Washing Of Cells Research Articles

AB0122 In Vivo Intraarticular Binding of A Biopolymeric Compound in the Course of Experimental Immune Complex Induced Arthritis, Evaluated as A Potential Tool for Targeting Inflamed Synovium

ObjectivesTo evaluate in an in vivo rat experimental model of antigen induced arthritis (AIA) the concentration and persistence of intraarticular (ia) binding of a biocompatible modified polysaccharide, composed of a...

Rapid formation of cell-particle complexes via dielectrophoretic manipulation for the detection of surface antigens

A rapid and simple method for the fabrication of the island patterns with particles and cells was applied to detect the presence of specific antigens on the cell surface. An upper interdigitated microband array (IDA) electrode was mounted on a lower substrate with the same design to fabricate a microfluidic-channel device for dielectrophoretic manipulation. The electrode grid structure was fabricated by rotating the upper template IDA by 90° relative to the lower IDA. A suspension of anti-CD33 modified particles and HL-60 cells was introduced into the channel. An AC electrical signal (typically 20V peak-to-peak, 100kHz) was then applied to the bands of the upper and lower IDAs, resulting in the formation of island patterns at the intersections with low electric fields. Immunoreactions between the antibodies immobilized on the accumulated particles and the CD33 present on the surface of the cells led to the formation of complexes comprising corresponding antigen-antibody pairs. Non-specific pairs accumulated at the intersection, which did not form complexes, were then dispersed after removal of the applied field. The time required for the detection of the formation/dispersion of the complexes is as short as 6min in the present procedure. Furthermore, this novel cell binding assay does not require pretreatment such as target labeling or washing of the unbound cells.

Copper export contributes to low copper levels and copper tolerance in Emiliania huxleyi

Previous studies have documented that the coccolithophore Emiliania huxleyi maintains lower cellular copper concentrations and is more tolerant to high copper than are many other species of marine algae. We confirm that in comparison to the diatom Thalassiosira pseudonana, E. huxleyi maintains substantially lower Cu levels while maintaining near maximal growth rates. Using the stable isotope 65Cu as a tracer, we show that a constitutive or rapidly induced mechanism for Cu efflux is present in Cu‐replete E. huxleyi cells. This was revealed during uptake experiments in which the accumulation of the isotopically enriched spike, as measured by the isotope fractionation, exceeded the total accumulated copper. Evidence of efflux was not observed during Cu uptake in T. pseudonana cells. In contrast, copper‐limited E. huxleyi cells exhibited no observable efflux during uptake over the same time period, though net uptake rates were similar to replete cells, suggesting that efflux is modulated by cellular copper needs. This Cu‐efflux mechanism is effective over a wide range of Cu concentrations, as E. huxleyi is able to maintain much lower cellular Cu levels than T. pseudonana during 4 h uptake experiments. Direct measurement of export from copper‐loaded cells provided further evidence of a very effective efflux mechanism in E. huxleyi, while loss of copper from T. pseudonana was considerably less. Improvements to cell washes to remove surface‐bound Cu were implemented.

Washing red cells after leucodepletion does not decrease human leukocyte antigen sensitization risk in patients with chronic kidney disease.

Standard leucodepleted blood transfusions can induce the production of human leukocyte antigen (HLA)-specific antibodies, which are associated with longer transplant waiting times and poorer graft outcomes. We hypothesized that additional washing of leucodepleted red cells might reduce antigenic stimulus by removal of residual leukocytes and soluble HLA. A retrospective review of HLA antibodies in children with chronic kidney disease stage 4-5 who had ≥ two HLA antibody screens between 2000 and 2009, pre- and post-transfusion, and were HLA antibody-negative at first testing. Patients were divided according to whether they received standard leucodepleted blood or "washed cells". To assess the efficacy of washing methods, total leukocytes were enumerated pre- and post- manual and automated washing of standard leucodepleted red cells that had been supplemented with whole blood to achieve measurable leukocyte levels pre-washing. A total of 106 children were included: 23 received no blood transfusions (group 1), six had washed cells only (group 2), 59 had standard transfusions only (group 3), and 18 had both standard and washed cells (group 4). Sensitization rates were 26, 17, 44, and 44 % in groups 1-4 (p = 0.32). Patients in groups 3 and 4 had more transfusions with red cells, platelets, and plasma products. There was no difference in HLA sensitization risk with washed or standard red cells on analysis of co-variance controlling for platelets and plasma transfusions. The red cell washing study showed no significant reduction in leukocytes using manual methods. Although there was a statistically significant reduction (33 %) from baseline pre-washing using the automated method, from 6.54 ± 0.84 × 10(6) to 4.36 ± 0.67 × 10(6) leukocytes per unit, the majority of leukocytes still remained. There was no evidence that using washed leucodepleted red cells reduced patient HLA sensitization rates. Washing leucodepleted red cells is unlikely to reduce the risk of HLA sensitization due to the limited effect on residual leukocytes.

