Washing Of Cells Research Articles

Cervicovaginal secretions protect from human papillomavirus infection: Effects of vaginal douching

ObjectiveCervicovaginal secretions (CVSs) are reported to protect against human papillomavirus (HPV) infection. Although vaginal douching is known to clear both viral inoculants and CVSs, its effect on CVSs in women with HPV infection is unknown. Materials and MethodsThe in vitro HPV pseudovirus infection system was used to test the protective activity of CVSs against HPV infection in samples collected before and after vaginal douching. To simulate different time points of vaginal douching in relation to viral exposure, the cell CVS reconstitute was washed after different viral exposure durations. ResultsIn the CVSs of premenopausal and postmenopausal women who did not perform douching, the CVSs inhibited HPV infection by 56.7 ± 1.8% and 53.6 ± 2.5%, respectively; in women who had performed douching, the CVSs inhibited HPV infection by only 31.2 ± 7.1%, which was significantly lower (p < 0.01). Cell washing effectively cleared 60–90% of the infectious load with the greatest activity occurring within 30 minutes after inoculation. In the presence of CVSs, a sustained inhibition of HPV infection existed for up to 8 hours after HPV exposure, and cell washing increased the clearance to up to 82–93% of the infectious load. ConclusionThis study confirms the protective activity of CVSs against HPV infection regardless of age. In this in vitro study, the net effect of douching was found to be beneficial.

Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum.

BackgroundReducing the production cost of, and increasing revenues from, industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to lack of an efficient method for deoxyribonucleic acid (DNA) transfer.ResultsThis work reports the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum ATCC 6013 together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. With proper selection of the replication origin and antibiotic-resistance marker, we initially electroporated methylated DNA into C. pasteurianum at a low efficiency of 2.4 × 101 transformants μg-1 DNA by utilizing conditions common to other clostridial electroporations. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency, up to 7.5 × 104 transformants μg-1 DNA, an increase of approximately three order of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection.ConclusionsC. pasteurianum ATCC 6013 can be electrotransformed at a high efficiency using appropriately methylated plasmid DNA. The electrotransformation method and tools reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

Optimization of the immunomagnetic selection in microcythemic donors enrolled for haploidentical transplantation

BackgroundImmunomagnetic cell selection (ICS) cells is increasingly used in allogeneic hematopoietic transplantation in order to reduce the T cells quantity. The aim of this study was to evaluate an protocol based on Ficoll method before ICS. Study design and methodsThe automated procedure was compared with the standard method. In the group 1 the cell processing involves the extraction of the buffy-coat by Ficoll before incubation with antibodies. This procedure was performed with the Sepax S-100 device. The efficacy of this automated procedure was compared with the group 2. In this group, the cell washing and the incubation were performed through the standard method. The CD34+ cells collected by apheresis (HPC-A) were selected with ICS. ResultsThe results obtained after Ficoll procedure, showed a total nucleated cells (TNCs) and CD34+ cells recovery of 85.73% (75.90–90.63; SD 4.25) and 79.31% (51.77–112.31; SD 18.40), respectively. The TNC and CD34+ cells recovery after the pre-incubation washing performed through the standard method, was 75.54% (38.36–97.76; SD 22.5) and 61.51% (30.87–81.79; SD 19.3), respectively. The CD34+ cells recovery after ICS was 79% (51.77–100; SD 18.40) and 44% (15.57–88.24; SD 25.91) in the group 1 and the group 2, respectively. This difference was statistically significant (p=0.001). ConclusionThe efficacy of the ICS which resulted to be higher in the group 1 compared to the group 2. Overall, our data suggest that the Ficoll procedure before incubation is suitable for the clinical routine in the ICS for haploidentical transplantation in patients affected by thalassemia.

