Washing Of Cells Research Articles

Multifunctional Supramolecular Assemblies with Aggregation-Induced Emission (AIE) for Cell Line Identification, Cell Contamination Evaluation, and Cancer Cell Discrimination.

It is undoubted the important role of cells in biology and medicine, but worldwide misidentified and cross-contaminated cell lines have caused much trouble in related fields. Herein, three kinds of supramolecular AIE (aggregation-induced emission) nanoassemblies were constructed by the host-guest interaction between tetraphenylethene (TPE) derivatives and cucurbit[8]uril (CB[8]). Based on the recognized mechanism of AIE, the TPE derivatives could achieve stronger fluorescence emission and higher fluorescence quantum yield after assembling with CB[8]. Moreover, the constructed supramolecular AIE complexes obtained well-confirmed nanostructures and exhibited different sizes and shapes. Correspondingly, they generated characteristic biological properties and fluorescence enhancement of cells. Inspired by the concept of Big Data Analysis, these fluorescence signals were further transformed into a unique fingerprint of cells via linear discriminant analysis. Immediately, we realized the veracious identification between a normal cell line, two cancer cell lines, and two metastasized cancer cell lines in a qualitative analysis. More importantly, it was well used to monitor the evaluation of cross-contaminated cells and the discrimination of cancer cells. As a proper bioapplication of ideal supramolecular nanomaterials, this system was easy to learn and apply, and the whole procedure was kept to 20 min, without cell disruption, centrifugation, or washing steps.

Rotea: a closed and automated instrument for efficient cell isolation, washing and conentration in cell therapy workflows

Background & Aim BACKGROUND: Processing and delivering cell and gene therapy products is of vital importance to patients and has presented numerous challenges. Rotea's counterflow centrifugation technology enables cell separation, washing, and concentration of many cell types and can be easily programmed to serve many additional workflows. The single use kit and instrument are able to capture and sustain billions of cells in a stable, fluidized bed with flexible inputs and output volumes as low as 5 mL. AIM We set out to evaluate Rotea's ability to isolate PBMCs from leukopaks, and wash and concentrate T cells, two common and vital processes in cell therapy workflows. We also investigated the input and output capabilities of the instrument. Methods, Results & Conclusion METHODS: Protocols were written using the Rotea Protocol Builder. Results from Rotea were compared to density gradient separation for PBMC isolations. T Cells for wash and concentrate protocols were expanded for 12 days in complete Optimizer media with Glutamax, L-Gluamine, HSA (Human Serum Albumin) and Il2. Before and after processing with Rotea, cells were counted and phenotyped using flow cytometry to determine the recovery and change in viability and marker expression. RESULTS Rotea performed consistent lymphocyte separation from leukapheresis products with greater than 80% recovery. Similar to manual isolation methods, cells isolated and cryopreserved using Rotea exhibit high viability and robust expansion in culture for over 14 days. Processed cells also maintain similar percentages of helper T Cells, cytotoxic T Cells and exhausted cells before and after processing. For T Cell wash and concentrate protocols, recovery was consistently above 90% and viability was virtually unchanged after processing. The distribution of CD4 and CD8 cells was also relatively unchanged. As many as 5 billion T cells were captured in a fluidized cell bed and outputs as low as 5 mL were performed without a decrease in cell recovery. CONCLUSION Rotea is a flexible, efficient system that can isolate cells and perform wash and concentrate protocols with high recovery of cells and maintenance of viability. Cells that undergo Rotea processing maintain their phenotype and readily expand in culture. Rotea has the ability to support multiple cell therapy workflows and the scalability to take a therapy from process development to manufacturing.

Acoustic Affinity Cell Selection: a non-paramagnetic scalable technology for T cell selection from unprocessed apheresis products

