Biophysical subsets of embryonic stem cells display distinct phenotypic and morphological signatures.

The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of "soft" cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.

Microfluidic Sorting of Cells by Viability Based on Differences in Cell Stiffness.

The enrichment of viable cells is an essential step to obtain effective products for cell therapy. While procedures exist to characterize the viability of cells, most methods to exclude nonviable cells require the use of density gradient centrifugation or antibody-based cell sorting with molecular labels of cell viability. We report a label-free microfluidic technique to separate live and dead cells that exploits differences in cellular stiffness. The device uses a channel with repeated ridges that are diagonal with respect to the direction of cell flow. Stiff nonviable cells directed through the channel are compressed and translated orthogonally to the channel length, while soft live cells follow hydrodynamic flow. As a proof of concept, Jurkat cells are enriched to high purity of viable cells by a factor of 185-fold. Cell stiffness was validated as a sorting parameter as nonviable cells were substantially stiffer than live cells. To highlight the utility for hematopoietic stem cell transplantation, frozen samples of cord blood were thawed and the purity of viable nucleated cells was increased from 65% to over 94% with a recovery of 73% of the viable cells. Thus, the microfluidic stiffness sorting can simply and efficiently obtain highly pure populations of viable cells.

Cellular enrichment through microfluidic fractionation based on cell biomechanical properties.

The biomechanical properties of populations of diseased cells are shown to have differences from healthy populations of cells, yet the overlap of these biomechanical properties can limit their use in disease cell enrichment and detection. We report a new microfluidic cell enrichment technology that continuously fractionates cells through differences in biomechanical properties, resulting in highly pure cellular subpopulations. Cell fractionation is achieved in a microfluidic channel with an array of diagonal ridges that are designed to segregate biomechanically distinct cells to different locations in the channel. Due to the imposition of elastic and viscous forces during cellular compression, which are a function of cell biomechanical properties including size and viscoelasticity, larger, stiffer and less viscos cells migrate parallel to the diagonal ridges and exhibit positive lateral displacement. On the other hand, smaller, softer and more viscous cells migrate perpendicular to the diagonal ridges due to circulatory flow induced by the ridges and result in negative lateral displacement. Multiple outlets are then utilized to collect cells with finer gradation of differences in cell biomechanical properties. The result is that cell fractionation dramatically improves cell separation efficiency compared to binary outputs and enables the measurement of subtle biomechanical differences within a single cell type. As a proof-of-concept demonstration, we mix two different leukemia cell lines (K562 and HL60) and utilize cell fractionation to achieve over 45-fold enhancement of cell populations, with high purity cellular enrichment (90% to 99%) of each cell line. In addition, we demonstrate cell fractionation of a single cell type (K562 cells) into subpopulations and characterize the variations of biomechanical properties of the separated cells with atomic force microscopy. These results will be beneficial to obtaining label-free separation of cellular mixtures, or to better investigate the origins of biomechanical differences in a single cell type.

Microfluidic cellular enrichment and separation through differences in viscoelastic deformation.

We report a microfluidic approach to separate and enrich a mixture of two cell types based on differences in cell viscoelastic behavior during repeated compressions and relaxation events. As proof of concept, we demonstrate that variations in viscoelasticity affect the flow trajectory of one type of leukemia cell line (K562) in relation to another leukemia cell line (HL60) as well as healthy leukocytes. These differences in cell trajectory can be utilized to enrich and sort K562 cells from HL60 cells and leukocytes. The microfluidic device utilizes periodic, diagonal ridges to compress and translate the cells laterally perpendicular to channel axis. The ridge spacing is tuned to allow relaxation of the K562 cells but not the HL60 cells or leukocytes. Therefore, the periodic compression laterally translates weakly viscous cells, while highly viscous cells respond to hydrodynamic circulation forces generated by the slanted ridges. As a result, cell sorting has strong dependency on cell viscosity. We use atomic force microscopy and high-speed optical microscopy to measure cell stiffness, cell relaxation rate constant, and cell size for all cell types. With properly designed microfluidic channels, we can optimize the enrichment of K562 cells from HL60 and leukocytes.

Stiffness dependent separation of cells in a microfluidic device.

Abnormal cell mechanical stiffness can point to the development of various diseases including cancers and infections. We report a new microfluidic technique for continuous cell separation utilizing variation in cell stiffness. We use a microfluidic channel decorated by periodic diagonal ridges that compress the flowing cells in rapid succession. The compression in combination with secondary flows in the ridged microfluidic channel translates each cell perpendicular to the channel axis in proportion to its stiffness. We demonstrate the physical principle of the cell sorting mechanism and show that our microfluidic approach can be effectively used to separate a variety of cell types which are similar in size but of different stiffnesses, spanning a range from 210 Pa to 23 kPa. Atomic force microscopy is used to directly measure the stiffness of the separated cells and we found that the trajectories in the microchannel correlated to stiffness. We have demonstrated that the current processing throughput is 250 cells per second. This microfluidic separation technique opens new ways for conducting rapid and low-cost cell analysis and disease diagnostics through biophysical markers.

A microfabricated platform for high-throughput unconfined compression of micropatterned biomaterial arrays.

High-throughput screening techniques for cellular response are often unable to account for several factors present in the in vivo environment, many of which have been shown to modulate cellular response to the screened parameter. Culture in three-dimensional biomaterials and active mechanical stimulation are two such factors. In this work, we integrate these microenvironmental parameters into a versatile microfabricated device, capable of simultaneously applying a range of cyclic, compressive mechanical forces to cells encapsulated in an array of micropatterned biomaterials. The fabrication techniques developed here are broadly applicable to the integration of three-dimensional culture systems in complex multilayered polymeric microdevices. Compressive strains ranging from 6% to 26% were achieved simultaneously across the biomaterial array. As a first demonstration of this technology, nuclear and cellular deformation in response to applied compression was assessed in C3H10T1/2 mouse mesenchymal stem cells encapsulated within poly(ethylene glycol) hydrogels. Biomaterial, cellular, and nuclear deformations were non-linearly related. Parametric finite element simulations suggested that this phenomenon was due to the relative stiffness differences between the hydrogel matrix and that of the encapsulated cell and nucleus, and to strain stiffening of the matrix with increasing compression. This complex mechanical interaction between cells and biomaterials further emphasizes the need for high-throughput approaches to conduct mechanically active experiments in three-dimensional culture.