METHOD AND DEVICE FOR CONTROLLING THE FILLING LEVEL IN A CHAMBER

Abstract

The invention relates to a method and device for controlling a filling level of a suspension of cells, cell derivatives, organelles, sub-cellular particles and/or vesicles within at least one chamber of a device for applying an electric field to the suspension. In order to avoid overfilling of the chamber if multiple electroporation cycles are performed and to achieve exact filling in an environment of unpredictable chamber volume, the amount of suspension filled into the chamber is dynamically limited in the course of several electroporation cycles by determining at least one change of the electrical resistance at the outlet port. The resistance between at least one electrode and a grounding electrode is measured during the filling procedure of each cycle at several points in time. Once a change of resistance is detected, the termination routine is initiated and the filling procedure is finally terminated. Exact filling of the chamber is thus ensured during each electroporation cycle so that enhanced reproductive electroporation performance can be guaranteed.

Claims

1. A method for controlling a filling level of a suspension of cells, cell derivatives, organelles, sub-cellular particles and/or vesicles within at least one chamber of a device for applying an electric field to the suspension, said device comprising at least a first and a second electrode, at least one inlet port disposed at one end of the chamber, and at least one outlet port disposed at the opposite end of the chamber, wherein the first electrode is disposed within the chamber at the inlet port and the second electrode is disposed within the chamber at the outlet port, and wherein the chamber further comprises at least one grounding electrode, said method comprising: a) Starting a filling procedure wherein the suspension is charged into the chamber through the inlet port; b) Measuring the electrical resistance within the chamber between the second electrode and the grounding electrode during the filling procedure at several points in time; and c) Initiating a termination routine comprising terminating the filling procedure, wherein the termination routine is initiated depending on at least one change of the electrical resistance between the second electrode and the grounding electrode.

2. The method according to claim 1, wherein the termination routine is initiated when the electrical resistance has reached a predetermined value.

3. The method according to claim 1, wherein the filling procedure is terminated if termination conditions are met, said termination conditions comprising: a first slope of a change of the resistance exceeds a first predetermined threshold, and a second slope of a change of the resistance reaches a second predetermined threshold, wherein the second threshold represents a slope that is lower than the slope represented by the first threshold.

4. The method according to claim 3, wherein the termination conditions further comprise that a third slope of a change of the resistance, which is determined after the second slope, is equal to or lies below the second threshold.

5. The method according to claim 3, wherein the termination routine comprises a deferred termination after it has been determined that the termination conditions are met, wherein the filling procedure is continued before final termination based on at least one preset parameter.

6. The method according to claim 5, wherein the preset parameter comprises a number of steps performed by a peristaltic pump.

7. The method according to claim 6, wherein the number of steps is calculated by the equation N_target=(N_p+N_sts)/2+P, wherein N_target is the calculated number of steps necessary to complete the filling procedure, N_p is the number of steps performed during the previous filling procedure, N_sts is the number of the current standard detection target steps, and P is an empirically determined number of steps.

8. The method according to claim 6, wherein the number of steps is calculated by the equation N_target=((N_now+S−R)+N_p)/2+P−N, wherein N_target is the calculated number of steps necessary to complete the filling procedure, N_now is the current number of steps, S is a preset number of steps, R is a preset number of steps correlating with the energy of an electrical voltage pulse, N_p is the number of steps performed during the previous filling procedure, P is an empirically determined number of steps, and N is the number of filling procedures multiplied by 20.

9. The method according to claim 1, wherein an initial delivering procedure is started before the filling procedure is started, said delivering procedure comprising: mixing the suspension of cells, cell derivatives, organelles, sub-cellular particles and/or vesicles with a substrate or probe at a mixing point upstream of the inlet port, and charging the mixture or the suspension through the inlet port into the chamber until a resistance drop is detected at the first electrode, wherein the electrical resistance is measured within the chamber between the first electrode and a grounding electrode during the initial delivering procedure at several points in time.

10. The method according to claim 9, wherein the resistance drop comprises a decrease of the electrical resistance in the range from 5 to 15 Ohms, preferably about 10 Ohms.

11. The method according to claim 9, wherein the initial delivering procedure further comprises: stopping the charging of the suspension into the chamber when the resistance drop is detected and retracting the suspension back to the mixing point where the suspension is mixed with the substrate or probe before the filling procedure is started.

12. The method according to claim 9, wherein the initial delivering procedure is only performed once before the first filling procedure is started.

13. The method according to claim 1, wherein the electric field is applied to the suspension within the chamber by supplying at least one voltage pulse through at least one electrode after the filling procedure is terminated and/or wherein filling the chamber with the suspension and subsequently supplying at least one voltage pulse is repeated for a predefined number of cycles.

