Earticle

The aggregability of red blood cells (RBCs) was determined by laser backscattering light analysis in a microfluidic channel. Available techniques for RBC aggregation often adopt a rotational Couette-flow using a bob-and-cup system for disaggregating RBCs, which causes the system to be complex and expensive. A disposable microfluidic channel and vibration generating mechanism were used in the proposed new detection system for RBC aggregation. Prior to measurement, RBC aggregates in a blood sample were completely disaggregated by the application of vibration-induced shear. With the present apparatus, the aggregation indexes of RBCs can be measured easily with small quantities of a blood sample. The measurements with the present aggregometer were compared with those of LORCA and the results showed a strong correlation between them. The aggregability of the defibrinogenated blood RBCs is markedly lower than that of the normal RBCs. The noble feature of this design is the vibration-induced disaggregation mechanism, which can incorporate the disposable element that holds the blood sample.

Pig embryonic stem cells (ESC) has been suggested to become important animal model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, the quality of cloned embryo and derivation rate of cloned blastocyst has been presented limits for derivation of cloned embryonic stem cell. In this study, we have tried to overcome these problems by aggregating porcine embryos. Zonafree reconstructed SCNT Embryos were cultured in micro-wells singularly (non-aggregated group) or as aggregates of three (aggregated groups) at the four cell stage. Embryo quality of the cloned embryos and attachment on feeder layer rate significantly increased in the aggregates. The aggregation of pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of pig cloned blastocyst and improvement in the percentage of attachment on the feeder layer of cloned embryos. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

최근 항암제로서 관심을 끌고있는 camptothecin을 생산하기 위한 희수(Camptotheca acuminata) 현탁세포 배양과 camptothecin의 생산을 촉진하는 연구를 수행하였다. 장기간 배양으로 발생되는 이차대사산물 생산능력의 변이가 관찰되어 이를 극복하고자 세포의 응집을 통한 camptothecin의 생산을 촉 진하고자 하였다. 세포의 응집은 hybrid 배지와 sucrose 4%일 때에 가장 많이 유도되었고, 이때 camptothecin의 생산량도 18.04×10-4 ㎎/L로 가장 높은 생산성을 가졌다. 또한, 세포의 응집을 유도하기 위한 일환으로 교반속도를 변화시켜 이에 대한 효과를 알아보았는데, 100rpm에서 가장 많은 세포응집이 유도되었으며, camptothecin의 생산 역시 19.4×10-4 ㎎/L로 가장 높은 결과를 나타내었다.

Suspension cultures of Camptotheca acuminata, which is known to produce the anticancer indole alkaloid camptothecin and its derivatives, were made to increase camptothecin production. The capability of camptothecin production in suspended cells is decreased by repeated subculturing. Aggregated cells produced more camptothecin than single cells. Optimal cell aggregation was achieved in hybrid medium supplemented with 4% sucrose. Aggregated cells in hybrid medium with 4% sucrose produced 18.04×10-4 ㎎/L of camptothecin. The control of shaking speeds was effective at inducing cell aggregation and camptothecin production. A shaking speed of 100 rpm was found optimum to increase the cell aggregation with a camptothecin production of 19.4×10-4 ㎎/L.

Cell aggregation during suspension culture causes obstruction to nutrient and oxygen transfer, leading to cease of the cell growth and eventually cell death. Approaches to reduce these cell aggregation issues were made both physically and chemically. Addition of anti-clumping agents, like dextran sulfate, in the cell culture media or decreasing concentrations of calcium ions, involving in cell aggregation, are well known chemical approaches.1),2) Physical approaches relate to hydrodynamics, supplying more power for stirring in the vessel. We focused on physical approaches in CHO cells to observe possible effects, which displayed positive result in chemical approaches previously. The shaker was controlled through 80-200 rpm to diversify the input power of the Erlenmeyer flask, on assay for antibody production was done to assume any cell damage and protease leakage following high rpm. When the rpm was raised in a rCHO-DG44 cell line where cell aggregation is severe, the maximum cell density increased, the mean cell diameter decreased, while the antibody production did not decrease. We also observed the cell aggregation following the rpm in another rCHO-DG44 cell line to find universal characteristics.

