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author:("Sun, shijie")
1.  Effect of the Polydispersity of RBCs on the Recovery Rate of RBCs during the Removal of CPAs 
In the process of removing cryoprotectants from cryopreserved blood, the theoretically optimal operating condition, which is based on the assumption that the distribution of red blood cells is uniform, is often used to reduce or even avoid the hypotonic damage to cells. However, due to the polydispersity of cells, the optimal condition is actually not reliable. In this study, based on the discrete concept developed in our previous work, the effect of the polydispersity on the recovery rate of cells in the dilution-filtration system was statistically investigated by assigning three random parameters, isotonic cell volume, cell surface area, and osmotically inactive cell volume, to cells in small units of blood. The results show that, due to the polydispersity, the real recovery rate deviates from the ideal value that is based on uniform distribution. The deviation significantly increases with the standard errors of cell parameters, and it can be also magnified by high cryoprotectant concentrations. Under the effect of polydispersity, the uniform distribution-based optimized blood or diluent flow rate is not perfect. In practice, one should adopt a more conservative blood or diluent flow rate so that the hypotonic damage to cells can be further reduced.
PMCID: PMC4279270  PMID: 25580156
2.  Theoretical optimization of the removal of cryoprotective agents using a dilution-filtration system 
In the cryopreservation of blood, removing cryoprotectants from the cryopreserved blood safely and effectively is always being focused on. In our previous work, a dilution-filtration system was proposed to achieve the efficient clearance of cryoprotectants from the cryopreserved blood.
In this study, a theoretical method is presented to optimize the diluent flow rate in the system to further reduce the osmotic damage to red blood cells (RBCs) and shorten the washing time necessary to remove cryoprotective agents (CPAs), based on a discrete mass transfer concept. In the method, the diluent flow rate is automatically adjusted by a program code in each cycle to maximize the clearance of CPAs, whereas the volume of RBCs is always maintained below the upper volume tolerance limit.
The results show that the optimized diluent flow rate can significantly decrease the washing time of CPAs. The washing time under the optimized diluent flow rate can be reduced by over 50%, compared to the one under the fixed diluent flow rate. In addition, the advantage of our method becomes more significant when the blood flow rate is lower, the dilution region volume is larger, the initial CPA concentration is higher, or the cell-swelling limit set by the system is smaller.
The proposed method for the dilution-filtration system is an ideal solution for not only guaranteeing the volume safety of RBCs but also shortening the washing time of CPAs. In practice, the optimization strategies provided here will be useful in the rapid preparation of cryopreserved blood for clinical use.
PMCID: PMC4148939  PMID: 25145611
Mass transfer; Cryoprotective agent; Osmotic damage; Red blood cell
3.  Distinct Functional Effects for Dynamin 3 During Megakaryocytopoiesis 
Stem Cells and Development  2011;20(12):2139-2151.
Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41+ and CD61+ MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.
PMCID: PMC3225063  PMID: 21671749
4.  A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures 
Biopreservation and Biobanking  2011;9(4):355-362.
Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs. To obtain these unknown parameters, membrane transport properties of the human UT-7/TPO megakaryocytic cell line were investigated using a microfluidic perfusion system. UT-7/TPO cells were immobilized in a microfluidic system on poly-D-lysine-coated glass substrate and perfused with various hyper-osmotic salt and CPA solutions at suprazero and subzero temperatures. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and the information was processed and analyzed using the Kedem–Katchalsky model to determine the membrane transport properties. The osmotically inactive cell volume (Vb=0.15), the permeability coefficient to water (Lp) at 37°C, 22°C, 12°C, 0°C, −5°C, −10°C, and −20°C, and dimethyl sulfoxide (DMSO; Ps) at 22, 12, 0, −10, −20, as well as associated activation energies of water and DMSO at different temperature regions were obtained. We found that MKs have relatively higher membrane permeability to water (Lp=2.62 μm/min/atm at 22°C) and DMSO (Ps=1.8×10−3 cm/min at 22°C) than most other common mammalian cell types, such as lymphocytes (Lp=0.46 μm/min/atm at 25°C). This information could suggest a higher optimal cooling rate for MKs cryopreservation. The discontinuity effect was also found on activation energy at 0°C–12°C in the Arrhenius plots of membrane permeability by evaluating the slope of linear regression at each temperature region. This phenomenon may imply the occurrence of cell membrane lipid phase transition.
PMCID: PMC3247705  PMID: 22232706
5.  In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art 
Transfusion medicine reviews  2010;24(1):33-43.
The exciting and extraordinary capabilities of stem cells to proliferate and differentiate into numerous cell types not only offers promises for changing how diseases are treated, but may also impact how transfusion medicine is practiced in the future. The possibility of growing platelets in the laboratory to some day supplement and/or replace standard platelet products has clear advantages for blood bank centers and patients. Due to the high utilization of platelets by patients undergoing chemotherapy or receiving stem cell transplants, platelet transfusion has steadily increased over the past decades. This trend is likely to continue as the number of adult and pediatric patients receiving stem cell transplants is also continuously rising. As a result of increased demand coupled with the short shelf-life of platelet concentrates, providing platelets to patients can stretch the resources of most blood centers, drive donor recruitment efforts, and on occasion platelet shortages can compromise the care of thrombocytopenic patients.
The purpose of this article is to review current scientific progress to develop in vitro strategies to manufacture platelets, with an emphasis on efforts to produce functional platelets in quantities that would be required for clinical transfusion. There are a number of publications indicating that human platelets can be obtained in vitro from the controlled differentiation of hematopoietic stem cells. However, the hemostatic quality of such manufactured platelets has not been confirmed and current technologies are inadequate to ensure satisfactory expansion and platelet biogenesis on an industrial scale. Nonetheless, these studies provide proof-of-principle that developing in vitro strategies to manufacture platelets is feasible and also provide a foundation for developing more sophisticated approaches to achieve this goal.
PMCID: PMC2790431  PMID: 19962573
Megakaryocyte; Stem cells; Megakaryocytopoiesis; Thrombocytopoiesis; ex vivo cultures; Platelet; Platelet biogenesis; Hematopoiesis

Results 1-5 (5)