14. A device (1) for applying an electric field to a suspension of cells, cell derivatives, organelles, sub-cellular particles and/or vesicles within at least one chamber, comprising: at least a first and a second electrode (20, 21), at least one inlet port (10, 11) disposed at one end (12) of the chamber (6), and at least one outlet port (7, 8) disposed at the opposite end (9) of the chamber (6), wherein the first electrode (20) is disposed within the chamber (6) at the inlet port (10, 11) and the second electrode (21) is disposed within the chamber (6) at the outlet port (7, 8), and wherein the chamber (6) further comprises at least one grounding electrode (5), characterized in that at least the second electrode (21) is a first sensor electrode for measuring the electrical resistance within the chamber (6) between the second electrode (21) and the grounding electrode (5).

15. The device according to claim 14, wherein the first electrode (20) is a second sensor electrode for measuring the electrical resistance within the chamber (6) between the first electrode (20) and the grounding electrode (5).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows a flow chart representing an exemplary suspension handling procedure of the method according to the invention.

[0031] FIG. 2 shows a flow chart representing an exemplary sequence of the initial delivering procedure (“priming”) of the method according to the invention.

[0032] FIG. 3 shows a flow chart representing an exemplary embodiment of the method according to the invention including a filling procedure and a termination routine.

[0033] FIG. 4 shows a diagram representing an exemplary course of the electrical resistance during a filling procedure measured by an electrode at the outlet port of a device according to the invention.

[0034] X axis shows the number of measurements taken (interval: about 33 steps of a stepper motor pump);

[0035] Y axis shows the electrical resistance measured [Ohm].

[0036] FIG. 5 shows a perspective view of the outer side of an exemplary embodiment of a device according to the invention.

[0037] FIG. 6 shows a plan view of the inner side of one component of the device according to FIG. 5.

[0038] FIG. 7 shows a plan view of the inner side of another component of the device according to FIG. 5.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0039] FIG. 1 shows an overview of an embodiment of the entire suspension handling process according to the invention. First, an initial delivering procedure (“priming”) is started before the chamber is filled with the suspension. The initial delivering procedure comprises mixing of the suspension with a substrate/probe at a mixing point upstream of the inlet port and charging the mixture through the inlet port into the chamber. This delivering procedure is only performed once before the first filling procedure is started. The initial delivering procedure is further described in detail with reference to FIG. 2. If priming has not been successful, the suspension handling process is terminated. If priming has been successful, the filling procedure is started. The filling procedure comprises charging the suspension/probe mixture into the chamber and controlling the filling level until the optimal filling level is reached. The filling procedure is further described in detail with reference to FIG. 3. Thereafter, electroporation is performed by applying one or more voltage pulse(s) to the mixture (“pulsing”). After pulsing, and if the preset number of cycles has not yet been reached, the chamber is cleared and the second (next) aliquot of the mixture is filled into the chamber for electroporation. The filling and pulsing process can be repeated several times, that is, for a preset number of cycles. If the preset number of cycles is reached, or the electrical resistance measured between two electrodes within the chamber is out of range for two consecutive measurements (“Error2”), the chamber is cleared and the suspension handling process is finally terminated.

[0040] FIG. 2 shows an exemplary sequence of the initial delivering procedure (“priming”). In order to be independent of the tubing length, the optical properties of the suspension and to avoid more complex measurements such as capacitive methods during the initial delivering procedure, the electrodes of the reaction chamber are used to observe liquid-based electrical changes inside the chamber to determine when the first fraction of the suspension has reached the reaction chamber. To this end, the initial delivering procedure (“priming”) is started by starting a first pump (the “upper pump” as referred to in FIG. 2, the first pump being a peristaltic, stepper motor pump in this example) which pumps a suspension of cells, cell derivatives, organelles, sub-cellular particles and/or vesicles out of a first reservoir to a mixing point and then to an inlet port of the reaction chamber. The measurement of the electrical resistance at the first electrode of the chamber (the “lowest (electrode) segment” as referred to in FIG. 2) is started when a preset number of steps of the first pump has been performed. For example, pumping may start with 1,000 steps before starting the resistance measurement. This is to be immune to small liquid fractions moving and affecting the resistance at the very beginning (see FIG. 4, “initial drop of resistance”). If the resistance measured at the first electrode drops below a predetermined threshold within a preset timeout period (e.g. 60 seconds), the first (“upper”) pump is stopped. For example, the resistance threshold value may be about 10 Ohms below the initially measured resistance value upon start of priming As soon as the measured resistance at the first electrode drops by 10 Ohms, the suspension shall be considered as detected. That is, in this “priming” process the resistance near the inlet port between the first electrode and a grounding electrode is measured. If the infinite electrical resistance of the empty electroporation chamber falls below the predetermined threshold, it can be reliably indicated that the suspension has reached the electrode closest to the inlet port.