Transcriptional profiling of BrazilianSaccharomyces cerevisiaestrains selected for semi-continuous fermentation of sugarcane must

Brazil played a pioneering role in the global establishment of the sugarcane bioethanol industry. The bioethanol fermentation process currently used in Brazil is unique due to the acid wash and recycling of yeast cells. Two, industrially adopted, wild yeast strains, CAT-1 and PE-2, have become the most widely used in Brazil. How these strains respond to the unique fermentation process is poorly understood. The improved performance of CAT-1 and PE-2 is hypothesised to be related to enhanced stress tolerance. This study presents a genome-wide analysis of the CAT-1 and PE-2 transcriptomes during a small-scale fermentation process that mimicked the industrial conditions. The common and unique transcriptional responses of the two strains to the Brazilian fermentation process were identified. Environmental stress response genes were up-regulated postfermenter feeding, demonstrating the impact of the prior acid wash and high glucose environment. Cell wall and oxidative stress tolerance were subsequently demonstrated to be enhanced for the industrial strains. Conversely, numerous genes involved in protein synthesis were down-regulated at the end of fermentation revealing the later impact of ethanol-induced stress. Subsequently, the industrial strains demonstrated a greater tolerance of ethanol and the disruption of endoplasmic reticulum homoeostasis. This increased ethanol tolerance was finally correlated with an increased unfolded protein response and increased HAC1 splicing.

LIM Kinase 1 (LIMK1) Interacts with Tropomyosin-related Kinase B (TrkB) and Mediates Brain-derived Neurotrophic Factor (BDNF)-induced Axonal Elongation

BDNF/TrkB signaling plays critical roles in axonal outgrowth of neurons, the process of which requires the remodeling of the cytoskeleton structure, including microtubules and filamentous actin. However, the mechanism by which BDNF/TrkB signaling regulates cytoskeleton reorganization is still unclear. Here, we identified a novel interaction between LIMK1 and TrkB, which is required for the BDNF-induced axonal elongation. We demonstrated that BDNF-induced TrkB dimerization led to LIMK1 dimerization and transphosphorylation independent of TrkB kinase activity, which could further enhance the activation and stabilization of LIMK1. Moreover, activated LIMK1 translocated to the membrane fraction and phosphorylated its substrate cofilin, thus promoting actin polymerization and axonal elongation. Our findings provided evidence of a novel mechanism for the BDNF-mediated signal transduction leading to axonal elongation.

Differential Effects of Protein Disulfide Isomerase (PDI) Inhibitors On the Expression of Tissue Factor Procoagulant Activity (TF PCA) by AML Blasts

