RESUMO
During the mitotic cycle, the rod-shaped fission yeast cells grow only at their tips. The newly born cells grow first unipolarly at their old end, but later in the cycle, the 'new end take-off' event occurs, resulting in bipolar growth. Photographs were taken of several steady-state and induction synchronous cultures of different cell cycle mutants of fission yeast, generally larger than wild type. Length measurements of many individual cells were performed from birth to division. For all the measured growth patterns, three different functions (linear, bilinear and exponential) were fitted, and the most adequate one was chosen by using specific statistical criteria, considering the altering parameter numbers. Although the growth patterns were heterogeneous in all the cultures studied, we could find some tendencies. In cultures with sufficiently wide size distribution, cells large enough at birth tend to grow linearly, whereas the other cells generally tend to grow bilinearly. We have found that among bilinearly growing cells, the larger they are at birth, the rate change point during their bilinear pattern occurs earlier in the cycle. This shifting near to the beginning of the cycle might finally cause a linear pattern, if the cells are even larger. In all of the steady-state cultures studied, a size control mechanism operates to maintain homeostasis. By contrast, strongly oversized cells of induction synchronous cultures lack any sizer, and their cycle rather behaves like an adder. We could determine the critical cell size for both the G1 and G2 size controls, where these mechanisms become cryptic. TAKE AWAY: Most individual fission yeast cells in steady-state cultures grow bilinearly. In strongly oversized fission yeast cells, linear growth dominates over bilinear. Above birth length thresholds, both the G1 and G2 size controls become cryptic.
Assuntos
Tamanho Celular , Mitose , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Homeostase , Mutação , Schizosaccharomyces/genéticaRESUMO
BACKGROUND: The isolation of lymphocytes - and removal of platelets (PLTs) and red blood cells (RBCs) - from an initial blood sample prior to culture is a key enabling step for effective manufacture of cellular therapies. Unfortunately, currently available methods suffer from various drawbacks, including low cell recovery, need for complex equipment, potential loss of sterility and/or high materials/labor cost. METHODS: A newly developed system for selectively concentrating leukocytes within precisely designed, but readily fabricated, microchannels was compared with conventional density gradient centrifugation with respect to: (i) ability to recover lymphocytes while removing PLTs/RBCs and (ii) growth rate and overall cell yield once expanded in culture. RESULTS: In the optimal embodiment of the new microfluidic approach, recoveries of CD3+, CD19+ and CD56+ cells (85%, 89% and 97%, respectively) were significantly higher than for paired samples processed via gradient-based separation (51%, 53% and 40%). Although the removal of residual PLTs and RBCs was lower using the new approach, its enriched T-cell fraction nevertheless grew at a significantly higher rate than the gradient-isolated cells, with approximately twice the cumulative cell yield observed after 7 days of culture. DISCUSSION: The standardization of each step of cellular therapy manufacturing would enable an accelerated translation of research breakthroughs into widely available clinical treatments. The high-throughput approach described in this study - requiring no ancillary pumping mechanism nor expensive disposables to operate - may be a viable candidate to standardize and streamline the initial isolation of lymphocytes for culture while also potentially shortening the time required for their expansion into a therapeutic dose.
Assuntos
Separação Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Filtração/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Linfócitos T/citologia , Transferência Adotiva/métodos , Plaquetas/citologia , Contagem de Células , Sobrevivência Celular , Células Cultivadas , Eritrócitos/citologia , HumanosRESUMO
Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA below the recommended levels, hence double washing is frequently required. Here, we describe a prototype of a simple, portable, disposable system capable of washing stored RBCs without centrifugation, while reducing IgA below 0.05 mg/dL in a single run. Samples from RBC units (n = 8, leukoreduced, 4-6 weeks storage duration) were diluted with normal saline to a hematocrit of 10%, and then washed using either the prototype washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics. The prototype of the washing system was able to process stored RBCs at a rate of 300 mL/hour, producing a suspension of washed RBCs with 43 ± 3% hematocrit and 86 ± 7% cell recovery. Overall, the two washing methods performed similarly for most measured parameters, lowering the concentration of free hemoglobin by >4-fold and total free protein by >10-fold. Importantly, the new washing system reduced the IgA level to 0.02 ± 0.01 mg/mL, a concentration 5-fold lower than that produced by conventional centrifugation. This proof-of-concept study showed that centrifugation may be unnecessary for washing stored RBCs. A simple, disposable, centrifugation-free washing system could be particularly useful in smaller medical facilities and resource limited settings that may lack access to centrifugation-based cell processors.
Assuntos
Preservação de Sangue/métodos , Centrifugação , Eritrócitos , Imunoglobulina A/sangue , Preservação de Sangue/instrumentação , Deformação Eritrocítica , Hemoglobinas/análise , Humanos , Hidrogéis , Membranas Artificiais , Estudo de Prova de ConceitoAssuntos
Preservação de Sangue/métodos , Eritrócitos/citologia , Hemoglobinas/isolamento & purificação , Potássio/isolamento & purificação , Centrifugação , Transfusão de Eritrócitos/métodos , Transfusão de Eritrócitos/normas , Hemoglobinas/análise , Humanos , Métodos , Potássio/sangue , Fatores de TempoRESUMO
Millions of blood components including red blood cells, platelets, and granulocytes are transfused each year in the United States. The transfusion of these blood products may be associated with adverse clinical outcomes in some patients due to residual proteins and other contaminants that accumulate in blood units during processing and storage. Blood products are, therefore, often washed in normal saline or other media to remove the contaminants and improve the quality of blood cells before transfusion. While there are numerous methods for washing and volume reducing blood components, a vast majority utilize centrifugation-based processing, such as manual centrifugation, open and closed cell processing systems, and cell salvage/autotransfusion devices. Although these technologies are widely employed with a relatively low risk to the average patient, there is evidence that centrifugation-based processing may be inadequate when transfusing to immunocompromised patients, neonatal and infant patients, or patients susceptible to transfusion-related allergic reactions. Cell separation and volume reduction techniques that employ centrifugation have been shown to damage blood cells, contributing to these adverse outcomes. The limitations and disadvantages of centrifugation-based processing have spurred the development of novel centrifugation-free methods for washing and volume reducing blood components, thereby causing significantly less damage to the cells. Some of these emerging technologies are already transforming niche applications, poised to enter mainstream blood cell processing in the not too distant future.
