Microfluidic perfusion shows intersarcomere dynamics within single skeletal muscle myofibrils

The sarcomere is the smallest functional unit of myofibrils in striated muscles. Sarcomeres are connected in series through a network of elastic and structural proteins. During myofibril activation, sarcomeres develop forces that are regulated through complex dynamics among their structures. The mechanisms that regulate intersarcomere dynamics are unclear, which limits our understanding of fundamental muscle features. Such dynamics are associated with the loss in forces caused by mechanical instability encountered in muscle diseases and cardiomyopathy and may underlie potential target treatments for such conditions. In this study, we developed a microfluidic perfusion system to control one sarcomere within a myofibril, while measuring the individual behavior of all sarcomeres. We found that the force from one sarcomere leads to adjustments of adjacent sarcomeres in a mechanism that is dependent on the sarcomere length and the myofibril stiffness. We concluded that the cooperative work of the contractile and the elastic elements within a myofibril rules the intersarcomere dynamics, with important consequences for muscle contraction.

A Microfluidic Perfusion Chamber Utilized in the Study of Biophysical Properties of Cell Membrane and Its Fluidic Evaluation

A microfluidic system is demonstrated here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell membrane transport properties. The system is comprised of microchannels, a cell immobilization chamber, an inlet and an outlet, and is made of poly(dimethylsiloxane) (PDMS) using softlithographic method. During experiments, mouse dendritic cells (mDCs), mixed with media of known concentrations, are quickly injected to the inlet of such microfluidic device, flow through a microchannel, and are then immobilized by a sieving structure, where kinetic images of cell volume response are captured by a CCD camera lively. The fluid keeps flowing due to the continuous suction from the outlet by a programmable syringe pump. Two sets of experiments have been performed: the cells are mixed with (1) solutions prepared in different concentrations of non-permeating solutes, and (2) solutions containing a permeating cryoprotective agent (CPA) plus non-permeating solute, respectively. Based on the captured images, both cell inactive volumes (Vb), permeability coefficients of water (Lp) and of CPA (Ps) through cell membranes of mDCs at different temperatures (10°C, 22°C, and 34°C) can be determined by least-squared curve fittings, respectively. A quantitative evaluation conducted using ImageJ will be performed in order to validate the microfluidic perfusion system, as well as help us understand the dynamic concentration changes around those immobilized cells. The use of this microfluidic perfusion system enables us to: 1) confine cells in a monolayer channel to prevent image ambiguity, 2) perform cell counting, 3) statistically study cell osmotic response and determine cell membrane transport properties, and (4) lower manufacturing costs.

Quantitative Measurements of Cryobiological Characteristics of Mouse Dendritic Cells and Its Evaluation Using Commercialized Coulter Counter

Quantitative measurements of cell osmotic behavior and membrane transport properties is critical for the development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system has been developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithographic techniques and is composed of inlets, outlets, micchannels and a perfusion chamber for trapping cells. In this study Dendritic cells (DCs) are using as a model to validate our microfluidic system with a commercialized Beckham Coulter Counter (Multisizer 3). DCs are antigen presenting cells that have been increasingly used in immunotherapy for the treatment of various diseases. Cryopreservation and banking of DCs is critical to facilitate flexible and effective immunotherapy treatment. Using mouse DCs (MDC), membrane transport properties were first investigated using our microfluidic perfusion system. Cells in the microfluidic system were perfused with 3x phosphate buffer solution. The kinetics of cell volume changes under the specific extracellular conditions were monitored by a digital camera and analyzed using a biophysical model to determine water and cryoprotectant transport properties of the cell membrane. DCs were later tested using Beckman Coulter Counter, where the kinetic osmotic behaviors of cells were quantified through the correlation between electric pulses and their corresponding cell sizes. It was shown from this study that the cryobiological characteristics of DCs determined using microfluidic perfusion system and Coulter Counter agreed well.