Gonghao Wang

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.

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.

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.

Stiffness Dependent Separation of Cells in a Microfluidic Device

Rapidly sorting and separating cells are critical for detecting diseases such as cancers and infections and can enable a great number of applications in bio-related science and technology. While a variety of techniques demonstrate separation by physical parameters such as size[1] and mass[2], inexpensive and easy to use methods are needed to separate cells by mechanical compliance. A number of pathophysiological states of individual cells result in drastic changes in stiffness in comparison with healthy counterparts. Mechanical stiffness has been utilized to identify abnormal cell populations in detecting cancer[3–5] and identifying infectious disease[4, 6]. Recently, microfluidic methods were developed to classify and enrich cell populations utilizing mechanical stiffness[7–9]. We demonstrate a new strategy to continuously and non-destructively separate cells into subpopulations of soft and stiff cells. In our microfluidic separation method, we employ a microchannel with the top wall decorated by a periodic array of rigid diagonal ridges (Fig. 1). The gap between the ridges and the bottom channel wall is smaller than the cell diameter, thus the cells are periodically compressed by the ridges. The difference in mechanical resistance to compression of cells gives rise to a stiffness-dependent force associated with cell passage through narrow constrictions formed by the consecutive channel ridges. This elastic force is directed normal to the compressive diagonal ridges and, therefore, deflects cells propelled by the flow in the lateral direction with a rate proportional to their compliance. In this paper, we employ this principle to separate modified lymphoblastic cells with dissimilar mechanical stiffness in high-throughput.Copyright