Cell culture monitoring system and dielectrophoresis cartridge

Abstract

The invention relates to a method and apparatus for measuring cell culture medium comprising: a monitoring device (3) for coupling to a culture tank (2) containing a cell culture medium (15); and a fluid circulation system (4) for fluidly coupling to the cell culture tank (2), the fluid circulation system comprising a dielectrophoresis cartridge (5) for connecting to the cell culture tank (2) via a supply conduit (14a) and a return conduit (14b), the dielectrophoresis cartridge comprising a base (20) and an electrode support (19) having an electrode (21) in or on the electrode support (19), the electrode being configured for traveling wave dielectrophoresis and interposed between a floor (22) of the electrode support (19) and the base (20) forming a measurement chamber therebetween. 6), wherein cells in a liquid medium flowing through the measurement chamber are subjected to a traveling wave dielectrophoretic force perpendicular to a direction of liquid flow in the measurement chamber, and the monitoring device (3) comprises a computing unit (9), an image capture system (7) connected to the computing unit (9), and a cartridge holder portion (28) for receiving the dielectrophoresis cartridge (5) such that the image capture system (7) can detect cells flowing through the measurement chamber (23 b).

Description

Technical Field

[0001] The present invention relates to a system for monitoring the culture of cells in a liquid medium.

Background Art

[0002] With the emergence of cell therapy and cell-based products, the need to accurately and timely control cell culture has increased. Cell culture may also be used, for example, in the bioproduction of antibodies and vaccines. In many steps of conventional culture processes, human intervention is required, especially for cell counting and cell viability measurement. Each intervention increases the risk of contamination and the final cost of treatment. The loss of treatment batches due to errors and contamination has a great impact on patients.

Summary of the Invention

Problems to be Solved by the Invention

[0003] In view of the above, an object of the present invention is to provide a cell culture monitoring system that enables accurate control of cell growth and reduces the risk of contamination at low cost.

Means for Solving the Problems

[0004] It is advantageous to provide a reliable cell culture monitoring system.

[0005] It is advantageous to provide a cell culture monitoring system that enables continuous or frequent analysis of the state of cells during culture at low cost and in a sterile state. By continuously measuring the viability, it is possible to detect cell culture diseases at an early stage.

[0006] The object of the present invention is achieved by providing a cell culture monitoring system according to claim 1.

[0007] The object of the present invention is achieved by providing a cell culture monitoring system according to claim 16.

[0008] The object of the present invention is achieved by providing a dielectrophoresis cartridge for a cell culture monitoring system according to claim 31.

[0009] Disclosed herein is a cell culture monitoring system comprising a monitoring device for coupling to a culture tank containing a cell culture medium and a fluid circulation system for fluidly coupling to the cell culture tank. The fluid circulation system comprises a dielectrophoresis cartridge for connection to the cell culture tank via a supply conduit and a return conduit. The dielectrophoresis cartridge comprises a base and an electrode support having electrodes arranged in an electrode plane X-Y within or on the electrode support. The electrodes are configured for traveling wave dielectrophoresis and comprise a measurement zone arranged above a measurement chamber formed between the electrode support and the floor of the base, which can be filled with cell culture medium from the cell culture tank. The monitoring device comprises a signal generator connected to the electrodes, a computing unit, an image capture system connected to the computing unit, and a cartridge holder part for receiving the dielectrophoresis cartridge such that the image capture system can detect cells within the measurement chamber.

[0010] In a first aspect of the present invention, the image capture system is configured to capture the displacement of cells in the cell culture medium within the measurement chamber in a direction Z orthogonal to the electrode plane X-Y to enable measurement of cell type and / or cell state.

[0011] In a second aspect of the present invention, the electrodes are insulated from the interior of the measurement chamber by an insulator layer, except for a measurement zone where the electrodes make non-insulated contact with the interior of the measurement chamber.

[0012] This specification also discloses a dielectrophoresis cartridge for connection to a cell culture tank, comprising a base and an electrode support having electrodes arranged in an electrode plane X-Y within or on the electrode support, the electrodes being configured for traveling wave dielectrophoresis and having a measurement zone disposed above a measurement chamber formed between the electrode support and the base floor of the base, the measurement chamber being formed between the electrode support and the base floor, and the electrodes being insulated from the interior of the measurement chamber by an insulator layer except for the measurement zone.

[0013] In an advantageous embodiment, the image capture system and the computing unit are configured to capture a plurality of slice-like images taken in the orthogonal direction Z, and the plurality of images are processed to determine the position and displacement of cells in the orthogonal direction Z.

[0014] In an advantageous embodiment, the image capture system comprises a microscope having a depth of field smaller than the height of the measurement chamber and an electrically adjustable lens for adjusting the focus to capture slices of images at different heights.

[0015] In an advantageous embodiment, the cells in the cell culture liquid medium in the measurement chamber are subjected to traveling wave dielectrophoresis forces in a plane parallel to the electrode plane X-Y, and the image capture system is configured to capture the displacement of the cells in the cell culture medium in the measurement chamber in the plane parallel to the electrode plane X-Y, enabling the measurement of cell types and / or cell states in relation to measurements based on the displacement of the cells in the orthogonal direction Z.

[0016] In an advantageous embodiment, the signal generator is configured to scan different electrode signal frequencies or apply a plurality of discrete different electrode signal frequencies during the measurement or over a series of measurements, and the image capture system and the computing unit are configured to measure the displacement of the cells for different electrode signal frequencies.

[0017] In an advantageous embodiment, the electrode support is made of a transparent polymer or glass, and at least the electrodes within the measurement zone may be made of a conductive transparent material, for example, a thin indium tin oxide (ITO) layer. However, the contact regions of the electrodes preferably comprise a gold layer or other conductive material that has corrosion resistance and ensures good electrical contact with the connectors.