Abstract 1112Acute myelogenous leukemia (AML) may be complicated by DIC. TF plays a critical role in AML-associated coagulopathy, and induction of apoptosis significantly increases TF PCA on leukemic blasts, mainly via phosphatidylserine (PS) membrane exposure. However, PDI, a thiol isomerase with oxidoreductase and chaperone activity, has also been implicated in cellular TF regulation. Particularly, PDI inhibitors have been shown to exert antithrombotic activity in animal models. Besides its predominant localization in the endoplasmic reticulum, PDI is present on cell surfaces, where it may represent a promising therapeutic target. We investigated the effect of PDI inhibitors on the expression of TF PCA by leukemic HL60 and THP1 cells to explore their potential as anticoagulant drugs for the prevention and/or treatment of AML-associated DIC. Using a fluorescence-based insulin reduction assay, we confirmed inhibition of recombinant human PDI by bacitracin and quercetin-3-rutinoside (also known as rutin and recently shown to be a specific PDI inhibitor) with IC50 values of 0.6 mM and 14 μM, respectively, showing >95% inhibition at 1 mM (bacitracin) and 50 μM (rutin). Significant insulin reductase activity was observed on HL60 cells, and this activity was inhibited by 75% and 49% using 1 mM bacitracin and 100 μM rutin, respectively, suggesting the presence of additional, PDI-independent thiol isomerase activity. Short-term treatment with 100 μM rutin for 15 min also inhibited TF PCA on HL60 cells by 37%. Importantly, the inhibitory effect of rutin on cell-associated PDI and TF activity was completely abolished by cell washing, confirming previous evidence that rutin is a reversible PDI inhibitor. When HL60 cells were exposed to rutin (100 μM) for 24 hrs, cell-associated TF PCA was increased 2.3-fold (P<0.01), an effect that was accompanied by enhanced PS exposure, as assessed by annexin V-FITC binding (positive cells, 32±11 vs. 10±4%; P<0.01), and increased PCA of cellular microparticles (MPs) isolated from culture supernatants, as evidenced by the thrombin generation parameters lag phase (LP, 14±1 vs. 19±4 min), peak thrombin (PT, 55±17 vs. 22±14 nM), and area under the curve (AUC, 1193±329 vs. 476±347 nM*min; P<0.01). Interestingly, treatment with 100 μM rutin also resulted in a 1.7-fold increase in total cellular TF antigen (P=0.07). The effects of long-term incubation with bacitracin (1 mM) were even more pronounced, involving an 8.3-fold and 4.6-fold increase in cell-associated TF PCA and total cellular TF antigen, respectively. PS exposure (45±9%) and shedding of procoagulant MPs (LP, 7±1 min; PT, 175±49 nM; AUC, 2756±402 nM*min) were also significantly increased. While neither short-term nor long-term exposure to rutin affected TF PCA on THP1 cells, co-incubation with rutin dose-dependently (10–100 μM) inhibited daunorubicin-induced TF PCA in this cell model, an effect that could not be explained by decreased PS exposure. Importantly, both the reaction pattern of HL60 and that of THP1 cells were reproduced ex vivo using myeloblasts from AML patients. In summary, our findings suggest a highly complex and context-dependent role of PDI in leukemic-cell TF PCA expression. While short-term exposure to rutin can reversibly inhibit both PDI and TF activity, long-term exposure may result in significantly increased cellular TF PCA and MP shedding, pointing to a possible role of PDI in PS homeostasis, cytoskeleton rearrangement, and/or TF recycling. In addition, induction of leukemic-cell apoptosis and necrosis by cytotoxic drugs, which is associated with an early loss in membrane integrity and enhanced accessibility of cytoplasmic enzymes, may involve an additional role of (intracellular) PDI in the efficient presentation of TF PCA by AML blasts. Disclosures:No relevant conflicts of interest to declare.

A method for the use of the radiotracer55Fe for microautoradiography and CARD-FISH of natural bacterial communities

Heterotrophic bacteria are key players in the biogeochemical cycle of iron (Fe) in the ocean, but the capability of different bacterial groups to access this micronutrient is ignored thus far. The aim of our study was to develop a protocol for the combined application of microautoradiography (MICRO) and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) using the radioisotope 55Fe. Among the different washing solutions tested, Ti-citrate-EDTA was the most efficient for the removal of extracellular 55Fe providing sufficiently low background values. We further demonstrate that the washing of cells with Ti-citrate-EDTA and the fixation with paraformaldehyde or formaldehyde do not induce leakage of intracellular 55Fe. Incubating natural bacterial communities collected from contrasting environments, the NW Mediterranean Sea and the Southern Ocean, with 55Fe revealed that 3-29% of bacterial cells were associated with silver grains. Combining microautoradiography with CARD-FISH, we demonstrate that the contribution of different bacterial groups to total 55Fe-incorporating cells was overall reflected by their relative contribution to abundance. An exception to this pattern was the proportionally higher contribution of Gammaproteobacteria, SAR86 and Alteromonas. Our study demonstrates the feasibility of MICRO-CARD-FISH using the radiotracer 55Fe and provides the first description of marine bacterial assemblages actively incorporating Fe.

