Electrical impedance tomography enabled bioreactor

Abstract

An EIT imaging-enabled bioreactor has a housing with inlet and outlet ports for flowing culture medium, a substrate bearing mammalian cells can be positioned in the housing. An electrode array, the electrodes each surfaced with precious metal, is exposed to an interior of the bioreactor. An EIT imaging device is coupled to the electrodes of the electrode array, to measure impedance data using multiple pairs of the electrodes of the electrode array as stimulus electrodes while measuring at other electrodes of the electrode array, the EIT-imaging device is configured to construct a three-dimensional map of impedance within the housing. A method of monitoring cell growth includes growing the cells on or in a substrate, using the electrode array to measure impedance data, and mapping impedance within the bioreactor at at least a first and a second time to observe changes in the impedance map with time.

Description

Atty Docket No. DRTC.P2346WO / 00609897 ELECTRICAL IMPEDANCE TOMOGRAPHY ENABLED BIOREACTOR RELATED APPLICATION

[0001] The present document claims priority to U.S. Provisional Patent Application 63 / 536,032 filed August 31, 2023. The entire contents of the aforementioned patent application are incorporated herein by reference. GOVERNMENT RIGHTS

[0002] This invention was made with government support under Grant No. T0086 awarded by the Advanced Regenerative Manufacturing Institute, and Contract No. W911NF- 17-3-0003 awarded by the Department of Defense. The government has certain rights in the invention. BACKGROUND

[0003] Cell culture is of increasing importance in biology and medicine. Most mammalian cells grow far better on a substrate than suspended in a culture medium. The substrate must, of course, be exposed to circulating, oxygenated, culture medium. Some other types of cells, including many used for production of drugs, alcohol, and other products can grow suspended in culture medium.

[0004] Electrical impedance imaging has been used to observe properties of mammalian tissues both in vivo and ex vivo but has not been used to observe progress of cell cultures on a substrate.

[0005] Much recent work has led to methods of inducing three-dimensional mammalian cell cultures to differentiate into various cell types, these differentiated cultures may at times resemble portions of specific organs or sections of specific tissues. It has been proposed that three-dimensional (3d) cell cultures may be able to produce differentiated tissues having specific organ-like shapes for transplantation. These cultures may be useful to prepare autologous skin, bone, and other grafts from a patient’s own stem cells, thereby avoiding immune-mediated rejection of the grafted tissues. Since cell shape and functions in 3D cultures may differ significantly from even the same cell type in monolayer culture, 3D cultures can also be of use in drug evaluation experiments because they may more closely mimic in-vivo drug metabolism and drug effects than monolayer cultures. LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897

[0006] 3D cultures typically are either grown with cells in gel suspension or into or growing on or in porous scaffolds or substrates, instead of with cells forming a single layer on a bottom of a culture dish.

[0007] Quality checking 3d tissue culture is typically destructive and provides data for only one time point.

[0008] One destructive method to check the quality and growth of three-dimensional cultures is to slice it into thin sections, mount these slices on slides and then stain them. Alternatively, cell proliferation assays can be used to determine cell count via the metabolic activity of the cells. Some of these techniques use cytotoxic reagents which are destructive to the cell culture. In general, previous proliferation assays designed for 2d cultures and have been found to be inaccurate when applied to 3d cultures having higher cell densities. DNA quantification also can be used to measure cell proliferation and is fairly accurate. However, DNA quantification is also destructive as it requires removing the cells from the scaffold, lysing the cells and measuring the quantity of DNA in the resulting liquid suspension.

