Janet Zoldan

A vector-free microfluidic platform for intracellular delivery.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

The influence of scaffold elasticity on germ layer specification of human embryonic stem cells

Mechanical forces are critical to embryogenesis, specifically, in the lineage-specification gastrulation phase, whereupon the embryo is transformed from a simple spherical ball of cells to a multi-layered organism, containing properly organized endoderm, mesoderm, and ectoderm germ layers. Several reports have proposed that such directed and coordinated movements of large cell collectives are driven by cellular responses to cell deformations and cell-generated forces. To better understand these environmental-induced cell changes, we have modeled the germ layer formation process by culturing human embryonic stem cells (hESCs) on three dimensional (3D) scaffolds with stiffness engineered to model that found in specific germ layers. We show that differentiation to each germ layer was promoted by a different stiffness threshold of the scaffolds, reminiscent of the forces exerted during the gastrulation process. The overall results suggest that three dimensional (3D) scaffolds can recapitulate the mechanical stimuli required for directing hESC differentiation and that these stimuli can play a significant role in determining hESC fate.

Effect of Scaffold Stiffness on Myoblast Differentiation

Successful tissue engineering requires optimization of scaffold stiffness for a given application and cell type. Here, we investigated the effect of scaffold stiffness on myoblast cells, demonstrating the ability of cells to affect and to sense their mechanical microenvironment. Myoblasts were cultured on composite three-dimensional poly-lactic acid (PLLA)/poly-lactic co glycolic acid (PLGA) porous scaffolds of varied elasticity. The elasticity was controlled by changing the ratio of PLLA versus PLGA in the scaffolds. Cell organization, myotube formation, and cell viability were affected by scaffold stiffness. PLLA-containing scaffolds (100% to 25% PLLA) provided stiffness that supported myotube formation, while neat PLGA scaffold failed to support myotube formation and cell viability. Furthermore, scaffold stiffness correlated to its size/area reduction upon culturing experiments, suggesting different shrinkage degree by cell forces. Inhibition of scaffold shrinking by affixing device resulted in spacious cell organization with normal cell morphology. This may suggest that scaffold shrinkage led to cellular degeneration and shape deformation. Our results indicate that compliant scaffolds are insufficient to withstand cell forces. On the other hand, excessively firm scaffold could not lead to parallel oriented myotube organization. Hence, optimal scaffold stiffness can be tailored by PLLA/PLGA blending to direct specific stages of myoblast differentiation and organization.

Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture.

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60-90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27-90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.

Engineering Three‐Dimensional Tissue Structures Using Stem Cells

The worldwide status of rapidly increasing demand for organ and tissue transplantation has promoted tissue engineering as a promising alternative, in particular the use of stem cells. Human embryonic stem cells (hESCs) have the advantage of differentiating to all cell types in the body and high proliferation capabilities. Considering their ability to organize into complex multi-cell-type structures during embryonic-like differentiation, hESCs can potentially provide a source of cells for tissue engineering applications and meet the growing demand for viable human tissue structures in therapeutic clinical application. This chapter describes first steps toward realizing this goal gathered from our experience in growing and differentiating hESCs in three dimensions.

Regenerated cellulose micro-nano fiber matrices for transdermal drug release

In this work, biobased fibrous membranes with micro- and nano-fibers are fabricated for use as drug delivery carries because of their biocompatibility, eco-friendly approach, and potential for scale-up. The cellulose micro-/nano-fiber (CMF) matrices were prepared by electrospinning of pulp in an ionic liquid, 1-butyl-3-methylimidazolium chloride. A model drug, ibuprofen (IBU), was loaded on the CMF matrices by a simple immersing method. The amount of IBU loading was about 6% based on the weight of cellulose membrane. The IBU-loaded CMF matrices were characterized by Fourier-transform infrared spectroscopy, thermal gravimetric analysis, and scanning electron microscopy. The test of ibuprofen release was carried out in an acetate buffer solution of pH5.5 and examined by UV-Vis spectroscopy. Release profiles from the CMF matrices indicated that the drug release rate could be determined by a Fickian diffusion mechanism.

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Gene Transfection for Stem Cell Therapy

Genetic engineering of stem cells is a strategy that holds promise for realizing the potential therapeutic benefits of stem cell therapy. Through precise control of the stem cell genome, stem cells can replace or repair damaged tissues as well as serve as a depot for the sustained delivery of therapeutic molecules. Various individual genes, genome editing techniques, and transfection agents have been studied and developed for use in stem cell gene transfection. The goal for this review is to introduce specific genes and editing techniques used in stem cell therapy. Diverse gene transfection agents such as liposomes, polymers, dendrimers, peptides, inorganic nanoparticles, and physical transfection agents are also discussed with particular focus on stem cell considerations.

Dielectric Spectroscopy of Anisotropic Polypropylene/Nylon‐66 Blends Containing Carbon Black

The relationships of the dielectric properties and structure of polypropylene (PP)/nylon (Ny) blends containing carbon black (CB) were studied. Dielectric anisotropy was found in blends exhibiting an oriented fibrillar Ny network covered by CB particles. In the Ny fibril direction the composites are conductive, while in the perpendicular direction they are insulating, as indicated by the different frequency dependence of the AC conductivity in the two orthogonal directions. However, once CB is located within both the Ny and the PP the dielectric behavior is isotropic. This was further confirmed by Cole‐Cole plots, which, for the first time, were found to fit numerical predictions of the “resistor‐capacitor” (RC) model. The CB network formed upon the surface of the Ny fibrils is denser and/or better structured than that formed within the Ny phase. Thus, the former can be envisioned as a 2D system, as suggested by the values of the scaling exponents of the AC conductivity and permittivity with frequency, which have a lower activation energy for charge transport. The dielectric measurements were found helpful in elucidating the CB network structure.

Synthetic microparticles conjugated with VEGF165 improve the survival of endothelial progenitor cells via microRNA-17 inhibition

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)—VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.