DE102010027154A1 - Measuring gas or gas mixture using cell culture with viable cells, comprises covering cells with liquid cell culture medium, measuring cells, directly contacting gas mixture with part of cells and determining physiological changes in cells - Google Patents

Abstract

Measuring a gas or a gas mixture (11) using a cell culture (3) with viable cells, comprises covering the cells in a cell culture with a liquid cell culture medium (10) to produce an initial state, subjecting the cells with the gas or the gas mixture for measuring, directly contacting at least partially undissolved gas or gas mixture with at least a part of the cells, determining the physiological changes in the cells by exposing to the gas or the gas mixture during the measuring step with at least one sensor (2). Measuring a gas or a gas mixture (11) using a cell culture (3) with viable cells, comprises covering the cells in a cell culture with a liquid cell culture medium (10) to produce an initial state, subjecting the cells with the gas or the gas mixture for measuring, directly contacting at least partially undissolved gas or gas mixture with at least a part of the cells, determining the physiological changes in the cells by exposing to the gas or the gas mixture during the measuring step with at least one sensor (2), where loading the cell culture with the gas or the gas mixture is simultaneously carried out to measure the physiological changes by the sensor. An independent claim is also included for a device for measuring the gas or the gas mixture, comprising at least one receiving unit (1) to receive a cell culture with viable cells, at least one sensor to measure the physiological changes in the cells by an exposure to the gas or the gas mixture, a gas inlet to supply the gas or the gas mixture to the cells in the cell culture, an inflow to supply the liquid cell culture medium for the receiving unit, a drain to discharge the liquid cell culture medium from the receiving unit, and a controlling means designed for a measuring the cell culture medium, which is at least partially removed from the receiving unit via the outlet for introducing the gas or the gas mixture into the receiving unit via the gas inlet, such that the gas is directly contacted with the cell culture for a period of time, and carrying out a measurement of the physiological changes of the cells using the sensor during that period.

Description

The measurement of the presence of hazardous, for example, toxic gases in a gas mixture can be carried out by means of various physical and chemical measuring methods. Typically, the concentration of one or a few specific gases is determined. Gases for which the measuring method is not designed are either not detected at all or lead to a falsified measurement result. This makes it difficult to test for, for example, toxic substances, since there is an unmanageable number of such substances.

As an advantageous alternative to the physical and chemical measurement methods, a measurement with living cells has therefore been found. In this, the reaction of the cells is observed on the gas mixture. The result is not or only with effort the exact composition of the gas mixture. However, the cells react to all gases that have a physiological effect on the cells. This makes it possible to detect the presence of, for example, toxic substances without knowing the specific toxic substances.

From the WO 2007/01669 A1 and the WO 2008/11876 A2 It is known to preserve the cells in a nutrient medium and at the same time to observe the reaction to those gases which dissolve in sufficient quantity in the nutrient medium. The disadvantage is that the solution behavior of some gaseous substances, however, is far below the limit for physiological changes. This is particularly disadvantageous if it is not known which substances could occur, that is, if their solubility can not be estimated beforehand.

It is also known to temporarily remove the cells for the measurement from the nutrient medium and to expose it directly to the gas mixture. The gas mixture then acts directly on the cells. Then the cells are again placed in the nutrient medium and subsequently a measurement of one or more parameters of the cells is made in order to produce the actual measurement result. The disadvantage here is that a distinction of the strength of certain effects on the cells is difficult. If cells have died, this can be determined after reintroduction into the nutrient medium, but not when or how fast the cells have died.

The invention has for its object to provide a device which avoids the disadvantages mentioned. Another object of the invention is to provide a measuring method which avoids the disadvantages mentioned. In particular, the device or the method should enable a measurement with improved accuracy.

This object is achieved by a measuring method having the features of claim 1. A further solution consists in the measuring device with the features of claim 4. The dependent claims relate to advantageous embodiments of the invention.