[0009] Electrical Impedance Sensing or Electrical Impedance Spectroscopy (EIS) (also known as ECIStm(Trademark of Applied Biophysics, Inc)), is a technique for monitoring single layers of cells grown directly on electrodes in a culture dish; in EIS cell growth of monolayer cultures is performed through electrical impedance sensing. EIS uses impedance measured over a spectrum to determine what cell characteristics are. EIT (electrical impedance tomography) as known in the art and applied to sensing tissues, on the other hand, uses tomographic techniques to build a three-dimensional conductivity or permittivity map of the measured space. This creates an image with spatial information rather than just bulk impedance data of a measured space. SUMMARY

[0010] In an embodiment, an electrical impedance tomography (EIT) imaging-enabled bioreactor has a housing within which a substrate bearing mammalian cells can be positioned; the housing having inlet and outlet ports adapted for flowing culture medium through an interior of the EIT-enabled bioreactor; and an electrode array with 16 or more electrodes, the electrodes each surfaced with precious metal and exposed to the interior of the bioreactor. The bioreactor is used with an electrical impedance imaging (EIT-imaging) device coupled to the electrodes of the electrode array, the EIT-imaging device adapted to measure impedance data using multiple pairs of the electrodes of the electrode array as stimulus electrodes while measuring at least voltage at other electrodes of the electrode array, 2 LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 and where the EIT-imaging device is configured to construct a three-dimensional map of impedance within the housing.

[0011] A method of monitoring cell growth of mammalian cells designated B includes: growing the mammalian cells on or in a substrate, the substrate being within an electrical impedance imaging (EIT)-enabled bioreactor having an electrode array with 16 or more electrodes, the electrodes each surfaced with precious metal and exposed to an interior of the bioreactor; measuring impedance data using multiple pairs of the electrodes of the electrode array as stimulus electrodes while measuring at least voltage at other electrodes of the electrode array; and mapping impedance within the bioreactor. BRIEF DESCRIPTION OF THE FIGURES

[0012] Fig.1 is a perspective drawing of a front view of an electrical impedance tomography-enabled bioreactor.

[0013] Fig.2 is a perspective drawing of a back view of an electrical impedance tomography-enabled bioreactor.

[0014] Fig.3 is an exploded drawing of the electrical impedance tomography-enabled bioreactor of Figs.1 and 2.

[0015] Figs.4, 5, and 6 are a connector side, an end view, and an electrode side, respectively, of an electrode array for the electrical impedance tomography-enabled bioreactor of Figs 1, 2, and 3.

[0016] Fig.7 illustrates the bioreactor in use in a cell culture system.

[0017] Fig.8 is a photograph illustrating the bioreactor with a decellularized bone scaffold in place for three-dimensional cell culture. The decellularized bone scaffold serves as a porous scaffold such that cells may grow on a surface of the scaffold and within pores of the scaffold. Scaffolds of other types may also be used, including gels and collagen-based scaffolds.

[0018] Fig.9 is a schematic drawing of an alternative embodiment of the bioreactor with a third port for gas and bubble removal.

[0019] Fig.10 is a schematic of a gas-lock device that may optionally be used with the bioreactor of Fig.9.

[0020] Fig.11 is a schematic illustration of an embodiment of an open-top tissue- culture cell with an electrode array upon which a substrate is placed for culture. LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897

[0021] Fig.12 illustrates an embodiment of an open-top tissue-culture cell with an electrode array in a fitting that may be positioned atop a substrate cultured cells for culture assessment.

[0022] Fig.13 is a frontal view of an alternative embodiment of a flow-through tissue-culture cell with a third port for bubble removal.

[0023] Fig.14 is a side view of the alternative embodiment of a flow-through tissue- culture cell of Fig.13.

[0024] Fig.15 is a frontal view of an alternative embodiment of a flow-through tissue culture cell with a gas trap for bubble removal. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] This invention is a cell culture bioreactor with electrical impedance tomography (EIT) electrodes embedded in the chamber space for non-invasive, label-free monitoring of cell growth, cell viability and scaffold infiltration of 3d tissue culture.

[0026] A primary feature of this invention is the integration of EIT for non-invasive monitoring of the cell culture. This is particularly useful for three-dimensional (3d) cultures.

[0027] EIT integrated into a bioreactor can non-invasively provide continuous observation of a cell culture.