Numerical modeling of ultrasonic particle manipulation for microfluidic applications

A numerical simulation methodology for ultrasonic particle/cell separation and cell washing processes is introduced and validated by comparing with the results from the literature. In this study, a finite element approach is used for modeling fluid flow in a microchannel and analytical relations are utilized for the calculation of the ultrasonic radiation forces. The solutions in acoustic and fluidic domains are coupled, and the particle separation under the influence of ultrasonic waves is numerically simulated. In order to simulate the cell washing process, diffusion and fluid dynamics solutions are coupled and solved. A Monte Carlo approach is chosen where statistical distributions are implemented in the simulations. Uniform distributions for the starting locations of particles/cells in the microchannel and normal distributions for the size of the particles are used in numerical simulations. In each case, 750 particles are used for the simulation, and the performance of separation process is evaluated by checking how many microparticles resulted in the targeted outlet channels. Channel geometries for the numerical simulations are adapted from the experimental studies in literature, and comparison between the reported experimental results and the numerical estimations is performed. It has been observed that the numerical estimations and experimental results from the literature are in good agreement, and the proposed methodology may be implemented as a design tool for ultrasonic particle manipulation for microfluidic applications.

Elastomeric Negative Acoustic Contrast Particles for Capture, Acoustophoretic Transport, and Confinement of Cells in Microfluidic Systems

We present a particle-based method for the immunospecific capture and confinement of cells using acoustic radiation forces. Ultrasonic standing waves in microfluidic systems have previously been used for the continuous focusing of cells in rapid screening and sorting applications. In aqueous fluids, cells typically exhibit positive acoustic contrast and are thus forced toward the pressure nodes of a standing wave. Conversely, elastomeric particles exhibit negative acoustic contrast and travel toward the pressure antinodes. We have developed a class of elastomeric particles that are synthesized in bulk using a simple nucleation and growth process, providing precise control over their size and functional properties. We demonstrate that the biofunctionalization of these particles can allow the capture and transport of cells to the pressure antinodes solely via acoustic radiation forces, which may enable new acoustics-based cell handling techniques such as the washing, labeling, and sorting of cells with minimal preparatory steps.

Biofilms: United we stand, divided we fall.

Bacteria have been into existence since the dawn of time. From the discovery of bacteria to the heyday of antibiotic discovery, our view of bacteria has shifted dramatically. It is now widely accepted that microbes rarely survive as solitary cells, but rather grow as biofilms. A biofilm is a surface-associated population of microbes that is embedded in a cement of extracellular compounds. This cement is known as matrix and synonymously as extracellular polymeric substance [1]. Biofilms can contain different types of microorganisms and are ubiquitous. Recently, viral biofilms have come into picture with the finding that in the case of HTLV-I, biofilms may be a major mechanism of its propagation and transmission. HTLV-1 infects 15–20 million people worldwide. Although most of the infected individuals are asymptomatic, 5–10 % develop T cell leukemia or inflammatory syndrome such as HTLV-1–associated myelopathy. Extracellular viral assemblies account for over 80 % of the infectious capacity of HTLV-1–infected cells. Both collagen and fibronectin are overexpressed in HTLV- 1 infected cells, collagen is enriched in viral assemblies but randomly distributed on uninfected cells. Although heparin washes do not completely remove extracellular viral assemblies, but the infectious capacity of cells is markedly reduced. Cell supernatant obtained after cell washes is infectious, although much less efficient than infected cells, indicating that the integrity of extracellular viral assemblies and their transfer at cell contacts are key for HTLV-1 transmission. It is likely that other viruses have also developed transmission strategies based on similar biofilm-like viral assemblies. As extracellular structures of a particular composition, viral biofilms might be potential targets for future antiviral therapy [1]. Apart from this, biofilms have been a cause of concern as microbial biofilms on surfaces cost billions of dollars yearly in equipment damage, product contamination, energy losses and medical infections. Nevertheless, biofilms can also be harnessed for constructive purposes like bioremediation of hazardous materials and waste sites, biofilteration of industrial waste water and formation of natural biological barriers to protect soil and ground water from contamination. They can also be seen as a source of bioactive agents as bacteria in biofilms produce such chemical weapons which individual bacteria cannot produce like violecin pigment which bacteria in marine biofilms release to combat the enemy (paralysing the attacker). Microbes are smarter than we thought and combating biofilms is a hard nut to crack as biofilms can require 100–1,000 times the concentration of an antibiotic to control an infection which is medically impractical to administer. Also, repeated use of anti-microbial agents can cause bacteria to develop increased resistance. This virtue is imparted by the ‘matrix’ which prevents drugs and stress from penetrating the biofilm as well as holds the cells together. Matrix also provides a means by which bacteria within a biofilm can communicate amongst themselves by producing specific signals and arousing an attack, a phenomenon known as quorum sensing, jamming these signals might provide a way to fight back. So, the solution to the problem lies in divide and rule policy. Enzymes such as Dispersin B and deoxyribonuclease degrade the matrix which holds the cells together and may play a role as anti-biofilm agents. Genes like ZapI have been found which have negative effect on matrix formation [2]. Some bacteriophages produce polysaccharide depolymerases that have the potential to degrade biofilm matrix [3]. Hence, the biofilm research should focus on bacteria in the biofilm rather than suspended bacteria in culture as the properties and the genetic regulation of the two are different since bacteria in biofilm behave differently from planktonic cells. Achieving the task may seem difficult but not impossible. The day shall come soon when the concept of biofilms will only be utilized for the benefit of mankind.

