An in vitro non-contact electrical stimulation and recording assembly and an operation method thereof

Abstract

The invention relates to a non-contact electrical stimulation and recording assembly designed for use in electrophysiological studies in cell culture laboratories carried out in the field of biological sciences, medicine and pharmacy and an operation method thereof. The fact that this assembly of the invention is able to perform a non-invasive stimulation and recording process increases the standardization and reliability of the studies to be performed. In addition, the cost of the electrical stimulation and recording assembly of the invention is also reduced.

Description

[0001] DESCRIPTION

[0002] AN IN VITRO NON-CONTACT ELECTRICAL STIMULATION AND RECORDING ASSEMBLY AND AN OPERATION METHOD THEREOF

[0003] Technical Field of the Invention

[0004] The invention is related to an in vitro non-contact electrical stimulation and recording assembly for use in research carried out in the fields of biological sciences, medicine and pharmacy, and to and an operation method thereof.

[0005] State of the Art

[0006] In vitro cell models are an experimental study method which is frequently used in research in the fields of biological sciences, medicine and pharmacy. Although this method has many advantages in terms of cost and ethics, it also has some limitations. One of these limitations is the difficulty of in vitro modeling of diseases caused by cell excitability. Some researchers try to overcome this problem by means of simple mechanisms they have developed with their own means, but this is not preferred because of both the decrease in the scientific reliability of the study and the fact that it requires a great effort on the part of the researcher. In addition, it is likely that these studies will create major problems in standardization.

[0007] In relation to the aforementioned problems encountered in the experimental study methods of in vitro cell models in the state of the art, effective solutions have been created through the assemblies produced by foreign companies (for example; multielectrode array (MEA) systems), but the high cost has made it almost impossible for researchers to use the aforementioned assemblies [8], [9],

[0013] . In addition, the fact that assemblies such as MEA systems do not allow the use of standard culture dishes used as consumables is another factor that increases the cost of the studies.

[0008] In the state of the art, the technique applied by MEA systems to perform the stimulation of cells can also have negative effects on the cells. The fact that electrical stimulation is carried out through the contact of the electrodes with the cells has an invasive effect on the cells, significantly reducing the standardization and reliability of the studies. In the state of the art, another problem encountered in MEA systems is that the electrode circuits embedded in the well deteriorate in repeated use and the system becomes unusable. In addition, the fact that the electrodes are embedded in the wells also complicates the sterilization process of the culture dishes. These two factors can lead to possible deviations, especially in the standardization of experiments.

[0009] In the state of the art, these problems encountered in MEA systems have been tried to be solved through simple mechanisms developed by researchers with their own means, or through the advanced mechanisms created by a limited number of companies. In the simple devices developed by the researchers themselves, only the stimulation of the cells was concentrated on, and the recording and analysis of electrical signals to be obtained from the cells and the environment could not be developed sufficiently due to the need for more facilities and capabilities. This has led to a decrease in the standardization and scientific validity of the studies. Also, it is very difficult to create in vitro models of diseases due to the low-technology functioning of these systems. However, there are still many studies in which the electrical stimulation of cells has been performed using gold, platinum, silver or bipolar concentric electrodes [2],[6],

[0010] ,

[0012] ,

[0017] ,

[0010] Given the advanced mechanisms created by some companies, it is seen that the most important systems that stand out are MEA systems. A limited number of companies in the world commercially produce the MEA laboratory system. These systems provide in- vitro analysis of neuronal network activity in cultures prepared on the surface consisting of microelectrodes. The electrical potentials that arise in the neural network are detected, amplified, recorded and analyzed by software

[0014] , Not only cell cultures but also brain organotypic cultures are studied with the systems and provide very valuable information

[0001] , [3] , [4] , [5] ,[7],

[0014] ,

[0015] ,

[0016] ,

[0011] Today, a passive MEA contains an average of 128 electrodes (60 electrodes / mm2), but recently the development of next-generation active MEA systems using CMOS (complementary metal-oxide-semiconductor) technology has been accelerated

[0011] . This technology provides stimulation patterns at very high resolution and offers precision in the control of neural activity. In CMOS technology, the number of electrodes can be expressed in thousands per square millimeter. Given the functioning of MEA systems in the state of the art, the first problem encountered is the invasive potential of the system. Electrical stimulation by direct contact with the cells using the electrodes embedded in the culture dish leads to invasive effects on the cells. In the stimulation to be made by the physical contact of the metal electrode with the membrane of the cells, it is inevitable that heterogeneous and asymmetrical effects will occur on the membrane. These effects include the triggering of repair processes due to a mechanical damage to the membrane close to a contact site and the observation of local deformations due to the physical and electrical forces on the bonds of molecules. All this leads to the fact that the cell type being studied diverges from physiological behavior and the consistency of the study data deteriorates. Another problem encountered is that in said contact systems, an equal effect on all cells does not occur; some cells are contacted by the electrode while some cells are not. This non-homogeneous invasive effect on cells significantly reduces the standardization and reliability of their operation.