[0018] However, in a variant, it is possible to have gold electrodes or other opaque conductive materials including within the measurement zone, and the opaque electrode lines may be considered or excluded for image acquisition of the cell chamber by an image processing algorithm, and the space between the electrodes enables the measurement of the position and displacement of the cells.

[0019] In an advantageous embodiment, the cartridge comprises at least two impedance measurement electrodes configured to measure the impedance (Zi) of the cells. The impedance measurement electrodes may be two of the electrodes used for traveling wave dielectrophoresis or may be impedance electrodes used only for impedance measurement.

[0020] In an advantageous embodiment, the dielectrophoresis cartridge comprises an outlet and an inlet configured to couple to flexible polymer tubes forming the supply conduit and the return conduit.

[0021] In an advantageous embodiment, the electrodes are formed on the inner surface of the electrode support defining the measurement chamber, and the inner surface has a contact portion extending to an electrode connection window formed in the base for inserting contacts into the spring contacts of the monitoring device, and the electrode connection window is hermetically separated from the measurement chamber.

[0022] In an advantageous embodiment, the measurement chamber comprises a raised floor and a lateral guide defining a gap between the floor and the electrode support.

[0023] In an advantageous embodiment, the electrodes comprise a measurement zone formed by one or more spiral conductive tracks.

[0024] In an advantageous embodiment, the electrodes consist of 4 to 10 electrodes, preferably 4 to 8 electrodes.

[0025] In an advantageous embodiment, the electrodes are arranged in two sets of measurement zones that are mirror-symmetrical.

[0026] In an alternative embodiment, the electrodes comprise at least two meshing electrodes.

[0027] In an advantageous embodiment, the cartridge holder part of the monitoring device comprises a cartridge holder slot configured to slidably insert the dielectrophoresis cartridge.

[0028] In an advantageous embodiment, the cartridge holder part comprises a positioning element that engages with a complementary positioning element in the dielectrophoresis cartridge to position and fix the dielectrophoresis cartridge in the measurement position.

[0029] In an advantageous embodiment, the positioning element comprises a spring protrusion or a spring resistance part either on the cartridge holder part or on the dielectrophoresis cartridge.

[0030] In an advantageous embodiment, the image capture system comprises a microscope connected to an image processing circuit of a computing unit configured for digital analysis of the trajectories of cells captured by the image capture system.

[0031] In an advantageous embodiment, the computing unit comprises a signal generator connected via a connector to the electrodes of a dielectrophoresis cartridge configured to generate a traveling wave dielectrophoresis signal in the measurement zone of the electrodes.

[0032] In an advantageous embodiment, the measurement chamber between the electrode and the floor ranges from 10 μm to 500 μm.

[0033] In an advantageous embodiment, the cell culture tank is separated from the monitoring device and comprises a fluid connector for connecting to a supply conduit and a return conduit connected to the dielectrophoresis cartridge.

[0034] Further objects and advantages of the present invention will become apparent from the claims, the detailed description, and the appended drawings.

Brief Description of the Drawings

[0035]

Figure 1

Figure 2a

Figure 2b

Figure 3a

Figure 3b

Figure 4

Figure 5a

Figure 5b

Figure 5d

Figure 5c

Figure 6a

Figure 6b

Figure 6c

Figure 6d

Figure 6e

Figure 7

Figure 8a

Figure 8b

Figure 8c

Figure 9

Figure 10a

Figure 10b

DETAILED DESCRIPTION OF THE INVENTION

[0036] Referring to the drawings, a cell culture monitoring system 1 according to an embodiment of the present invention includes a monitoring device 3, a cell culture tank 2, and a fluid circulation system 4 for transporting a cell culture medium containing cells to be observed between the cell culture tank and the monitoring device.

[0037] The monitoring device 3 includes an image capture system 7, a spectrometer 8, a computing unit 9, and a cartridge holder unit 28 for receiving the dielectrophoresis cartridge 5 of the fluid circulation system 4.

[0038] The fluid circulation system 4 includes a dielectrophoresis cartridge 5 and conduits 14a, 14b that interconnect the dielectrophoresis cartridge 5 with the cell culture tank 2. The fluid circulation system 4 may be an attachment part or a forming part of the monitoring device 3, or in other modified forms, it may be attached onto the cell culture tank and further include a pump 6 that may be electrically connected to the monitoring device to control the pump. In a preferred embodiment, the pump may be attached onto the monitoring device and advantageously may be in the form of a peristaltic pump. At least a part of the supply conduit 14a includes a flexible part of a tube attached to a peristaltic pump for delivering the cell culture medium in a sterile state.

[0039] The dielectrophoresis cartridge 5 and the supply conduit 14a and the return conduit 14b connected to the cell culture tank 2 of the fluid circulation system advantageously form a closed circuit in which the fluid of the cell culture medium 15 contained in the cell culture tank 2 is circulated through the dielectrophoresis cartridge 5 and returned to the cell culture tank. In a modified form, the fluid circulation system may further include an outlet conduit coupled to the drain 23d to remove dead (i.e., apoptotic) cells separated from liver cells or to separate different cell phenotypes due to different trajectories within the dielectrophoresis cartridge. The fluid connector 18 may be connected to the cell culture tank via a luer lock connection known per se in the field of fluid connection, or may be interconnected by other means. The fluid connector 18 enables the connection of flexible tubes, particularly the tank supply tube and the return tube, to the connector.

