Patent Grant
11573221
Tumor Treating Fields (TTFields) are low intensity electric fields in the intermediate frequency range that target solid tumors by disrupting mitosis. When treating patients, TTFields are delivered via transducer arrays made from a plurality of ceramic disks with a high dielectric constant (as described, for example, in U.S. Pat. No. 8,715,203, which is incorporated herein by reference). The transducer arrays capacitively couple the electric field into the patient's body.
The Inovitro™ system supplied by Novocure is an existing system for studying TTFields in vitro. The Inovitro™ system includes ceramic culture dishes constructed to capacitively couple electric field into the cell culture while the cell cultures are maintained at a controlled temperature. This is used to simulate the capacitive coupling of the electric fields into a patient's body.
One aspect of the invention is directed to a first apparatus for applying electric fields to a sample and for observing the sample using an inverted microscope while the sample is illuminated by a light source. The inverted microscope has a stage and an objective. This first apparatus comprises a bottom panel, ceramic sidewalls, a plurality of electrodes, and a plurality of electrical conductors. The bottom panel has a transparent region. The ceramic sidewalls are affixed to the bottom panel such that the ceramic sidewalls and the bottom panel cooperate to form a container for holding the sample, wherein the ceramic sidewalls are affixed to the bottom panel at a position that surrounds the transparent region, and wherein the ceramic sidewalls have at least one outer surface. The plurality of electrodes are disposed on the at least one outer surface of the ceramic sidewalls at positions selected so that when the sample is positioned in the container, application of a voltage between the plurality of electrodes induces an electric field through the sample. Each of the plurality of electrical conductors provides electrical contact with a respective one of the plurality of electrodes. The bottom panel, the transparent region, and the ceramic sidewalls are sized and shaped to facilitate positioning of the container on the stage of the inverted microscope so that when the sample is positioned in the container, light emanating from the light source is free to travel along an optical path that passes through the sample, through the transparent region, and into the objective of the inverted microscope. And each of the plurality of electrodes and each of the plurality of electrical conductors is positioned with respect to the transparent region so as not to interfere with the optical path.
In some embodiments of the first apparatus, the ceramic sidewalls are cylindrical.
Some embodiments of the first apparatus further comprise at least one thermistor mounted to the ceramic sidewalls at a position that does not interfere with the optical path. Some embodiments of the first apparatus further comprise at least one thermistor mounted to the bottom panel at a position that does not interfere with the optical path.
In some embodiments of the first apparatus, the ceramic sidewalls are mounted to the bottom panel using an adhesive.
In some embodiments of the first apparatus, the ceramic sidewalls are mounted to the bottom panel using a screw mount configured to squeeze the ceramic sidewalls and the bottom panel together. Some of these embodiments further comprise an O-ring disposed between the ceramic sidewalls and the bottom panel.
In some embodiments of the first apparatus, the ceramic sidewalls are mounted to the bottom panel using an O-ring that has (a) an outer diameter that matches the inner diameter of a Petri dish and (b) an inner diameter that matches the outer diameter of the cylindrical sidewalls, and the cylindrical sidewalls are jammed into the O-ring to provide an interference fit.
In some embodiments of the first apparatus, the entire bottom panel is transparent.
Some embodiments of the first apparatus further comprise at least one thermistor mounted to the container at a position that does not interfere with the optical path. In these embodiments, the entire bottom panel is transparent, the ceramic sidewalls are cylindrical, and the second direction is roughly perpendicular to the first direction.
In some embodiments of the first apparatus, the plurality of electrodes comprises at least four electrodes disposed on the at least one outer surface of the ceramic sidewalls at positions selected so that when the sample is positioned in the container, (a) application of a voltage between a first subset of the at least four electrodes induces an electric field in a first direction through the sample, and (b) application of a voltage between a second subset of the at least four electrodes induces an electric field in a second direction through the sample. The plurality of electrical conductors comprises at least four electrical conductors, and each of the at least four electrical conductors provides electrical contact with a respective one of the at least four electrodes. In some of these embodiments, the second direction is roughly perpendicular to the first direction.