Calcium is a critical parameter which is required for influencing the interaction of both cell-cell and cell-matrix. Therefore extracellular calcium has strong potentiation effects on cell adhesion and cell aggregation for diverse cell types in vitro.1) Cell aggregation during cell culture causes obstruction to nutrient and oxygen transfer, leading to cease of the cell growth and eventually cell death. It has been reported that low calcium levels in serum-free media reduces aggregation of HEK293 cells2) and chondrocytes.1) and in the preceding study, we observed the effect of calcium on cell growth and aggregation of Chinese Hamster Ovary(CHO) cells. In preexisting commercial media which the concentration of calcium is high, cell aggregation occurred and cell growth was low. On the other hand, when the concentration of calcium was relatively low, the cell growth was higher and the cells average diameter maintained uniformly. In this study, a same CHO-DG44 cell line, but selected by a different route was used in suspension culture to observe how the optimum concentration of calcium differed when the aggregation appearances are more severe. We also observed how the phenomenon of aggregation changed by addition of substances like dextran sulfate which reduces cell aggregation.

Leiomyosarcoma (LMS) is a mesenchymal malignancy with a complex karyotype. Despite accumulated evidence, the factors contributing to the development of LMS are unclear. Here, we investigated the role of tight-junction protein 1 (TJP1), a membrane-associated intercellular barrier protein during the development of LMS and the tumor microenvironment. We orthotopically transplanted SK-LMS-1 cells and their derivatives in terms of TJP1 expression by intramuscular injection, such as SK-LMS-1 Sh-Control cells and SK-LMS-1 Sh-TJP1. We observed robust tumor growth in mice transplanted with LMS cell lines expressing TJP1 while no tumor mass was found in mice transplanted with SK-LMS-1 Sh-TJP1 cells with silenced TJP1 expression. Tissues from mice were stained and further analyzed to clarify the effects of TJP1 expression on tumor development and the tumor microenvironment. To identify the TJP1-dependent factors important in the development of LMS, genes with altered expression were selected in SK-LMS-1 cells such as cyclinD1, CSF1 and so on. The top 10% of highly expressed genes in LMS tissues were obtained from public databases. Further analysis revealed two clusters related to cell proliferation and the tumor microenvironment. Furthermore, integrated analyses of the gene expression networks revealed correlations among TJP1, CSF1 and CTLA4 at the mRNA level, suggesting a possible role for TJP1 in the immune environment. Taken together, these results imply that TJP1 contributes to the development of sarcoma by proliferation through modulating cell-cell aggregation and communication through cytokines in the tumor microenvironment and might be a beneficial therapeutic target.

Fucoidan, a sulfated polysaccharide found in brown algae, possesses various biological activities including anti-inflammatory and anti-cancer effects. In this study, we investigated the effects of fucoidan on the cell growth and morphology of human osteosarcoma MG-63 cells and found that fucoidan induces cell aggregation and apoptosis in osteosarcoma cells when the cells are treated with fucoidan upon seeding before becoming stably attached to the plates. Typical characteristics of apoptotic cells such as chromatin condensation and nuclear fragmentation were observed in fucoidan-treated cells. The number of annexin V-stained cells was increased by fucoidan treatment in a dose-dependent manner. Consistent with these results, cell viability and growth rate were decreased and cell spreading was inhibited in the presence of fucoidan, leading to rounded morphological changes of the cells. Our results demonstrate that fucoidan treatment results in cell aggregation through F-actin accumulation at the rounded cell cortex and apoptosis in osteosarcoma MG-63 cells.

The extent of red blood cell (RBC) aggregation has widely been accepted as a reliable indicator of inflammatory processes. The ability to measure RBC aggregation indexes in capillary blood samples may simplify the application of the test and may bring advantages in its clinical usage as well as application in field studies. This study compared RBC aggregation indexes measured in venous and capillary blood samples using a newly developed capillary tube aggregometer. Although strong correlations between aggregation indexes measured in capillary and venous samples were detected, the indexes measured in venous samples were higher compared to capillary samples, thus indicating a bias. The possible reason for this bias is proposed to be related to the difference in oxygen saturation of venous and capillary samples and therefore altered the light absorption spectrums. Our results indicate that capillary blood samples can be used to estimate RBC aggregation in venous blood samples, however the bias should be taken into account.

The aggregation characteristics of red blood cells (RBCs) are known as important factors in the microvascular flow system, and increased RBC aggregation has been observed in various pathological diseases, such as thrombosis and myocardial infarction. This paper describes a simple microfluidic device for measuring the RBC aggregation by integrating a microfluidic slit rheometry and laser-backscattering technique. While a decreasing-pressure mechanism was applied to the microfluidic rheometry, a syllectogram (the light intensity versus time) showed an initial increase and a peak caused by the high shear stress-induced disaggregation, immediately followed by a decrease in the light intensity due to RBC aggregation. The critical shear stress (CST) corresponding to the peak intensity was examined as a new index of the RBC aggregation characteristics. The CST of RBCs increased with increasing aggregation-dominating protein (fibrinogen) in the blood plasma. The essential feature of this design was the combination of the rheometric-optic characterization of RBC aggregation with a microfluidic chip, which may potentially allow cell aggregation measurements to be easily carried out in a clinical setting.

