Electrical Cell-substrate Impedance Sensing (ECIS)法を用いた培養細胞の微細挙動の定量的評価法 - 細胞-細胞間隙と細胞-電極間隙の評価 -

We have proposed a mathematical model for the micro-dynamics for cultured cells measured with ECIS system for the detection of nanometer-order dynamics of cells cultured on a small gold electrode and could separately evaluate cell-to-cell distance (A) and cell-to-substrate distance (h). For wide applications of this method, we constructed mathematical models which express cell-to-electrode impedances for some kinds of confluent conditions. Based on this mathematical model, we defined new parameters S(A) and S(h) in order to evaluate cell-to-cell distance and cell-to-substrate distance. As the application, we investigated the effect of X-irradiation to bovine aortic endothelial cell (BAEC). We analyzed the micro-dynamics of cells from the impedance of BAEC before and after X-irradiation. It was proved that the stimulation of 100 Gy X-irradiation to the BAEC resulted in the large scale of increase in the cell-to-cell distances (A), and the slight increase in the cell-to-substrate distances (h) accompany with continuous fluctuations.[背景] Electrical Cell-substrate Impedance Sensing (ECIS)は培養細胞の電気的計測により,その微細挙動を評価する工学的手法である｡我々はECISを用いてこの微細挙動を細胞-細胞間隙と細胞-電極間隙に分離して推定することが可能な数学的モデルを提案してきた｡本研究ではこの数学的モデルを使用して,Ⅹ線を照射した牛大動脈内皮細胞(bovine aortic endothelial cell : BAEC)の微細挙動の経時変化を評価した｡ [方法と結果] ECISシステム(Model 1600R Applied BioPhysics)を用いてBAECのコンフルエント到達前後で計測を行い,数学的モデルを構築した｡このモデルは細胞の微細挙動を検出するために重要な周波数レンジである1-10kHzにおいて測定結果とよく一致し,Cole-Cole円弧則に従う｡さらに細胞-細胞間距離Aの増減に対応する校正定数S(A)と細胞-電極間距離hの増減に対応する校正定数S(h)を導入し,ベクトルインピーダンスの変化に対応した値を算出することで細胞の微細挙動を評価することとした｡次に本法によりX線(150kV, 100Gy)を照射したBAECの微細挙動を評価した｡Ⅹ線照射細胞では時間経過と共に抵抗成分の変化が支配的なインピーダンスの減少が確認された｡この現象はS(A)の大きな増加とS(h)の微小な減少をもたらした｡このパラメータの変化は細胞間隙が拡大したことを示しており,Ⅹ線照射による細胞内損傷により細胞密度が低下したと考えられた｡ [結論] 本法は培養細胞の微細動態の変化を細胞-細胞間隙と細胞-電極間隙に分けてリアルタイムに定量評価することが可能であり,各臓器の細胞レベルでの薬物の治療効果や Ⅹ線に対する耐性の評価に適応できるものと考える

Continuous downstream process of monoclonal antibody developed based on the process analysis/understanding and its validation

As downstream process (DSP) of monoclonal antibody (mAb) includes several batch chromatography steps, and other operations such as virus inactivation (VI), it is important to consider how each operation is carried out in order to make an efficient continuous or pseudo-continuous DSP. Our consortium* developed a continuous DSP (CDSP) of mAb under the AMED** project “Development of platform technologies for the continuous manufacturing of biopharmaceuticals”, and successfully carried out several runs at our GMP facility and laboratory. In this paper, we will present how our CDSP was developed based on the process analysis and understanding, and the purification results along with several important issues to be addressed in the future [1]. For the first “capture” step, a 2-column “periodic counter-current chromatography (PCCC)” with protein A chromatography columns was chosen. The efficiency of PCCC is strongly dependent on the operating conditions, which should be determined properly [2, 3]. Please click Additional Files below to see the full abstract

Flow-through chromatography as a continuous and integrated purification method

Continuous manufacturing is expected to improve the efficiency and economics of protein and other bio-product production processes. However, it is not easy to design and operate the continuous process especially for downstream processing as many unit operations (chromatography and membrane filtration) are involved. An operation method known as flow-through chromatography (FTC) is considered to be an efficient purification method as the flow is continuous. In FTC, a target bio-product is eluted from the chromatography column without adsorption whereas contaminants are strongly bound. Usually two different modes of chromatography are needed in order to remove various kinds of contaminants. Two FTC columns have to be connected in order to build the integrated continuous process. This is not an easy task since the mobile phase properties (pH, salt, buffer ions) are different for the two columns. Please click Additional Files below to see the full abstract

Flow-velocity programmed chromatography as an alternative method for increasing the efficiency of continuous- or integrated-chromatography processes

Solvent (mobile phase) programming is most commonly employed for controlling adsorption/desorption in chromatography (linear gradient elution or stepwise elution). For gas separation, temperature- or pressure-swing adsorption is frequently used. Although flow-velocity is another important parameter, which affects both the dynamic adsorption capacity (DBC) and the resolution, it is seldom used as a programmed operating variable. The one exception is the standard 4-zone simulated moving bed (SMB) chromatography, in which the flow-velocities of the 4-zones are different. Several researchers have already shown that DBC can be increased by using two different flow velocities. However, a rational method for determining the optimum flow velocity program has not been established. Moreover, application of this method to periodic counter-current (PCC) chromatography or connected flow-through chromatography (FTC) has not been attempted yet. In this study, we have developed a flow-velocity gradient method for analyzing the breakthrough curves of proteins in ion-exchange or protein A chromatography (Figure 1). The data were obtained at various different gradient slopes. The obtained curves were analyzed based on a model considering mass transfer (pore diffusion) and non-linear isotherm. Then, numerical simulations were carried out in order to find the optimum flow-velocity program for improving the efficiency. This method was further applied to PCC and FTC (Figure 2). The effect of flow programming on productivity and cost reduction has also been examined in both batch and continuous configuration in capture chromatography of mAbs by simulation of the process models. Experimental verification was also carried out using monoclonal antibody samples in the filtered cell culture liquid. Please click Additional Files below to see the full abstract

THE INFLUENCES OF THE BUNT IMAGE UPON BALL MEETING BAT

In today’s baseball, a bunt is considered as an important tactic. Therefore the aim of this study was to define the kinematic characteristics of bunt comparing between experts and beginners. Five expert players and five healthy male students participated in this study. Subjects bunted 10 times. 3-D coordinate was determined by DLT METHOD. Using these variables, the variance of distance between the bat-head and forehead, and the variance of bat-angle were researched and analyzed by z-test (