Cell Sorting

This paper presents a color object recognition scheme which proceeds in three sequential steps: segmentation, features extraction and classification. We mainly focus on the first and the third steps here. A color watershed using global and local criteria is first described. A color contrast value is defined to select the best color space for segmenting color objects. Then, an architecture of binary neural networks is described. Its properties relies on the simplification of the recognition problem, leading to a noticeable increase in the classification rate. We conclude with the abilities of such a recognition scheme and present an automated cell sorting system.

The accumulation of cytoplasmic polyhydroxyalkanoates (PHAs) and the heterogeneity of bacterial populations were analysed by flow cytometry and SYTO-13 and Nile red staining in rhamnolipid-producing Pseudomonas aeruginosa cultures grown in waste frying oil as carbon source. A combination of SYTO-13 and Nile red fluorescence with cytometric forward and side scatter values may allow increases in the final production of polyhydroxyalkanoates

Integration and miniaturisation in electronics has undoubtedly revolutionised the modern world. In biotechnology, emerging lab-on-a-chip (LOC) methodologies promise all-integrated laboratory processes, to perform complete biochemical or medical synthesis and analysis encapsulated on small microchips. The integration of electrical, optical and physical sensors, and control devices, with fluid handling, is creating a new class of functional chip-based systems. Scaled down onto a chip, reagent and sample consumption is reduced, point-of-care or in-the-field usage is enabled through portability, costs are reduced, automation increases the ease of use, and favourable scaling laws can be exploited, such as improved fluid control. The capacity to manipulate single cells on-chip has applications across the life sciences, in biotechnology, pharmacology, medical diagnostics and drug discovery. This thesis explores multiple applications of optical manipulation within microfluidic chips. Used in combination with microfluidic systems, optics adds powerful functionalities to emerging LOC technologies. These include particle management such as immobilising, sorting, concentrating, and transportation of cell-sized objects, along with sensing, spectroscopic interrogation, and cell treatment. The work in this thesis brings several key applications of optical techniques for manipulating and porating cell-sized microscopic particles to within microfluidic chips. The fields of optical trapping, optical tweezers and optical sorting are reviewed in the context of lab-on-a-chip application, and the physics of the laminar fluid flow exhibited at this size scale is detailed. Microfluidic chip fabrication methods are presented, including a robust method for the introduction of optical fibres for laser beam delivery, which is demonstrated in a dual-beam optical trap chip and in optical chromatography using photonic crystal fibre. The use of a total internal reflection microscope objective lens is utilised in a novel demonstration of propelling particles within fluid flow. The size and refractive index dependency is modelled and experimentally characterised, before presenting continuous passive optical sorting of microparticles based on these intrinsic optical properties, in a microfluidic chip. Finally, a microfluidic system is utilised in the delivery of mammalian cells to a focused femtosecond laser beam for continuous, high throughput photoporation. The optical injection efficiency of inserting a fluorescent dye is determined and the cell viability is evaluated. This could form the basis for ultra-high throughput, efficient transfection of cells, with the advantages of single cell treatment and unrivalled viability using this optical technique.

We discuss two dieren t types of Cellular Automata (CA): lattice-gas- based cellular automata (LGCA) and the cellular Potts model (CPM), and describe their applications in biological modeling. LGCA were originally developed for modeling ideal gases and uids. We describe several extensions of the classical LGCA model to self-driven biological cells. In partic- ular, we review recent models for rippling

The present study was undertaken to characterize primary epithelial cultures obtained from human skin explants as experimental systems for studies of the differentiation process. When human skin explants were incubated at 34-35 degrees C, fibroblastic growth was strongly inhibited, whereas the epithelial growth proceeded unchanged. The lateral growth of the epithelial cells could be divided into two phases - a migratory and a proliferative one. Only cultures incubated at 35 degrees C or below completed the morphological differentiation process before sloughing, whereas no qualitative difference in protein synthesis was observed between cultures incubated at temperatures from 33-37 degrees C. Cultured epidermal cells were labelled with 3H-thymidine and analysed by flow cytometry and cell sorting. Cells sorted from the S- and G2-phase populations were further analysed by autoradiography and a considerable heterogeneity as to the nuclear labelling was disclosed. A large fraction of S-p...

To defend the host from malignancies, the immune system can spontaneously raise CD8(+) T-cell responses against tumor antigens. Investigating the functional state of tumor-reactive cytolytic T cells in cancer patients is a key step for understanding the role of these cells in tumor immunosurveillance and for evaluating the potential of immunotherapeutic approaches of vaccination against cancer. In this study we identified a subset of circulating tumor-reactive CD8(+) T lymphocytes, which specifically secreted IFN-gamma after exposition to autologous tumor cell lines in stage IV metastatic melanoma patients. Additional phenotypic characterization using multicolor flow cytometry revealed that a significant fraction of these cells were CD45RA(+)CCR7(-), a phenotype that has been proposed recently to characterize cytolytic effectors potentially able to home into inflamed tissues. In the case of an HLA-A2-expressing patient, the antigen specificity of this population was identified by us...

Satellite cells are myogenic stem cells responsible for the postnatal regeneration of skeletal muscle. Here we report the successful in vitro induction of Pax7-positive satellite-like cells from mouse embryonic stem (mES) cells. Embryoid bodies were generated from mES cells and cultured on Matrigel-coated dishes with Dulbecco's modified Eagle medium containing fetal bovine serum and horse serum. Pax7-positive satellite-like cells were enriched by fluorescence-activated cell sorting using a novel anti-satellite cell antibody, SM/C-2.6. SM/C-2.6-positive cells efficiently differentiate into skeletal muscle fibers both in vitro and in vivo. Furthermore, the cells demonstrate satellite cell characteristics such as extensive self-renewal capacity in subsequent muscle injury model, long-term engraftment up to 24 wk, and the ability to be secondarily transplanted with remarkably high engraftment efficiency compared to myoblast transplantation. This is the first report of transplantable, functional satellite-like cells derived from mES cells and will provide a foundation for new therapies for degenerative muscle disorders.

Precisely manipulating and sorting live cells on a lab on a chip is still a major challenge. This article shows how to use dielectrophoresis for cell sorting. The authors also describe a prototype CMOS chip with a sensor-actuator array, row-column addressing logic and readout circuitry. In this article, we examine the new microelectronic technology that gives scientists the ability to monitor, sort, and analyze vast populations of cells and interact with each cell individually. A microelectronic platform called a lab on a chip (LoC) allows precise manipulation of cells with no effect on their phenotypes. The motivation for developing this technology is that investigations in recent years have shown that a few cells changing their behavior unexpectedly can induce deadly diseases such as cancer. Current LoC design and manufacturing techniques are spawning new biotechnology methods with potential for research, diagnosis, and therapy.