[0028] Bioreactors are used in tissue engineering to create a controlled environment for the culturing of cells. Our invention targets immobilized 3d cultures such as bone or cartilage tissue growing on a structured scaffold but could be adapted to suspension monitoring as well. The electrodes enable non-invasive EIT monitoring of tissue culture progression such as cell infiltration into the scaffold, cell proliferation, and cell viability. The progress of growth and quality of three-dimensional (3d) cell cultures is typically assessed by destructive means because the majority of cell monitoring tools in laboratories are designed for two-dimensional (2d) culture systems, not for 3d cell cultures. Electrical Impedance Tomography

[0029] Electrical impedance tomography (EIT) has been used to identify tumors in tissues based upon differences in electrical impedance between tissue types. EIT of tissues is performed by measuring impedances of tissues using alternating current (AC) stimulus at multiple frequencies; measuring impedances of tissues is performed contacting tissue with typically four or more electrodes, then driving currents between two stimulus electrodes while measuring voltages and phase between two or more sensing electrodes while gathering 4 LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 data. EIT systems may swap stimulus and sensing electrodes, having these electrodes do double-duty, while gaining additional data. Further, systems may have arrays of many sensing electrodes measured simultaneously and may repeat measurements using different electrode pairs as stimulus electrodes; measurements are also repeated at different frequencies of the AC stimulus. Recorded measurement data are then analyzed. EIT data gathering amounts to, in pseudocode: For F(frequency) = (first frequency .. last frequency) do For Stim(stimulus electrode pair) = (first stimulus electrode pair .. last electrode pair) do For SE(sense electrode) = (first sense electrode .. last sense electrode) do Measurement(F, Stim, SE) = measured voltage at sense electrode SE; End End End

[0030] Analysis of EIT measurement data of tissues often involves setting up a voxel-based model of the tissue with impedances as a parameter for each voxel, then fitting impedances at each voxel to the measurements. The impedance parameters for each voxel of the model may be displayed as a map of impedance throughout the tissue. Impedance parameters can include impedance, conductivity, permittivity at individual frequencies, the magnitude and phase of the admittivity at individual frequencies, and / or multi-frequency spectral impedance parameters derived from fitting parameterized curves to the recorded permittivity, conductivity, or impedance spectra at at least one, and typically more than one, frequencies. EIT Monitoring of 3D Culture Growth

[0031] In embodiments, we apply EIT to monitoring growth of tissue cultures. As shown below, we have developed a bioreactor in which 3D mammalian tissue cultures can grow and where a voxel-based model having impedance parameters for each voxel is then used to generate a three-dimensional EIT map of impedances in a culture chamber. The EIT map of impedances can nondestructively illustrate cell growth and culture shape including illustrating where cell growth occurs in or on a three-dimensional substrate.

[0032] In some embodiments, we generate an initial EIT map of impedances in the culture chamber after placing a seeded substrate in the chamber and filling the chamber with culture media. A current EIT map of impedances is generated after incubation. A difference LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 EIT map may then be generated by subtracting the initial map of impedances from the current EIT map to illustrate cell growth in the chamber or bioreactor. In an alternative embodiment, a sequence of such impedance maps is generated to show cell growth over time. A Prototype Bioreactor

[0033] In an embodiment, the bioreactor 100 (Fig.1, 2, and 3) has a clear front faceplate 102 attachable by screws 106 to a front side of a body 104, the body defining an inner culture chamber. The body has an inlet and an outlet cell media port 108, 110. While barbed fittings are shown on the inlet and outlet ports, we have also experimented with Luer- Lock fittings, and expect other port fittings known in the art to work. The faceplate is sealed to the body with a gasket 112 that in an embodiment is an “O” ring. On a back side of the body, an electrode array printed circuit 116 is sealed to the body with a rear gasket and fitted with a connector, the connector of the electrode array printed circuit being accessible through an opening in a back cover plate 114, the back cover plate also attached to the body by screws. The faceplate, wall of the body, and electrode array printed circuit 116 together form a housing. The electrode array printed circuit is electrically couples each electrode 120 of the electrode array to a separate pin of the connector. The electrodes of the electrode array printed circuit are disposed on an interior of the bioreactor space formed by the faceplate, electrode array printed circuit, and body, so that cell growth media fed through the inlet cell media port can reach the electrode array and any substrate or scaffold attached thereto.