Another aspect of the invention is directed to a second apparatus for optically observing a sample while applying electric fields to the sample. This second apparatus comprises a fluid tight container; first, second, third, and fourth electrodes; and first, second, third, and fourth electrical conductors. The fluid-tight container is shaped and dimensioned for holding the sample, and the container has a transparent bottom panel and ceramic sidewalls affixed to the transparent bottom panel, wherein the ceramic sidewalls have at least one outer surface. The first electrode is disposed at a first position on the at least one outer surface of the ceramic sidewalls. The second electrode is disposed at a second position on the at least one outer surface of the ceramic sidewalls, wherein the second position is opposite to the first position, so that application of an AC voltage between the first electrode and the second electrode induces an AC electric field through the sample in a first direction. The third electrode is disposed at a third position on the at least one outer surface of the ceramic sidewalls. The fourth electrode is disposed at a fourth position on the at least one outer surface of the ceramic sidewalls, wherein the fourth position is opposite to the third position, so that application of an AC voltage between the third electrode and the fourth electrode induces an AC electric field through the sample in a second direction. The first electrical conductor is arranged to route electricity between a first electrical terminal and the first electrode along a path that circumvents the transparent bottom panel. The second electrical conductor is arranged to route electricity between a second electrical terminal and the second electrode along a path that circumvents the transparent bottom panel. The third electrical conductor is arranged to route electricity between a third electrical terminal and the third electrode along a path that circumvents the transparent bottom panel. And the fourth electrical conductor is arranged to route electricity between a fourth electrical terminal and the fourth electrode along a path that circumvents the transparent bottom panel.
In some embodiments of the second apparatus, the first electrical terminal, the second electrical terminal, the third electrical terminal, and the fourth electrical terminal are all disposed in a single electrical connector. In some embodiments of the second apparatus, the ceramic sidewalls are cylindrical.
Some embodiments of the second apparatus further comprise at least one thermistor mounted to the ceramic sidewalls.
In some embodiments of the second apparatus, the ceramic sidewalls are mounted to the bottom panel using an adhesive. In some of these embodiments, the container has an O-ring disposed between the ceramic sidewalls and the bottom panel, and the ceramic sidewalls are mounted to the bottom panel using a screw mount configured to squeeze the ceramic sidewalls and the bottom panel together.
In some embodiments of the second apparatus, the ceramic sidewalls are mounted to the bottom panel using an O-ring that has (a) an outer diameter that matches the inner diameter of a Petri dish and (b) an inner diameter that matches the outer diameter of the cylindrical sidewalls, and the cylindrical sidewalls are jammed into the O-ring to provide an interference fit.
In some embodiments of the second apparatus, the second direction is roughly perpendicular to the first direction.
Various embodiments are described in detail below with reference to the accompanying drawings, wherein like reference numerals represent like elements.
One limitation of the Inovitro™ system is that the construction of the ceramic culture dishes and the circuitry for applying electrical signals to the electrodes on the dishes makes it impossible to perform live-cell microscopy experiments on cells while those cells are being subjected to TTFields. The embodiments described below overcome this disadvantage.
To allow light to flow through the sample for the purpose of microscopy experiments or other imaging techniques, the dish-like apparatus has a bottom panel 20 with a transparent region. In the illustrated embodiment, the entire bottom panel 20 is transparent. However, in alternative embodiments, only a portion of the bottom panel 20 may be transparent. In the illustrated embodiment, the floor of a glass Petri dish 10 serves as the bottom panel 20. Preferably, the transparent region does not introduce optical distortions. In those embodiments that use the floor of a glass Petri dish to serve as the bottom panel, Ibidi® dishes may be used to minimize optical distortions.
Ceramic sidewalls 30 (visible in
The bottom panel 20, the transparent region of the bottom panel 20, and the ceramic sidewalls 30 are sized and shaped to facilitate positioning of the container on the stage of the inverted microscope so that when the sample is positioned in the container, light emanating from the light source is free to travel along an optical path that passes through the sample, through the transparent region of the bottom panel 20, and into the objective of the inverted microscope.