In the method according to the invention for measuring a gas or a gas mixture by means of a cell culture with living cells, the cells of the cell culture are covered by a liquid cell culture medium to produce an initial state. This step may take place near or far before an actual measurement or repeated. Furthermore, the cells are subjected to the gas or gas mixture in a subsequent measuring step, with the gas or gas mixture in this case coming into direct contact with at least part of the cells, at least partially unresolved. In other words, the gas or gas mixture is brought to the cells in such a way that they are exposed directly to the gas, so that the gas is not or only partially present in dissolved form. During the measuring step, physiological changes of the cells are determined by the application of the gas or gas mixture with at least one sensor. The exposure of the cell culture with the gas or gas mixture is carried out at the same time for measuring the physiological changes by the sensor.

• – mindestens eine Aufnahmeeinheit zur Aufnahme einer Zellkultur mit lebenden Zellen,
• – mindestens einen Sensor zur Messung von physiologischen Veränderungen der Zellen durch die Beaufschlagung mit dem Gas oder Gasgemisch,
• – einen Gaseinlass zur Zuführung des Gas oder Gasgemischs zu den Zellen der Zellkultur,
• – einen Zufluss zur Zuführung eines flüssigen Zellkulturmediums zur Aufnahmeeinheit,
• – einen Abfluss zur Abführung des flüssigen Zellkulturmediums von der Aufnahmeeinheit, und
• – Steuerungsmittel auf.

The device according to the invention for measuring a gas or a gas mixture has:

• At least one receiving unit for receiving a cell culture with living cells,
• At least one sensor for measuring physiological changes in the cells by exposure to the gas or gas mixture,
• A gas inlet for supplying the gas or gas mixture to the cells of the cell culture,
• An inflow for supplying a liquid cell culture medium to the receiving unit,
• A drain for removing the liquid cell culture medium from the receiving unit, and
• - Control means on.

The control means are configured to at least partially remove the cell culture medium via the outflow from the receiving device for a measuring step, to introduce the gas or gas mixture into the receiving device via the gas inlet and thus to bring it into direct contact with the cell culture for a determinable period of time and during the period to measure the physiological changes of the cells by the sensor.

Advantageously, it can be achieved that even during the admission of the cells of the cell culture with the gas or gas mixture already possible physiological changes of the cells are recognizable and evaluable. This therefore allows a determination of the kinetic profile of the effect of the gas or gas mixture and therefore advantageously provides an increased information content. It therefore requires no elaborate endpoint determinations or test methods that damage the cells in the detection of cell physiological change lasting. Preferred measurement methods are impedance measurements to characterize the morphology and optical measurement methods such as fluorescence or chemiluminescence. Thus, an actual real-time measurement is made possible for the first time, since it is not necessary to carry out further tests after the cells have been exposed to the gas to be examined.

At the same time, it is advantageous over known measuring methods that solubility of the gases to be investigated in water or another liquid medium is not necessary. The measuring method is therefore particularly advantageous for the measurement of insoluble or poorly soluble gases such as carbon dioxide.

It is also possible that, in addition to the measurement of physiological changes in the cells during exposure to the gas or gas mixture, a measurement is made or continued even after the application of the gas. In other words, a measurement is also carried out when the cells have returned to the cell culture medium. This may be advantageous, for example, if the oxygen consumption of the cells is permanently or temporarily changed by exposure to the gas or gas mixture. This in turn is preferably also measured when the cells are in the cell culture medium.

The cells used are preferably animal, plant, insect or human cells, in particular V79, N2A, A594, HFBE or HUVE. The sensors in turn preferably measure chemical or electrical parameters. It can also be used sensors for optical measurement.

It is advantageous to introduce the cell culture in a receiving device, in which also the cell culture medium is introduced. The cell culture medium serves as a nutrient solution and thus ensures the preservation of the living cells. Subsequently, the cell culture medium is at least partially removed from the receiving device via a drain for a measurement. The gas or gas mixture is further introduced via a gas inlet into the receiving device and thus brought into direct contact with the cell culture. The introduction of the gas or gas mixture can take place before, at the same time or after the removal of the cell culture medium. As a result, the gas or gas mixture acts more or less strongly on the cells, which may have physiological effects depending on the gas or gas mixture. At the same time, the physiological effects, if present, are recorded by means of the sensor or the sensors. Subsequently, the receiving device is expediently filled to restore the initial state via an inflow with cell culture medium. It is also possible, for example, to replace the cells after the measuring step.

