Controlled cell adhesion using a biocompatible anchor for membrane-conjugated bovine serum albumin/bovine serum albumin mixed layer.

We report here a method for controlling cell adhesion, allowing simple yet accurate cell detachment from the substrate, which is required for the establishment of new cytometry-based cell processing and analyzing methods. A biocompatible anchor for membrane (BAM) was conjugated with bovine serum albumin (BSA) to produce a cell-anchoring agent (BAM-BSA). By coating polystyrene substrates with a mixture of BAM-BSA and BSA, controlled suppression of the substrate's adhesive properties was achieved. Hook-shaped nanoneedles were used to pick up cells from the substrate, while recording the cell-substrate adhesion force, using an atomic force microscope (AFM). Due to the lipid bilayer targeting property of BAM, the coated surface showed constant adhesion forces for various cell lines, and controlling the BAM-BSA/BSA ratio enabled tuning of the adhesion force, ranging from several tens of nano-Newtons down to several nano-Newtons. Optimized tuning of the adhesion force also enabled the detachment of cells from BAM-BSA/BSA-coated dishes, using a shear flow. Moreover, the method was shown to be noncell type specific and similar results were observed using four different cell types, including nonadherent cells. The attenuation of cell adhesion was also used to enable the collection of single cells by capillary aspiration. Thus, this versatile and relatively simple method can be used to control the adhesion of various cell types to substrates.

Non-invasive characterization of mouse embryonic stem cell derived cardiomyocytes based on the intensity variation in digital beating video.

The interest in cardiomyocytes derived from differentiation of embryonic stem (ES) cells or induced pluripotent stem (iPS) cells is increasing due to their potential for regenerative therapeutics and as a pharmaceutical model of drug screening. Characterization of ES or iPS derived cardiomyocytes is challenging and inevitable for the intended usage of such cells. In this paper we have outlined a novel, non-invasive method for evaluating in vitro beating properties of cardiomyocytes. The method is based on the analysis of time dependent variation in the total pixel intensities in derivative images obtained from the consecutive systolic and diastolic frames from the light microscopic video recordings of beating tissue. Fast Fourier transform (FFT) yielded the frequency domains for these images. The signal to noise ratio for the analysis met the Rose criterion. We have successfully applied our method for monitoring mouse ES cell (mESC) derived cardiac muscle cells to determine the initiation of beating, organization and maturation of beating tissue, calculating the beating rhythms in terms of beating interval or frequency and the strength of beating. We have shown the successful application of our image analysis method in direct monitoring of the responses of differentiated cardiomyocytes towards caffeine hydrate, p-hydroxyphenylacetamide and calcium chloride dehydrate - respectively as positive, neutral and negative inotropic agents. This non-invasive method of characterization will be useful in studying the response of these cells to various external stimulations, such as differentiation promoting agents or treatments, as well as in preliminary drug screening in a quick and inexpensive manner without needing much expertise.

Oscillating high-aspect-ratio monolithic silicon nanoneedle array enables efficient delivery of functional bio-macromolecules into living cells.

Delivery of biomolecules with use of nanostructures has been previously reported. However, both efficient and high-throughput intracellular delivery has proved difficult to achieve. Here, we report a novel material and device for the delivery of biomacromolecules into live cells. We attribute the successful results to the unique features of the system, which include high-aspect-ratio, uniform nanoneedles laid across a 2D array, combined with an oscillatory feature, which together allow rapid, forcible and efficient insertion and protein release into thousands of cells simultaneously.

Characterization of a new keratin-degrading bacterium isolated from deer fur.

A keratin-degrading bacterium was isolated from soil containing deer fur. An axenic culture of the keratin-degrading bacterium was obtained in liquid culture using a keratin enrichment technique. The isolated bacterium was gram negative and catalase- and oxidase-positive. Transmission electron microscopic observations showed that the bacterium was rod-shaped, 1.0-1.3 microm long and 0.7 microm in diameter. Phylogenetic analysis of 16S rDNA revealed that the new isolate has only 90.6% homology with Stenotrophomonas nitritireducens. Hence, this new bacterium was designated as Stenotrophomonas sp. D-1. The optimum temperature was determined to be 20 degrees C for maximum growth and keratinolytic enzyme production. Amino acid data, obtained after treating keratin powder with the supernatant culture, suggest that the major free amino acids resulting from keratin degradation are phenylalanine, tyrosine and valine. In addition, native chicken feather was degraded completely at 20 degrees C in 2.5 d by this bacterium.

Single-cell microarray for analyzing cellular response.

Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.