Methadone concentrations in blood, plasma, and oral fluid determined by isotope-dilution gas chromatography–mass spectrometry

Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography-mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate-buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N = 5) were 1.27 and 1.21, respectively. In clinical samples from patients (N = 46), the concentrations of MTD in plasma and whole blood were highly correlated (r = 0.92, p < 0.001) and mean (median) plasma/blood distribution ratios were 1.43 (1.41). The correlations between MTD in OF and plasma (r = 0.46) and OF and blood (r = 0.52) were also statistically significant (p < 0.001) and the mean OF/plasma and OF/blood distribution ratios were 0.55 and 0.77, respectively. The MTD concentration in OF decreased as salivary pH increased (more basic). These results will prove useful in clinical and forensic medicine when MTD concentrations in alternative specimens are compared and contrasted.

Simple Detection of Surface Antigens on Living Cells by Applying Distinct Cell Positioning with Negative Dielectrophoresis

We report the fabrication of two different cell patterns based on negative dielectrophoresis (n-DEP) and apply it to simple and rapid distinction of cells with specific surface antigens from a cell population. The DEP device for cell manipulation comprised a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO-interdigitated band array (ITO-IDA) electrode modified with an antibody. Cells immediately accumulated on the surface in the gap area between both bands of the ITO-IDA electrode by n-DEP upon AC voltage between the upper ITO and both lower bands. Switching of the applied band electrode voltage resulted in the removal of accumulated cells to form another pattern because of the formation of a different electric field pattern in the device. Modifying the ITO-IDA surface with the antibody inhibited the removal of the cells with a specific surface antigen for irreversible capture by immunoreactions during the first accumulation. In this study, we targeted the CD33 surface antigen expressed on human promyelocytic leukemia cells (HL-60). The time required for the assay was substantially short: 60 s for forcing and 60 s for separating the unbound cells. Furthermore, the present method does not require pretreatment such as target labeling or washing of unbound cells. Moreover, the use of the swing technique considerably improved cell binding to the antibody-modified surface for cells with a specific surface antigen. The distinct integration of cells with n-DEP in the high conductivity medium provided higher cell binding efficiency compared to that obtained in our previous study (Hatanaka, H.; Yasukawa, T.; Mizutani, F. Anal. Chem., 2011, 83, 7207-7212) without loss of rapidity and simplicity.

In vitro comparison of two different methods of cell washing

The storage of red blood cells (RBC) results in increased concentrations of plasma free hemoglobin, potassium, glucose, and lactate, among other undesirable substances. These concentrations continue to increase as RBC products age and can cause deleterious effects to the patient. In the setting of cardiac surgery, the autotransfusion devices are routinely used to wash blood that is shed from the surgical site. These devices could also be used to wash stored RBC units obtained from the blood bank. The objective of this study was to compare the product created by washing a unit of RBCs with the AutoLog autotransfusion device in the operating room to the washed products from a standard cell washer in the blood bank. Eleven outdated RBC units (stored for >42 days at 4 °C) were split in half. One half was washed using the Medtronic AutoLog device; the other half was washed using the blood bank's Cobe 2991 Cell Processor. Analytes were measured on samples from the unwashed parent unit and from the washed daughter units. The parameters measured included hematocrit, free hemoglobin, lactate, lactate dehydrogenase (LDH), potassium, glucose, and pH. When compared to the original untreated RBCs, the glucose, lactate, and potassium levels were decreased when washed in an autotranfusion device. Additionally, the free hemoglobin and LDH levels were significantly lower with the Medtronic Autolog cell saver than in the COBE 2991 Cell Processor. Washing the RBC donor units in an autotransfusion device prior to transfusion can effectively attenuate the increases seen in glucose, potassium, free hemoglobin, and LDH associated with RBC storage lesion.