[0040] The supply conduit 14a may further include a perforated tube 17 that is immersed in the cell culture medium 15 and preferably extends to the bottom of the cell culture tank. The holes in the tube 17 may include more holes towards the bottom of the tank and gradually fewer holes towards the top of the tank, such that the inlet resistance is arranged to decrease towards the bottom of the tank. This ensures that the suction pressure is substantially evenly distributed so that the cell culture medium throughout the height of the cell culture tank is reliably drawn into the supply tube for uniform sampling throughout the entire height. A weight may be provided at the bottom of the perforated tube and a float at the top of the tube to ensure that all holes are immersed in the liquid. However, other tube holding and positioning means may be provided. Further, the perforated tube may have various shapes, such as a "corkscrew" shape, to increase the uniformity of horizontal sampling. The cell culture vessel may further include a mixing system, such as a rotor or a magnetic bar stirrer (not shown), to equalize cell dispersion in the medium.

[0041] A valve may be provided in the fluid connector 18 to enable recirculation of the cell culture medium within the supply return conduit, either to circulate within a closed circuit without passing through the culture tank or to change the valve settings such that fresh cell culture medium drawn from the cell culture tank is fed into the supply conduit. The function of the valve may depend on the analysis being performed. For example, when the supply conduit and the return conduit are connected to each other, the sample medium may be recirculated multiple times through the dielectrophoresis cartridge for measurement, for example, to increase the sensitivity of the measurement, or fresh cell culture medium may be fed into the supply conduit and returned to the cell culture tank for a single pass through the dielectrophoresis cartridge.

[0042] In some cases where the sample being measured is discarded and not returned to the cell culture medium, a valve may be provided to switch the return conduit to a waste container (not shown).

[0043] According to an advantageous embodiment of the invention, the dielectrophoresis cartridge 5 comprises a base 20 and an electrode support 19. The base 20 may advantageously be made of a polymer material, which in some embodiments may advantageously be a transparent polymer material such as ABS (acrylonitrile-butadiene-styrene copolymer). The base may advantageously be molded, for example, by injection molding or by additive manufacturing techniques (such as 3D printing).

[0044] The electrode support 19 may be made of a polymer material, but is preferably made of glass and has conductive electrodes on the glass that can be made by various deposition and patterning techniques known per se, such as chemical vapor deposition, lithography, printing, and other known metal layer deposition techniques. In an advantageous embodiment, the electrode support 19 is a part that is formed separately from the base and is assembled to the base, for example, by adhesion, ultrasonic bonding, or welding. However, it is also possible to form the base, the support, and the electrodes as a single part via additive manufacturing techniques.

[0045] The base 20 comprises a fluid connector part 24 having an inlet 24a and an outlet 24b, and a microfluidic circuit formed in the base and having a flow path interconnecting the inlet 24a and the outlet 24b. The base further comprises an electrode connection window 22 that enables contact with the contact part 21b of the electrode 21.

[0046] The microfluidic circuit 23 comprises an inlet channel 23a connected to the inlet 24a and flowing into the measurement chamber 23b, and a return channel 23c flowing from the measurement chamber 23b to the outlet 24b. The measurement chamber 23b may advantageously comprise a raised floor 26 that defines the flow path height between the base 20 and the electrode support 19. This ensures that a clearly defined gap for the fluid to flow through the measurement chamber is formed below the measurement zone 21a of the electrode 21 located above the measurement chamber. The height between the electrode and the floor 26 in the measurement chamber 23 is preferably in the range of 10 μm to 200 μm.

[0047] Cells in the liquid flowing through the measurement chamber 23b are subject to a traveling-wave dielectrophoretic force F depending on the state of the cells. twDEP Determining the state of cells using dielectrophoretic electrodes is a concept known per se. In conventional systems, generally, cells in a liquid medium are displaced by dielectrophoresis, and such displacement indicates the state of the cells. Dead cells are either not displaced as much or do not experience the traveling-wave dielectrophoretic force, whereby living cells experience the dielectrophoretic force and translate parallel across the electrodes. When a phase-shifted signal is applied to the aligned electrodes separated by a gap, a force parallel to the X-Y plane in which the electrodes are arranged, called the traveling-wave dielectrophoretic force F twDEP is applied to the cells depending on the state of the cells. The traveling-wave dielectrophoretic force is characterized by the following equation known per se.

[0048]

Equation

[0049] However, the imaginary part of the Clausius-Mossotti (CM) coefficient [f CM in the above equation varies depending on the cell characteristics and the applied frequency of the phase-shifted signal on the electrodes. FIG. 10a shows a plot of the traveling-wave dielectrophoretic force for different cell models as a function of the applied frequency of the phase-shifted signal on the electrodes.

[0050] As shown in FIG. 8b, the coplanar electrodes also generate a non-uniform electric field in the direction orthogonal to the plane of the electrodes, thereby generating a dielectrophoretic force F in the direction Z that is orthogonal to the plane X-Y of the electrodes and to the direction of the traveling-wave dielectrophoretic force that translates the cells parallel across the electrodes. DEP The orthogonal dielectrophoretic force F DEP depends on the real part of the CM coefficient.

[0051]

Equation

[0052] Figure 10b shows a plot of the real part of the CM coefficient. The force acting on the cells depends on the cell characteristics. By tracking the displacements of both the horizontal (X-Y plane) cell population and the vertical (orthogonal direction Z) cell population, these cell populations can be distinguished from each other, and the state of the cells can be distinguished between live cells and dead cells, and in some cases between normal live cells and diseased live cells. The tracking of cell displacements in both the coplanar direction and the orthogonal direction with respect to the plane X-Y of the electrode 21 is performed by the image capture system 7.

[0053] The image capture system 7 includes a microscope 7 that captures the horizontal position and displacement of the cells in a plane parallel to the plane X-Y of the electrode 21.