Comparison of chemical washing and physical cell-disruption approaches to assess the surface adsorption and internalization of cadmium by Cupriavidus metallidurans CH34

Bacterial biosorption of heavy metals is often considered as a surface complexation process, without considering other retention compartments than cell walls. Although this approach gives a good description of the global biosorption process, it hardly permits the prediction of the fate of biosorbed metals in the environment. This study examines the subcellular distribution of cadmium (Cd) in the metal-tolerant bacterium Cupriavidus metallidurans CH34 through the comparison of an indirect chemical method (washing cells with EDTA) and a direct physical method (physical disruption of cells). The chemical washing approach presented strong experimental biases leading to the overestimation of washed amount of Cd, supposedly bound to cell membranes. On the contrary, the physical disruption approach gave reproducible and robust results of Cd subcellular distribution. Unexpectedly, these results showed that over 80% of passively biosorbed Cd is internalized in the cytoplasm. In disagreement with the common concept of surface complexation of metals onto bacteria the cell wall was poorly reactive to Cd. Our results indicate that metal sorption onto bacterial surfaces is only a first step in metal management by bacteria and open new perspectives on metal biosorption by bacteria in the environment, with implications for soil bioremediation or facilitated transport of metals by bacteria.

Single-use centrifugation solution for volume reduction and cell washing process in cell therapy manufacturing

Current bioprocessing technologies have limited applications in cell therapy manufacturing as they are optimized for the manufacture of recombinant proteins. For example, most cell harvest technologies are efficient in removing cells but not in maximizing viable cell density, viability, washing, and recovery; parameters that are most important for cell therapy manufacturing. Most commonly cells are retained by either centrifugation or filtration based devices. These approaches are often problematic. Conventional centrifugation based devices cause stress and nutrient deprivation to the cells in the pellet which results in low viability, while filtration based devices often suffer from issues such as clogging, retention of cell debris, and shear stress on cells. We have developed the first single-use fluidized-bed centrifugation systems that are completely closed. Our results show that kSep systems can concentrate diverse types of cells with high recovery while maintaining high viability. Additionally, kSep systems can remove cell debris, light particulate impurities, all while significantly reducing aggregation of cells. We have designed these systems without any rotary seals (providing reduced sterility risk) or filters (for reduced issues from clogging). Once captured and concentrated, the cells can efficiently be washed, manipulated, and harvested. This technology is a breakthrough for applications requiring maintenance of cellular integrity during processing. Results show >90% recovery of cells with unchanged viability and 99.9% removal of contaminants. Our automated systems are currently being used for cell therapy, recombinant proteins, and vaccine manufacturing processes.