RESUMO
BACKGROUND: The use of centrifugation-based approaches for processing donated blood into components is routine in the industrialized world, as disparate storage conditions require the rapid separation of 'whole blood' into distinct red blood cell (RBC), platelet, and plasma products. However, the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. The objective of this study was to evaluate a proof-of-concept system for whole blood processing, which does not employ electromechanical parts, is easily portable, and can be operated immediately after donation with minimal human labor. METHODS AND FINDINGS: In a split-unit study (n = 6), full (~500mL) units of freshly-donated whole blood were divided, with one half processed by conventional centrifugation techniques and the other with the new blood separation system. Each of these processes took 2-3 hours to complete and were performed in parallel. Blood products generated by the two approaches were compared using an extensive panel of cellular and plasma quality metrics. Comparison of nearly all RBC parameters showed no significant differences between the two approaches, although the portable system generated RBC units with a slight but statistically significant improvement in 2,3-diphosphoglyceric acid concentration (p < 0.05). More notably, several markers of platelet damage were significantly and meaningfully higher in products generated with conventional centrifugation: the increase in platelet activation (assessed via P-selectin expression in platelets before and after blood processing) was nearly 4-fold higher for platelet units produced via centrifugation, and the release of pro-inflammatory mediators (soluble CD40-ligand, thromboxane B2) was significantly higher for centrifuged platelets as well (p < 0.01). CONCLUSION: This study demonstrated that a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation-particularly for applications in remote or resource-limited settings, or for patients requiring highly functional platelet product.
Assuntos
Doadores de Sangue , Sangue , Manejo de Espécimes , Centrifugação , HumanosRESUMO
BACKGROUND AND PURPOSE: Iodinated contrast media (Xenetix®, Ultravist®, Omnipaque®, Visipaque® and Iomeron®) used for computed tomography (CT) may decrease fibrinolysis by recombinant tissue plasminogen activator (rt-PA). We hypothesized that receiving iodinated contrast media before rt-PA may impair thrombolysis as measured by a new model system. METHODS: Whole blood from Wistar Kyoto rats (nâ=â10) was obtained and allowed to form blood clots. Thrombolysis was performed by placing individually the prepared clots into 15âmL tubes and adding 5âmL saline buffer, 100µg rt-PA and a different contrast media; adjusting the quantity of iodine to either 30âmg or 60âmg. The thrombolytic efficacy was quantified by measuring the optical density (OD415) of the supernatant at different time points, namely at 0, 30, 60, and 90âmin. RESULTS: There was a significant decrease in clot lysis efficiency observed in presence of iodine containing contrast media comparing to positive control group. Moreover, when the quantity of iodine was increased from 30âmg to 60âmg; the dissolution rate downturned with additional â¼50%. CONCLUSION: In conclusion, our study suggests that high dose of iodine potentially could negatively affect the efficiency of the thrombolytic therapy performed by rt-PA.
Assuntos
Angiografia por Tomografia Computadorizada/métodos , Meios de Contraste/farmacologia , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Meios de Contraste/uso terapêutico , Fibrinolíticos , Humanos , Masculino , Ratos , Ativador de Plasminogênio Tecidual/uso terapêuticoRESUMO
BACKGROUND: During hypothermic storage, a substantial fraction of red blood cells (RBCs) transforms from flexible discocytes to rigid sphero-echinocytes and spherocytes. Infusion of these irreversibly-damaged cells into the recipient during transfusion serves no therapeutic purpose and may contribute to adverse outcomes in some patients. In this proof-of-concept study we describe the use of hypotonic washing for selective removal of the irreversibly-damaged cells from stored blood. MATERIALS AND METHODS: Stored RBCs were mixed with saline of various concentrations to identify optimal concentration for inducing osmotic swelling and selective bursting of spherical cells (sphero-echinocytes, spherocytes), while minimising indiscriminate lysis of other RBCs. Effectiveness of optimal treatment was assessed by measuring morphology, rheological properties, and surface phosphatidylserine (PS) exposure for cells from several RBCs units (n=5, CPD>AS-1, leucoreduced, 6 weeks storage duration) washed in hypotonic vs isotonic saline. RESULTS: Washing in mildly hypotonic saline (0.585 g/dL, osmolality: 221.7±2.3 mmol/kg) reduced the fraction of spherical cells 3-fold from 9.5±3.4% to 3.2±2.8%, while cutting PS exposure in half from 1.48±0.86% to 0.59±0.29%. Isotonic washing had no effect on PS exposure or the fraction of spherical cells. Both isotonic and hypotonic washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) substantially, and improved the ability of stored RBCs to perfuse an artificial microvascular network by approximately 25%, as compared with the initial sample. DISCUSSION: This study demonstrated that washing in hypotonic saline could selectively remove a significant fraction of the spherical and PS-exposing cells from stored blood, while significantly improving the rheological properties of remaining well-preserved RBCs. Further studies are needed to access the potential effect from hypotonic washing on transfusion outcomes.