Development of a downscale sedimentation field flow fractionation device for biological event monitoring.

Classically described as a macroscale size-density based method, Sedimentation field flow fractionation (SdFFF) has been successfully used for cell sorting. The goal of this study was to develop a new SdFFF device for downscale applications, in particular for oncology research to rapidly monitor chemical biological event induction in a cell line. The development of a downscale SdFFF device required reduction of the separation channel volume. Taking advantage of a newly laboratory designed apparatus, channel volume was successfully decreased by reducing both length and breadth. To validate the apparatus and method, we used the well-known model of diosgenin dose-dependent induction of apoptosis or megakaryocytic differentiation in HEL cells. After a minute scale acquisition of a reference profile, the downscale device was able to perform fast, early, significant and reproducible monitoring of apoptosis and differentiation, two important biological mechanisms in the field of cancer research.

Analysis of relationship between cell cycle stage and apoptosis induction in K562 cells by sedimentation field-flow fractionation.

Recently, sedimentation field-flow fractionation (SdFFF) was used to study the specific kinetics of diosgenin-induced apoptosis in K562 cells. Here, we propose a new SdFFF cell separation application in the field of cancer research concerning the correlation between induction of a biological event (i.e. apoptosis) and cell status (i.e. cell cycle position). SdFFF isolated subpopulations depending on the cell cycle position allowing the study of apoptosis kinetics and extent. Results showed that cells in G0/G1 phases (F3 cells) underwent significant and earlier apoptosis than cells in the active part of the cell cycle (S/G2/M phases). Results shed light on the correlation between differences in apoptosis kinetics and cell cycle stage when exposure to the inducer began. SdFFF monitoring and size measurement also led to the description of different subpopulations demonstrating complex variations in density between fractions associated with differences in biological processes.

Study of the phenotypic relationship in the IMR-32 human neuroblastoma cell line by sedimentation field flow fractionation.

Neuroblastoma (NB) is the most common childhood solid tumor. Although spontaneous regression can occur in patients <1-year old, 70% of patients over the age of 1 year have a high-risk and difficult-to-treat NB. Cell type heterogeneity is observed either in the morphological appearance of NB tumors or in cell lines isolated from tumor specimens. NB consists of two principal neoplastic cell types: i) neuroblastic or N-type (undifferentiated cells); and ii) stromal or S-type (differentiated cells). As NB cells seem to have the capacity to differentiate spontaneously in vivo and in vitro, their heterogeneity could affect treatment outcome, in particular the response to apoptosis induced by chemotherapy. Therefore, it is important to understand the underlying process governing changes in differentiation in order to improve treatment response and NB patient outcome and the neoplastic population in IMR-32 represented a good model for such a study. Results showed that this cell line was extremely heterogeneous and highly variable in its stage of differentiation and we demonstrated that sedimentation field flow fractionation (SdFFF) permitted the isolation of 2 N-phenotypes and contributed to the understanding of the IMR-32 cell population dynamics. The first N-phenotype forms a pool of quiescent undifferentiated cells while the second one was able to proliferate (incorporation of BrdU) and also give rise to adherent S-type cells (PSA-N-CAM+ and N-CAM+). The results could also suggest a close interaction between these different cellular phenotypes to create the IMR-32 cell lineage.

Sedimentation field flow fractionation to study human erythroleukemia cell megakaryocytic differentiation after short period diosgenin induction.

Anti-cancer differentiation therapy could be one strategy to stop cancer cell proliferation. We propose a new sedimentation field flow fractionation (SdFFF) cell separation application in the field of cancer research. It concerns the study of megakaryocytic differentiation processes after a short exposure to an inducting agent (diosgenin). Washout process and early dual SdFFF separation--removing the influence of diosgenin and decreasing the influence of undifferentiated cells--resulted in the preparation of an enriched population to study the mechanism and kinetics of megakaryocytic differentiation. A short exposure to diosgenin was able to induce complete differentiation leading to maximal maturation which ended naturally after 192h incubation without the influence of a secondary effect of diosgenin. The study of isolated undifferentiated cells also showed that no resistance to diosgenin was observed. This result suggested different sensitivities to differentiation induction, and SdFFF cell separation would be of great interest to explore this phenomena.