Detection of PAX8 and p53 is beneficial in recognizing metastatic carcinomas in pelvic washings, especially in cases with suspicious cytology

ObjectiveThe aim of this study is to evaluate the detection of paired box gene 8 (PAX8) and p53 with immunohistochemistry in pelvic washing cell block sections. MethodsA total of 92 cases were used in this study, which were assigned to three groups according to the cytopathology files. The first group with positive cytology including endometrial and ovarian carcinomas comprised 32 cases. The second group with suspicious cytology for endometrial or ovarian carcinomas consisted of 29 cases. The third group with negative cytology (regarded as mesothelial cells) included 31 cases. The pelvic washing cell blocks underwent immunohistochemistry to detect PAX8 and p53 expression. ResultsImmunoreactivity for PAX8 was found in 75% (24/32) of the cases in the group with positive cytology, in 6.9% (2/29) of the cases with suspicious cytology, and in none of the 31 cases with negative cytology (sensitivity: 75%; specificity: 100%; p<0.05). p53 expression was detected in 37.5% (12/32) of the cases in the first group, in 3.4% (1/29) of the cases in the second group, and in none of the cases in the third group (sensitivity: 37.5%; specificity: 100%; p<0.05). Moreover, the combined expression of PAX8 and p53 showed the same result as the single expression of p53 in the three groups. ConclusionThe detection of PAX8 and p53 is beneficial in recognizing metastatic carcinomas in pelvic washings, especially in cases with suspicious cytology, which additionally supports the Müllerian origin of these carcinomas.

Use of lissamine green before conjunctival impression cytology and flow cytometry in patients with sjögren’s syndrome

AbstractPurpose Lissamine Green (LG) is a vital dye that stains damaged epithelial cells in ocular surface disease. Conjunctival Impression Cytology (CIC) is used with flow cytometry, to examine surface epithelial cells. We were concerned that, if used prior to CIC, cell uptake of LG might affect the fluorescent signal during flow cytometry. We therefore studied whether the use of LG before CIC, could cause fluorescence interference.Methods CIC was performed with two autoclaved Supor semicircular filters applied to the lateral bulbar conjunctiva of 6 patients with Sjögren's Syndrome. LG was eluted from a standard strip with a drop of anaesthetic and delivered into the inferior fornix. Two further filters were applied to the medial bulbar conjunctiva. Cells were recovered by agitation for 1 minute, washed, stained and analysed by flow cytometry.Results When compared with samples taken before LG instillation, cells isolated from all 6 samples taken after LG instillation, and excited at 630nm, showed an increase in fluorescence at 665nm. Some increased fluorescence was also noted at 680nm but was within the limits of spectral compensation. There was no observable effect of LG when cells were excited at 488nm.Conclusion Cell uptake of LG is detected by flow cytometry, predominantly in the 665nm channel when excited at 630nm. This persists despite cell washing. Whilst this excludes the use of fluorochromes that emit within this spectrum, the use of fluorochromes excited or emitting at other wavelengths, is feasible after LG staining.

Glycosylated hemoglobin is a good indicator of blood glucose status in Persian cats

The purposes of this study were to determine normal value of glycated hemoglobin in Persian cats and to investigate its relation to fasting plasma glucose. Blood samples were collected from 67 clinically healthy adult Persian cats (32 males and 35 females). After separation and washing of red blood cells, hemolysate was prepared and subjected to weak cation exchange chromatography for determination of glycosylated hemoglobin. Glucose was measured in fasting plasma samples (after 10–12 h of fasting) using glucose oxidase method. Glycosylated hemoglobin % in the studied cats was 1.56 ± 0.47 in males and 1.61 ± 0.52 in females. Fasting plasma glucoses were 88.3 ± 10.2 and 90.8 ± 11.5 mg/dl in males and females, respectively. Glycosylated hemoglobin % and plasma glucose strongly correlated together (r = 0.79, p < 0.001). Three cats with persistent high fasting plasma glucose showing glycosylated hemoglobin percentage of 2.6, 2.8, and 2.9 % which exceeded the upper limit of the normal value obtained in this study were found. We concluded that glycosylated hemoglobin % is a good indicator of fasting plasma glucose, and its determination can be considered as a method for diabetes screening in Persian cats. Considering three cats with persistent high fasting plasma glucose and glycosylated hemoglobin percent, we concluded that glycosylated hemoglobin percent could be a good biochemical test for screening or diagnosis diabetes in cats.