[0034] In alternative embodiments, the printed circuit may incorporate analog multiplexers so that there may be a greater number of electrodes than pins on the connector

[0035] A 25-electrode version of the printed circuit (Figs.4, 5, and 6) has precious metal plated electrodes (represented by circles on Fig.6) on a contact side. The electrodes 120 may be plated with a low-toxicity, corrosion-resistant, metal such as hard, electroplated, gold, platinum or silver because many other metals would corrode when exposed to salts in culture media or kill cells. In alternative embodiments, the electrodes are coated with a conductive plastic such as Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) to both protect the electrodes from corrosion and protect growing cells from copper ions leached from the electrodes; and in other alternative embodiments the electrodes are fabricated from a nonmetallic conductive material such as carbon. Other than the exposed electrodes, the surface of the contact side of the printed circuit within the rear gasket area is coated with a nontoxic, nonconductive, masking material. We anticipate that other numbers and patterns of electrodes may be used, ranging from 16 to 32 electrodes when using our 6 LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 existing electrical impedance measuring devices, to 64 electrodes when using two of our existing measuring devices, or even more electrodes in future embodiments. Our electrical impedance measuring devices couple through a ribbon cable and connector 118 to the electrodes of the electrode array of the electrode array printed circuit and can use each electrode both as a stimulus electrode for applying weak currents while measuring voltages at a range of frequencies from 1kHz through 1MHz, and in particular embodiments 1kHz, 10KHz, 100kHz, and 1MHz, to the culture medium, and as a sense electrode for measuring phase and voltage at the applied frequency.

[0036] While the bioreactor could be used with an EIT-imaging device that dedicates certain pairs of electrodes as stimulus electrodes through which the weak currents are applied, while dedicating other electrodes as sense electrodes, our EIT-imaging devices are adapted to use a pair of electrodes as stimulus electrodes, and to use the same pair of electrodes as sense electrodes while using another pair of electrodes as stimulus electrodes, thereby using most or all electrodes as both stimulus and sense electrodes.

[0037] We anticipate there may be larger bioreactors with even more electrodes in future systems.

[0038] The prototype bioreactor is configured for use with a matrix or substrate that can be attached to the printed circuit, in a particular embodiment, the substrate is an 8 millimeter (mm) diameter Epibone ® (Trademark of Epibone, Jersey City, New Jersey) decellularized bovine bone scaffold two millimeters thick upon which mammalian cells are seeded for growth. We anticipate other shapes and sizes of scaffolds, and substrate materials besides decellularized bone, such as but not limited to gels and collagen scaffolds, may also be used particularly when growing tissue for transplantation.

[0039] As the chamber body is 3-D printable, and the printed circuit can be prepared in many layouts of electrodes, the system is easily reconfigurable for a wide variety of substrate sizes and shapes.

[0040] In an alternative embodiment, adaptable for growing cells on a substrate having a particular substrate shape as needed for transplant, an electrode array can be printed on a chamber wall conforming to the particular substrate shape so as to precisely image cell growth on and into the substrate.

[0041] When growing 3D cell cultures, a substrate is affixed within the bioreactor. In the example of Fig.8, a decellularized bone scaffold 802 is in place in the bioreactor 800 for three-dimensional cell culture. LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897

[0042] Gas bubbles may interfere with EIT measurements. In an alternative embodiment 900, as illustrated in Fig.9, the bioreactor has a third port 902 for gas-removal; in some embodiments this embodiment is operated with an axis through the third port oriented vertical, and a slight upslope 906 to the upper edge of the cavity in the bioreactor body 904 aids bubble extraction through third port 902.

[0043] In some embodiments using the bioreactor of Fig.9, a gas-lock such as that illustrated in Fig.10 is coupled to third port 902.

[0044] Fig.11 is a schematic illustration of an embodiment of an open-top tissue- culture cell culture plate 1104 with an electrode array 1108 in each cell, such as cell 1106, upon which a substrate is placed for culture. EIT may be used to measure cell growth in or on a substrate positioned on the electrode array.