In a preferred embodiment of the invention, the sensor forms at least part of the bottom of the receiving unit and a surface of the sensor serving for the measurement faces the cell culture. In other words, in the embodiment of the invention, the cells of the cell culture come to rest directly on the functional surface of the sensor. As a result, the coupling between sensor and cells is optimal.

It is expedient if the receiving device and the sensors or the sensor can be sterilized, d. H. are sufficiently stable to heat, pressure and / or solvents.

In a further preferred embodiment of the invention means are provided for adjusting a dilution of the gas or gas mixture before or during the introduction into the receiving device. If the gas or gas mixture is diluted prior to the application of the cells, it can be avoided for certain gases too strong reaction of the cells and so a better, because longer lasting signal can be achieved or even the survival of the cells are made possible.

It is advantageous if one or more sensors are provided for parameters of the gas or gas mixture. These can then be advantageously used, for example, to determine the humidity and / or temperature of the gas or gas mixture.

Furthermore, a device for moistening the gas or gas mixture is preferably present. Thus, the gas or gas mixture can be moistened before or during the introduction into the receiving device.

Particularly preferred is a device for controlling the temperature of at least a part of the device is provided. In this way, advantageously, the part of the device, for example the area around the receiving device, can be kept at a largely constant temperature. Of the preferred temperature range is 5 ° C to 65 ° C, preferably 15 ° C to 45 ° C. This reduces the influence of the surrounding temperature on the measurement results. In addition, condensation can be avoided.

Preferred, but by no means limiting embodiments of the invention will now be explained in more detail with reference to the drawing. The features are shown schematically and corresponding features are marked with the same reference numerals. Hereby show in detail

1 a cross-section of an open device for online measurement of physiological changes in a cell culture when exposed to gaseous substances with separate distribution and connector,

2 a cross section of a closed device for online measurement of physiological changes in a cell culture,

3 a cross section of a closed device for online measurement of physiological changes of a cell culture with a sheath,

4 Components for a device for the online measurement of physiological changes in a cell culture including the fogging device.

This in 1 illustrated embodiment includes as a receiving unit for cells a culture vessel 1 with a number of components as well as a loading device 15 , which also includes a number of other components. In this case, the loading device 15 opposite the culture vessel 1 slidably disposed in the vertical direction.

The culture vessel 1 includes a substrate 12 a sensor chip. On the substrate 12 of the sensor chip are several planar (electro) chemical and physical sensors 2 formed so that in this embodiment, on a chip area of 0.7088 cm ² five proton activity sensors, five oxygen sensors and an impedance sensor housed and connected to operating circuits. The sensor chip is fastened with a lever, not shown, on the contacts of the operating circuit. Furthermore, in the culture vessel 1 on the physical sensors 2 a cell culture 3 educated. The sensors form this 2 the bottom of the culture vessel 1 so that the cells of the cell culture 3 attach directly to the sensors.

The loading device 15 has a distributor 5 in, in the present embodiment, an inflow 8th and two outlets 9 are bored. About the inflow 8th and the drains 9 becomes the cell culture 3 in the culture vessel 1 with a cell culture medium 10 overlaid, which serves as a nutrient solution, for example. In other embodiments, multiple inflows 8th and / or at least two outlets 9 be provided. The initial state in which the cell culture medium 10 the cells of the cell culture 3 covered, is in 1 shown.

Before being exposed to cell culture 3 with a gas 11 is about the two outlets 9 the cell culture medium 10 aspirated to the surface of the cell culture 3 to get rid of a liquid supernatant. Via a gas inlet 6 becomes the gas to be examined for a defined period of time 11 into the distribution section 5 pumped or flowed by pressure gradient.

The resulting state is in 2 shown. In this case, the loading device 15 closer to the culture vessel 1 postponed, so that on the one hand, a suction of the cell culture medium 10 over the drains 9 is possible and on the other a gas-tight closure of the culture vessel 1 at least to the extent that the gas is produced 11 can be flowed. For this purpose, the distance between the cell culture is preferred 3 and the distributor 5 from the original length of 1 to 5 cm, preferably 2 to 3 cm, significantly reduced.