[0054] According to one aspect of the present invention, the image capture system 7 is configured to capture the position and displacement of the cells in the orthogonal direction Z. An important advantage of this feature is that the displacement of the cells subjected to the dielectrophoretic force in the orthogonal direction can be measured so as to distinguish the cell type and / or cell state before it becomes possible to distinguish the cell type and / or cell state by measuring the displacement in a plane parallel to the plane X-Y of the electrode. Therefore, a more rapid measurement can be performed. Also, this measurement can be used together with the measurement in a plane parallel to the plane X-Y of the electrode to improve the accuracy and reliability of the measurement of the characteristics of the cell type and / or cell state.

[0055] In an advantageous embodiment, scanning different electrode signal frequencies during the measurement using the image capture system or applying a plurality of discrete different electrode signal frequencies makes it possible to improve the identification of both the type of cells observed by accurately measuring the characteristics over a certain frequency range and the state of those cells. Figures 10a and 10b show that different cell types and cell states result in different CM / frequency plots that can be used to distinguish the cell type and state.

[0056] Measurement of cell displacement in a plane parallel to the X-Y plane of the electrode can be performed simultaneously with, in sequence with, or independently of the measurement of cell displacement in the orthogonal Z direction.

[0057] In one embodiment, microscope 7 may include a small depth of field and an electrically adjustable lens configured to view through the culture sample and capture the position of cells in the orthogonal Z direction. The electrically adjustable lens may be configured to capture a plurality of slice-type images taken in the Z direction, and these images may be processed to determine the position and displacement of cells in the orthogonal Z direction. High-speed Z slicing capture and reconstruction enables calculation of the cell sample volume, provides more information about the sample, and enables more accurate detection and characterization of the object. When viewing a cell focused by adjusting the microscope lens, the shape of the cell can be accurately determined. The focus can be further adjusted to compensate for any mechanical errors in the orthogonal Z-axis when inserting the dielectrophoresis cartridge 5 into the monitoring device 3. Positioning errors in the X-Y plane can be compensated by selecting the region of interest in the camera sensor. The field of view of the camera is larger than the measurement zone.

[0058] Thus, the system can measure the elevation of cells relative to the plane of electrode 21 by analyzing the slices at which the cells are focused.

[0059] Electrode 21 may advantageously be made of a conductive transparent material, such as a thin indium tin oxide (ITO) layer, whereby the microscope can view the interior of measurement chamber 23b through transparent electrode support 19 and capture an image of the cells at any position above the electrode.

[0060] Within the scope of the present invention, in an image capture system, instead of using an optical microscope, a light sheet microscope or a confocal microscope can be used, and such microscope systems are known per se and need not be described herein.

[0061] As shown in FIGS. 7a and 7b, according to an aspect of the present invention, the electrode 21 is insulated from the inside of the measurement chamber 23b in contact with the liquid medium, except for the measurement zone 21a, by an insulator layer, for example, a layer of silicon dioxide SiO2. The measurement zone 21a of the electrode is not insulated so as to avoid voltage drop and signal distortion, and thus to generate an optimal electric field. The contact 21b located outside the measurement chamber is also not insulated.

[0062] When the electrode is insulated around the measurement zone 21a, it is also possible to extend the distance between the contact 21b aligned with the image capture system and the measurement zone 21a without adversely affecting the dielectrophoretic signal of the portion of the electrode interconnecting the measurement zone and the contact.

[0063] As schematically shown in FIG. 8b, the impedance Zi of the cell can advantageously be measured using two parallel impedance measurement electrodes 21', 21". The impedance measurement electrodes 21', 21" may be two of the electrodes used for traveling wave dielectrophoresis, or may be independent electrodes used only for impedance measurement. The cell position is detected using an image capture system, and while the position is detected by the image capture system, by the controlled operation of the pump 6 and / or by applying the traveling wave dielectrophoretic force F twDEP by controlling the flow of the liquid in the measurement chamber, it can be arranged between the impedance measurement electrodes 21', 21". This can be very useful for performing measurements on a selected single cell sample.

[0064] Referring to FIGS. 7a and 6c, a large-area traveling-wave dielectrophoresis (twDEP) zone can be realized using a spiral electrode according to one embodiment. As described above, in order to avoid signal attenuation or distortion, an insulating layer may be added on top everywhere except at the measurement zone 21a and the contact 21b.

[0065] According to one embodiment of the present invention, as shown in FIG. 7b, a plurality of electrodes 21 may simply be arranged side by side and placed in parallel within the measurement zone 21a connected to individual non-spiral tracks.

[0066] Referring to FIG. 7c, according to another embodiment, the electrode 21 has four electrodes 21i, 21ii, 21iii, 21iv for generating four different signals that mesh with each other, and the four electrodes are placed on an insulatingly separated layer. The four layers may be constructed, for example, as a stack of a first conductive layer, a first insulating layer, a second conductive layer, and then a second insulating layer, whereby two of the electrodes 21i, 21iii are on the first conductive layer but separated, and the other two electrodes 21ii, 21iv are on the second conductive layer but separated.

[0067] Referring to FIG. 7d, according to another embodiment, the levitation of cells in the orthogonal direction Z can be measured using two signals generated by two electrodes 21v, 21vi. The maximum levitation force occurs when the two signals are 180° out of phase. In this embodiment, the discrimination of cell types and cell states may be performed only by measuring the position and displacement of cells in the orthogonal direction Z using an image capture system.

[0068] While the position and displacement of cells due to traveling-wave dielectrophoresis are being measured by the image capture system, the liquid in the measurement chamber may be stationary or substantially stationary, or may be subject to a fluid flow, thereby showing a component in the liquid flow direction LF from the inlet to the outlet in the measurement chamber as shown in FIG. 8a, as well as the traveling-wave dielectrophoresis force F twDEPThere may be a case where it shows a lateral parallel movement T caused by [reason not specified]. The movement direction of the cells is captured by the image capture system 7 and analyzed by the computing unit 9. The advantages of the parallel movement by the simultaneous fluid flow and dielectrophoresis are that the vertical component enables very accurate and easy measurement of the cell state to distinguish normal cells from dead cells, and also enables very accurate and easy measurement of the cell state that affects the dielectrophoretic force. This measurement may be performed together with the measurement of the displacement in the orthogonal direction Z in order to increase the accuracy and reliability of the distinction between cell types and different states of each cell.

