WO2007000247A1

WO2007000247A1 - Measuring cell and method carried out using said measuring cell for determining the degree of dissociation of biological cells induced by electroporation

Info

Publication number: WO2007000247A1
Application number: PCT/EP2006/005712
Authority: WO
Inventors: Martin Sack; Hansjoachim Bluhm
Original assignee: Forschungszentrum Karlsruhe Gmbh
Priority date: 2005-06-24
Filing date: 2006-06-14
Publication date: 2007-01-04
Also published as: DE102005029414A1; EP1893998A1; DE102005029414B4

Abstract

The invention relates to a measuring cell for determining the degree of dissociation of biological cells induced by electroporation, which cell is adapted for resistive/capacitative measuring or for inductive measuring. The inventive method using said measuring cell comprises the following steps: The measuring cell is subjected to a time-variable current and a time-variable voltage by means of a signal generator. The current through the measuring cell and the voltage applied to the measuring cell are measured at a selected frequency or a selected frequency range and the phase angle between the measured current and voltage is determined using an electronic signal processing device.

Description

Measuring cell and thus performed method for determining the degree of digestion of biological cells by electroporation

The invention relates to a measuring cell and a method that can be carried out therewith for determining the degree of digestion of biological cells by electroporation by measuring the electrical conductivity of a mass flow of biological cells / cell assemblies guided in a tube / channel in a transport liquid.

In the electroporation of plant cells in large mass flows, it makes sense to monitor the degree of electroporation in the process at the flowing Elektroporationsgut in order to obtain a control variable for the energy-optimal setting of the system.

In / 1 / a method for measuring the degree of electroporation is described, in which the change in the ohmic resistance in the lower frequency range is evaluated in relation to the ohmic resistance in the upper frequency range. The measurement is carried out via a four-electrode system, which is inserted into the cell tissue to be measured.

The piercing method is only suitable for sampling and is therefore unsuitable for continuously supplying measured values for a plant control.

The measurement at two different frequencies including amplitude determination is circuitry more complex than the determination of the phase angle at only one frequency. For the phase angle determination, a time measurement between the zero crossings of current and voltage is sufficient, while a current and voltage measurement is required for the amplitude determination. For sugar beet, the upper frequency range is a few megahertz, the frequency of maximum phase shift is about 50 kHz. At this frequency, the lead inductances at moderate line lengths do not significantly affect the measurement result, with an impedance correction appearing necessary in the megahertz range.

The object of the invention is to provide an industrial-scale measuring cell and a process suitable for industrial use with which the degree of digestion of large mass flows caused by electroporation of biological cells or cell assemblies guided in a transport fluid is reached and from this a control and / or regulating signal for optimal energetic guiding of an electroporation system can be derived. The object is achieved by a measuring cell according to the features of claim 1 and a measuring method which can be carried out in accordance with the method steps of claim 7.

The measuring cell for determining the degree of digestion of biological cells consists according to claim 1 of a structure which can be flowed through for measuring at least a partial flow of the mass flow. The measuring cell is constructed either for a resistive / capacitive measurement and then consists of two oppositely positioned electrodes, the pair of measuring electrodes, between which flows at least part of the mass flow. Or it is constructed for an inductive measuring and then consists of at least one coil in a simple solenoidal form, which at least partially comprises the cross-section of the mass flow. In the case of several coils, the coils of the measuring cell are arranged coaxially with each other.

The degree of digestion of the cells is determined from the decrease in the measured phase angle compared to a comparison measurement on untreated cells. Therefore, the method for determining the digestion degree caused by electroporation is more biological Cells via the measurement of the electrical conductivity of a mass flow of biological cells / cell aggregates guided in a tube / channel in a transport liquid according to claim 7 from the following steps:

The meter is set by a signal generator under a time-varying current and a time-varying voltage. This can be done either by means of a voltage source or a power source. The current through the measuring cell and the voltage across the measuring cell are measured at a selected frequency or a selected frequency range and the phase angle or the course of the phase angle in the frequency range between the current through the measuring cell and the voltage across the measuring cell with an electronic signal processing device determined , The phase angle or its frequency-dependent profile is displayed and documented as a measure of the degree of digestion of the cells, but also derived therefrom a signal for controlling and regulating the device for electroporation. The fundamental measurement error due to a proportion of current flowing past the cells through the transport liquid surrounding the cells is compensated for by calculating this current component from the degree of filling, the conductivity of the transport liquid and the geometry data of the measurement path in a computer of the signal processing device and subtracting it from the measurement current in the correct phase becomes.