Even though the trade-off between the benefits and risks of blood transfusion has been discussed for the last several decades, it requires further understanding of the rheological changes in stored blood that include the alteration of red blood cell (RBC) aggregation. The RBC aggregation of stored blood in its autologous plasma was monitored through the storage period (35 days). The critical shear stress, as a measure of RBC aggregation, was determined by using a microfluidic aggregometer. Blood was processed into a blood bag containing the anticoagulant CPDA1 and stored at $4^{\circ}C$. It was subjected to assays after zero, seven, 14, and 35 days. The critical shear stress for stored blood did not change up to 14 days of storage but exhibited a significant decrease after 35 days of storage. These results were identical to those of the conventional aggregation index (AI). Also, in the alteration of RBC aggregation for blood storage, the effect of the plasma factor was slightly stronger than that of the cellular factor. Through the present study, the critical shear stress as a new measure of RBC aggregation may help to monitor and control the quality of blood storage.

The aggregation characteristics of red blood cells (RBCs) were measured using a newly developed light-transmission slit rheometer. Conventional methods of RBC disaggregation such as the rotational Couette system were replaced with a pressure-driven slit flow system with a vibrational mechanism. Using a vibration generator, one can disaggregate the RBC aggregates stored in the slit. While shear stress decreases exponentially, instantaneous pressure and the transmitted light intensity were measured over time. Applying an abrupt shearing flow after disaggregation caused a rapid elongation of the RBCs followed by loss of elongation with the decreasing shear stress. While the shear stress is further decreasing, the RBCs start to re-aggregate and the corresponding transmitted intensity increases with time, from which the aggregation indices can be obtained using a curve-fitting program.

The disaggregation and displacement of stacked red blood cell (RBC) are known to be dependent on the electric state of hemoglobin and the strength and gradient of external magnetic field. The present study has been investigated the dependence of the change in aggregation and mobility of RBCs on the intensity and time duration of pulsed magnetic field (PMF), in addition to stimulus area. The magnetic field intensities are 0.07, 0.19, and 0.27 T, pulse time of 0.102 ms and pulse repetition rate was 1 Hz. Live blood analysis was used in order to quantitatively estimate the mobility and morphology of RBC exposed to PMF stimulus. The optimal time and intensity of PMF stimulus were 10 minutes and 0.27 T. The difference in the stimulus area did not show much. The continuance of the effect of PMF stimulus was observed to be 2 hours. Also it was verified the movement of the RBCs was accelerated for steady exposure of PMF stimulus. In order to identify a link between disaggregation in rouleau-formed RBCs and PMF stimulus, it is necessary to investigate the correlation between PMF intensity and the stimulus time, the mobility of RBCs, and the number of stacked RBCs.

The present study investigated the effect of transverse vibration on the hemorheological characteristics of blood using a newly designed pressure-scanning capillary viscometer. As vibration was applied, aggregated blood cells (rouleaux) were disaggregated. The range of vibration frequency and amplitude are $0{\sim}100\;Hz$ and $0{\sim}0.8\;mm$, respectively for a capillary diameter 0.84 mm. As vibration increased, blood viscosity initially increased and tended to decrease. In order to delineate the unexpected results, the present study proposed two counteracting mechanisms of vibration related with red blood cell (RBC) aggregation affecting hemo-rheological properties. One is the reduction of RBC aggregation due to vibration causing an increase of blood viscosity. The other is forced cell migration due to the transverse vibration, which in turn forms a cell-free layer near the tube wall and causes a decrease of flow resistance.

Calcium ions are involved in the regulation of diverse cellular processes. Fourteen genes encoding calcium binding proteins have been identified in Dictyostelium. CBP7, one of the 14 CBPs, is composed of 169 amino acids and contains four EF-hand motifs. Here, we investigated the roles of CBP7 in the development and cell migration of Dictyostelium cells and found that high levels of CBP7 exerted a negative effect on cells aggregation during development, possibly by inhibiting chemoattractant-directed cell migration. While cells lacking CBP7 exhibited normal development and chemotaxis similar that of wild-type cells, CBP7 overexpressing cells completely lost their chemotactic abilities to move toward increasing cAMP concentrations. This resulted in inhibition of cellular aggregation, a process required for forming multicellular organisms during development. Low levels of cytosolic free calcium were observed in CBP7 overexpressing cells, which was likely the underlying cause of their lack of chemotaxis. Our results demonstrate that CBP7 plays an important role in cell spreading and cell-substrate adhesion. cbp7 null cells showed decreased cell size and cell-substrate adhesion. The present study contributes to further understanding the role of calcium signaling in regulation of cell migration and development.

