Patent DE-102005048187-A1 (DE)
Title: Mechanism for the determination of paracellular and transcellular permeabilities of cell cultures, comprises filter mechanism, fluorescent marker, fluorescence photometer, level measurer, pump, impedance spectrometer and computer system
Inventor: SCHNITTLER HANS-JOACHIM
IPC: C12M1/34
Filed: 2005-10-02
Abstract: The mechanism for the determination of paracellular and transcellular permeabilities of cell cultures, comprises transwell filter mechanism (2), fluorescent marker, fluorescence photometer (3), level measurer (4), pump (5), impedance spectrometer (7) and computer system (9). The filter mechanism comprises the cell culture for the analysis of transcytosis processes in the cell culture by means of a measurement of the input concentration and of the input volume of the marker in a liquid column that loads the cell culture with hydrostatic pressure. The mechanism for the determination of paracellular and transcellular permeabilities of cell cultures, comprises transwell filter mechanism (2), fluorescent marker, fluorescence photometer (3), level measurer (4), pump (5), impedance spectrometer (7) and computer system (9). The filter mechanism comprises the cell culture for the analysis of transcytosis processes in the cell culture by means of a measurement of the input concentration and of the input volume of the marker in a liquid column that loads the cell culture with hydrostatic pressure. The fluorescence photometer is used for the determination of a respective initial concentration of the cell culture of the marker diffusing through the cell culture. A pressure sensor is used for the regulation of the liquid column. The level measurer with another pressure sensor is used for the measurement of a liquid quantity diffusing through the cell culture. The pump is used for the maintenance of an open loop liquid cycle (6), which stands in connection with the filter mechanism. The impedance spectrometer is used for the measurement of the electrical transendothelial resistance of the cell culture. The computer system is used for the control and evaluation of the measurement, and for the display of the permeabilities as functions of the quantities such as input concentration, initial concentration, input volume of the liquid, output volume of the liquid by means of a program-oriented mass balance. The filter mechanism exhibits upper and lower compartments between a downwardly-porous, small-volumed chamber with a filter insert. The cell culture is present in the form of a layer of inter-connected cells. The chamber to the liquid column is arranged with a ring electrode assigned to the upper compartment and to an electrode plate turned away from the liquid column, and is assigned to the lower compartment for the measurement of the transendothelial resistance with the help of the impedance spectrometer. The chamber is applicable in the upper compartment and is connected to the lower compartment over the open loop liquid cycle. The upper compartment possesses a supply line in the area of the liquid column for the controlled metering of the marker, which mixed with water rests on the cell culture. The pressure sensor, which constantly examines the liquid column height- and signal-wise, is connected with the computer system over a line. The fluorescence photometer exhibits detection unit comprising a power supply unit, to which a light source of light is attached. Lens is arranged after an excitation filter, which together form an excitation arrangement. A flow cuvette is arranged on a holding device with a supply and a discharge. An evaluation mechanism is integrated in the computer system. The liquid with the intended marker is present in the flow cuvette. The fluorescence photometer is equipped with supply- and signal reception leads or the connection of the light source and for the connection of the evaluation mechanism. The fluorescence photometer is provided with detection units and the holding device is used for flow cuvettes in centrally symmetrical arrangement, where the holding device possesses a base for holding the flow cuvettes and associated partitions. A rotatable holder is connected with a computer-controlled drive system capable of switching the direction of rotation. A measuring sector is formed by partitions and a flow cuvette present between them. The fluorescence photometer is connected with the holding device for the flow cuvettes. The flow cuvettes are stationarily fixed and are aligned next to one another in a row and the fluorescence photometer is movably arranged relatively linear and parallel to the row arrangement of the flow cuvettes. The detector is connected to the evaluation mechanism over a lead and to a semipermeable mirror, which is present between the excitation filter and the lens. In the fluorescence photometer, screens are inserted into the paths of rays. Skew angles are optimally adjusted the flow cuvettes that are black dyed for the substantial decrease of the scattered light and/or, for improvement of the wavelength-dependent fluorescence signal/noise-ratio. The flow cuvettes are made from quartz glass, with which a high signal/noise-ratio is attainable. A volume measuring capillary with another liquid column, is assigned to the level measurer and is connected with a valve-operatable discharge. A discharge volume is transferred as a measured variable to the computer system. The volume measuring capillary serves for the quantification of the liquid volume takes place through the pressure sensor, which is coupled with the volume measuring capillary. The impedance spectrometer is attached to the transwell filter arrangement over electrical junction wires. The computer system is electrically connected over lines with the impedance spectrometer, the fluorescence photometer, the level measurer, and the pressure sensor and with the pump. The computer system represents a self-regulation/control unit or is integrated as the primary component in a unit. The pump is designed as a multi-channel roller pump for multiple measuring chambers, where each measuring chamber is assigned to one of the closed loop liquid cycles and the impedance spectrometer and the computer system are singly present. The fluorescent marker is a fluorescein isothiocyanate albumin or tetramethylrhodamine dextran, where the marker to be supplied differ from one another in their molecular weight or quality. An independent claim is included for a method for determination of paracellular and transcellular permeabilities of cell cultures.