Background & Aim Acoustic Cell Processing is a unique acousto-fluidics platform technology for minimal shear manipulation of cells using ultrasonic standing waves. The platform has broad applications in the field of cell and gene therapy, e.g., cell concentration and washing, cell culturing, microcarrier/cell separation, acoustic affinity cell selection and label-free cell selection. The acoustic radiation force exerted by the ultrasonic standing wave on the suspended cells in combination with fluid drag forces and gravitational forces is used to manipulate the cells and achieve a certain cell processing unit operation, e.g., separate, concentrate, or wash. The technology is single-use, continuous, and can be scaled up, down or out. It therefore allows for a flexible and modular approach that can be customized to process a desired cell count, cell culture volume or cell concentration within a given required process time. Utilizing its proprietary multi-dimensional standing wave platform, MilliporeSigma has been developing the Acoustic Affinity Cell Selection (AACS) system for closed and automated Cell and Gene Therapy manufacturing, e.g., CAR-T immunocellular therapies. The AACS technology is a scalable acoustic affinity cell selection method using acoustic (non-paramagnetic) affinity beads for positive or negative cell selection. A multi-dimensional acoustic standing wave is then used to separate the affinity bead-cell complexes from the unbound cells, thereby completing the process of a negative or positive cell selection. Methods, Results & Conclusion In this work the AACS system has been used to capture CD4+ and CD8+ cells from unprocessed apheresis products. The AACS column and acoustic section (Fig. 1A) allow for a continuous flow of the initial cell population (pre-labeled with biotinylated antibodies) while acoustically retaining the acoustic affinity particles in the column. The affinity particles are functionalized with Neutravidin and thus capture the target cells that are kept inside the column, while the non-target cells are washed out of the column. The small scale 5mL column has a total target cell capture capacity of 1.8B cells (60mL Leukopak volume), of which 83% are retained in the column at a 92% purity (Fig. 1B). The AACS scale up system can process a full apheresis product (300-450mL) in less than 2h.

Transfusion Use and Hemoglobin Levels by Blood Conservation Method After Cardiopulmonary Bypass

Guidelines recommend modified ultrafiltration (MUF) and cell washing for blood conservation after cardiopulmonary bypass (CPB), although information on outcomes is lacking. This research compared online MUF (ultrafiltration of the patient's entire circulating volume) with off-line MUF (ultrafiltration of the residual CPB volume) and centrifugation (cell washing of the residual CPB volume). This prospective cohort study enrolled 99 consecutive patients, grouped by method (group I, online MUF, n= 35; group II, off-line MUF, n= 30; group III, centrifugation, n= 34). Primary outcome was transfusion by 18 hours. Secondary outcomes were 18-hour hemoglobin levels, fluid balance (weight change), and biomarker levels indicating coagulation and organ function. By 18 hours, 22.9%, 6.7%, and 14.7% of group I, II, and III patients, respectively, had undergone transfusion (P= .19). Percentage weight gain differed by group (group I, 5.7%; group II, 1.3%; group III, 4.5%; P<.0001). Baseline to 18-hour hemoglobin change also differed by group, with the group I increase significantly exceeding that of group II (P= .002) but not differing from group III (P= .36). After adjustment for European System for Cardiac Operative Risk Evaluation II (EuroSCORE), weight gain, and transfusion, only the group II to III difference remained significant (P= .002). Online MUF does not appear to offer a reduction in blood transfusion over other methods. Although patients undergoing online MUF had greater improvement in baseline to 18-hour hemoglobin compared with patients undergoing off-line MUF, this benefit appeared attributable to fluid shifting. Off-line MUF was associated with the least frequent transfusions. Although online MUF does not appear to reduce blood transfusion, larger prospective randomized controlled studies are required for confirmation.

Comparison of cell salvage with one and two suction devices during cesarean section in patients with placenta previa and/or accrete: a randomized controlled trial.

BackgroundCell salvage has recently been recommended for obstetric use in cases with a high risk of massive hemorrhage during cesarean section (CS). However, limited data are available to support the use of one suction device to collect lost blood. This study aimed to investigate the volume of red blood cells (RBCs) salvaged and the components of amniotic fluid (AF) in blood salvaged by one suction device or two devices during CS in patients with placenta previa and/or accrete.MethodsThirty patients with placenta previa and/or accrete undergoing elective CS in the Women's Hospital of Zhejiang University School of Medicine were recruited for the present study from November 1, 2017 to December 1, 2018. The patients were randomly assigned to one of the two groups according to an Excel-generated random number sheet: Group 1 (n = 15), in which only one suction device was used to aspirate all blood and AF, and Group 2 (n = 15), in which a second suction device was mainly used to aspirate AF before the delivery of the placenta. Three samples of blood per patient (pre-wash, post-wash, and post-filtration) were collected to measure AF components. The salvaged RBC volumes were recorded. Continuous data of pre-wash, post-wash, and post-filtration samples were analyzed by using one-way analysis of variance with Tukey's test for multiple comparisons, or Kruskal-Wallis test with Dunn test for multiple comparisons. Comparisons of continuous data between Group 1 and Group 2 were conducted using Student's t test or Mann-Whitney U test.ResultsThe salvaged RBC volume was significantly higher in Group 1 than that in Group 2 (401.6 ± 77.2 mL vs. 330.1 ± 53.3 mL, t = 4.175, P < 0.001). In both groups, squamous cells, lamellar bodies, and fat were significantly reduced by washing (all P<0.001) and squamous cells were further reduced by filtering (P < 0.001). Squamous cells were found in six post-filtration samples (three from each group). Lamellar bodies and fat were completely removed by filtering. Insulin-like growth factor binding protein 1, alpha-fetoprotein, albumin, lactate dehydrogenase, and potassium were significantly reduced post-wash (all P < 0.05), with no further significant reduction after filtration in either group (all P > 0.05). The mean percentage of fetal RBCs post-filtration was (1.8 ± 0.8)% with a range of 1.0% to 3.5% and (1.9 ± 0.9)% with a range of 0.7% to 4.0% in Groups 1 and 2, respectively, showing no significant difference between the two groups (U = 188.5, P = 0.651).ConclusionCell salvage performed by one suction device could result in higher volume of salvaged RBCs and can be used safely for CS in patients with placenta previa and/or accrete when massive hemorrhage occurs.Trial registration numberChiCTR-INR-17012926, http://www.chictr.org.cn/ Chinese Clinical Trial Registry.