[0012] Another most important problem encountered with MEA systems in the state of the art is the costs of the systems. The production of electrode circuits used in MEA systems in the state of the art with gold electrodeposition on glass increases the cost of these systems considerably due to the fact that they contain nanotechnological components. In addition, the fact that the system uses a contact-based stimulation and record analysis method prevents the use of standard cell culture dishes in studies and necessitates the use of special culture dishes embedded in wells specially prepared for the system. The use of special culture dishes embedded in wells specially prepared for the system is an extra cost problem created by the system to a user.

[0013] In the state of the art, another problem that indirectly causes the formation of MEA systems is that special culture dishes containing electrodes embedded in the well and determined as consumables by the manufacturers are available in limited numbers due to their cost. Due to the limited number of culture dishes available, the researchers use these culture dishes in repeated trials; however, these systems are not suitable for repetitive experiments. The reason for this is that sterilization cannot be performed at the desired levels due to the degradation of the electrode circuits in repeated trials and the adhesion of the cells to the electrodes embedded in the well in the culture medium. Description of Figures

[0014] Fig. 1. is a representative view of cell stimulation of multi-electrode arrays (MEA).

[0015] Fig. 2. shows stimulation and recording techniques performed in culture medium, A) contact system, B) Non-contact system

[0016] Fig. 3. shows replaceable electrode heads (5) to which the electrodes are attached for use in culture dishes (7) containing different numbers of wells

[0017] Fig. 4. is a representative view of the in vitro non-contact electrical stimulation and recording assembly of the invention.

[0018] Fig. 5. is an overall structure of the electrodes (8) on the electrode head (5) to which the electrodes are attached.

[0019] Fig. 6. shows an overall structure of the electrodes (8) on the replaceable electrode head (5) to which the electrodes are attached, and a representative view of the distance sensor (6) which allows to stop at an appropriate distance from the bottom of a culture.

[0020] Description of the References in the Figures

[0021] 1. x-axis movement mechanism

[0022] 2. y-axis movement mechanism

[0023] 3. z-axis movement mechanism

[0024] 4. Electronic circuit

[0025] 5. Electrode head

[0026] 5.1 2x2 electrode head

[0027] 5.2 4x4 electrode head

[0028] 5.3 8x8 electrode head

[0029] 6. Distance sensor

[0030] 7. Culture dish

[0031] 8. Electrode

[0032] 9. Insulated body

[0033] 10. Non-insulated tip 11. System interface

[0034] 12. Processor device

[0035] 13. Microcontrol module

[0036] 14. Excitation circuit module

[0037] 15. Measuring circuit module

[0038] 16. Sterilization chamber

[0039] 17. Cells adhered to the surface

[0040] 18. Electrode contact interface

[0041] 19. Culture dish seat

[0042] 20. Culture liquid

[0043] 21. Cultured cells

[0044] 22. Recording signals

[0045] 23. Stimulation signals and movement circuit control

[0046] 24. Controller of stepper motors of electrode head and signal amplifying circuits

[0047] Summary and Objectives of the Invention

[0048] The invention discloses a non-contact electrical stimulation and recording assembly provided for use in electrophysiological studies in cell culture laboratories carried out in the field of biological sciences, medicine and pharmacy and an operation method thereof. The non-invasive stimulation and recording ability of the electrical stimulation and recording device of the invention increases the standardization and reliability of the studies to be performed. In addition, the cost of the electrical stimulation and recording assembly of the invention is also reduced.

