A method for isolating contractile smooth muscle cells from cryopreserved tissue.

Multiple techniques have been developed to isolate contractile smooth muscle cells (SMCs) from tissues with varying degrees of success. However, most of these approaches rely on obtaining fresh tissue, which poses logistical challenges. In the present study, we introduce a novel protocol for isolating contractile SMCs from cryopreserved smooth muscle (SM) tissue, thereby enhancing experimental efficiency. This protocol yields abundant viable, spindle-shaped, contractile SMCs that closely resemble those obtained from fresh samples. By analyzing the expression of contractile proteins, we demonstrate that both the isolated SMCs from cryopreserved tissue represent more accurately fresh SM tissue compared with cultured SMCs. Moreover, we demonstrate the importance of a brief incubation step of the tissue in culture medium before cell dissociation to achieve contractile SMCs. Finally, we provide a concise overview of our protocol optimization efforts, along with a summary of previously published methods, which could be valuable for the development of similar protocols for other species.NEW & NOTEWORTHY We report a successful protocol development for isolating contractile smooth muscle cells (SMCs) from cryopreserved tissue reducing the reliance on fresh tissues and providing a readily available source of contractile SMCs. Our findings suggest that SMCs isolated using our protocol maintain their phenotype better compared with cultured SMCs. This preservation of the cellular characteristics, including the expression of key contractile proteins, makes these cells more representative of fresh SM tissue.

Vortex chip incorporating an orthogonal turn for size-based isolation of circulating cells.

Size-based label-free separation of rare cells such as CTCs is attractive due to its wider applicability, simpler sample preparation, faster turnaround and better efficiency. Amongst such methods, vortex-trapping based techniques offer high throughput but operate at high flow velocities where the resulting hydrodynamic shear stress is likely to damage cells and compromise their viability for subsequent assays. We present here an orthogonal vortex chip which can carry out size-differentiated trapping at significantly lower (38% of previously reported) velocities. Composed of entry-exit channels that couple orthogonally to a trapping chamber, fluid flow in such configuration results in formation of a vortex which selectively traps larger particles above a critical velocity while smaller particles get ejected with the flow. We call this phenomenon the turn-effect. Critical velocities and optimal architectures for trapping of cells and particles of different sizes are characterized. We explain how shear-gradient lift, centrifugal and Dean flow drag forces contribute to the turn-effect by pushing particles into specific vortex orbits in a size- and velocity-dependent fashion. Selective trapping of human breast cancer cells mixed with whole blood at low concentration is demonstrated. The device shows promising results for gentle isolation of rare cells from blood.