Mysterious Ca2+-independent muscular contraction: déjà vu

The permeabilized cells and muscle fibres technique allows one to study the functional properties of mitochondria without their isolation, thus preserving all of the contacts with cellular structures, mostly the cytoskeleton, to study the whole mitochondrial population in the cell in their natural surroundings and it is increasingly being used in both experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibres are very different from those in isolated mitochondria in vitro. In the present study, we have analysed the data showing the dependence of this parameter upon the muscle contractile state. Most remarkable is the effect of recently described Ca(2+)-independent contraction of permeabilized muscle fibres induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous Ca(2+)-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibres that result in a disorganization of mitochondrial arrangement, leading to a significant increase in affinity for ADP. These data show that Ca(2+)-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore the use of their inhibitors is strongly advised at the permeabilization step with careful washing of fibres or cells afterwards. A possible physiologically relevant relationship between Ca(2+)-regulated ATP-dependent contraction and mitochondrial functional parameters is also discussed.

Reply to Ji et al.

[1] Boks RH, Golab HD, Takkenberg JJ, Bogers AJ. Washing of irradiated red blood cells in paediatric cardiopulmonary bypass: is it clinically useful? A retrospective audit. Eur J Cardiothorac Surg 2012;41:283–6. [2] Swindell CG, Barker TA, McGuirk SP, Jones TJ, Barron DJ, Brawn WJ et al. Washing of irradiated red blood cells prevents hyperkalaemia during cardiopulmonary bypass in neonates and infants undergoing surgery for complex congenital heart disease. Eur J Cardiothorac Surg 2007; 31: 659–64. [3] Keidan I, Amir G, Mandel M, Mishali D. The metabolic effects of fresh versus old stored blood in the priming of cardiopulmonary bypass solution for pediatric patients. J Thorac Cardiovasc Surg 2004;127: 949–52. [4] Liu J, Ji B, Feng Z, Zhao J, Li C, Li B et al. The effect of preprocessing stored red blood cells on neonates undergoing corrective cardiac surgery. ASAIO J 2007;53:680–3. [5] Cholette JM, Henrichs KF, Alfieris GM, Powers KS, Phipps R, Spinelli SL et al. Washing red blood cells and platelets transfused in cardiac surgery reduces postoperative inflammation and number of transfusions: results of a prospective, randomized, controlled clinical trial. Pediatr Crit Care Med 2011; doi: 10.1097/PCC.0b013e31822f173c.

Optimized Processing of Growth Factor Mobilized Peripheral Blood CD34+ Products by Counterflow Centrifugal Elutriation

Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory. Each fraction was analyzed for cell count, viability, and blood cell differential. Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions. With modification of the basic protocol, we were able to collect all of the CD34+ cells in a single fraction. The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34, washed using the Elutra device, and transferred directly to a magnetic bead selection device for further purification. CD34+ cell purities from the column were extremely high (98.7 ± 0.9%), and yields were typical for the device (55.7 ± 12.3%). The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.

Abstract 4046: Kinetic quantification of caspase-3/7 mediated apoptosis using live-cell imaging