Russian Pioneers of Therapeutic Hemapheresis and Extracorporeal Hemocorrection: 100‐Year Anniversary of the World's First Successful Plasmapheresis

On 15 February 2013 (2 February on the Julian Calendar) we celebrated the 100-year anniversary of the world's first successful experimental plasmapheresis. Scientific research projects in this field were carried out by the Department of Infectious Disease, Russian Imperial Medical Surgical Academy located in Saint-Petersburg. Doctor of Medical Sciences and Professor Vadim A. Yurevich was a Principal Investigator for this research, which in 1913 resulted in the discovery of a new way of treatment. The results were published in Russki Vratch (Russian Physician) Journal no. 18 (1914) - V.A. Yurevich and N.K. Rosenberg "For the Question Regarding Washing of Blood Outside of the Body and the Vitality of Red Blood Cells". There was no terminology offered for this medical innovation at that time. Plasma removal was performed not solely, but in combination with washing of blood cells returned to the patient. Nowadays this combination is still considered to be more effective than separate plasmapheresis. According to the published experimental protocols this new treatment was done on 15 February (2 February on the Julian Calendar or "old style"). One year later in 1914 a famous researcher, John Abel and coauthors, repeated a separate plasma removal treatment with retransfusion of the blood cells and suggested the term "plasmapheresis", which is now official. The article entitled "Plasma Removal With Return of Corpuscles (Plasmapheresis)", written by Abel was published 3 months later than the article by Professor Yurevich. In 1924, Dr Ivan P. Mikhailovskiy repeated experiments by Yurevich and Rosenberg in vivo on a dog model, confirmed the clinical efficiency and developed the methodology in his article "Washing of Blood In Vivo, the Methodology, Problems, and Importance for the Treatment of Toxic Conditions."

Optimized Internalization of Fluorescently Labeled Biomolecules into Live Bacteria

We have recently developed a method for delivering short DNA fragments and proteins labeled with bright and stable organic fluorophores into live E. coli. Cells are electroporated with a high-voltage pulse, recovered in a rich medium and thoroughly washed to remove any non-internalized fluorescence. Internalized molecules can be tracked at the single-molecule level, and both single-cell and single-molecule FRET can be measured.We have explored the effect of electroporation voltage on the internalization efficiency of short DNA fragments and proteins, and have found a linear relationship. Hence, an appropriate voltage can be selected depending on the application of interest, such as whether single-molecule or ensemble measurements are desired. In addition, we have optimized the buffer and salt conditions for electroporation, in terms of maximizing internalization efficiency whilst preserving protein integrity.Various conditions have been tested for cell washing, including the use of salt and detergent in the washing buffers. The medium used for cell recovery after electroporation has also been noted to affect the efficiency of cell washing. A significant improvement in the removal of non-internalized fluorescence has been achieved by cell filtration. In the case of proteins prone to aggregation, cell filtration has also been found to remove any high-molecular weight species from the cell suspension.Finally, we have found free dye to be internalized at much higher efficiency than the labeled biomolecules, and hence it is important to remove any contaminating free dye from the samples used for electroporation. We have optimized the methodology for determining and minimizing the amount of free dye in our samples, and have obtained samples that show less than 1% free dye contamination, which is at the level of background autofluorescence.

Bovine lactoferrin enhances proliferation of human peripheral blood lymphocytes and induces cytokine production in whole blood cultures.

Lactoferrin belongs to the immunoregulatory milk proteins involved in iron metabolism as well in providing innate immunity to newborns. The protein has been the subject of numerous clinical studies. The aim of this investigation was to evaluate the effects of bovine lactoferrins (bLF), differing in source and iron content, on spontaneous proliferation of human peripheral blood mononuclear cells (PBMC) and cytokine production by human whole blood cultures. The following bLF preparations were used: partially iron saturated or devoid of iron bLF from milk and bLF from colostrum. The study was conducted on 12 healthy volunteers (men, 20-24years old). The effects of bLFs on the proliferation of PBMC in four-day cultures was studied at 50-0.6µg/mL concentration range and the rate of proliferation was determined using the MTT colorimetric method. TNF αand IL-6 levels, induced by the bLFs in 24 hwhole blood cultures, were measured by ELISA. The lactoferrins stimulated autologous proliferation of human peripheral blood mononuclear cells (PBMC) in adose-dependent manner, with acomparable efficacy. This stimulation occurred both in the constant presence of bLFs in the cultures and also upon preincubation of PBMC with bLFs with subsequent exhaustive wash of cells. Only bLF from colostrum induced production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in cultures of whole blood cells. This phenomenon took place predominantly at concentration of 50µg/mL. The results showed potent stimulation of the proliferative response of PBMC by bovine lactoferrin, associated with the induction of proinflammatory cytokines only in the case of colostral bLF.This observation may be of importance when high doses of bLF are used in therapy and by designing diet supplementation with this protein.