In the dependent claims 2 to 6 properties / constructions of the measuring cell are specified:

According to claim 2, the measuring cell is connected to a signal generator to be operated with a time-varying current and a time-varying voltage.

According to claim 3, the measuring cell is manageable. For this purpose, the two measuring electrodes, between which at least a part of the mass flow flows without obstruction when measuring, mounted in a dielectric frame / tube in which they face each other in a defined position with respect to the longitudinal axis. This construction will held for example via a mounted on the frame / tube lever in the mass flow, so that the cross-section of the measuring cell in the flow cross-section of the mass flow is.

A fixed installation of the measuring electrodes of the measuring device is described in claim 4. The measuring cell is also of resistive / capacitive design and forms a section of the transport tube / channel. In the dielectric channel wall of this section, the two measuring electrodes are installed / recessed in such a way that they do not form a flow resistance for the passing mass flow. With regard to the flow / longitudinal axis in the tube / channel, the two measuring electrodes face each other with their forehead in mirror image. The two measuring electrodes may be pin-shaped or plate-shaped and follow with their exposed surface of the channel contour steadily or smoothly.

In claim 5, the measuring cell is also described as manageable and constructed for inductive measuring. For this purpose, it consists of a coil or two coaxial coils, an excitation and a measuring coil, which can be held in the mass flow or can, so that the cross section of the measuring cell is located in the flow cross-section.

The stationary installation of the inductive measuring cell is described in claim 6. The inductive measuring cell forms a portion of the transport tube / channel and consists for this purpose of a coil or of two mutually coaxial coils, an excitation and a measuring coil. The cross section of the measuring cell then covers the flow cross section of the mass flow in any case.

In the subordinate to the method claim 7 method claims 8 to 13, various steps are described, with which the method can be operated depending on the. In claim 8, the measured value of the degree of digestion for controlling and regulating the processing time and / or the required energy is included and the operating parameters determining the degree of digestion during electroporation, such as electric field strength, pulse length, number of pulses per volume element, temperature, number of passes through the cell disruption reactor, storage time of the cell suspension between two electroporation passages or between electroporation and extraction, according to a characteristic field based on the measurement of the Digestion set. The measurement, continuous or intermittent, takes place over at most the cross section of the mass flow, the cell suspension of biological cells or such cell aggregates and the carrier liquid takes place (claim 9).

It is stated in claim 10 that in order to measure the conductivity of the mass flow, a periodic waveform of the current / voltage from the signal generator is applied to the measuring device. A sine wave involved in the waveform with at least strong occurrence of the phase shift is evaluated for measurement.

In order to measure the phase shift, a pulse-shaped, aperiodically or periodically damped curve profile is used according to claim 11. By conversion into the frequency domain with usual time frequency transformation method, preferably by means of Fast Fourier Transformation, FFT, a frequency with the strongest occurrence of the phase shift or a narrowband frequency range with a strong occurrence of the phase shift is evaluated for measurement.

In claim 12 it is described that the same electrodes are used for the resistive / capacitive coupling of the measuring signal as for the electroporation and the measurement is carried out in electroporation pauses. The measuring cell is then an immediate part of the electroporation path. The switching between the signal generator and the measuring device then takes place via a switching device or coupling unit. The method can be refined according to claim 13, when measured at the electroporation at the same time at several points. It is then possible to make a statement about the electroporation course along the measuring section at several measuring points.

According to claim 14, instead of a measuring pulse, the electroporation pulse itself can also be used for the measurement. For this purpose, this pulse must have a sufficiently large frequency components in the frequency range sensitive to the phase measurement.