[0041] If the first pump is stopped due to a detection of a sufficient resistance drop and no separate substrate/probe feed is selected, the initial delivering procedure is terminated and the filling procedure is started (see FIG. 3). However, if no resistance drop is detected at the first electrode, the first (“upper”) pump is stopped in this case as well and the routine is terminated with error indication. If the first pump is stopped due to a detection of a sufficient resistance drop and a separate substrate/probe feed is established, the suspension is retracted to the mixing point by reversing the running direction of the first pump. At the mixing point the suspension is mixed with the substrate or probe. To this end, a second pump is started (the “lower pump” as referred to in FIG. 2, the second pump being a peristaltic, stepper motor pump in this example) so as to pump the substrate or probe from a second reservoir to the mixing point. Mixing the suspension with the probe upstream of the inlet port and measuring the resistance upon entry of the mixture into the chamber ensures that the suspension is perfectly prepared for electroporation before it is introduced into the chamber. Incomplete mixture due to varying tubing length and different optical properties of the suspension can thus be avoided. Preferably, at least one optical sensor is disposed upstream of the mixing point at the tube connecting the second reservoir with the mixing point in order to detect the liquid comprising the substrate or probe. If no liquid is optically detected within a preset timeout period, the second “lower” pump is stopped and the routine is terminated with error indication. But if the liquid comprising the substrate/probe is detected, the second “lower” pump is stopped as well and the tubing connecting the mixing point with the inlet port is then filled with the mixture by both pumps running simultaneously until it reaches the inlet port (the “cartridge entrance”” as referred to in FIG. 2). The initial delivering procedure is then terminated and the filling procedure is started (see FIG. 3).

[0042] FIG. 3 shows an exemplary embodiment of the filling procedure and termination routine according to the invention, while FIG. 4 shows a typical resistance characteristic during the filling procedure. According to the invention filling of the reaction chamber is controlled by monitoring the electrical resistant characteristic in the chamber at the outlet port (second electrode). Upon start of the filling procedure, at least one pump (e.g. first (“upper”) and/or second (“lower”) pump according to FIG. 2) pumps a certain volume of the suspension/mixture into the chamber. For example, if a peristaltic (stepper motor) pump is used, the pump is set to a predetermined number of motor steps (N_p). For the first filling (cycle) N_p is set to a default value and for all further fillings (cycles) N_p is set to a predetermined value based on values of previous fillings.

[0043] During the entire filling procedure the resistance value of the second electrode at the outlet port of the reaction chamber is measured continuously. Once the resistance falls below a predetermined value (limit “threshold T1”), the termination routine is initiated. If the system determines that a first slope of the change in resistance exceeds a first predetermined threshold (T2), the system waits for the slope to decrease again until a second slope of the change in resistance reaches a second predetermined threshold (T3) for at least two consecutive measurements. If these criteria are being met then the pump (which is at N_now steps) continues to rotate for another fix amount of steps (S). Since the effect of volume reduction is also affected by the energy of the voltage pulse delivered, this goes into the pump correction by reducing the target number of steps by R.

[0044] Extensive experiments have shown that variations in the correlation of resistance and filling level can be sufficiently reduced by setting the target pump steps for the current filling (if it is not the first filling cycle) to:

(Number of steps from previous filling “N_p”+current standard detection target steps)/2+a fix value (P).

[0045] These experiments have still shown a deviation from the perfect filling amount that constantly increases from filling to filling. This is addressed by adding further steps N to the filling that are a product of a constant value and the current filling cycle:

N_target=((N_now+S−R)+N_p)/2+P−N

[0046] After these additional pump steps have been performed, filling of the chamber is terminated. In addition, the number of steps for the pump cannot be higher than the steps from the previous filling (if it is not the first filling cycle). Accordingly, if a preset maximum number of pump steps (N_max) is reached, filling of the chamber is terminated.

[0047] T1 can be, e.g., set to 500 Ohm;

[0048] T2 can be, e.g., set to 50 Ohm/400 pump steps (system specific);

[0049] T3 can be, e.g., set to 20 Ohm;

[0050] S can be, e.g., set to 850;

[0051] R is set to a number of steps correlating with the voltage pulse energy;

[0052] P is an empirically determined value (e.g. 200);

[0053] N is set to the number of filling cycles, for example, multiplied by 20;

[0054] N_now is the current number of steps;

[0055] N_p is the previous combined number of steps (first pump+second pump);

[0056] N_target is the target number of steps (first pump+second pump combined) to complete the current filling.