[0045] Fig.12 illustrates an embodiment of an open-top tissue-culture cell plate 1100 having 6 cavities with an electrode array in a fitting 1102 that may be positioned atop a substrate cultured cells for culture assessment.

[0046] An alternative embodiment of a flow-through tissue-culture cell body 1300 (Fig.13, 14) has inlet and outlet ports 1302, 1304 for nutrient fluid and at least one opening 1306 for mounting the electrode array of Figs.4, 5, and 6; two openings 1306 for electrodes are shown. This embodiment also has a third port 1308 for bubble removal above a bubble settling chamber 1310.

[0047] Fig.15 is a frontal view of an alternative embodiment 1500 of a flow-through tissue culture cell resembling that of Figs.13 and 14 with a gas trap 1502 for bubble removal replacing third gas-removal port 1308. Operation:

[0048] This bioreactor has an internal chamber with walls lined with biocompatible electrodes. The 3d cell culture / scaffold is placed in the chamber space and immobilized by attaching it to either the bioreactor body or the electrode array printed circuit. The chamber is closed and is constantly perfused with cell growth medium through the inlet and outlet cell media ports via a pump and media reservoir with oxygenator as illustrated in Fig.7.

[0049] The electrodes are either periodically or continuously connected to hardware that can perform electrical impedance imaging observations by injecting low amplitude AC currents at 1kHz, 10kHz, 100kHz, 1MHz, or other frequencies between 1kHz to 1MHz between selected pairs of electrodes while simultaneously recording voltage differentials 8 LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 between other electrode pairs; our electrical impedance imaging hardware can use each electrode both as stimulus electrodes and sense electrodes. Typically, during each observation session we obtain data at multiple frequencies while using multiple selected pairs of electrodes as stimulus electrodes and recording measurements of voltages and phase from all electrodes on the printed circuit other than the pair of electrodes being driven. This data is passed to a computer that, with a finite element mesh model of the internal chamber space, can create an image of the interior space based on either interior conductivity or permittivity. Conductivity and permittivity can reflect desired tissue culture attributes such as cell proliferation, scaffold infiltration and cell viability. EIT data may be collected at one frequency or over multiple frequencies to provide spectral information.

[0050] We observe changes in impedance as cells grow on the substrate, or as cells die. Results:

[0051] We have tested the biocompatibility of a multiple electrode array designs made with standard printed circuit board fabrication methods with a u251 cell line. We have also performed testing of current injection parameters that are compatible with cell growth (i.e., current amplitude and current frequency). We have verified the ability of the system to perform difference imaging over time in the chamber space; this essentially creates an initial 3D map of either permittivity or conductivity and a current 3D map of either permittivity or conductivity, then subtracts the initial 3D map from the current 3D map to create a 3D map of changes in permittivity or conductivity that reflects cell growth in the bioreactor, including cell growth in or on the substrate. We have designed and fabricated prototypes of our EIT enabled bioreactor using a mix of printed circuit boards, laser cutting, and 3d printing. We have performed electrical impedance spectroscopy (EIS) tests of cell suspensions of multiple concentrations to study the spectral characteristics of cell density. We have performed high contrast phantom studies in the bioreactor chamber. Indeed, we find that it may be best to obtain spectral impedance data at many frequencies between 1kHz and 1MHz to best map impedance in the three-dimensional volume within the bioreactor.

[0052] We have produced data at multiple growth times while growing osteosarcoma cells in addition to u251 glioblastoma cells, but note the system is adaptable to a wide variety of human and animal cells.

[0053] EIT data provided from an EIT integrated bioreactor provides a lot of impedance data that needs to be connected to the variables of interest, such as cell 9 LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 proliferation, cell viability, scaffold infiltration, etc.; this data varies with time and changes in impedance data with time can be evaluated over time to monitor growth on the substrate within the chamber. This can be accomplished by computer algorithms, machine learning, or a variety of statistical computational methods. While there exists a large data set for each EIT measurement, the relationships between impedance and tissue properties can be calibrated for each tissue type and scaffold shape. Combinations

[0054] The various features discussed herein can be combined in multiple ways. Among combinations anticipated by the inventors are:

[0055] An electrical impedance tomography (EIT) imaging-enabled bioreactor designated A including: a housing within which a substrate bearing mammalian cells can be positioned; the housing having inlet and outlet ports adapted for flowing culture medium through an interior of the EIT-enabled bioreactor; and an electrode array with 16 or more electrodes, the electrodes each surfaced with precious metal and exposed to the interior of the bioreactor.