By comparing the degree of digestion before and after the electroporation reactor, as it allows the arrangement of two measuring devices according to claim 15, the operating point of the system can be adapted to the present before the electroporation passage degree of digestion of the cells. The adaptation is done by means of a characteristic field, as described in claim 8.

Summarized :

Only the phase angle between current and voltage across a cell structure at a frequency with the greatest possible phase shift for determining the degree of electroporation is measured and evaluated.

A method for correcting a parallel resistance of the suspension water by means of additional conductivity and filling degree measurement is specified. To minimize energy, a waiting period between electroporation and extraction is maintained. The control is performed on the basis of a characteristic field.

The proposed electroporation meter is a conductivity meter which measures the complex conductivity of a cell suspension at the frequency at which the phase shift between current and voltage is ideally greatest and, as described above, evaluates this phase shift. With the continuous measurement of the degree of electroporation in large mass flows the energy-optimized system control is possible. By installing the measuring device before the

Electroporation distance can be concluded from the initial degree of digestion of the cells. For example, the cells of frosted sugar beets are already open-minded. The minimum energy consumption is given by waiting time and setting of the optimal operating point according to the characteristic field.

The invention will be described in more detail with reference to the drawing and the example of the e- lektroporation of sugar beet cubes. The

Figures show, on the one hand, the schematic structure of the measuring instrument and, on the other hand, experimental results at the end. It shows:

FIG. 1 shows the resistive / capacitive measuring cell;

FIG. 2 shows the inductive transformer measuring cell;

FIG. 3 shows the inductive measuring cell;

FIG. 4 shows the equivalent circuit diagram of the cell tissue;

FIG. 5 shows the course of the complex impedance;

FIG. 6 shows the complex impedance at 50 kHz;

Figure 7a R _s , R _p depending on the waiting time 2 min;

Figure 7b C, φ depending on the waiting time 2 min;

FIG. 8a R _{s /} R _p as a function of the waiting time 12 min;

Figure 8b C, φ depending on the waiting time 12 min.

FIG. 1 schematically shows the situation for the resistive / capacitive measurement. The mass flow is indicated by the arrow as the direction of flow in the pipe 7. In the tube 7, the two measuring electrodes 1 are exposed so that they do not form a flow obstacle, but directly touch the passing cell suspension. This can be embedded in the pipe wall plates or rods. The two electrodes 1 are connected to the voltage source 3, the signal generator. The complex impedance 2 is representative of that of the electrical measuring circuit. For current and voltage measurement, the electrodes are connected to the evaluation unit 4. The current measurement takes place indirectly at the shunt 5. FIG. 2 shows in the same schematic way the inductive type of measurement with only one coil 6, the transformer coupling which comprises the tube 7 with the mass flow flowing therein and thus does not form a flow obstacle. Current and voltage measurement and evaluation are as described for Figure 1. The further inductive variant with two coils 6, one for the signal reception (left in the picture) for voltage measurement and the other as a transmission coil for the indirect current measurement on the shunt 5, Figure 3. Both coils 6 include the tube 7 also and do not form a flow obstacle.

The complex impedance of an association of intact biological cells shows a capacitive component in the middle frequency range, while at low and high frequencies the impedance 2 is almost ohmic. To describe this frequency-dependent electrical behavior, a simplistic equivalent circuit diagram of the biological cell tissue is used (FIG. 4). In this equivalent circuit, the capacitance C represents the effective capacity of the cell membranes, the parallel resistance R _P the effective ohmic resistance of the cell membranes and the series resistance R _s the effective resistance of the cell interior. The sum of R ₅ and R _P acts in the lower frequency range and only Rp in the upper frequency range.

In the case of electroporated cells, the capacitive component of the impedance in the middle frequency range decreases sharply, at the same time the ohmic resistance decreases in the lower frequency range. If the complex impedance of an intact cell grouping is measured in the middle frequency range, there is a phase shift between the measured voltage and the measured current due to the capacitive impedance component in the middle frequency range. From the decrease in the phase angle with increasing degree of electroporation, it is thus possible to deduce the degree of electroporation. FIG. 5 confirms the informative value of the detection of the phase shift for the degree of electroporation. The magnitude impedance | Z | and the phase angle φ are plotted against the frequency f before and after the electroporation by the equivalent circuit diagram (FIG. 4). The largest phase angle occurs at about 50 kHz.