Sedimentation field flow fractionation monitoring of bimodal wheat starch amylolysis.

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this study, we investigated the capacity of sedimentation field flow fractionation (SdFFF) to monitor the amylolysis of a bimodal starch population: native wheat starch. Results demonstrated a correlation between fractogram changes and enzymatic hydrolysis. Furthermore, SdFFF was used to sort sub-populations which enhanced the study of granule size distribution changes occurring during amylolysis. These results show the interest in coupling SdFFF with particle size measurement methods to study complex starch size/density modifications associated to hydrolysis. These results suggested different applications such as the association of SdFFF with structural investigations to better understand the specific mechanisms of amylolysis or starch granule structure.

Pre-apoptotic sub-population cell sorting from diosgenin apoptosis induced 1547 cells by Sedimentation Field-Flow Fractionation. The effect of channel thickness on sorting performance.

Apoptosis is one of the most important phenomena in cell biology. Pre-apoptotic cells, defined as cells engaged in early stages of apoptosis, could be used as a cellular tool to study apoptosis pathways. The human 1547 osteosarcoma cell line and diosgenin (a plant steroid) association was selected as an in vitro cellular apoptosis model. In a previous study, using this model, we demonstrated that SdFFF monitored apoptosis induction as early as 6h after incubation. In this study, we investigated the capacity of Sedimentation Field-Flow Fractionation (SdFFF) to sort an enriched population of pre-apoptotic cells from 1547 cells incubated for 6 h with 40 microM diosgenin. In that way, two different separation devices which differed especially in channel thickness, 125 and 175 microm, were used and compared. Results showed, for the first time, that SdFFF is an effective method to obtain an enriched pre-apoptotic sub-population. These results suggest, as a new application, that SdFFF could be an included tool in the study of apoptotic mechanisms or the kinetic action of apoptotic drugs.

Megakaryocyte cell sorting from diosgenin-differentiated human erythroleukemia cells by sedimentation field-flow fractionation.

Anticancer differentiation therapy could be one strategy to stop cancer cell proliferation. Human erythroleukemia (HEL) cell line, incubated with 10 microM diosgenin, underwent megakaryocytic differentiation. Thus, the association diosgenin/HEL could be used as a model of chemically induced cellular differentiation and anticancer treatment. The goal of this work was to determine the capacity of sedimentation field-flow fractionation (SdFFF) to sort megakaryocytic differentiated cells. SdFFF cell sorting was associated with cellular characterization methods to calibrate specific elution profiles. As demonstrated by cell size measurement methods, cellular morphology, ploidy, and phenotype, we obtained an enriched, sterile, viable, and functional fraction of megakaryocytic cells. Thus, SdFFF is proposed as a routine method to prepare differentiated cells that will be further used to better understand the megakaryocytic differentiation process.

Sedimentation field flow fractionation monitoring of rice starch amylolysis.

Enzymatic starch granule hydrolysis is one of the most important reactions in many industrial processes. In this work, we investigated the capacity of SdFFF to monitor the native rice starch amylolysis. In order to determine if fractogram changes observed were correlated to granule biophysical modifications which occurred during amylolysis, SdFFF separation was associated with particle size distribution analysis. The results showed that SdFFF is an effective tool to monitor amylolysis of native rice starch. SdFFF analysis was a rapid (less than 10 min), simple and specific method to follow biophysical modifications of starch granules. These results suggested many different applications such as testing series of enzymes and starches. By using sub-population sorting, SdFFF could be also used to better understand starch hydrolysis mechanisms or starch granule structure.

