Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-ß-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients.

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS), the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility, gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors, important regulators of cell/tissue functions in vivo, influence the survival and growth of human embryonic stem cells. Thus, this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening.

Product study of the photolysis of ketene and ethyl ethynyl ether at 193.3 nm.

The product distributions of the excimer laser photolysis of ketene (CH2CO) and ethyl ethynyl ether (C2H5OCCH) at lambda = 193.3 nm (ArF) were studied using a time-of-flight mass spectrometer (TOFMS) as an analytical tool. Ketene was photolyzed in bath gases consisting of mixtures of He and H2/D2 at various mixing ratios at constant total pressures of 4 Torr and temperature of about 300 K. Singlet methylene (1CH2) produced in the photolysis of ketene was almost instantaneously converted either to triplet methylene (3CH2) or to methyl radicals in collisions with He and H2 or D2. By extrapolating the methyl and methylene signals to zero time after photolysis, initial concentrations of these radicals were obtained. Analyzing the initial 3CH2 and CH3 concentrations as functions of hydrogen-to-helium ratios as well as simulating the observed traces of reactant and product species resulted in 1CH2 + CO (66 +/- 8)%, as the main product channel of the ketene photolysis with smaller contributions from HCCO + H (17 +/- 7)% and 3CH2 + CO (6 +/- 9)%. Hydrogen atoms, acetylene, ethylene, ethyl, and ketenyl radicals, and small amounts of ketene were observed as primary products of the ethyl ethynyl ether photolysis. Quantification of C2H2, C2H4, C2H5, and CH2CO product leads to a HCCO yield of (91 +/- 14)%.

Product study of the reaction of CH3 with OH radicals at low pressures and temperatures of 300 and 612 K.

The product distribution for the title reaction was studied using our time-of-flight mass spectrometer (TOFMS) connected to a tubular flow reactor. The methyl and hydroxyl radicals were produced by an excimer laser pulse (lambda = 193 nm) photolyzing acetone and nitrous oxide in the presence of excess water or hydrogen. Helium was used as the bath gas; the total density was held constant at 1.2 x 10(17) cm(-3). At 300 K the observations were consistent with singlet methylene ((1)CH(2)) and water as the main product channel with a small contribution of methanol. In contrast, at about 610 K three channels-formaldehyde isomers and methanol in addition to (1)CH(2) + H(2)O-are formed with similar yields. When acetone-d(6) was used, the production of both CHDO and CD(2)O was observed, indicating that two different formaldehyde-producing channels are operating simultaneously. These experimental results are compared with RRKM and master equation calculations on the basis of the properties of the methanol potential energy surface from a recent ab initio study.