[0069] Electroporation is a technique used to improve cell transfection. According to another aspect of the present invention, the dielectrophoretic zone in the measurement chamber may be used for this purpose. The generated electric field (amplitude-dependent) increases the permeability of the cell membrane and promotes the integration of vectors (e.g., viruses) into cells. When different-sized microorganisms (e.g., viruses and cells) can be moved at different speeds through dielectrophoresis, collisions occur, so the integration of the virus is amplified. Therefore, the traveling-wave dielectrophoretic force generated in the measurement chamber can be used to move the microorganisms laterally in both directions to cause multiple collisions.

[0070] In another embodiment, as schematically shown in FIG. 9, it is possible to have two outflow channels. The first outflow channel corresponds to the return flow channel 23c, and the other outflow channel corresponds to the drainage channel 23d, where non-viable cells are removed from the fluid flow and viable cells return to the cell culture medium.

[0071] The dielectrophoresis cartridge 5 that enables continuous or semi - continuous analysis of cell viability collaborates with a closed - circuit connection that exits from and returns to a cell culture tank using a peristaltic pump or a shuttle pump (or other pump types without an actuator that contacts the liquid medium). On one hand, it ensures a sterile liquid circuit and at the same time enables automatic analysis of the state of cells in the medium at low cost. The dielectrophoresis cartridge and the cell culture tank are further separated from the monitoring device 3 in a sterile state and can be disposed of at low cost and easily, while the monitoring device is reused without the need for special treatment.

[0072] The dielectrophoresis cartridge 5 may be coupled to flexible tubes forming a supply conduit 40a and a return conduit 40b and removably inserted into a slot of the cartridge holder portion 28 of the monitoring device 3. While the dielectrophoresis cartridge 5 is in a predetermined position within the cartridge holder portion 28, the image capture system 7 and the spectrometer 8 are positioned above the measurement chamber 23b so as to be able to capture the movement of cells flowing into the measurement chamber and detect the characteristics of the fluid. The cartridge is provided with a transparent window formed at least above the measurement chamber in the measurement chamber. The transparent window may be formed, for example, in the form of a glass layer by the electrode support 19, but is also visible through the transparent polymer window of the base 20.

[0073] In some variants, the light source 13 may be positioned on the opposite side of the cartridge holder portion with respect to the image capture system 7.

[0074] The spectrometer 8 may be used to capture the characteristics of the fluid, while the image capture system may be used to detect cells in the liquid and capture the movement of cells in the measurement chamber.

[0075] The computing unit 9 connected to the spectrometer 8 and the image capture system 7 is configured using an algorithm to count cells, analyze the trajectories of the cells, and determine the cell viability from that analysis. The computing unit comprises a signal generator 12 connected to the electrodes 21 to generate traveling wave dielectrophoresis signals. An impedance meter 11 may further be connected to the computing unit 9, and the impedance meter measures the electrical impedance of the liquid flowing through the measurement chamber. The impedance meter Zi may comprise two spaced electrodes 21', 21" immersed in the medium flowing through the cartridge 5.

[0076] As can be best seen in FIG. 7a, according to an advantageous embodiment, the plurality of electrodes may form a pair of mirror image spirals. In the illustrated implementation, there are eight electrodes, four on each spiral. The spirals in the illustrated embodiment have a substantially rectangular form, but may have an elliptical or round form. In an advantageous embodiment, there may be fewer electrodes, for example, the electrodes may be six or four.

[0077] However, in one embodiment (not shown), only a single spiral of the plurality of electrodes may be present.

[0078] This spiral measurement section of the electrodes advantageously reduces the number of electrodes, while allowing a traveling wave dielectrophoresis signal to be applied over a sufficiently large width to cause an easily measurable translocation of the living cells.

[0079] Reducing the number of electrodes advantageously makes it possible to reduce the number of electrodes to be contacted, and the contact portion 21b extends and spreads outwards to have a larger width, providing a sufficient contact surface area to the supplementary terminal 31a of the electrical connector 31 in the cartridge holder portion 28 of the monitoring device. As can be best seen in FIGS. 5d and 5c, the connector 31 comprises a spring-mounted contact that is elastically pressed against the metallized pad of the electrode connection portion 21b when the dielectrophoresis cartridge 5 is fully inserted into the cartridge holder portion 28.

[0080] The cartridge holder part 28 is provided with a cartridge holder long hole 29 into which the dielectrophoresis cartridge can be completely inserted at the measurement position, whereby a positioning element 30 in the form of a projection 30a received in a corresponding recess 30b at the base 20 of the dielectrophoresis cartridge holds and positions the dielectrophoresis cartridge within the cartridge holder long hole 29. The positioning element 30b may be a spring attached to the cartridge holder part 28 or may be rigid, whereby elastic compliance is provided by the material of the dielectrophoresis cartridge 5 and optionally by providing elastic guides and recesses on the cartridge holder part 28 that engage projections on the dielectrophoresis cartridge.

[0081] The monitoring device may be provided with a manually or electrically actuated ejector 33 having a pusher mechanism (only schematically shown) for ejecting the cartridge from the cartridge holder long hole 29 or for assisting in ejecting the cartridge from the cartridge holder long hole 29.