Background: Extracorporeal circulation (ECC) can induce alterations in blood viscoelasticity and cause red blood cell (RBC) aggregation. In this study, the authors evaluated the effects of pump flow pulsatility on blood viscoelasticity and RBC aggregation. Methods: Mongrel dogs were randomly assigned to two groups: a nonpulsatile pump group (n=6) or a pulsatile pump group (n=6). After ECC was started at a pump flow rate of 80 mL/kg/min, cardiac fibrillation was induced. Blood sampling was performed before and at 1, 2, and 3 hours after ECC commencement. To eliminate bias induced by hematocrit and plasma, all blood samples were adjusted to a hematocrit of 45% using baseline plasma. Blood viscoelasticity, plasma viscosity, hematocrit, arterial blood gas analysis, central venous $O_2$ saturation, and lactate were measured. Results: The blood viscosity and aggregation index decreased abruptly 1 hour after ECC and then remained low during ECC in both groups, but blood elasticity did not change during ECC. Blood viscosity, blood elasticity, plasma viscosity, and the aggregation index were not significantly different in the groups at any time. Hematocrit decreased abruptly 1 hour after ECC in both groups due to dilution by the priming solution used. Conclusion: After ECC, blood viscoelasticity and RBC aggregation were not different in the pulsatile and nonpulsatile groups in the adult dog model. Furthermore, pulsatile flow did not have a more harmful effect on blood viscoelasticity or RBC aggregation than nonpulsatile flow.

Background: Since ginsenosides exert an anti-thrombotic activity, blood flow-improving effects of DK-MGAR101, an extract of mountain ginseng adventitious roots (MGAR) containing various ginsenosides, were investigated in comparison with an extract of Korean Red Ginseng (ERG). Methods: In Sprague-Dawley rats orally administered with DK-MGAR101 or ERG, oxidative carotid arterial thrombosis was induced with FeCl₃ (35%), and their blood flow and occlusion time were measured. To elucidate underlying mechanisms, the cytoprotective activities on rat aortic endothelial cells (RAOECs) exposed to hydrogen peroxide (H₂O₂) were confirmed. In addition, the inhibitory activities of DK-MGAR101 and ERG on agonist-induced platelet aggregation, thromboxane B₂ production, and ATP granule release from stimulated platelets as well as blood coagulation were analyzed. Results: DK-MGAR101 containing high concentrations of Rb1, Rg1, Rg3, Rg5, and Rk1 ginsenosides (55.07 mg/g) was more effective than ERG (ginsenosides 8.45 mg/g) in protecting RAOECs against H₂O₂ cytotoxicity. DK-MGAR101 was superior to ERG not only in suppressing platelet aggregation, thromboxane B₂ production, and granule release, but also in delaying blood coagulation, FeCl₃-induced arterial occlusion, and thrombus formation. Conclusions: The results indicate that DK-MGAR101 prevents blood vessel occlusion by suppressing platelet aggregation, thrombosis, and blood coagulation, in addition to endothelial cell injury.

The rapid progress in wireless communication technology and proliferation of multimedia applications, including voice over WiFi (VoWiFi), demand exploration of innovative approaches to enhance the network performance and quality of service. We propose a technique for enhancing the cell capacity of wireless local area network (WLAN) access point that provides VoWiFi service in the sixth-generation WLAN standard. The proposed technique uses the aggregate media access control protocol data unit (A-MPDU) for frame aggregation with constant bit rate (CBR) traffic in the WiFi 6 standard (i.e., IEEE 802.11ax). On the other hand, the retransmission of voice packets substantially deteriorates the VoWiFi cell capacity. We compare the results obtained from the use of WiFi 6 with currently existing WLAN standards, such as IEEE 802.11b/g/n/ac. This comparison focuses on distributed coordination function interframe spacing (DIFS) and arbitration interframe spacing (AIFS) using CBR traffic. Using our technique, we can increase the VoWiFi cell capacity for CBR traffic by 24.25% and 25.20% when using DIFS and AIFS, respectively, while considering the A-MPDU frame aggregation technique.