Abstract TP460: Regulatory T-Cells and CD4+ T Helper Cell Subsets Are Differentially Regulated and Express Distinct Activation States After Aneurysmal Subarachnoid Hemorrhage (SAH) and During Post-SAH Complications

Background: Aneurysmal subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. Devastating post-SAH complications after aneurysm treatment lead to poor clinical outcome. Current research suggests critical role of inflammation during early and delayed brain injury phases over which these complications arise. T helper cells can polarize to multiple functionally unique subsets. Here, we investigate different CD4+ T cell subsets during these brain injury phases after SAH and their dynamics during post-SAH complications. Methods: Anticoagulated peripheral venous blood was obtained from 15 SAH patients on days 1 and 7, and once from healthy controls. After erythrocyte lysis and single cell wash, 1 million cells were stained with different anti-human mouse monoclonal antibodies and were acquired on BD LSR Fortessa. Lymphocytes were gated based on low side scatter and high CD45 expression. Different CD3+CD4+ T cell subsets were characterized by differential cell surface expression of CXCR3 and CCR6 into Th1, Th2, Th17, whereas Tregs by CD25 hi and CD127 lo . SAH patients were dichotomized based on presence or absence of different post-SAH complications (hydrocephalus, seizures, CVS, cerebral ischemia) to assess association of T cell subsets with these complications. Results: Total CD4+ T cells were significantly increased after SAH. Interestingly, Th2 cells were significantly decreased and Th17 cells increased on day 7 compared to day 1 after SAH. However, regulatory T-cells were significantly increased on both assessment days compared to controls. The analysis of activation states was done by CD38 and HLA-DR expression. Th1 and Treg cells were significantly increased on day 1 in SAH patients who developed seizures and CVS, respectively. HLA-DR + CD38 + Th2 cells significantly increased on day 1 in SAH patients who developed hydrocephalus, whereas HLA-DR - CD38 - Th1 cells significantly increased on day 1 in patients with infections. HLA-DR - CD38 - Treg cells were significantly reduced on day 1 and day 7 in patients developing cerebral ischemia . Conclusion: CD4+ T cell subsets and Treg cells display dynamic patterns after SAH, and show a distinct pattern of polarization and activation states in specific post-SAH complications.

自動血球洗浄装置「セルウォッシャーUltraCW II」の基礎的性能評価

自動血球洗浄装置「セルウォッシャーUltraCW II」の基礎的性能評価

Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable.Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes.Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells.Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

Structural investigation of the capsular polysaccharide from a clinical isolate of Fusobacterium necrophorum subspecies necrophorum biotype a strain LA 81–617

A capsular polysaccharide (CPS) from Fusobacterium necrophorum subspecies necrophorum biotype A strain LA 81–617 was isolated from a saline cell wash and purified by gel and anion-exchange chromatography. The structure of the CPS was studied by one- and two-dimensional 1H and 13C NMR spectroscopy techniques in combination with electrospray ionization mass spectrometry (ESI-MS). The CPS was found to resemble the bacterial cell-wall murein polysaccharide backbone, consisting of N-acetylglucosamine and N-acetylmuramic acid (MurNAc) components, with the following structure:-[-4-β-MurNAc-4-β-GlcNAc-]n-with N-acetyl-1,6-anhydro-β-muramic acid at the reducing end. This is the first report on the structure of F. necrophorum capsular polysaccharide.

