. The hydrogel maintains the water content and concurrently acts as a slow-release carrier. The concentration gradient of growth factor induces cell migration and assembly into toroidal aggregates. An optimum cell concentration resulted in the toroidal (doughnut-like) tissue after 12 hours. The harvested cell toroids showed rapid closure of the inner opening when treated with the growth factor. We also present a geometric growth model to describe the shape of the toroidal tissue over time. In analogy to the classical two-dimensional scratch assay, we propose that the cell toroids reported here open up new possibilities to screen drugs affecting cell migration in three dimensions.

Liquid marbles as bioreactors for the study of three-dimensional cell interactions.

Liquid marble as a bioreactor platform for cell-based studies has received significant attention, especially for developing 3D cell-based assays. This platform is particularly suitable for 3D in-vitro modeling of cell-cell interactions. For the first time, we demonstrated the interaction of olfactory ensheathing cells (OECs) with nerve debris and meningeal fibroblast using liquid marbles. As the transplantation of OECs can be used for repairing nerve injury, degenerating cell debris within the transplantation site can adversely affect the survival of transplanted OECs. In this paper, we used liquid marbles to mimic the hostile 3D environment to analyze the functional behavior of the cells and to form the basis for cell-based therapy. We show that OECs interact with debris and enhanced cellular aggregation to form a larger 3D spheroidal tissue. However, these spheroids indicated limitation in biological functions such as the inability of cells within the spheroids to migrate out and adherence to neighboring tissue by fusion. The coalescence of two liquid marbles allows for analyzing the interaction between two distinct cell types and their respective environment. We created a microenvironment consisting of 3D fibroblast spheroids and nerve debris and let it interact with OECs. We found that OECs initiate adherence with nerve debris in this 3D environment. The results suggest that liquid marbles are ideal for developing bioassays that could substantially contribute to therapeutic applications. Especially, insights for improving the survival and adherence of transplanted cells.

A lab-on-a-chip device for investigating the fusion process of olfactory ensheathing cell spheroids.

Understanding the process of fusion of olfactory ensheathing cell spheroids will lead to improvement of cell transplantation therapies to repair spinal cord injuries. The successful fusion of transplanted spheroids will enable alternative transplantation strategies to be developed for in vivo applications. This paper describes the use of a microfluidic device to trap and fuse olfactory ensheathing cell spheroids. The velocity, the pressure distribution in the device were simulated numerically to predict the trapping location. The simulation predicted the optimum flow rates for trapping the spheroids in the later experiments. Simulated particle trajectories were verified experimentally with tracing of fluorescent micro particles. The fusion process of the spheroids was investigated over a period of 48 hours. The microfluidic platform presented here can be used for testing potential drugs that can promote the fusion process and improve the transplantation therapy.

An electromagnetic cell-stretching device for mechanotransduction studies of olfactory ensheathing cells.

Olfactory ensheathing cells (OECs) are primary candidates for cell transplantation therapy to repair spinal cord injury (SCI). However, the post transplantation survival of these cells remains a major hurdle for a success using this therapy. Mechanical stimuli may contribute to the maintenance of these cells and thus, mechanotransduction studies of OECs may serve as a key benefit to identify strategies for improvement in cell transplantation. We developed an electromagnetic cell stretching device based on a single sided uniaxial stretching approach to apply tensile strain to OECs in culture. This paper reports the design, simulation and characterisation of the stretching device with preliminary experimental observations of OECs in vitro. The strain field of the deformable membrane was investigated both experimentally and numerically. Heterogeneity of the device provided an ideal platform for establishing strain requirement for the OEC culture. The cell stretching system developed may serve as a tool in exploring the mechanobiology of OECs for future SCI transplantation research.

Floating mechanism of a small liquid marble.

Flotation of small solid objects and liquid droplets on water is critical to natural and industrial activities. This paper reports the floating mechanism of liquid marbles, or liquid droplets coated with hydrophobic microparticles. We used X-ray computed tomography (XCT) to acquire cross-sectional images of the floating liquid marble and interface between the different phases. We then analysed the shape of the liquid marble and the angles at the three-phase contact line (TPCL). We found that the small floating liquid marbles follow the mechanism governing the flotation of solid objects in terms of surface tension forces. However, the contact angles formed and deformation of the liquid marble resemble that of a sessile liquid droplet on a thin, elastic solid. For small liquid marbles, the contact angle varies with volume due to the deformability of the interface.

Abuse potential and adverse cognitive effects of mitragynine (kratom).

Mitragynine is the major psychoactive alkaloid of the plant kratom/ketum. Kratom is widely used in Southeast Asia as a recreational drug, and increasingly appears as a pure compound or a component of 'herbal high' preparations in the Western world. While mitragynine/kratom may have analgesic, muscle relaxant and anti-inflammatory effects, its addictive properties and effects on cognitive performance are unknown. We isolated mitragynine from the plant and performed a thorough investigation of its behavioural effects in rats and mice. Here we describe an addictive profile and cognitive impairments of acute and chronic mitragynine administration, which closely resembles that of morphine. Acute mitragynine has complex effects on locomotor activity. Repeated administration induces locomotor sensitization, anxiolysis and conditioned place preference, enhances expression of dopamine transporter- and dopamine receptor-regulating factor mRNA in the mesencephalon. While there was no increase in spontaneous locomotor activity during withdrawal, animals showed hypersensitivity towards small challenging doses for up to 14 days. Severe somatic withdrawal signs developed after 12 hours, and increased level of anxiety became evident after 24 hours of withdrawal. Acute mitragynine independently impaired passive avoidance learning, memory consolidation and retrieval, possibly mediated by a disruption of cortical oscillatory activity, including the suppression of low-frequency rhythms (delta and theta) in the electrocorticogram. Chronic mitragynine administration led to impaired passive avoidance and object recognition learning. Altogether, these findings provide evidence for an addiction potential with cognitive impairments for mitragynine, which suggest its classification as a harmful drug.

Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles.

We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.

Deformation of a floating liquid marble.

A rigid spherical particle floating on a liquid is a known problem with well-defined solutions. Under the combined effect of gravity and surface tension, the rigid particle deforms the liquid surface. However, in the case of a floating soft particle such as a liquid marble, not only the liquid surface but also the particle itself deforms. In this paper, we investigate the deformation of a floating liquid marble and characterise its height as well as aspect ratio. The experimental results show that theoretical models for a rigid spherical particle suit well for small liquid marbles. Larger marbles require an oblate liquid spheroid model. We will discuss the limitations of the two models and characterise the deformation of these marbles.