The Utility Of Minimal Residual Disease (MRD) Assessment At First Relapse: Results From The BSBMT/Ukmf Myeloma X (Intensive) Trial

IntroductionMinimal residual disease (MRD) assessment by multicolour flow cytometry (MFC) following upfront therapy is a powerful prognostic tool in multiple myeloma (MM) and there is growing evidence supporting its role as an outcome measure in clinical trials. There is however no published data assessing its utility in the relapse setting. Our aim was to test the utility of MFC at first relapse following a second autologous transplant (ASCT2) or weekly cyclophosphamide (C-weekly) consolidation following re induction chemotherapy in patients entered into the BSBMT/UKMF Myeloma X trial. Patients and MethodsEligible MM patients relapsing after a prior ASCT were enrolled and received a bortezomib-containing re-induction regimen (PAD, Bortezomib, Doxorubicin and Dexamethasone) before being randomized to either ASCT2 or C-weekly. The primary endpoint was time-to-progression (TTP) with secondary endpoints of overall survival and overall response rate (to PAD and randomized intervention). Bone marrow aspirate samples were analyzed by MFC at trial entry to establish neoplastic phenotype and then at specified time-points following treatment. After red cell lysis and washing, samples were labelled with CD27-FITC, CD56-PE, CD19-PerpCPCy5.5, CD38-PE-Cy7, CD138-APC and CD45-APC-Cy7. A minimum of 500,000 events were acquired on a FacsCanto II cytometer and data was analyzed using a standardized gating strategy. Sensitivity of the assay is 0.01% of leucocytes. Response assessment (after re-induction, 100 days post-ASCT2 or 30 days post C-weekly) and disease progression were determined using IMWG criteria. Unfavourable genetic abnormalities were defined as FGFR3/IGH, IGH/MAF and TP53 deletion by interphase FISH. Results297 patients with a median age of 61 (range 38 -75) were entered into the study and 174 randomized from April 2008 to November 2012: ASCT2 n=89, C-weekly n=85. Of the whole trial cohort, MFC analysis was available in 224 at presentation, 162 at completion of re-induction therapy, 46/89 at D100 post ASCT2 and 50/85 at D30 post C-weekly.In an intention to treat analysis of all available randomized patients, achieving MRD negativity was highly predictive for improved TTP: All patients (MRD+ 12mns 95%CI 10-13 vs. MRD- 24mns 95CI NR; logrank P=0.0003), ASCT arm (MRD+ 15mns 95%CI 11-19 vs. MRD- 24mns 95%CI 16-42; logrank P=0.0227) & C-weekly arm (MRD+ 11mns 95%CI 8-12 vs. MRD- 15mns 95%CI 9-NR) logrank P=0.0107).MRD status was also assessed in conjunction with IMWG conventional categorical response assessment. CR was demonstrable in 39% of patients post ASCT2 and 22% of patients following C-weekly. The presence of MRD predicted outcome in those patients achieving a CR and the most favourable outcome was seen in CR/MRD- patients (<CR/MRD+ 11 mns (95%CI 9-12 mns), <CR/MRD- 16 mns (95%CI 4-29 mns), CR/MRD+ 10 mns (95%CI 7-24 mns) and CR/MRD- 25 mns (95%CI 15-41 mns); logrank p<0.0001). [Display omitted] MRD status was also assessed according to cytogenetic risk group as defined by interphase FISH. Adverse cytogenetics were present in 19% of patients. Achieving MRD negativity was associated with an improved outcome in patients with both favourable and adverse cytogenetic profiles: Adverse; MRD+ 10 mns (95%CI 8-11 mns), MRD- 23 mns (95%CI 16-25 mns), Favourable; MRD+ 11 mns (95%CI 9-13 mns), MRD- 16mns (95%CI 10-NR mns); logrank p= 0.0058. ConclusionThese data clearly demonstrate that MRD assessment by MFC is a sensitive predictor of outcome in the first relapse setting, irrespective of the consolidation therapy given. MRD assessment predicts outcome in patients achieving CR and is applicable to patients with both favourable and adverse cytogenetics. This data is comparable to that published by us and others in the context of primary therapy and suggests that MRD assessment is an appropriate outcome measure in relapsed MM. Disclosures:Williams:Janssen: Honoraria, Speakers Bureau. Snowden:Janssen: Honoraria, Research Funding, Speakers Bureau.