The gas to be examined 11 enters through the gas inlet 6 a the culture vessel 1 one. The gas inlet 6 opposite is a gas outlet 7 , Through this the gas enters 11 out again. On the path between the gas inlet 6 and the gas outlet 7 branches in the distribution section 5 a connector 4 from. The connector 4 is with the housing of the culture vessel 1 sealed gas-tight. The gas 11 diffused through the connector 4 in the culture vessel 1 and covers the cell culture 3 , The cell culture 3 reacts - depending on the gas 11 - with physiological changes on the direct contact with the gas 11 , These physiological changes are made by the sensors 2 detected and via an operating circuit to an evaluation 21 forwarded.

This is the gas 11 in this example in a laboratory measuring environment with synthetic air as carrier gas 24 mixed and with a gas humidifier 16 brought to a relative humidity of at least 60%. The gas, which is the loading device 15 over the gas outlet 7 leaves, becomes an exhaust tank 23 fed. In other measuring environments, for example, the dilution and / or forwarding into the exhaust gas tank 23 be omitted. Alternatively or in addition to dilution may be a device be provided to the volume flow of the gas 11 that reaches and delves the cells to settle.

The connector 4 , For example, designed as a piece of tubing, preferably has a flexible, smooth, inert, sterilizable and gas-impermeable material, in particular silicone, rubber, fluororubber, polyethylene or polyethylene terephthalate. The distribution piece 5 in turn, preferably has a solid, smooth, inert, sterilizable, gas-impermeable and corrosion-resistant material, in particular stainless steel, glass or ceramic. The distribution piece 5 is on the connector 4 attached.

• – das Zellkulturmedium 10 wird über zumindest einen der Abflüsse 9 teilweise oder komplett aus dem Kulturgefäß 1 entfernt, und
• – das zu untersuchende Gas 11 über zumindest einen Gaseinlass 6 in das Kulturgefäß 1 eingebracht und gelangt somit in direkten Kontakt mit der Zellkultur 3,
• – der Sensor 2 oder die Sensoren 2 messen die Veränderungen physiologischer Parameter der Zellkultur 3,
• – eine Auswerteelektronik erfasst die Signale, die die Sensoren 2 aufgrund der Änderungen erzeugen und wertet diese aus, und
• – das Kulturgefäß 1 wird über den Zufluss 8 wieder mit Zellkulturmedium 10 gefüllt.

A measurement thus runs in this example according to the following scheme:

• - the cell culture medium 10 is about at least one of the outflows 9 partially or completely from the culture vessel 1 removed, and
• - the gas to be analyzed 11 via at least one gas inlet 6 in the culture vessel 1 introduced and thus comes into direct contact with the cell culture 3 .
• - the sensor 2 or the sensors 2 measure changes in physiological parameters of cell culture 3 .
• - A transmitter detects the signals that the sensors 2 because of the changes, it generates and evaluates, and
• - the culture vessel 1 is about the inflow 8th again with cell culture medium 10 filled.

As a result, the measurement can be repeated, for example as a regular measurement cycle. In this case, there is a regular exchange of cell culture medium 10 and the gas 11 instead of. Since the cells after the exchange of cell culture medium 10 and the gas 11 the gas 11 can be exposed directly, as a gas 11 or corresponding gas mixture also a gas 11 or more gases 11 be present, which are not or slightly soluble in water. The duration of a measurement cycle can be varied within wide ranges, for example, the cells between 10 seconds and 24 hours or more with the gas 11 be charged.

Become several sensors 2 are preferred, such sensors that affect each other virtually not chemically and physically to get the most meaningful and independent signals.

It is also beneficial if the feeds 8th , the processes 9 , the gas inlets 6 , the gas outlets 7 and the culture vessel 1 autoclavable and sterilizable.

3 shows a further embodiment. In this embodiment, the distribution piece 5 and the connector 4 integrated into each other. The connector 5 is then without example, a hose element directly on the culture vessel 1 postponed. In this case they are the gas inlets 6 and the gas outlets 7 in a sheath element 13 integrated, the part of the distribution 5 is. The jacket 13 preferably comprises an inert, sterilizable, gas impermeable and corrosion resistant material, in particular, silicone, rubber, fluororubber, polyethylene, polyethylene terephthalate, stainless steel, glass or ceramic.

4 shows a system of several measuring units 25a ... c the way they are in the 1 to 34 are shown. These are integrated in the example shown in a laboratory system.