[0056] An EIT-enabled bioreactor designated AA including the EIT-enabled bioreactor designated A, further including an electrical impedance imaging (EIT-imaging) device coupled to the electrodes of the electrode array, the EIT-imaging device adapted to measure impedance data using multiple pairs of the electrodes of the electrode array as stimulus electrodes while measuring at least voltage at other electrodes of the electrode array, and where the EIT-imaging device is configured to construct a three-dimensional map of impedance within the housing.

[0057] An EIT-enabled bioreactor designated AB including the EIT-enabled bioreactor designated A or AA, where the EIT-imaging device is adapted to use a first pair of electrodes as stimulus electrodes while measuring impedance data from a second pair of electrodes, and to measure impedance data using the first pair of electrodes as sense electrodes while using a second pair of electrodes as stimulus electrodes.

[0058] An EIT-enabled bioreactor designated AC including the EIT-enabled bioreactor designated A, AB or AA, where the EIT-imaging device is adapted to measure the impedance data at at least one frequency between 1kHz and 1MHz.

[0059] An EIT-enabled bioreactor designated AD including the EIT-enabled bioreactor designated A, AB, AC or AA, where the EIT-imaging device is adapted to measure the impedance data at a plurality of frequencies between 1kHz and 1MHz. LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897

[0060] An EIT-enabled bioreactor designated AE including the EIT-enabled bioreactor designated A, AB, AC, AD or AA, further including a substrate adapted for tissue culture and fixed within the housing.

[0061] An EIT-enabled bioreactor designated AE including the EIT-enabled bioreactor designated A, AB, AC, AD, AE or AA, where the electrode array is formed to conform to a shape of the substrate.

[0062] An EIT-enabled bioreactor designated AF including the EIT-enabled bioreactor designated A, AB, AC, AD, AE or AA, where the EIT-imaging device is configured to monitor changes in the impedance over time.

[0063] An EIT-enabled bioreactor designated AG including the EIT-enabled bioreactor designated A, AB, AC, AD, AE, AF or AA, wherein the three-dimensional map of impedance within the housing comprises a map of conductivity or permittivity.

[0064] An EIT-enabled bioreactor designated AH including the EIT-enabled bioreactor designated A, AB, AC, AD, AE, AF, AG or AA, further comprising a third port adapted for gas bubble removal from the bioreactor.

[0065] A method of monitoring cell growth of mammalian cells designated B includes:

[0066] growing the mammalian cells on or in a substrate, the substrate being within an electrical impedance imaging (EIT)-enabled bioreactor having an electrode array with 16 or more electrodes, the electrodes each surfaced with precious metal and exposed to an interior of the bioreactor; measuring impedance data using multiple pairs of the electrodes of the electrode array as stimulus electrodes while measuring at least voltage at other electrodes of the electrode array; and mapping impedance within the bioreactor.

[0067] A method of monitoring cell growth of mammalian cells designated BA including the method designated B and further including observing changes in the impedance map over time.

[0068] A method of monitoring cell growth of mammalian cells designated BB including the method designated B or BA and further including wherein the mammalian cells are disposed on, and grow on or within pores of, a porous substrate.

[0069] A method of monitoring cell growth of mammalian cells designated BC including the method designated B, BA, or BB and wherein the impedance data is acquired at at least one frequency between 1kHz and 1MHz. LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897

[0070] A method of monitoring cell growth of mammalian cells designated BD including the method designated B, BA, BB, or BC and wherein the impedance data is measured at a plurality of frequencies between 1kHz and 1MHz.