Separation Of In Vitro-Derived Megakaryocytes and Platelets Using Spinning Membrane Filtration

In vitro-derived platelets could supplement volunteer donations for transfusions, while also overcoming problems associated with storage and patient alloimmunization. While platelets generated in vitro from hematopoietic stem and progenitor cells (HSPCs) exhibit similar functional capacity to normal peripheral blood platelets, further advances are needed for this technology to become clinically relevant. One challenge is how to efficiently purify platelets from their precursors, megakaryocytes (Mks), as well as other cell types that arise during heterogeneous differentiation of HSPCs in culture. Recent studies on platelet production from 2D culture have used successive centrifugation to pellet large cells, then platelets, but this multi-step method is subject to error-prone manual manipulation and is unlikely to generate a pure platelet population. As an alternative, we are developing a rapid, one-step approach to separate platelets from larger cells using spinning membrane filtration. Furthermore, because Mk maturation and subsequent platelet release have been shown to occur asynchronously in culture, we have explored how this separation technique could be used to separate platelets and cells at several time points during culture. In this way, one could harvest and prevent pre-activation of platelets generated early in culture, while reseeding immature Mks to generate proplatelets and platelets later in culture.To first characterize the method, 3 replicative experiments were performed wherein phorbol 12-myristate 13-acetate (PMA)-stimulated (up to 32N) CHRF megakaryoblastic cells were suspended in PAS-V platelet additive solution and mixed with apheresis platelets collected using the Amicus Separator. The mixture of CHRF cells and platelets was processed on a cell washing device consisting of a polycarbonate spinning membrane with 4-µm cylindrical pores. Cells that passed through the membrane (“platelet fraction”) or were retained by the membrane (“cell fraction”) were collected in separate containers. The separation process efficiently recovered platelets in the platelet fraction, while CHRF cells were excluded. Platelets recovered in the platelet fraction showed minimal pre-activation, and CHRF cells recovered in the cell fraction had a similar distribution of viable and apoptotic cells compared to the input population. The ploidy distribution of recovered CHRF cells was also very similar to the input population, suggesting that both smaller and larger cells were eluted from the membrane. Finally, when the recovered CHRF cells were transferred to an adhesive culture surface, they formed proplatelet-like structures similarly to unprocessed cells.To then apply the method, mobilized peripheral blood CD34+ HSPCs were differentiated to Mks, which formed proplatelets and released platelets in culture. The mixture of in vitro-derived Mks and platelets was then processed on the cell washing device. Similar to the experiments with the CHRF Mk line, primary Mks were excluded from the platelet fraction, and the ploidy distribution of the recovered Mks was similar to the input Mks. Recovered Mks formed fewer proplatelets when reseeded into culture and exhibited increased spreading on the culture surface compared to proplatelet-forming cells observed prior to processing. Since proplatelet formation has been shown to be negatively regulated by Mk contraction caused by engagement of activated integrins, we are exploring whether decreasing the shear rate experienced by Mks during processing may reduce integrin activation and improve proplatelet formation after reseeding. Platelet fraction recovery of CD41+CD42b+ in vitro-derived platelets was lower than that observed with apheresis platelets, and may be attributed to the fact that these larger preplatelets had greater difficulty passing through the 4-µm pores. Recovered in vitro-derived platelets spread in the presence of thrombin similarly to unprocessed in vitro-derived platelets. We are currently exploring how changes in the membrane pore size and shear rate may improve the platelet yield. [Display omitted] Disclosures:Radwanski:Fresenius Kabi: Employment. Wegener:Fresenius Kabi: Employment. Min:Fresenius Kabi: Employment.Pre-WashPost-Wash: Platelet fractionPost-Wash: Cell fractionPlatelets% Recovery---91 ± 48 ± 4% CD62P+, resting15 ± 813 ± 6---CHRF cells% Recovery---<0.174 ± 9% Viable50 ± 3---50 ± 7% Apoptotic29 ± 6---26 ± 7Mean ploidy6.3 ± 0.3---6.3 ± 0.6Table shows average ± SD for 3 experiments with apheresis platelets and CHRF cells.

