Delivery of polyethylenimine/DNA complexes assembled in a microfluidics device.

Polyethylenimine (PEI) and plasmid DNA (pDNA) complexes (PEI/pDNA) are nonviral vectors for gene delivery. The conventional method for producing these complexes involves bulk mixing (BM) of PEI and DNA followed by vortexing which at low N/P ratios results in large particle size distribution, low cytotoxicity, and poor gene transfection, while at high N/P ratios it results in small particle size and better gene transfection but high cytotoxicity. To improve size control, gene transfection efficiency, and cytotoxicity, in this study, we used a microfluidic hydrodynamic focusing (MF) device to prepare PEI/pDNA complexes at N/P = 3.3 and 6.7. We used bulk mixing as control, mouse NIH 3T3 fibroblast cells and mouse embryonic stem (mES) cells as model cell lines, plasmid encoding green fluorescent protein (pGFP) and secreted alkaline phosphatase (pSEAP) as the reporter gene, and commercially available Lipofectamine 2,000 as a positive control. The complexes were characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), and zeta potential (zeta) measurement. Confocal laser scanning microscopy (CLSM) and fluorescent labeling techniques were used to visualize the complex size distribution, complexation uniformity, and cellular distribution. The results showed that MF produced complexes were smaller and more uniformly complexed and had higher cell viability and improved exogenous gene expression.

Bioassembly of three-dimensional embryonic stem cell-scaffold complexes using compressed gases.

Tissues are composed of multiple cell types in a well-organized three-dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue-engineered constructs should have well-defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two-dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well-defined microstructure were constructed into a multilayer cell-scaffold complex using low pressure carbon dioxide (CO(2)) and nitrogen (N(2)). The mouse ES cells in the assembled constructs were viable, retained the ES cell-specific gene expression of Oct-4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO(2) was replaced with N(2). The compressed gas-assisted bioassembly of stem cell-polymer constructs opens up a new avenue for tissue engineering and cell therapy.

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds.

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.

Assembly of three-dimensional polymeric constructs containing cells/biomolecules using carbon dioxide.

Using low-pressure carbon dioxide (CO2), we demonstrated a novel and versatile approach to assembling polymeric constructs in the presence of cells and/or biomolecules in an aqueous environment. By regulating the CO2 pressure, the assembly was completed at biologically permissive temperatures with excellent preservation of the original structures. We further demonstrated that mammalian cells can survive the CO2-assisted bioassembly process (37 degrees C, 1.38 MPa, approximately 1 h). Human mesenchymal stem cells from bone marrow (hMSCs) exhibited the same cell morphology and proliferation potential as the untreated control. Mouse embryonic stem cells (mESCs) maintained ES-specific Oct-4 gene expression and differentiation potential after CO2 treatment as well. This method highlights the ability to construct multiple biodegradable polymeric scaffolds with well-defined architecture, on which various types of cells were grown, into a predesigned three-dimensional complex. In addition, protein and DNA bioactivity can be preserved in the context of a CO2-assisted assembly. This CO2-assisted bioassembly method provides for a manufacturing platform that, thus far, has been lacking in the fields of tissue engineering, cell-based biochips, cell therapy, and drug delivery.

Adipose tissue model using three-dimensional cultivation of preadipocytes seeded onto fibrous polymer scaffolds.

A better understanding of the mechanism of adipose tissue differentiation is of paramount importance in the development of therapeutic strategies for the treatment and prevention of obesity and type 2 diabetes mellitus. Optimal results using tissue culture models can be expected only when the in vitro adipocyte resembles adipose tissue in vivo as closely as possible. In this study, we used tissue-engineering principles to develop a three-dimensional (3-D) culture system to mimic the geometry of adipose tissue in vivo. Mouse preadipocyte 3T3-L1 cells were seeded onto nonbiodegradable fibrous polyethylene terephthalate scaffolds and differentiated with a hormone cocktail consisting of insulin, dexamethasone, isobutylmethylxanthine, and fetal calf serum. Cell morphology, growth, differentiation, and function were studied by immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay, and oil red O staining. Cells grown on 3-D fibrous scaffolds were differentiated in situ by hormone induction with high efficiency (approximately 90%) as shown by scanning electron microscopy. Immunocytochemistry, immunoblot analysis, and RT-PCR revealed that the 3-D constructs expressed adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, leptin, adipsin, aP2, adiponectin, GLUT4, and resistin. Adipocytes matured on 3-D constructs secreted leptin at levels even greater than that of fully differentiated adipocytes in 2-D conventional cell cultures. Finally, adipocyte-specific phenotypic function was demonstrated by accumulation of neutral lipids in larger fat droplets. In conclusion, preadipocytes grown on 3-D matrices acquire morphology and biological features of mature adipocytes. This new culture model should have significant utility for in vitro studies of adipocyte cell biology and development.