[0049] The primary object of the invention is to eliminate the invasive effect in the existing multielectrode array (MEA) systems. In order to eliminate the invasive effects of the system, the electrodes were moved onto the cells and the cells were stimulated through the growth medium. In this way, both the electric field is spread homogeneously to the medium and the negative effects that may occur on the cells due to the direct contact of the electrodes with the cell are eliminated. The fact that the stimulation can be carried out in a non-contact and non-invasive way by taking advantage of the conductive properties of the growth medium is also a technique that better reflects the physiological conditions. Another object of the invention is to provide a low-cost experimental electrical stimulation and recording assembly. In the experimental electrical stimulation and recording assembly of the invention, the need for special culture dishes with electrodes embedded in the culture dish is eliminated by immersing the electrodes in the growth medium, thereby allowing researchers to use standard culture dishes and reducing the extra cost to be incurred. Another situation in which cost-related problems are tried to be overcome is the presence of an automatic movement mechanism that can move the electrodes in the inventive assembly on the wells. In this way, the necessity of a separate electrode for each well is eliminated.

[0050] The invention provides an electrical stimulation system in which standardization deviations are prevented for use in research carried out in the fields of biological sciences, medicine and pharmacy. Since the electrodes are not embedded in the well surfaces, standard disposable culture dishes are sufficient for the experiments, and only sterilization of the electrode head is required. However, the culture wells with electrodes in contact systems have to be sterilized after the study, and exposing them to the repetitive sterilization process leads to the deterioration of the cell adhesion surfaces in the aforementioned assemblies and affects the standardization of the studies. Since the system of the invention is not affected by the aforementioned problem, it can be used in a consistent and much larger number of studies and therefore is low cost. In addition, thanks to the replaceable electrode head included in the system of the invention, alcohol sterilization is easily carried out and possible standardization deviations are prevented by the sterilization provided. In addition to the aforementioned sterilization, there is also a sterilization chamber in the in vitro noncontact electrical stimulation and recording assembly. The sterilization chamber contains an Ultraviolet (UV) light source and emits UV rays.

[0051] Detailed Description of the Invention

[0052] The invention relates to a non-contact electrical stimulation and recording assembly provided for use in electrophysiological studies in cell culture laboratories carried out in the field of biological sciences, medicine and pharmacy and an operation method thereof. The non-invasive stimulation and recording ability of the electrical stimulation and recording device of the invention increases the standardization and reliability of the studies to be performed. In addition, the cost of the electrical stimulation and recording assembly of the invention is also reduced.

[0053] The in vitro non-contact electrical stimulation and recording assembly of the invention comprises:

[0054] • a replaceable electrode head (5) with a distance sensor (6) and electrode (8), which allows to stop at an appropriate distance from the bottom of a culture,

[0055] • an x-axis movement mechanism (1) which moves an electrode head (5) so as to be parallel to a plane on which the culture dishes are located,

[0056] • a y-axis movement mechanism (2) which moves an electrode head (5) so as to be parallel to a plane on which the culture dishes (7) are located,

[0057] • a z-axis movement mechanism (3) which moves an electrode head (5) so as to be perpendicular to a plane on which the culture dishes (7) are located,

[0058] • an electronic circuit (4) capable of performing a direct and alternating current stimulation and an amplification of the signals measured thereby,

[0059] • an electrode head (5) containing an insulated body (9), an uninsulated tip (10) and electrode (8),

[0060] • a distance sensor (6) which allows to stop at an appropriate distance from the bottom of a culture,

[0061] • a system interface (11 ) which performs stimulation and recording parameters as well as circuit controls,

[0062] • a processor device (12), which makes recording at a microvolt level,

[0063] • a microcontrol module (13) which moves the electrodes at coordinates x,y,z on the wells and controls the stepper motors,

[0064] • an excitation circuit module (14) which provides electrical impulses transmitted to the electrodes at certain parameters via the interface,

[0065] • a measuring circuit module (15) which records the electrical signals obtained at the microvolt level by means of the electrodes,

[0066] • a sterilization chamber (16) which sterilizes the electrode head (5).

[0067] Said electrode (8) is made of gold, platinum, a conductive metal, or an alloy material. In addition, said culture dishes (7) may have 6, 12, 24, 48 or 96 wells. In addition, said sterilization chamber (16) contains an ultraviolet (UV) light source, and the sterilization chamber emits UV rays. In addition, said processor device (12) is a computer. Said electrode head (5) herein contains 4-64 electrodes (8).

[0068] The operation method of in vitro non-contact electrical stimulation and recording assembly comprises the following steps of: i. preparing a cell culture consisting of the excitable cells in culture dishes with wells (7), ii. attaching a sterile electrode head (5) compatible with the culture dish (7) selected to the in-vitro experimental electrical stimulation and recording assembly, iii. setting the stimulation and recording parameters for the planned experiment on the system interface (11 ), iv. inserting the culture dish (7) into a seat on the system v. performing a depth calibration on the z-axis by means of the distance sensor (6) on the system electrode (8), vi. operating the experimental electrical stimulation assembly in a cell culture incubator or Class-ll laminar flow cabinet under non-sterile conditions or in cases where it will be sterile, depending on the conditions of the study.