[0082] The image capture system 7 may include an optical microscope 12 coupled to a digital image capture system that enables digital processing of optical images. However, in alternative forms, other image capture systems may be used as follows. - The phase contrast imaging method that uses a phase contrast microscope as an imaging system to enhance the contrast of the image and improve the quality of cell recognition. - A confocal microscope as an imaging system for increasing the resolution of the image. Thereby, the confocal image enables the reconstruction of a 3D model of the cell that improves the quality of cell characteristic evaluation. - The light sheet microscopy method can be used to create a 3D image of the internal flow path. The light sheet microscopy method provides more information regarding cell morphology.

[0083] In the measurement chamber 23b, lateral guides 27 may be provided on each side of the measurement chamber portion to determine the exact height of the measurement chamber, i.e., the gap between the electrode support 19 and the floor of the measurement chamber.

[0084] The electrode support 19 may be mounted within the recess 25 of the base 20 that enables protection of the electrode support 19.

[0085] Thus, the dielectrophoresis cartridge 5 can be easily inserted into the cartridge holder slot 29 and accurately positioned firmly within the cartridge holder slot, and at the same time contact can be established by the spring contact 31a of the connector 31 that is pressed against the electrode connection portion 21b through the electrode connection window 22 of the base 20.

[0086] Thus, the dielectrophoresis cartridge can be connected to supply and return conduits to a separately provided culture tank and can be easily coupled to a monitoring device to analyze cells semi - continuously or continuously, for example, during a two - week period in which cells grow in a culture medium.

[0087] Sterile separation from the monitoring device of the closed - loop configuration and the fluid circulation system enables automatic analysis of cells by an image capture system connected to a computing unit without the need for manual intervention, and enables cells to grow particularly safely, in a sterile state, and at low cost in the culture medium.

[0088] One of the main uses of the present invention is to monitor cell cultures aseptically during the expansion phase (e.g., within 2 weeks). The present invention may be connected to a monitoring device and provides a sterile single-use disposable kit that is disposed of after first use. Using a disposable kit connected to a monitoring device in a closed loop, the system is capable of performing continuous or semi-continuous analysis of the cell culture over the entire time of the culture. The measurement data may be made available via a communication network to remotely follow in real-time the state of the cell culture. For other phases than the expansion phase, for example, these phases including the logarithmic phase, stationary phase, and death phase may be interesting to monitor, for example, for bioproduction. Dielectrophoresis can detect cells in an early apoptotic state. Thus, the transition to the death phase can be predicted.

[0089] The operation of the system may include the following aspects. A sample is extracted from the cell culture tank and flows through a dielectrophoresis cartridge. An image capture system capable of magnification records cells passing through the measurement (observation) zone that is observed through the transparent window of the cartridge, through the base, or alternatively through the electrode support. In the observation zone, traveling wave dielectrophoresis may be used to manipulate the cells. Different cell populations can also be distinguished and sorted.

[0090] Optical spectroscopy and impedance spectroscopy of the medium allow further parameters such as metabolite content to be monitored. The data generated by these measurements may be analyzed to provide information regarding the cell culture status. For example, Raman spectroscopy may be used, and thereby the electrode support may be provided with a functional coating that enhances detection using, for example, surface-enhanced Raman scattering (SERS) measurement techniques. The functional coating on the electrode support may specifically include coatings configured to detect metabolites such as glucose, lactose, and other cell metabolites.

[0091] Cell density may be measured using an image capture system, followed by image analysis in a computing unit. The volume corresponding to the observed zone is known. Two dimensions (x and y) can be calculated using the projection model of an optical microscope. The measurement chamber height is known from the mechanical design, and thus, counting can be automatically performed using an image recognition algorithm.

[0092] Cell viability can be measured by analyzing the trajectories of cells using an image capture system, using traveling wave dielectrophoresis across the entire electrode and / or dielectrophoresis in the orthogonal direction. The viability of each cell can be evaluated according to the displacement of the cell (trajectory in the X-Y direction and / or displacement in the Z direction). By correlating this with image analysis, the exact viability of each cell type can be determined.

[0093] Cell phenotypes can be distinguished based on the displacement of cells generated by dielectrophoretic forces. The size, membrane, and dielectric properties of cells play a role in dielectrophoretic forces. Optical properties such as shape, absorption, nuclear size, granularity, membrane thickness, etc. may be extracted from an image processing algorithm executed in a signal processing unit, which may be an embedded computer or a distributed external system (cloud network), and can enhance the reliability of cell discrimination. Different cell types can be clustered along the electrode by applying different signal patterns. Different signal configurations (phase, amplitude, time) may be executed, and the same cell type may be regrouped using the feedback of the image capture system and / or a reinforcement learning method. A similar method can also be used for sorting.

[0094] Once the cells can be distinguished, it becomes possible to observe whether a certain population of cells grows faster than other populations or grows to the extent that it harms the required cells. The culture conditions for the required cells (nutrition, temperature, diluted observation, pH, metabolite content) can be improved using the collected data and its analysis. Unwanted cells and other particles (bacteria, viruses, etc.) can also be sorted during monitoring.

[0095] In addition to providing information on the culture state, the data provided by the spectrometer and impedance meter can be combined with other data provided by the system (viability, cell population, etc.) and data from other devices stored in the communication network and used. Patterns can be found using algorithms (e.g., machine learning), and predictions can be made for the current culture. The data of multiple monitoring records can be collected and analyzed in a cloud computing network or distributed devices.

