PubMed · 13255866
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F M DIETRICH. 1955. [Lederberg single-cell isolation method].. https://pubmed.ncbi.nlm.nih.gov/13255866/
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BACKGROUND: Simple, rapid tissue processing that preserves macromolecules will enhance translational research capabilities. Traditional fixative-based approaches for specimen preservation are ideal for histologic evaluation but are not conducive to molecular studies of nucleic acids and protein. Tissue cryosections preserve macromolecule integrity, but the process is labor intensive and technically challenging. To the authors' knowledge to date, an alternative method capable of retrieving cells while providing adequate histologic detail yet preserving macromolecule integrity has been lacking. In the current study, the authors evaluated the utility of using manual exfoliation of clinical tissue samples as a means of obtaining cells for molecular analysis. This technique possesses the advantages of fixed and frozen tissue sections without their drawbacks. This simple, rapid, nonfixative based technique is capable of preparing cells from human clinical material for further isolation without compromising the preservation of macromolecules in the tissue. METHODS: Cells from a variety of clinical resection specimens from solid tumors were directly scraped from the tissue samples using the edge of a glass microscope slide and smeared onto another slide for cytologic evaluation. The manually exfoliated cells were evaluated microscopically for cytologic quality and cellular quantity. Pure cell populations were procured by laser capture microdissection (LCM) with subsequent extraction of nucleic acids and proteins. The integrity and suitability of the recovered nucleic acids and proteins for molecular analysis were evaluated using the polymerase chain reaction (PCR), reverse transcriptase-PCR, and reverse-phase protein microarray, respectively. RESULTS: Manual exfoliation permits the selection of homogeneous cell populations by LCM based on well established cytologic characteristics. DNA and mRNA, of comparable quality to frozen sections, can be amplified from the manual exfoliation cells. Proteins of similar quality can be recovered using this technique and quantitated via reverse-phase protein microarray. CONCLUSIONS: Molecular macromolecules of high quality and sufficient quantity can be retrieved from human clinical samples using manual exfoliation and LCM to procure specific cell populations. The manual exfoliation technique does not destroy the original tissue source, thereby allowing subsequent formalin tissue fixation. The technique of manual exfoliation in conjunction with LCM can enable the molecular profiling of a sampled selected cell population. Because it does not destroy the original tissue, histologic correlation can be combined with molecular profiling.
We present the first known implementation of a passive, scalable architecture for trapping, imaging, and sorting individual microparticles, including cells, using a positive dielectrophoretic (p-DEP) trapping array. Our array-based technology enables "active coverslips" where, when scaled, many individually held cells can be sorted based upon imaged spatial or temporally variant characteristics. Our design incorporates a unique "ring-dot" p-DEP trap geometry organized in a row/column array format. This trap design, implemented in a two-level metal process, provides strong and highly spatially localized holding fields enabling single-cell capture for all traps in the array. We release individual trapped microparticles during sorting using a passive transistor-independent approach where we electrically ground the row and column electrodes associated with specific traps in the array. The demand for chip-to-world electrical connections in our arrays scales proportionally with the square root of the number of traps in a given array, delivering a substantial improvement over prior designs. We demonstrate capture, holding, and release operations with both beads and cells in small arrays of this new architecture.
Cross-flow microfiltration is an important step in separating Baker's yeast (Saccharomyces cerevisiae) from aqueous suspension in many processes. However the permeate flux often declines rapidly due to colloidal fouling of membranes and concentration polarisation. The present work explores the possibility of maintaining acceptable permeate flux by co-current sparging of gas along with the feed, which would scour away colloidal deposits and reduce concentration polarisation of membranes. In this work, both washed and unwashed yeast were used to study the effect of washing to reduce protein fouling of membranes. It was found that permeate flux increased by 45% for liquid throughput of 75 kg/h for a feed concentration of 2.0 kg/m3 of washed yeast as compared with unwashed yeast suspension without gas sparging. For washed yeast suspension, the increase in gas flow rate from 0.5 lpm to 1.5 lpm (30 l/h to 90 l/h) had beneficial effect on permeate flux. It is concluded that in the present case, the gas flow rate should be less than or equal to the liquid flow rate for enhancement of permeates flux.