In some preferred embodiments (including the illustrated embodiment), the ceramic sidewalls 30 are formed from a single cylindrical tube. In these embodiments, the ceramic sidewalls 30 will have a single cylindrical outer surface. In alternative embodiments, ceramic sidewalls 30 with different shapes may be used (e.g. square or octagonal). In these embodiments, the ceramic sidewalls 30 will have two or more outer surfaces. For example, in those embodiments where the square ceramic sidewalls are used, the ceramic sidewalls will have four outer surfaces.
In some embodiments, the ceramic sidewalls 30 are mounted to the bottom panel 20 using an adhesive (e.g., biocompatible glue or cement).
In alternative embodiments, the ceramic sidewalls 30 are mounted to the bottom panel 20 using a screw mount configured to squeeze the ceramic sidewalls 30 and the bottom panel 20 together. For example, the upper housing 70 can connect to a Petri dish 10 using a threaded screw-mount connection which includes a set of external threads (not shown) on the upper housing 70 and a corresponding set of internal threads (not shown) on the Petri dish 10. In these embodiments, an O-ring 75 is preferably positioned between the ceramic sidewalls 30 and the bottom panel 20 of the Petri dish 10 such that the O-ring 75 is compressed when the upper housing 70 is screwed into the Petri dish 10. The O-ring 75 seals liquids into the volume defined by the bottom panel 20 and the cylindrical sidewalls 30. The upper housing 70 has an opening 72 through which samples can be inserted into the Petri dish 10.
In alternative embodiments, instead of using a screw mount, an O-ring 75 that has (a) an outer diameter that matches the inner diameter of the Petri dish 10 and (b) an inner diameter that matches the outer diameter of the cylindrical sidewalls 30 is used, and the cylindrical sidewalls 30 are jammed into the O-ring 75 to provide an interference fit. The 35 mm m-dish made by Ibidi GmbH (ibid.com) is suitable for use as the Petri dish 10 in this embodiment.
Note that in those embodiments that use the floor of a glass Petri dish 10 to serve as the bottom panel 20, the vertical walls of the Petri dish 10 are disposed radially beyond the ceramic sidewalls 30.
The height of the ceramic sidewalls 30 may be varied to allow different amounts of media to be placed within each container as well as to accommodate possible inserts (e.g. Boyden inserts). In alternative embodiments, tall containers may be obtained by grafting a second cylinder made of a biocompatible material (e.g., glass or polycarbonate) to the top of a short ceramic cylinder. Positioning the ceramic cylinder at the bottom of the container facilitates the application of TTFields to bottom of the container where cells are plated.
Optionally, tubing 90 is provided to allow for media replacement without the need to remove the dish 10 from the stage of the microscope (not shown) while maintaining sterile conditions. Although only a single piece of tubing 90 is depicted in
As best seen in
The electrodes 41-44 and the region of the ceramic sidewalls 30 beneath the electrodes 41-44 form capacitive electrodes through which the electric field is coupled into the sample (i.e. the cell culture). The advantage of using a ceramic with a high relative permittivity is that the impedance of the electrodes can be kept low whilst maintaining the walls at a thickness that ensures the mechanical rigidity of the dish-like apparatus.
In the illustrated embodiment and as best seen in
Application of an AC voltage between a first subset of electrodes consisting of electrodes 41 and 42 induces an electric field in a first direction through the sample. Application of an AC voltage between a second subset of electrodes consisting of electrodes 43 and 44 induces an electric field in a second direction through the sample. When the electrodes are arranged as depicted in
A plurality of electrical conductors 50 is provided, and each of the plurality of electrical conductors 50 provides electrical contact with a respective one of the plurality of electrodes 41-44 and routes electricity between a given one of those electrodes 41-44 and a respective corresponding electrical terminal. In embodiments that have at least four electrodes, at least four electrical conductors 50 are provided, and each of the at least four electrical conductors 50 provides electrical contact between a respective one of at least four electrical terminals and a respective one of the at least four electrodes 41-44. The conductors 50 may be implemented using individual wires, ribbon cables, flex circuits, etc. Each of the conductors 50 may be connected to the electrodes 41-44 using any appropriate approach including but not limited to soldering, electrical connectors, etc. In some embodiments, each of the electrical terminals is disposed in a single electrical connector 52 (shown in
Each of the plurality of electrodes 41-44 and each of the plurality of electrical conductors 50 is positioned with respect to the transparent region of the bottom panel 20 so as not to interfere with the optical path described above. For example, in those embodiments where the entire bottom panel 20 is transparent, each of the plurality of electrical conductors 50 may traverse a path that circumvents the entire transparent bottom panel 20. The conductors are used for applying electric fields to a sample that is positioned in the container. For example, an AC voltage between 50 and 500 kHz may be applied across the conductors that are wired to the first electrode pair 41-42 and then across the conductors that are wired to the second electrode pair 43-44 in an alternating and repeating sequence. This will cause electric fields with different directions to be generated in the samples that are located in the container in a corresponding alternating and repeating sequence. In alternative embodiments, the voltages may be applied across different combinations of the electrodes 41-44 in a different sequence to provide alternative field shapes or directions.
