Single-cell analysis for bioprocessing

Cell population heterogeneity has attracted great interest for understanding the individual cellular performances in their response to external stimuli and in the production of targeted products. Physical characterization of single cells and analysis of dynamic gene expression, synthesized proteins, and cellular metabolites from one single cell are reviewed. Advanced techniques have been developed to achieve high-throughput and ultrahigh resolution or sensitivity. Single cell capture methods are discussed as well. How to make use of cellular heterogeneities for maximizing cellular productivity is still in the infant stage, and control strategies will be formulated after the causes for heterogeneity have been elucidated.

Blobs versus bars:psychophysical evidence supports two types of orientation response in human color vision

The classic hypothesis of Livingstone and Hubel (1984, 1987) proposed two types of color pathways in primate visual cortex based on recordings from single cells: a segregated, modularpathway that signals color but provides little information about shape or form and a second pathway that signals color differences and so defines forms without the need to specify their colors. A major problem has been to reconcile this neurophysiological hypothesis with the behavioral data. A wealth of psychophysical studies has demonstrated that color vision has orientation-tuned responses and little impairment on form related tasks, but these have not revealed any direct evidence for nonoriented mechanisms. Here we use a psychophysical method of subthreshold summation across orthogonal orientations for isoluminant red-green gratings in monocular and dichoptic viewing conditions to differentiate between nonoriented and orientation-tuned responses to color contrast. We reveal nonoriented color responses at low spatial frequencies (0.25-0.375 c/deg) under monocular conditions changing to orientation-tuned responses at higher spatial frequencies (1.5 c/deg) and under binocular conditions. We suggest that two distinct pathways coexist in color vision at the behavioral level, revealed at different spatial scales: one is isotropic, monocular, and best equipped for the representation of surface color, and the other is orientation-tuned, binocular, and selective for shape and form. This advances our understanding of the organization of the neural pathways involved in human color vision and provides a strong link between neurophysiological and behavioral data. © 2013 ARVO.

Acanthamoeba polyphaga trophozoite binding of representative fungal single cell forms

Acanthamoeba polyphaga trophozoites bind yeast cells of Candida albicans isolates within a few hours, leaving few cells in suspension or still attached to trophozoite surfaces. The nature of yeast cell recognition, mediated by an acanthamoebal trophozoite mannose binding protein is confirmed by experiments utilizing concentration dependent mannose hapten blocking. Similarly, acapsulate cells of Cryptococcus neoformans are also bound within a relatively short timescale. However, even after protracted incubation many capsulate cells of Cryptococcus remain in suspension, suggesting that the capsulate cell form of this species is not predated by acanthamoebal trophozoites. Further aspects of the association of Acanthamoeba and fungi are apparent when studying their interaction with conidia of the biocontrol agent Coniothyrium minitans. Conidia which readily bind with increasing maturity of up to 42 days, were little endocytosed and even released. Cell and conidial surface mannose as determined by FITC-lectin binding, flow cytometry with associated ligand binding analysis and hapten blocking studies demonstrates the following phenomena. Candida isolates and acapsulate Cryptococcus expose most mannose, while capsulate Cryptococcus cells exhibit least exposure commensurate with yeast cellular binding or lack of trophozoites. Conidia of Coniothyrium, albeit in a localized fashion, also manifest surface mannose exposure but as shown by Bmax values, in decreasing amounts with increasing maturity. Contrastingly such conidia experience greater trophozoite binding with maturation, thereby questioning the primacy of a trophozoite mannose-binding-protein recognition model.

The development of an in vivo model of drug-induced terminal differentiation of Leukaemic cells

The technique of growing human leukaemic cells in diffusion chambers was developed to enable chemicals to be assessed for their ability to induce terminal differentiation. HL-60 promyelocytic leukaemia cell growth, in a lucite chamber with a Millipore filter, was optimised by use of a lateral incision site. Chambers were constructed using 0.45um filters and contained 150ul of serum-free HL-60 cells at a density of 1x106 cells/ml. The chambers were implanted into CBA/Ca mice and spontaneous terminal differentiation of the cells to granulocytes was prevented by the use of serum-free medium. Under these conditions there was an initial growth lag of 72 hours and a logarithmic phase of growth for 96 hours; the cell number reached a plateau after 168 hours of culture in vivo. The amount of drug in the plasma of the animal and in chambers that had been implanted for 5 days, was determined after a single ip injection of equitoxic doses of N-methylformamide, N-ethylformamide, tetramethylurea, N-dibutylformamide, N-tetramethylbutylformamide and hexamethylenebisacetamide. Concentrations of both TMU and HMBA were obtained in the plasma and in the chamber which were pharmacologically effective for the induction of differentiation of HL-60 cells in vitro, that is 12mM TMU and 5mM HMBA. A 4 day regime of treatment of animals implanted with chambers demonstrated that TMU and HMBA induced terminal differentiation of 50% and 35%, respectively, of the implanted HL-60 cells to granulocyte-like cells, assessed by measurement of functional and biochemical markers of maturity. None of the other agents attained concentrations in the plasma that were pharmacologically effective for the induction of differentiation of the cells in vitro and were unable to induce the terminal differentiation of the cells in vivo.

Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al.

Discrimination of healthy and cancer cells of the bladder by metabolic state, based on autofluorescence

Bladder cancer is among the most common cancers worldwide (4th in men). It is responsible for high patient morbidity and displays rapid recurrence and progression. Lack of sensitivity of gold standard techniques (white light cystoscopy, voided urine cytology) means many early treatable cases are missed. The result is a large number of advanced cases of bladder cancer which require extensive treatment and monitoring. For this reason, bladder cancer is the single most expensive cancer to treat on a per patient basis. In recent years, autofluorescence spectroscopy has begun to shed light into disease research. Of particular interest in cancer research are the fluorescent metabolic cofactors NADH and FAD. Early in tumour development, cancer cells often undergo a metabolic shift (the Warburg effect) resulting in increased NADH. The ratio of NADH to FAD ("redox ratio") can therefore be used as an indicator of the metabolic status of cells. Redox ratio measurements have been used to differentiate between healthy and cancer breast cells and to monitor cellular responses to therapies. Here, we have demonstrated, using healthy and bladder cancer cell lines, a statistically significant difference in the redox ratio of bladder cancer cells, indicative of a metabolic shift. To do this we customised a standard flow cytometer to excite and record fluorescence specifically from NADH and FAD, along with a method for automatically calculating the redox ratio of individual cells within large populations. These results could inform the design of novel probes and screening systems for the early detection of bladder cancer.

Characterization of microvesicles released from human red blood cells

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

Mixing theory for culture and harvest in bioreactors of human mesenchymal stem cells on microcarriers

The use of human mesenchymal stem cells (hMSCs) in regenerative medicine is a potential major advance for the treatment of many medical conditions, especially with the use of allogeneic therapies where the cells from a single donor can be used to treat ailments in many patients. Such cells must be grown attached to surfaces and for large scale production, it is shown that stirred bioreactors containing ~200 μm particles (microcarriers) can provide such a surface. It is also shown that the just suspended condition, agitator speed NJS, provides a satisfactory condition for cell growth by minimizing the specific energy dissipation rate, εT, in the bioreactor whilst still meeting the oxygen demand of the cells. For the cells to be used for therapeutic purposes, they must be detached from the microcarriers before being cryopreserved. A strategy based on a short period (~7 min) of very high εT, based on theories of secondary nucleation, is effective at removing >99% cells. Once removed, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. This approach is shown to be successful for culture and detachment in 4 types of stirred bioreactors from 15 mL to 5 L.