Abstract Apoptosis, the biological process by which cells undergo programmed cell death, is required for normal tissue maintenance and development. However, aberrations in apoptotic signaling networks are implicated in numerous human diseases including neurodegeneration and cancer. Stimulation of either the extrinsic or intrinsic apoptotic pathways triggers a signaling cascade that typically results in the activation of executioner caspase-3. Numerous in vitro assays have been designed to measure the activation of caspase-3. The majority of these assays utilize reagent substrates that incorporate the DEVD motif which is recognized by both activated caspases 3 and 7. Such assays result in single, user-defined time point measurements of caspase-3/7 activity and typically require multiple wash steps or cell lifting prior to data collection; potentially resulting in the loss of cells or critical data in experiments where cells undergo apoptosis at different rates according to treatment conditions. In this study, we optimized an assay system incorporating a bi-functional reagent containing the DEVD motif linked to a DNA dye for use on the IncuCyte FLR imaging system. When added to cell culture growth medium, this relatively inert, non-fluorescent molecule crosses the cell membrane where it is available to be a substrate for activated caspase-3/7. The reaction product is a green fluorescent DNA dye that labels the nuclei of cells undergoing apoptosis. Our data show that this strategy can quantitatively measure the kinetic activation of caspase-3/7 through extrinsic stimulation of human tumor derived MDA-MB-231 breast adenocarcinoma cells, HeLa cervical adenocarcinoma cells, A549 lung epithelial carcinoma cells, and HT 1080 fibrosarcoma cells following treatment with TNFα and cycloheximide. Moreover, we exemplify the use of this strategy in 96-well plate screening protocols using both HT 1080 and MDA-MB-231 cells to measure the induction of caspase-3/7 activity in the presence of staurosporine, a well-known general protein kinase inhibitor and inducer of caspase-3/7 mediated apoptosis with Z’ values α0.6. We provide evidence that this stable, kinetic, mix-and-read assay can provide both quantitative and qualitative assessments of caspase-3/7 activity using fluorescent images and high-definition phase contrast images, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4046. doi:1538-7445.AM2012-4046

Diffusion-based extraction of DMSO from a cell suspension in a three stream, vertical microchannel.

Cells are routinely cryopreserved for investigative and therapeutic applications. The most common cryoprotective agent (CPA), dimethyl sulfoxide (DMSO), is toxic, and must be removed before cells can be used. This study uses a microfluidic device in which three streams flow vertically in parallel through a rectangular channel 500 µm in depth. Two wash streams flow on either side of a DMSO-laden cell stream, allowing DMSO to diffuse into the wash and be removed, and the washed sample to be collected. The ability of the device to extract DMSO from a cell stream was investigated for sample flow rates from 0.5 to 4.0 mL/min (Pe = 1,263-10,100). Recovery of cells from the device was investigated using Jurkat cells (lymphoblasts) in suspensions ranging from 0.5% to 15% cells by volume. Cell recovery was >95% for all conditions investigated, while DMSO removal comparable to a previously developed two-stream device was achieved in either one-quarter the device length, or at four times the flow rate. The high cell recovery is a ~25% improvement over standard cell washing techniques, and high flow rates achieved are uncommon among microfluidic devices, allowing for processing of clinically relevant cell populations.

Cell Washing Centrifuges

Cell Washing Centrifuges

Thawed autologous peripheral blood stem cells require modified quantification methods for hematopoietic progenitor cell evaluation

The aim of this study was first, to analyze the post-thaw progenitor assays usually performed on peripheral blood stem cell autografts and second, to achieve standardization with improved flow cytometric and CFU-GM assays. In the first part of the study (n=79), recovery and Intraclass Correlation Coefficient (ICC) of total nucleated cells, CD34 and CFU-GM were analyzed before and after cryopreservation. In the second part (n=20), evaluation methods were modified : the washing step was suppressed in the flow cytometric method and 500 CD34 were plated compared to 4×10(4) total nucleated cells in the CFU-GM assay. The recovery rates were analyzed and the CFU-GM results were regarded as reliable when 30-100 colonies were observed, according to the manufacturer recommendation. The analysis of the first part showed an ICC that was perfect for total nucleated cells (0.93), substantial for CD34 (0.67) and fair for CFU-GM (0.25). Median CD34 recovery was 112.6% (29.9-222%). The CFU-GM median recovery was 31.7% (0.19-142%) leading to reliable results for 27 grafts. In the second part, the median CD34 recovery was 85.75% (54-99%). No recovery over 100% was observed. The CFU-GM assay led to 18 out of 20 evaluable autografts when 500 CD34 were seeded, compared to 10 out of 20 when total nucleated cell were seeded. Avoiding cell washing in the flow cytometric method limited the overestimate of the CD34 percentage. Plating 500 thawed CD34 improved reliability of the results and allowed a better standardization of the assay.