In a suspension of the complex impedance of the cell assemblies is the ohmic resistance of the suspension water in parallel. This resistance varies with the conductivity of the suspension water and the density of the suspension. The current component through this resistor causes a higher active-current component in the impedance measurement and thus a reduction of the measured phase angle. However, this influence can be computationally compensated if the degree of filling and conductivity of the suspension water are known. These two measured variables are already recorded in the process for other control and monitoring purposes. For compensation, for example, the filling rate, conductivity of the suspension water and geometric data of the measuring section of the current component is calculated by the parallel resistor and subtracted in phase from the measuring current. If the system operates with a high filling density and / or constant conductivity of the suspension water (regulation of the conductivity is provided by the factory), it may be necessary to use a phase angle correction. U. also be waived. Laboratory experiments on beet cubes show that it makes sense for energy-optimized operation to apply a small number of pulses in the electroporation reactor and to store the electroporation material for a certain time before extracting the ingredients. In this mode of operation, the elec- troporation material is not yet fully digested upon exiting the electroporation reactor. On the basis of a previously determined characteristic field, in which the time course of the degree of digestion in dependence on the influencing parameters, such as temperature, pulse parameters, etc., is plotted, then the control of the electroporation reactor can take place. Previously, laboratory measurements were made on sugar beet cubes. To this end cubic pieces with an edge length of 5.5 cm were cut from sugar beets and in a homogeneous electric field with an aperiodically attenuated field strength profile at a peak field strength of 6 kV / cm and a pulse width value of 1.3 μs with 5 pulses at intervals of 3 seconds paced. A voltage of approx. 3 V was applied from a function generator before the pulsing and then over a longer period of time, and voltage, current through the sample and phase angle between current and voltage were continuously measured at the frequencies 500 Hz, 50 kHz and 5 MHz. Subsequently, a conversion into the values R ₅ , Rp and C of the simple equivalent circuit diagram took place. The experiments showed that in the course of the waiting time after the pulsing the phase angle, and R _P decreased, while the calculated capacity C increased. R _s remained almost constant (Figures 7 and 8). The experiments were stopped after different waiting times and the beet cubes cut as quickly as possible and 15 min. pressed. Fig. 6 shows the complex resistance measured at 50 kHz. First, 5 pulses were applied at intervals of 3 seconds (gray bar) and the samples were then pressed after different waiting times (colored bars) (pressing time: 15 min). Tab. 1 shows the pressing results in comparison to the phase angle at the beginning of the pressing. Since, due to the preparatory work for the pressing, the phase angle could only be measured up to approx. 5 min before the pressing, the expected phase angle was extrapolated on the basis of the curves. The degree of digestion determined by pressing correlates with the decrease of the phase angle within the scope of natural scattering and measurement uncertainties.

The test results also show that the waiting time between electroporation and pressing off plays a decisive role in the level of digestion. The diagrams Fign. 7 and fig. 8 show the decrease of the resistance R _P and the decrease of the phase angle after single pulses. For this purpose, waiting times of about 2 minutes (figure 7) and about 12 minutes (figure 8) were recorded between the individual pulses. held. Clearly the pulse-dependent decrease of R _P and φ can be recognized on the second pulse. Only after each 1st pulse was no decrease in these parameters visible, sometimes a slight increase in Rp.

Tab. 1: Pressing results in comparison to the phase angle at 50 kHz

LIST OF REFERENCE NUMBERS

1. measuring electrode

2. Impedance

3. Voltage source

4th evaluation unit

5th shunt

6. coil

7. pipe

Claims

Claims:

1. Measuring cell for determining the degree of digestion of biological cells by electroporation by measuring the complex electrical conductivity of a mass flow in a tube / channel in a transport liquid from biological cells / cell aggregates, wherein the cross section of the measuring cell during measurement at least over a part of the flow cross section of Mass flow is, characterized in that: the measuring cell is constructed either for a resistive / capacitive measuring and then consists of two in a defined position opposite measuring electrodes, or is constructed for an inductive measuring and consists of at least one coil, wherein in the case of multiple coils , the coil axes coincide.