The laboratory system includes a supply of synthetic air 24 and a gas humidifier 16 , These supply a distribution system 20 with humidified synthetic air as carrier gas. A gas container 22 is also to the distribution system 20 connected and supplies the test gas. The distribution system 20 supplies the three measuring units 25a ... c with the pure carrier gas or a mixture of the carrier gas and the test gas. Furthermore, there are for each of the measuring units 25a ... c a container 18 for subsequent delivery of the cell culture medium 10 and an evaluation unit 21 , Furthermore, an exhaust gas tank 23 and a cell culture medium waste container 19 intended.

The laboratory system allows a parallel measurement at the three measuring units 25a ... c, where the cell culture medium 10 can be renewed regularly and a zero point measurement of pure humidified synthetic air is possible. However, a measurement with the cell sensor is also possible outside of a laboratory system.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2007/01669 A1 [0003]
• WO 2008/11876 A2 [0003]

Claims (10)

Method for measuring a gas or a gas mixture ( 11 ) by means of a cell culture ( 3 ) with living cells, in which - the cells of the cell culture ( 3 ) for the preparation of a starting state by a liquid cell culture medium ( 10 ), - the cells in a subsequent measuring step with the gas or gas mixture ( 11 ), the gas or gas mixture ( 11 ) at least partially unsolved in direct contact with at least a part of the cells, - during the measuring step with at least one sensor ( 2 ) physiological changes of the cells by exposure to the gas or gas mixture ( 11 ), characterized in that the application of the cell culture ( 3 ) with the gas or gas mixture ( 11 ) at the same time as the measurement of physiological changes by the sensor ( 2 ) is carried out. A method according to claim 1, wherein the cells used are animal, plant, insect or human cells, in particular V79, N2A, A594, HFBE or HUVE. Process according to claim 1, in which - the cell culture ( 3 ) in a receiving device ( 1 ), - the cell culture medium ( 10 ) in the receiving device ( 1 ), - subsequently the cell culture medium ( 10 ) via an outflow ( 9 ) at least partially from the receiving device ( 1 ), - the gas or gas mixture ( 11 ) via a gas inlet ( 6 ) in the receiving device ( 1 ) and thus in direct contact with the cell culture ( 3 ), - subsequently the receiving device ( 1 ) via an inflow ( 8th ) with cell culture medium ( 10 ) is filled. Method according to claim 3, wherein a measurement of the physiological changes by the sensor ( 2 ) is also carried out when the cells of the cell culture ( 3 ) through the cell culture medium ( 10 ) are covered. Device for measuring a gas or a gas mixture ( 11 ), comprising: - at least one receiving unit ( 1 ) for receiving a cell culture ( 3 ) with living cells, - at least one sensor ( 2 ) for measuring physiological changes of the cells by exposure to the gas or gas mixture ( 11 ), - a gas inlet ( 6 ) for supplying the gas or gas mixture ( 11 ) to the cells of the cell culture ( 3 ), - an inlet for supplying a liquid cell culture medium ( 10 ) to the recording unit ( 1 ), - a drain for the removal of the liquid cell culture medium ( 10 ) from the receiving unit ( 1 ), - control means, which are designed, for a measuring step, the cell culture medium ( 10 ) over the drain ( 9 ) at least partially from the receiving device ( 1 ), the gas or gas mixture ( 11 ) via the gas inlet ( 6 ) in the receiving device ( 1 ) and thus for a definable period in direct contact with the cell culture ( 3 ) and during the period a measurement of the physiological changes of the cells by the sensor ( 2 ). Device according to claim 5, in which the sensor ( 2 ) at least a part of the bottom of the receiving unit ( 1 ) and a measuring surface of the sensor ( 2 ) of the cell culture ( 3 ) is facing. Apparatus according to claim 5 or 6, comprising means for adjusting a dilution of the gas or gas mixture ( 11 ) before or during the introduction into the receiving device ( 1 ). Device according to one of claims 5 to 7 with a device for humidifying the gas or gas mixture ( 11 ) before or during the introduction into the receiving device ( 1 ). Device according to one of claims 5 to 8 with at least one sensor for determining parameters of the gas or gas mixture ( 11 ), in particular for the determination of moisture and / or temperature. Device according to one of claims 5 to 9, comprising means for controlling the temperature of at least a part of the device.

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