Statistical validation of microcells reuse for sulfur determination in diesel fuel and gasoline–ethanol blends by MWDXRF spectrometry

The authors’ aims is to evaluate the reuse of microcells in a test method for sulfur in diesel fuel and gasoline–ethanol blends by monochromatic wavelength dispersive X-ray fluorescence spectrometry. An experimental procedure has been developed for cleaning and reassembling the used cells. Results derived from new cells were compared with those from after the first washing and after the second washing cells. Based on paired t-test, no statistical differences were observed between the data sets, validating the proposed procedure, besides that the recycled cells overcome the commercial ones in term of cost.

An acute lytic cell death induced by xanthohumol obstructed ROS detecting in HL-60 cells

Serum is an important component in cell culture medium. It also possesses potent antioxidant properties. Therefore, the conventional protocols for detecting reactive oxygen species (ROS) in cultured cells with fluorescent probes include washing and suspending cells with serum-free buffers, such as PBS. This transient serum deprivation is essential for the ROS detecting. Unfortunately, it may also cause unexpected results, which push us to choose more optimal experiment conditions. In the present study, we found an acute lytic cell death induced by xanthohumol (XN), which obstructed ROS detecting in human leukemia cell line HL-60 cells. XN induced ROS burst, caused cell swelling, membrane permeability increase, LDH release, and ultimately an acute lytic cell death and cell rupture. These effects could be alleviated by the antioxidant N-Acetyl-L-cysteine (NAC). Apoptosis, pyroptosis or necroptosis were not observed in this process. Results also indicated that 2% serum addition had already completely scavenged ROS induced by 10 μM XN. Taken together, it is strongly suggested to detecting ROS in a serum-free medium when studying where and how ROS generated in cells. The concentration at the ROS maximum point (10 μM XN in this study) can be selected as the optimal concentration.

DNA Purification Cell Lysis and Wash Step Modifications for Low-Template DNA Sample Processing.

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low-template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re-extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low-template DNA sample processing are not enough to justify the risk associated with additional manipulation.

Streptococcal Pyrogenic Exotoxin A-Stimulated Monocytes Mediate Regulatory T-Cell Accumulation through PD-L1 and Kynurenine

Bacterial superantigens (SAgs) are exotoxins that promote a fulminant activation of the immune system. The subsequent intense release of inflammatory cytokines often results in hypotension, shock, and organ failure with high mortality rates. In the current paradigm, the direct and simultaneous binding of SAgs with T-cell receptor (TCR)-bearing Vβ regions and conserved structures on major histocompatibility complex class II (MHC class II) on antigen-presenting cells (APCs) induces the activation of both cell types. However, by crosslinking MHC class II molecules, APCs can be activated by SAgs independently of T lymphocytes. Recently, we showed that streptococcal pyrogenic exotoxin A (SPEA) of Streptococcus pyogenes stimulates an immunogenic APC phenotype with upregulated costimulatory molecules and inflammatory cytokines. Additionally, we revealed that SPEA triggers immunosuppressive programs in monocytes that facilitate the accumulation of regulatory T cells (Tregs) in in vitro monocyte/CD4+ T-cell cocultures. Immunosuppressive factors include anti-inflammatory interleukin 10 (IL-10), co-inhibitory surface molecule programmed cell death 1 ligand 1 (PD-L1), and the inhibitory indoleamine 2,3-dioxygenase (IDO)/kynurenine effector system. In the present study, we investigated the underlying mechanism of SPEA-stimulated monocyte-mediated accumulation of Tregs. Blood-derived monocytes from healthy donors were stimulated with SPEA for 48 h (SPEA-monocytes). For the evaluation of SPEA-monocyte-mediated modulation of CD4+ T lymphocytes, SPEA was removed from the culture through extensive washing of cells before adding allogeneic CD3/CD28-activated T cells. Results: In coculture with allogeneic CD4+ T cells, SPEA-monocytes mediate apoptosis of CD4+Foxp3− lymphocytes and accumulation of CD4+Foxp3+ Tregs. PD-L1 and kynurenine are critically involved in the mediated cell death because blocking both factors diminished apoptosis and decreased the proportion of the CD25+/Foxp3+ Treg subpopulation significantly. Upregulation of PD-L1 and kynurenine as well as SPEA-monocyte-mediated effects on T cells depend on inflammatory IL-1β. Our study shows that monocytes activated by SPEA mediate apoptosis of CD4+Foxp3− T effector cells through PD-L1 and kynurenine. CD4+Foxp3+ T cells are resistant to apoptosis and accumulate in SPEA-monocyte/CD4+ T-cell coculture.