2. Measuring cell according to claim 1, characterized in that the resistive / capacitive or inductive measuring cell is connected to the frequency-specific measurement of the phase shift between current and voltage to a signal generator.

3. Measuring device according to claim 2, characterized in that the resistive / capacitive measuring cell is manageable and for this purpose its two measuring electrodes are incorporated in a dielectric, durable in the mass flow frame / tube.

4. Measuring device according to claim 2, characterized in that the resistive / capacitive measuring cell forms a section of the transport tube / channel, in the inner wall of which the two measuring electrodes are embedded / incorporated and with respect to the mirror face the flow / longitudinal axis.

5. Measuring device according to claim 2, characterized in that the inductive measuring cell can be handled and for this purpose consists of a coil or of two mutually coaxial coils, an exciter and a measuring coil, which can be held in the mass flow.

6. Measuring device according to claim 2, characterized in that the inductive measuring cell forms a portion of the transport tube / channel and for this purpose consists of a coil or of two mutually coaxial coils, an excitation and a measuring coil, which is flowed through by the mass flow or become.

7. A method for determining the degree of digestion of biological cells causing by electroporation on the measurement of the electrical conductivity of a conducted in a tube / channel in a transport liquid mass flow of biological cells / cell assemblies with a measuring cell described in claims 3 and 4 or 5 and 6, consisting from the steps:

the measuring cell is set by means of a signal generator under a time-variable current and a time-variable voltage,

- The current through the measuring cell and the voltage across the measuring cell are measured at a selected frequency or a selected frequency range and determines the phase angle between the measured current and voltage with an electronic signal processing device and display and documentation of the state of Elektroporationsguts and control and regulation of Elektroporationsanlage reused, - The fundamental measurement error due to a proportion of current flowing past the cells through the transport liquid surrounding the cells, is compensated by the fact that this proportion of current from the degree of filling, the conductivity of the transport liquid and the geometry data of the measuring section calculated in a computer of the signal processing device and in-phase of the measuring current is subtracted.

8. The method according to claim 7, characterized in that the measured value of the degree of digestion for controlling and regulating the processing time and / or the required energy is included and determining the degree of digestion during electroporation operating parameters, such as electric field strength, pulse length, number of pulses per volume element, Temperature, number of passes through an electroporation reactor, storage time of the cell suspension between two reactor runs or between electroporation and extraction, to be set according to a characteristic field on the basis of the measurement of the degree of digestion.

9. The method according to claim 8, characterized in that the continuous or intervalwise measurement takes place over at most the cross section of the mass flow.

10. The method according to any one of claims 7 to 9, characterized in that for measuring the conductivity of the mass flow, a periodic waveform of the current / voltage from the signal generator is applied to the measuring device, wherein participating in the waveform sinusoidal vibrations with strongest occurrence of the phase shift is evaluated for measurement.

11. The method according to any one of claims 7 to 9, characterized in that a pulse-shaped, aperiodic or periodically damped waveform is used for the measurement, wherein by conversion from the time in the frequency range, a frequency with the strongest occurrence of the phase shift or a narrowband frequency range with strong Appearance of the phase shift is evaluated for measurement.

12. The method according to claim 7, characterized in that for the resistive / capacitive coupling of the measuring signal, the same electrodes are used as for electroporation and the measurement is carried out in Elektroporationspausen.

13. The method according to any one of claims 7 to 12, characterized in that the determination of the degree of digestion is carried out at at least one point of a Elektroporationsstrecke.

H.Verfahren according to claim 11, characterized in that the electroporation pulse itself is used for the measurement.

15. The method according to claims 7 to 11, characterized in that a measurement immediately before the inlet of a E lektroporationsstrecke and a further measurement immediately after the outlet thereof is carried out and from the difference / the difference, a control / regulating signal for setting the Elektroporationsanlage for the complete ZeIl- digestion of the process material is derived.