Description of Symbols

[0096] 1 Cell culture monitoring system 3 Monitoring device 7 Image capture system 12 Microscope 13 Light 8 Spectrometer 9 Computing unit 10 Signal generator 11 Impedance meter 28 Cartridge holder part 29 Cartridge holder slot 30 Positioning element 30a Spring protrusion 31 Connector 31a Electrical terminal 33 Ejector 4 Fluid circulation system 5 Dielectrophoresis cartridge 20 Base 22 Electrode connection window 23 Microfluidic circuit 23a Inflow path 23b Measurement chamber 26 Raised floor 27 Lateral guide 23c Return flow path 23d Drainage path 23e Auxiliary inflow path 24b Outlet (return) 24a Inlet (supply) 30b Positioning recess 25 Support mounting recess 19 Electrode support 21 Electrode 21a Measurement zone 21b Contact point 32 Insulator layer 14a Supply conduit 14b Outlet / return conduit 16 Tank supply / return fluid connection 17 Perforated tube 18 Fluid connector 18a Supply connection 18b Return connection 6 Pump 2 Cell culture tank 15 Cell culture medium

Claims

1. 1. A cell culture monitoring system (1) comprising: a monitoring device (3) for coupling to a culture tank (2) containing a cell culture medium (15); and a fluid circulation system (4) for fluidly coupling to the cell culture tank (2), the fluid circulation system comprises a supply conduit (14a) and a return conduit (14b), and a dielectrophoresis cartridge (5) for connection to the cell culture tank (2) via the supply conduit (14a) and the return conduit (14b); The dielectrophoresis cartridge comprises a base (20) and an electrode support (19) having electrodes (21) arranged in an electrode plane XY within or on the electrode support (19); the electrodes are configured for traveling wave dielectrophoresis and comprise a measurement zone (21 a) arranged above a measurement chamber (23 b) formed between the electrode support (19) and a floor (26) of the base (20) forming a measurement chamber therebetween; the monitoring device (3) comprises a signal generator (12) connected to the electrodes, a computing unit (9), an image capture system (7) connected to the computing unit (9), and a cartridge holder portion (28) for receiving the dielectrophoresis cartridge (5) so that the image capture system (7) can detect cells in the measurement chamber (23b); The cell culture monitoring system (1) is configured to capture displacements of cells in the cell culture medium in the measurement chamber in an orthogonal direction Z relative to the electrode plane XY to enable measurement of cell type and / or cell state.

2. 2. The system of claim 1, wherein the image capture system and the computing unit are configured to capture a plurality of slice-type images taken in the orthogonal direction Z, and the plurality of images are processed to determine a position of a cell and a displacement of the cell in the orthogonal direction Z.

3. 3. The system of claim 2, wherein the image capture system comprises a microscope (7) having a depth of field smaller than the height of the measurement chamber and an electrically adjustable lens for adjusting the focus to capture image slices at different heights.

4. 2. The system of claim 1, wherein cells in the liquid cell culture medium in the measurement chamber are subjected to a traveling wave dielectrophoretic force in a plane parallel to the electrode plane XY, and wherein the image capture system is configured to capture the displacement of cells in the cell culture medium in the measurement chamber in the plane parallel to the electrode plane XY to enable measurement of a cell type and / or a cell state in association with the measurement based on the displacement of the cells in the orthogonal direction Z.

5. 5. The system of claim 4, wherein the signal generator is configured to scan through different electrode signal frequencies or apply a plurality of discrete different electrode signal frequencies during a measurement or over a series of measurements, and the image capture system and the computing unit are configured to measure cell displacement for the different electrode signal frequencies.

6. 2. The system of claim 1, wherein the electrode support (19) is made of a transparent polymer or glass, and the electrodes are made of a conductive transparent material, which is a thin indium tin oxide (ITO) layer.

7. 2. The system of claim 1, wherein the electrode (21) is insulated from the interior of the measurement chamber by an insulating layer, except for the measurement zone (21a) and the contacts (21b).

8. 2. The system of claim 1, wherein the cartridge comprises at least two impedance measuring electrodes (21', 21") configured to measure the impedance (Zi) of a cell.

9. 2. The system of claim 1, wherein the electrode (21) is formed on an inner surface of the electrode support (19) that defines the measuring chamber (23b), the inner surface having a contact portion (21b) that extends to an electrode connection window (22) formed in the base (20) for inserting a contact into a complementary spring contact (31a) of the monitoring device, the electrode connection window (22) being hermetically separated from the measuring chamber (23b).

10. 2. The system of claim 1, wherein the measuring chamber (23b) comprises a raised floor (26) and lateral guides (27) that define a gap between the floor (26) and the electrode support (19).

11. 2. The system of claim 1, wherein the electrode (21) comprises one or more spiral conductive tracks in two mirror-symmetric sets, a portion of which forms the measurement zone (21a).

12. The system according to claim 1, wherein the electrode comprises at least two meshed electrodes (21i, 21ii, 21iii, 21iv, 21v, 21vi).

13. The system according to claim 1, wherein the cartridge holder portion (28) of the monitoring device (3) comprises a cartridge holder slot configured to slidably insert the dielectrophoresis cartridge.

14. The system according to claim 1, wherein the cartridge holder portion (28) comprises a positioning element (30) that engages a complementary positioning element within the dielectrophoresis cartridge to position and fix the dielectrophoresis cartridge in a measurement position.

15. The system according to claim 1, wherein the gap between the electrode and the floor (26) in the measurement chamber (23) ranges from 10 μm to 500 μm.

16. A cell culture monitoring system (1) comprising a monitoring device (3) for coupling to a culture tank (2) containing a cell culture medium (15), and a fluid circulation system (4) for fluidly coupling to the cell culture tank (2), wherein the fluid circulation system comprises a supply conduit (14a) and a return conduit (14b), and a dielectrophoresis cartridge (5) for connecting to the cell culture tank (2) via the supply conduit (14a) and the return conduit (14b), wherein the dielectrophoresis cartridge comprises a base (20) and an electrode support (19) having electrodes (21) disposed in an electrode plane X - Y within or on the electrode support (19), wherein the electrodes are configured for traveling wave dielectrophoresis and comprise a measurement zone (21a) disposed above a measurement chamber (23b) formed between the electrode support (19) and the floor (26) of the base (20) that forms the measurement chamber therebetween, wherein the monitoring device (3) comprises a signal generator (12) connected to the electrodes, a computing unit (9), an image capture system (7) connected to the computing unit (9), and a cartridge holder portion (28) for receiving the dielectrophoresis cartridge (5) such that the image capture system (7) can detect cells within the measurement chamber (23b). The electrode (21) is insulated from the interior of the measurement chamber by an insulator layer, except for the measurement zone (21a), in a cell culture monitoring system (1).