[0071] A method of monitoring cell growth of mammalian cells designated BE includes the method designated BD wherein the electrode array is shaped to conform to a shape of the substrate.

[0072] Changes may be made in the above methods and systems without departing from the scope hereof. It should thus be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the present method and system, which, as a matter of language, might be said to fall there between. LEGAL\72016934\3

Claims

Atty Docket No. DRTC.P2346WO / 00609897 CLAIMS What is claimed is:

1. An electrical impedance tomography (EIT) imaging-enabled bioreactor comprising: a housing within which a substrate bearing mammalian cells can be positioned; the housing having inlet and outlet ports adapted for flowing culture medium through an interior of the housing; and an electrode array with 16 or more electrodes, the electrodes exposed to the interior of the bioreactor.

2. The EIT imaging-enabled bioreactor of claim 1, further comprising an electrical impedance imaging (EIT-imaging) device coupled to the electrodes of the electrode array, the EIT-imaging device adapted to measure impedance data using multiple pairs of the electrodes of the electrode array as stimulus electrodes while measuring at least voltage at other electrodes of the electrode array, and where the EIT-imaging device is configured to construct a three-dimensional map of impedance within the housing.

3. The EIT imaging-enabled bioreactor of claim 2 where the EIT-imaging device is adapted to use a first pair of the electrodes as stimulus electrodes while measuring impedance data from a second pair of the electrodes, and to measure impedance data using the first pair of the electrodes as sense electrodes while using the second pair of the electrodes as stimulus electrodes.

4. The EIT imaging-enabled bioreactor of claim 2 where the EIT-imaging device is adapted to measure the impedance data at at least one frequency between 1kHz and 1MHz.

5. The EIT imaging-enabled bioreactor of claim 4 where the EIT-imaging device is adapted to measure the impedance data at a plurality of frequencies between 1kHz and 1MHz.

6. The EIT imaging-enabled bioreactor of claim 5 further comprising a substrate adapted for tissue culture within the housing. LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 7. The EIT imaging-enabled bioreactor of claim 2 where the electrode array is formed to conform to a shape of the substrate.

8. The EIT imaging-enabled bioreactor of claim 7 where the EIT-imaging device is adapted to measure the impedance data at a plurality of frequencies between 1kHz and 1MHz.

9. The EIT imaging-enabled bioreactor of claim 6 or 8 where the EIT-imaging device is configured to monitor changes in the impedance over time.

10. The EIT imaging-enabled bioreactor of claim 2 wherein the three-dimensional map of impedance within the housing comprises a map of conductivity, permittivity, or a parameter derived from conductivity or permittivity at at least one frequency.

11. The EIT imaging-enabled bioreactor of claim 1, 2, 3, 4, 5, 6, 7, 8, or 10, further comprising a third port adapted for gas bubble removal from the bioreactor.

12. The EIT imaging-enabled bioreactor of claim 11, the electrodes each surfaced with precious metal.

13. A method of monitoring cell growth of mammalian cells comprising: growing the mammalian cells on or in a substrate, the substrate being within an electrical impedance imaging (EIT)-enabled bioreactor having an electrode array with 16 or more electrodes, the electrodes exposed to an interior of the bioreactor; measuring impedance data using multiple pairs of the electrodes of the electrode array as stimulus electrodes while measuring at least voltage at other electrodes of the electrode array; and mapping impedance within the bioreactor.

14. The method of claim 13 further comprising observing changes in the impedance map over time.

15. The method of claim 13 wherein the mammalian cells are disposed on, and grow on or within pores of, a porous substrate. LEGAL\72016934\3Atty Docket No. DRTC.P2346WO / 00609897 16. The method of claim 13 wherein the impedance data is acquired at at least one frequency between 1kHz and 1MHz.

17. The method of claim 13 wherein the impedance data is measured at a plurality of frequencies between 1kHz and 1MHz.

18. The method of claim 13 where the electrode array is shaped to conform to a shape of the substrate.

19. The method of claim 13, 14, 15, 16, or 18 where the electrodes are each plated with a precious metal. LEGAL\72016934\3

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