SdFFF monitoring of cellular apoptosis induction by diosgenin and different inducers in the human 1547 osteosarcoma cell line.

Apoptosis is one of the most important phenomena of cellular biology. Sedimentation field flow fractionation (SdFFF) has been described as an effective tool for cell separation, respecting integrity and viability. Because SdFFF takes advantage of intrinsic properties of eluted cells (size, density, shape or rigidity), we investigated the capacity of SdFFF in monitoring the early and specific biophysical modifications which occurred during cellular apoptosis induction. Then, we used, as an in vitro cellular apoptosis model, the association between human 1547 osteosarcoma cells and diosgenin, a plant steroid known to induce apoptosis. Four other molecules were studied: hecogenin, tigogenin, staurosporine and MG132. Our results demonstrated a correlation between SdFFF elution profile changes (peak shape modification and retention ratio evolution) and effective apoptosis induction. For the first time, we demonstrated that SdFFF could be used to monitor apoptosis induction as early as 6 h incubation, suggesting different applications such as screening series of molecules to evaluate their ability to induce apoptosis, or sorting apoptotic cells to study apoptosis pathway.

Mouse embryonic stem cell sorting for the generation of transgenic mice by sedimentation field-flow fractionation.

Mouse embryonic stem (ES) cells are an important tool for generation of transgenic mice and genetically modified mice. A rapid and efficient separation of ES cells that respects cell integrity, viability, and their developmental potential while also allowing purified ES fraction collection under sterile conditions might be of great interest to facilitate the generation of chimeric animals. In this study, we demonstrated for the first time the effectiveness of a sedimentation field-flow fractionation (SdFFF) cell sorter to provide, with a characteristic DNA content, a purified ES cell fraction and with a high in vivo developmental potential to prepare transgenic mice by generation of chimeras having a high percentage of chimerism.

Sedimentation field flow fractionation purification of immature neural cells from a human tumor neuroblastoma cell line.

The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, usable and purified immature neural cell fraction without inducting cell differentiation.

Deferred standards, an on-line qualification, validation and system stability probe for chromatographic assay.

Specific programming of automated HPLC systems allows total on-line qualification, validation and stability monitoring using the concept of deferred standards. Setting up such a process for routine analyses in an automated HPLC system requires specific autosampler programming as well as specific monitoring software. With an autosampler, a double injection procedure is programmed, the first introducing the sample, and the second, a few minutes deferred, the deferred control standard. Two additional compounds are therefore added to the sample before and during the chromatographic process: the intemal standard for sample quantification and the deferred standard for system control. Specific methodologies are described of how to obtain classical quantitative analysis information as well as system qualification validation stability information. Experiments were performed to develop specified methodologies to monitor the quality of quantitative analysis during the life of the column by using the deferred standard concept to probe the effects of column ageing on separation characteristics.

Visna virus-induced cytopathic effect in vitro is caused by apoptosis.

Visna-Maedi virus (VMV), an ungulate lentivirus, causes a natural infection in sheep. In vitro, VMV infection and replication lead to strong cytopathic effects with subsequent death of host cells. We investigated, in vitro, the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with VMV, by employing diverse strategies to detect its common end-stage alterations. We demonstrated that VMV-infection in sheep choroid plexus cells (SCPC), is associated with apoptosis, characterized by morphological changes such as condensation of chromatin and the appearence of apoptotic bodies. DNA fragmentation was documented by TUNEL assay. Although the mechanism by which VMV activates this cell suicide program is not known, we examined the activation of caspases, the family of death-inducing proteases that resulted in cleavage of several cellular substrates. To study the role of caspases in VMV-induced apoptosis, we focused on several protease targets: procaspase-3 and procaspase-1. During VMV-infection, SCPC display active caspase-3 and no caspase-1 activity. In conclusion, our results suggest that VMV infection, in vitro, induces cell death of SCPC by a mechanism that can be characterized by many of the properties most closely associated with apoptotic cell death.