[0069] In an embodiment of the invention, the operation method of the in vitro non-contact electrical stimulation and recording assembly comprises the following process steps of: i. preparing a cell culture consisting of the excitable cells in 6, 12, 24, 48 and 96-well culture dishes (7), ii. attaching a sterile electrode head (5) compatible with the culture dish (7) selected to the in-vitro experimental electrical stimulation and recording assembly, iii. setting the stimulation and recording parameters for the planned experiment on the system interface (11 ), iv. inserting the culture dish (7) into a seat on the system, v. performing a depth calibration on the z-axis by means of the distance sensor (6) on the system electrode (8), vi. operating the experimental electrical stimulation assembly in a cell culture incubator or Class-ll laminar flow cabinet under non-sterile conditions or in cases where it will be sterile, depending on the conditions of the study.

[0070] One of the technical effects of the in vitro non-contact electrical stimulation and recording assembly of the invention differs from similar systems in the state of the art is that the electrical stimulation and recording of the cell can be performed in a non- invasive way without contacting with the cells. The non-invasive non-contact stimulation provided by the system of the invention ensures the negative effects of the devices that perform stimulation through the contact of the electrodes with the cell to be minimized. Another specific technical effect of the assembly of the invention is the existence of an automatic movement mechanism that carries the electrodes. The movement of the electrodes between the wells (X and Y-axes) and on the well (Z-axis) is ensured by the mechanism. This feature eliminates the necessity of electrodes that need to be applied to each well to stimulate the cells. Said movement mechanism includes an X-axis movement mechanism (1 ) which moves the electrode head (5) so as to be parallel to a plane on which the culture dishes (7) are located, a Y-axis movement mechanism (2) which moves the electrode head (5) so as to be parallel to a plane on which the culture dishes (7) are located, and a Z-axis movement mechanism (3) which moves the electrode head (5) so as to be perpendicular to a plane on which the culture dishes (7) are located. Machine learning integrated into the system is another important feature that distinguishes the system from the existing systems. The in vitro non-contact electrical stimulation and recording assembly of the invention matches the electrophysiological map obtained with the signals measured from the electrodes (8) with the histological images obtained from the microscope using image processing algorithms, thereby making the signals two (2) times more meaningful and playing a role in finding active connections between cells. This makes an extra contribution to the in vitro modeling of diseases caused by cell excitability, making it possible for the studies to be carried out to be more scientific and reproducible. On the other hand, the system is enabled to determine the type of cells in the culture medium (i.e. excitator or inhibitor) by means of the machine learning. With all these advantages of image processing with machine learning, it will be possible to carry out many studies such as examining the effects of therapeutic agents to be tested in different cell pathways, in vitro modelling of diseases such as epilepsy, and drug interactions. Also, many diseases such as spinal cord injuries, in which synaptic connections are studied, can be studied on cell culture together with the system of the invention. One of the specific technical effects of the in vitro non-contact electrical stimulation and recording assembly of the invention is a replaceable electrode head (5). The replaceable electrode head (5) provides a solution to the sterilization problem in repetitive experiments and allows the electrodes (8) to be easily replaced in case of degradation.

[0071] Another component of the in vitro non-contact electrical stimulation and recording assembly of the invention, which can be expressed as a specific technical effect, is the sterilization chamber (16). This chamber, where replaceable electrode heads (5) can be easily placed, can be used not only when UV sterilization is required, but also as an additional application after alcohol sterilization of the electrodes.

[0072] The assembly of the invention is in accordance with standard 6, 12, 24, 48 or 96-well culture dishes (7).

[0073] The non-contact stimulation and recording assembly is controlled by a system interface (11 ) that is implemented via an open source software on a computer. The desired amplitudes and frequencies are transmitted to the stimulation electrode (8) via the system interface (11), and the recording electrodes measure the electrical field in the environment and transmit output to the system interface (11).

[0074] The measurements taken with the assembly of the invention are processed in a processor device (computer) environment and the electrical field changes in the wells containing cell cultures are analyzed.

[0075] The automatic movement mechanism which moves the electrode head (5) is achieved in a controlled manner through the processor device (computer) by means of stepper motors.

[0076] The stimulation and registration of the electrodes (8) are carried out in a non-invasive manner without contact with the cells through the growth medium.