A Crude Extract Preparation and Optimization from a Genomically Engineered Escherichia coli for the Cell-Free Protein Synthesis System: Practical Laboratory Guideline.

With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in vitro protein synthesis environment by escaping a physical living boundary. The cell extract plays an essential role in this in vitro format. Here, we propose a practical protocol and method for Escherichia coli-derived cell extract preparation and optimization, which can be easily applied to both commercially available and genomically engineered E. coli strains. The protocol includes: (1) The preparation step for cell growth and harvest, (2) the thorough step-by-step procedures for E. coli cell extract preparation including the cell wash and lysis, centrifugation, runoff reaction, and dialysis, (3) the preparation for the CFPS reaction components and, (4) the quantification of cell extract and cell-free synthesized protein. We anticipate that the protocol in this research will provide a simple preparation and optimization procedure of a highly active E. coli cell extract.

Successful management of catastrophic peripheral vascular hemorrhage using massive autotransfusion and damage control surgery in a dog

To describe a case of massive transfusion using unwashed, non-anticoagulated, nonsterile autologous blood in a dog with catastrophic hemorrhage from a peripheral vessel during orthopedic surgery. A damage control surgical strategy was also employed. A 6-year-old, 48kg neutered male Labrador Retriever experienced massive hemorrhage after transection of a large blood vessel while undergoing femoral head and neck osteotomy. Blood was collected from clean, but not sterile, suction canisters and clots were skimmed off. The blood was then transfused back to the dog using a standard in-line blood filter. Approximately 58% of the dog's blood volume was autotransfused in less than 2 hours, thereby meeting the criteria for massive transfusion. Surgery was aborted after hemostasis was achieved by ligation of the vessel and packing of the surgical site. Two units of fresh frozen plasma were administered postoperatively due to the development of a coagulopathy. Hemoglobinuria developed but resolved within 18 hours. Three days later, completion of the surgical procedure was performed without incident. The dog was discharged 4 days after the initial surgery. Marked swelling of the affected limb developed, but resolved after the sixth day. No other significant complications developed. In this case report, the authors describe the successful management of catastrophic hemorrhage with autotransfusion performed in the absence of sterile collection, cell washing, or anticoagulation. Although not ideal, autotransfusion under these conditions can be lifesaving in situations of massive hemorrhage. This case also highlighted the employment of a damage control surgical strategy.

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

Acoustofluidic separation of cells and particles

Acoustofluidics, the integration of acoustics and microfluidics, is a rapidly growing research field that is addressing challenges in biology, medicine, chemistry, engineering, and physics. In particular, acoustofluidic separation of biological targets from complex fluids has proven to be a powerful tool due to the label-free, biocompatible, and contact-free nature of the technology. By carefully designing and tuning the applied acoustic field, cells and other bioparticles can be isolated with high yield, purity, and biocompatibility. Recent advances in acoustofluidics, such as the development of automated, point-of-care devices for isolating sub-micron bioparticles, address many of the limitations of conventional separation tools. More importantly, advances in the research lab are quickly being adopted to solve clinical problems. In this review article, we discuss working principles of acoustofluidic separation, compare different approaches of acoustofluidic separation, and provide a synopsis of how it is being applied in both traditional applications, such as blood component separation, cell washing, and fluorescence activated cell sorting, as well as emerging applications, including circulating tumor cell and exosome isolation.

PF779 VIABILITY OF HEMATOPOETIC STEM CELLS FOLLOWING STORAGE AT −80OC WITH 5% DIMETHYLSULFOXIDE AND HEMATOLOGIC RECOVERY IN TRANSPLANTED MYELOMA PATIENTS