[0057] FIG. 5 shows the outer side of an exemplary embodiment of a device 1 according to the invention and FIGS. 6 and 7 each show the inner side of one component of the device 1 according to FIG. 5. The device 1 comprises base members 2, 30, each base member 2, 30 representing a component of the device 1 which is assembled of two components (base members 2 and 30) that are attached to each other. At their outer side, the base members 2, 30 are each provided with connectors 31 for connecting conduits to the ports 7, 8, 10, 11 of a curved reaction chamber 6. One or more reservoirs for the suspension/probe to be processed and one or more containers for processed suspension can be connected to the connectors 31 via suitable conduits. The suspension can be charged into and discharged from the chamber 6 by means of a pumping element, e.g., a vacuum pump or a peristaltic pump or the like, which may be connected to the suspension circuit between the reservoir(s)/ container(s) and the connectors 31. In order to render the device 1 compatible with common conduits and pumping systems, the connectors 31 can be Luer slip or Luer lock connectors.

[0058] The base member 30 further comprises a multitude of conductive areas 32 for providing electric connection to the electrodes 4, 5 in the chamber 6. The conductive areas 32 may comprise an electrically conductive polymer, in particular a polymer doped with electrically conductive material or an intrinsically conductive polymer. The conductive areas 32 are designed to provide an electrical connection between the electrodes 4, 5 and at least one electric contact point 33. In this embodiment the conductive areas 32 are holes in the base member 30 which are at least partially filled with the electrically conductive material. The conductive areas 32 are electrically coupled with at least one electric contact point 33 via at least one conductive path, e.g., copper tracks on a layer of the base member (not shown). The electric contact point 33 can be contacted by at least one electric contact, so as to provide direct or indirect electric connection to a power source and/or voltage pulse generator.

[0059] The base members 2, 30 each include a curved recess 3 which is provided with four electrodes 4, 5. According to a preferred embodiment of the device 1 the chamber 6 may comprise at least two segments, each segment comprising at least two electrodes, wherein the grounding (counter) electrode is a common electrode of at least two segments. That is, three of the electrodes are segment electrodes 4 while one electrode is a counter electrode 5. The base member 2 represents one component of the device 1 which is assembled of two components that are attached to each other, wherein at least the inner sides of these components are designed inversely. That is, the base member 2 and the base member 30 have mirror-inverted inner sides that are attached to each other so that the recesses 3 of base members 2, 30 form a chamber 6 for holding a suspension of cells, cell derivatives, organelles, sub-cellular particles and/or vesicles. In this chamber 6 an electric field can be applied to the cells, cell derivatives, organelles, sub-cellular particles and/or vesicles, e.g., for transferring biologically active molecules such as nucleic acids or proteins into the cells, cell derivatives, organelles, sub-cellular particles and/or vesicles. To this end, the electrodes 4, 5 of base member 2 and the corresponding electrodes 4, 5 of base member 30 establish electrode pairs, wherein the segment electrodes 4 of base member 2 and the oppositely arranged counter electrode 5 of base member 30 establish three electrode pairs while the counter electrode 5 of base member 2 and the three oppositely arranged segment electrodes 4 of base member 30 also establish three electrode pairs. In this configuration the counter electrode 5 of base member 2 and the counter electrode 5 of base member 30 are each common electrodes of three segments so that the chamber 6 comprises six segments, wherein each segment is provided with one segment electrode 4 and an area of one common counter electrode 5.

[0060] Two ports 7, 8 are disposed at one end 9 of the chamber 6 and two ports 10, 11 are disposed at the opposite end 12 of the chamber 6. At the “lower” end 12, one port of ports 10, 11 can be used as inlet port for charging the chamber 6 and the other port of ports 10, 11 can be either used as outlet port for discharging the chamber 6 or as additional outlet port for clearing the chamber 6. At the opposite end 9, one port of ports 7, 8 can be used as outlet port for discharging the chamber 6 and the other port of ports 7, 8 can be used as additional outlet port, either for discharging the chamber 6 or, e.g., for venting the chamber 6. Accordingly, in this exemplary embodiment, each end 9, 12 is provided with two ports 7, 8, 10, 11 through which the chamber 6 can be filled with the suspension and/or through which the suspension can be purged out of the chamber 6. Depending on the flow direction, one end of the chamber comprises at least one inlet port while the opposite end of the chamber comprises at least one outlet port. For example, if at least one of ports 10, 11 is used as inlet port and at least one of ports 7, 8 is used as outlet port, the electrode 4 next to the inlet port 10, 11 at end 12 is, by definition, a first electrode 20 (second sensor electrode) and the electrode 4 next to the outlet port 7, 8 at end 9 is, by definition, a second electrode 21 (first sensor electrode). Accordingly, in this exemplary embodiment, the electrical resistance for controlling the filling procedure is measured between the second electrode 21 of base member 30 and the grounding electrode 5 of base member 2. The measurement of the electrical resistance for controlling the initial delivering procedure is measured between the first electrode 20 of base member 2 and the grounding electrode 5 of base member 30. However, any other electrode configuration and/or chamber design can be realized as long as it is suitable to accomplish the method according to the invention as described above.