[0077] The stimulation and recording are carried out by means of the electrodes (8) via the embedded stimulator and recording circuits.

[0078] The stimulation model can be determined depending on the request of a relevant user. Stimulation can be applied in the form of an anodal and cathodal flow. The stimulation option can be changed by means of a switch, or during the determination of the program parameters. Although it is estimated that the voltages to be transmitted during stimulation will be in the range of 50-100 mV, it is thought to cover voltages between 1 -1000 mV. It is predicted that the records to be obtained will be at mV scale.

[0079] Noise and distortions that may occur in the measurements taken with the system of the invention are minimized.

[0080] References

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Claims

CLAIMS n in vitro non-contact electrical stimulation and recording assembly, characterized in that it comprises:• a replaceable electrode head (5) with a distance sensor (6) and electrode (8), which allows to stop at an appropriate distance from the bottom of a culture,• an x-axis movement mechanism (1 ) which moves an electrode head (5) so as to be parallel to a plane on which the culture dishes are located,• a y-axis movement mechanism (2) which moves an electrode head (5) so as to be parallel to a plane on which the culture dishes (7) are located,• a z-axis movement mechanism (3) which moves an electrode head (5) so as to be perpendicular to a plane on which the culture dishes (7) are located,• an electronic circuit (4) capable of performing a direct and alternating current stimulation and an amplification of the signals measured thereby,• an electrode head (5) containing an insulated body (9), an uninsulated tip (10) and electrode (8),• a distance sensor (6) which allows to stop at an appropriate distance from the bottom of a culture,• a system interface (11) which performs stimulation and recording parameters as well as circuit controls,• a processor device (12), which makes recording at a microvolt level,• a microcontrol module (13) which moves the electrodes at coordinates x,y,z on the wells and controls the stepper motors,• an excitation circuit module (14) which provides electrical impulses transmitted to the electrodes at certain parameters via the interface,• a measuring circuit module (15) which records the electrical signals obtained at the microvolt level by means of the electrodes,• a sterilization chamber (16) which sterilizes the electrode head (5).

2. A stimulation and recording assembly according to Claim 1 , characterized in that said electrode (8) is made of gold, platinum, a conductive metal, or an alloy material.

3. A stimulation and recording assembly according to Claim 1 , characterized in that said sterilization chamber (16) contains an Ultraviolet (UV) light source.

4. A stimulation and recording assembly according to Claim 1 , characterized in that said electrode head (5) contains 4-64 electrodes (8).

5. Use of a stimulation and recording assembly according to any of the claims 1 -4 for in vitro modeling of cell excitability-induced diseases, determination of the type of cells in culture medium whether excitatory or inhibitory, the creation of an in vitro model of epilepsy and detection of spinal cord injuries.

6. An operation method of in vitro non-contact electrical stimulation and recording mechanism, characterized in that it comprises the following process steps of: i. preparing a cell culture consisting of the excitable cells in culture dishes with wells (7), ii. attaching a sterile electrode head (5) compatible with the culture dish (7) selected to the in-vitro experimental electrical stimulation and recording assembly, iii. setting the stimulation and recording parameters for the planned experiment on the system interface (11), iv. inserting the culture dish (7) into a seat on the system v. performing a depth calibration on the z-axis by means of the distance sensor (6) on the system electrode (8), vi. operating the experimental electrical stimulation assembly in a cell culture incubator or Class-ll laminar flow cabinet under non-sterile conditions or in cases where it will be sterile, depending on the conditions of the study.

7. An operation method according to Claim 6, characterized in that it comprises the following process steps of: i. preparing a cell culture consisting of the excitable cells in 6, 12, 24, 48 and 96-well culture dishes (7), ii. attaching a sterile electrode head (5) compatible with the culture dish (7) selected to the in-vitro experimental electrical stimulation and recording assembly, iii. setting the stimulation and recording parameters for the planned experiment on the system interface (11), iv. inserting the culture dish (7) into a seat on the system, v. performing a depth calibration on the z-axis by means of the distance sensor (6) on the system electrode (8), vi. operating the experimental electrical stimulation assembly in a cell culture incubator or Class-ll laminar flow cabinet under non-sterile conditions or in cases where it will be sterile, depending on the conditions of the study.

8. An operation method according any one of Claim 6 or 7, characterized in that said electrode (8) is made of gold, platinum, a conductive metal, or an alloy material.

9. An operation method according to any one of Claim 6 or 7, characterized in that said electrode head (5) contains 4-64 electrodes (8).

Images