Background:Dimethylsulfoxide (DMSO) is a major intracellular cryoprotectant, used for cryopreservation of stem cells. It is toxic to both cells and patients at temperatures above 0oC. Reduction of this effect is achieved by either washing of cells after thawing or by reduction of DMSO during freezing and storage. The latter requires addition of extracellular cryoprotectants to the freezing media.Aims:We assessed the effect of low DMSO concentration and different hematocrits of the frozen cells on cell viability and hematologic recovery in patients, transplanted for multiple myeloma.Methods:Cells were non‐programmed frozen and stored at −80oC in a cryoprotectant solution achieving final concentrations of 5% DMSO, 3.6% of hydroxyethyl starch (HES, Weight average molecular weight 450 000 Da) and 3% of human serum albumin. The cell concentration in the frozen product for the first 77 patients (84 transplantations) varied between 100x106 and 250x106 cells/ml. In an attempt to reduce the amount of DMSO infused, for the rest of the patients (n = 172; 192 transplantations) we further decreased the volume of the freezing suspension by removal of the entire plasma.The average age of the transplanted patients was 55 (35 ‐ 71). The cells were bedside thawed at 37oC water bath. The average cell dose was 2,95x106/kg (1,3 – 9,2x106/kg).Results:Viability of the stem cells following thawing assessed by trypan blue exclusion was 95,34% (70–99). The hematocrit of the frozen cells had no effect on cell viability (94,80%(low) vs 95,52%(high)). The major complaints, if any, during stem cell infusion were coughing and an increase in nausea and vomiting induced by the prior conditioning.The average time for hematological recovery was 11,66 days (between 9 and 19) for the neutrophils, and 12,17 (between 9 and 20) days for the platelets. There was no significant difference in viability and hematologic recovery (11,86 and 12,60 vs 11,59 and 12,02) between patients receiving cells frozen with low or high hematocrit.Summary/Conclusion:Dimethylsulfoxide, despite its cryoprotective properties, is toxic for stem cells at temperatures above zero C and induces many side effects (cardiac, neurologic, respiratory, etc.) in the patients. To reduce those side effects we use lower DMSO concentration, high hematocrit resulting in lower volume of the frozen cell suspension, thus reducing the final quantity of DMSO to be infused to the patients. This does not affect the cell viability or the hematologic recovery of patients after transplantation. Our easily performed method for unprogrammed freezing of stem cells with final DMSO concentration 5% at −80oC is safe, well tolerated, and provides cryopreservation, which allows high viability and stable cell engraftment, while reducing the undesired side effects of DMSO.

Development of an automated on-line purification HPLC single cell-ICP-MS approach for fast diatom analysis

The most challenging part in performing a single cell ICP-MS (sc-ICP-MS) approach is the sample preparation, in particular the reduction of the ionic background. This step is, in many cases, time-consuming and required for each sample separately. Furthermore, sc-ICP-MS measurements are mostly carried out “manually”, given the fact that present systems are not allowing for an automated change of samples. Thus, within this work, we developed an approach based on a HPLC system coupled on-line with sc-ICP-MS via a set of switching valves as well as an in-line filter for automated cell washing. This set-up enables the ionic background removal as well as analysis of single cells completely automated without any manual sample pretreatment. Our approach was applied for the analysis of the single celled diatom species Cyclotella meneghiniana, a marine diatom species, on the basis of 24Mg and facilitates testing in 11 min per sample, requiring only around 10,000 cells in a volume of 10 μL and approx. 10 mL of a 5% MeOH/95% deionized water (v/v) mixture. Even at extremely saline culturing media concentrations (up to 1000 mg L−1 magnesium) our on-line approach worked sufficiently allowing for distinction of ionic and particulate fractions. Furthermore, a set of diatom samples was analyzed completely automated without the need for changing samples manually. So, utilizing this approach enables analyzing a high quantity of samples in a short time and therefore in future the investigation of ecotoxicological effects is simplified for example in terms of metal accumulation by taking biovariability into account.

Closed and automated cell processing using roteaTM to advance cell therapy

Background & Aim The manufacturing cost and complexity has been one of the biggest obstacles for delivering cell therapy products. Here we present RoteaTM, a compact closed automation system with counterflow centrifuge technology, which enables gentle cell separation, wash, and concentration with controllable input and output volumes. The 10ml chamber on the single use kit can sustain billions of cells as a fluidized bed and can be discharged multiple times if needed. The kit can be easily connected to other closed systems for upstream and downstream applications, for example Finesse bioreactor system, making Rotea a flexible modular instrument to fit in different workflows. Methods, Results & Conclusion Using human primary T cells as a model, we have demonstrated that across different input and output settings, Rotea contributes superior processing time, recovery, and viability for cell wash, concentration, and buffer exchange as compared to manual centrifugation. More importantly, Rotea is capable of facilitating lymphocyte separation from leukopheresis products, as well as bead removal after expansion, and therefore could shorten manufacturing time of cell therapy products.