The dish-like apparatus described herein is useful for various assays such as: watching the evolution of cellular structures in response to TTFields; using fluorescent dyes, GFP-tagged proteins, or other labeled proteins; scanning frequencies to determine the most effective frequency; measuring cells' sensitivity assays to different TTFields intensities; measuring the diameter of cells; measuring migration rates and directions during TTFields application; determining TTFields' effect on cell invasion using a Boyden chamber inserted into the container; determining TTFields' effect intracellular on different structures/molecules within the cell; and determining TTFields' effect on cell grown in 3D structure (e.g. microspheres) using specific inserts which maintain and support the 3D structures (e.g. agarose mesh).
As best seen in
Turning now to
When applying TTFields to cell cultures, Ohmic losses in the cell culture heat the cell culture medium. The thermistor or thermistors 60 are used to monitor the temperature in the container. The electric field intensity and/or the ambient temperature can then be controlled to maintain the desired temperature in the cell culture.
One example of a temperature control algorithm that is suitable for use when applying TTFields is provided below. The data from the thermistors attached to the ceramic sidewalls 30 is transferred to a processor (not shown). The processor compares the current temperature of the hottest thermistor with the temperature recorded in the previous 20 measurements and provides a prediction regarding the temperature to be reached within the next 20 measurements in the current settings. The predication is based on the following equations:
D=[(T(n)−T(n−20))] Equation 1:
T(n+20)=Tn+D Equation 2:
Where:
Based upon the predicted temperature the algorithm determines the change in the electric fields intensity by changing the output currents, thus allowing for the temperature within the dish-like apparatus to reach the target temperature TTARGET with minimal overshooting. The degree of change in the output current is based upon the following scheme:
1. If (TTARGET−0.4)≤T(n+20)≤TTARGET→don't change current
2. If Tn<(TTARGET−0.4) and D>0.3→don't change current
3. If Tn<(TTARGET−0.4) and D*20>0.8→don't change current
4. If 1-3 are false, change current as follows:
Where:
This algorithm takes into account the differences between the temperature of the ceramic sidewalls 30 and the ambient temperature and provides accurate estimation of the temperature within the container (e.g., ±1° C.). The precise estimation of the temperature within the container is based upon extensive temperature measurements performed using the thermistors attached to the ceramic sidewalls 30. Optionally, temperature probes inserted into the media within the container may be used to supplement the temperature measurements obtained using the thermistors.
The algorithm also ensures that the temperature increase within the container from room temperature to the target temperature will take at least 25 minutes to follow the temperature rate increase in control dishes when transferred from room temperature to an incubator set to the target temperature.
In some preferred embodiments, temperature measurements are made every 1-5 seconds (e.g., every 3 seconds); the prediction algorithm starts every 2-10 seconds (e.g., every 6 seconds), and the values of the constants K1-K4 are as follows:
In some preferred embodiments the maximum measured load current is 546 mA; the maximum measured output voltage is 210 V; and the algorithm works with the digital trimmer steps, with a minimal current of 25 steps (e.g., corresponding to 54.6 mA) and a maximum current of 250 steps (e.g., corresponding to 546 mA).
While the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.
This application claims the benefit of U.S. Provisional Application 62/448,152, filed Jan. 19, 2017, which is incorporated herein by reference in its entirety.
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