17. The system according to claim 16, wherein the insulator layer contains silicon dioxide.

18. The system according to claim 16, wherein the electrode support (19) is made of a transparent polymer or glass, and the electrode is at least partially made within the measurement zone of a conductive transparent material.

19. The system according to claim 18, wherein the conductive transparent material is a thin indium tin oxide (ITO) layer.

20. The system according to claim 16, wherein the cartridge comprises at least two impedance measurement electrodes (21', 21") configured to measure the impedance (Zi) of cells.

21. The electrode (21) is formed on the inner surface of the electrode support (19) that defines the measurement chamber (23b), and the inner surface has a contact portion (21b) that extends to an electrode connection window (22) formed in the base (20) for inserting a contact into the compensating spring contact (31a) of the monitoring device. The electrode connection window (22) is hermetically separated from the measurement chamber (23b). The system according to claim 16.

22. The measurement chamber (23b) comprises a raised floor (26) and a lateral guide (27) that defines a gap between the floor (26) and the electrode support (19). The gap between the electrode and the floor (26) in the measurement chamber (23) is in the range of 10 μm to 500 μm, according to the system of claim 16.

23. The system according to claim 16, wherein the electrode (21) comprises one or more helical conductive tracks in two mirror-symmetrical sets, with a part forming the measurement zone (21a).

24. The system according to claim 16, wherein the electrode comprises at least two meshed electrodes (21i, 21ii, 21iii, 21iv, 21v, 21vi).

25. The system according to claim 16, wherein the image capture system is configured to capture the displacement of cells in the cell culture medium within the measurement chamber in a direction Z orthogonal to the electrode plane X - Y, enabling the measurement of cell types and / or cell states.

26. The image capture system and the computing unit are configured to capture a plurality of slice-type images taken in the orthogonal direction Z, and the plurality of images are processed to determine the position and displacement of cells in the orthogonal direction Z. The system according to claim 16.

27. Cells in the cell culture liquid medium in the measurement chamber are subjected to traveling wave dielectrophoretic force in a plane parallel to the electrode plane X - Y, and the image capture system captures the displacement of cells in the cell culture medium in the measurement chamber in the plane parallel to the electrode plane X - Y, and is configured to enable measurement of cell types and / or cell states in relation to measurements based on the displacement of the cells in the orthogonal direction Z. The system according to claim 16.

28. The signal generator is configured to scan different electrode signal frequencies or apply a plurality of discrete different electrode signal frequencies during a measurement or over a series of measurements, and the image capture system and the computing unit are configured to measure the displacement of cells for different electrode signal frequencies. The system according to claim 27.

29. The cartridge holder part (28) of the monitoring device (3) comprises a cartridge holder slot configured to slidably insert the dielectrophoresis cartridge. The system according to claim 16.

30. The cartridge holder part (28) comprises a positioning element (30) that engages with a complementary positioning element in the dielectrophoresis cartridge to position and fix the dielectrophoresis cartridge in the measurement position. The system according to claim 16.

31. A dielectrophoresis cartridge (5) for connecting to a cell culture tank (2), comprising a base (20) and an electrode support (19) having electrodes (21) arranged in an electrode plane X - Y within or on the electrode support (19), the electrodes being configured for traveling wave dielectrophoresis and comprising a measurement zone (21a) disposed above a measurement chamber (23b) formed between the electrode support (19) and the floor (26) of the base (20) that forms the measurement chamber therebetween. A dielectrophoresis cartridge (5), wherein the electrodes (21) are insulated from the interior of the measurement chamber by an insulator layer, except for a measurement zone (21a).

32. 32. The cartridge of claim 31, wherein the insulator layer comprises silicon dioxide.

33. 32. A cartridge according to claim 31, wherein the electrode support (19) is made of a transparent polymer or glass, and the electrodes are made at least partly in the measurement zone of a conductive transparent material.

34. 34. The cartridge of claim 33, wherein the conductive transparent material is a thin indium tin oxide (ITO) layer.

35. 32. Cartridge according to claim 31, wherein the cartridge comprises at least two impedance measuring electrodes (21', 21") configured to measure the impedance (Zi) of a cell.

36. 32. The cartridge of claim 31, wherein the electrode (21) is formed on an inner surface of the electrode support (19) that defines the measuring chamber (23b), the inner surface having a contact portion (21b) that extends to an electrode connection window (22) formed in the base (20) for inserting a contact into a complementary spring contact (31a) of a monitoring device, the electrode connection window (22) being hermetically separated from the measuring chamber (23b).

37. 32. The cartridge of claim 31, wherein the measuring chamber (23b) comprises a raised floor (26) and lateral guides (27) that define a gap between the floor (26) and the electrode support (19), the gap between the electrode and the floor (26) in the measuring chamber (23) being in the range of 10 μm to 500 μm.

38. 32. A cartridge according to claim 31, wherein the electrodes (21) comprise one or more spiral conductive tracks in two sets of mirror images, a portion of which forms the measurement zone (21a).

39. 32. A cartridge according to claim 31, wherein the electrodes comprise at least two interdigitated electrodes (21i, 21ii, 21iii, 21iv, 21v, 21vi).

Images