Quality of red blood cells washed using an automated cell processor with and without irradiation

Sterile washing of red blood cells (RBCs) and use of an additive solution permits longer postwash storage. The effect of irradiation during this extended storage time is unclear. RBCs were washed 14 days after collection using an automated cell processor and stored in saline-adenine-glucose-mannitol. To determine how long washed and irradiated RBCs could be stored, units were irradiated 1, 4, 5, and 7 days after washing and in vitro quality was assessed. Determined limits of postwash storage time for washed and washed and irradiated RBCs were validated. Quality assessment included percent recovery, hemoglobin (Hb), hemolysis, extracellular K(+) , and adenosine triphosphate. Immunoglobulin (Ig)A levels were measured in the nonirradiated arm. RBCs irradiated 1 and 4 days after washing had unacceptably high hemolysis by Day 7 postwash, not meeting the acceptance criterion (<0.8% hemolysis in 98% of units with 95% confidence). Therefore, a 48-hour maximum storage time after irradiation was chosen. Storage limits tested in the validation phase were as follows: washing on Day 14 and subsequent storage for 7 days (washed RBCs) and washing on Day 14, irradiation on Day 19, and subsequent storage for 48 hours (washed and irradiated RBCs). All units met criteria for Hb, hematocrit, hemolysis, and sterility for washed RBCs. However, RBCs were washed with less than 2 L of saline, and IgA levels in 27 of 40 units were too high to be suitable for transfusion to IgA-deficient recipients. The extended expiry for washed and washed and irradiated RBCs met requirements for all indications except transfusion to IgA-deficient recipients.

Myocilin Mediates Myelination in the Peripheral Nervous System through ErbB2/3 Signaling

The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.

Development of Fluorogenic Probes for Quick No-Wash Live-Cell Imaging of Intracellular Proteins

We developed novel fluorogenic probes for no-wash live-cell imaging of proteins fused to PYP-tag, which is a small protein tag recently reported by our group. Through the design of a new PYP-tag ligand, specific intracellular protein labeling with rapid kinetics and fluorogenic response was accomplished. The probes crossed the cell membrane, and cytosolic and nuclear localizations of PYP-tagged proteins without cell washing were visualized within a 6-min reaction time. The fluorogenic response was due to the environmental effect of fluorophore upon binding to PYP-tag. Furthermore, the PYP-tag-based method was applied to the imaging of methyl-CpG-binding domain localization. This rapid protein-labeling system combined with the small protein tag and designed fluorogenic probes offers a powerful method to study the localization, movement, and function of cellular proteins.

High-Throughput Microfluidic Single-Cell Digital Polymerase Chain Reaction

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

A non-toxic fluorogenic dye for mitochondria labeling

BackgroundMitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases. MethodsA novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye. ResultsAcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7. ConclusionsA simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity. General SignificanceThe probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging.

Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine

Entamoeba histolytica, which causes amebic colitis and liver abscess, is considered a major enteric pathogen in residents and travelers to developing countries where the disease is endemic. Interaction of this protozoan parasite with the intestine is mediated through the binding of the trophozoite stage to intestinal mucin and epithelium via a galactose and N-acetyl-d-galactosamine (Gal/GalNAc) lectin comprised of a disulfide linked heavy (ca. 180kDa) and light chain (ca. 35kDa) and a noncovalently bound intermediate subunit (ca. 150kDa). Our efforts to develop a vaccine against this pathogen have focused on an internal 578 amino acid fragment, designated LecA, located within the cysteine-rich region of the heavy chain subunit because: (i) it is a major target of adherence-blocking antibodies of seropositive individuals and (ii) vaccination with his-tagged LecA provides protection in animal models. We developed a purification process for preparing highly purified non-tagged LecA using a codon-optimized gene expressed in Escherichia coli. The process consisted of: (i) cell lysis, collection and washing of inclusion bodies; (ii) solubilization and refolding of denatured LecA; and (iii) a polishing gel filtration step. The purified fragment existed primarily as a random coil with β-sheet structure, contained low endotoxin and nucleic acid, was highly immunoreactive, and elicited antibodies that recognized native lectin and that inhibited in vitro adherence of trophozoites to CHO cells. Immunization of CBA mice with LecA resulted in significant protection against cecal colitis. Our procedure yields sufficient amounts of highly purified LecA for future studies on stability, immunogenicity, and protection with protein-adjuvant formulations.