Apparatuses and methods for in-SITU biomass monitoring in fixed bed bioreactors using interdigitated electrodes

Abstract

A cell culture bioreactor is provided that enables in-situ biomass sensing using interdigitated electrodes. The bioreactor includes a cell culture vessel having at least one inlet, at least one outlet, and an interior reservoir disposed between the at least one inlet and the at least one outlet. The bioreactor also includes a fixed bed disposed in the interior reservoir and having a substrate for culturing cells adhered to a surface thereof. The bioreactor further includes at least one biomass sensor, which includes a pair of electrode structures, each having a plurality of interdigitated electrode branches, where the pair of electrode structures are disposed within the fixed bed. The biomass sensor also includes an impedance analyzer electrically coupled to the pair of electrode structures, wherein the biomass sensor can detect a change in impedance between the pair of electrode structures based on changes in the microenvironment surrounding the interdigitated electrodes.

Description

Attorney Docket No.: SP24-268 APPARATUSES AND METHODS FOR IN-SITU BIOMASS MONITORING IN FIXED BED BIOREACTORS USING INTERDIGITATED ELECTRODESCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U. S. C. §119 of U. S.Provisional Application Serial No. 63 / 736,378 filed on December 19, 2024 and U. S, Provisional Application Serial No. 63 / 725,730 filed on November 27, 2024, the content of which are relied upon and incorporated herein by reference in their entirety.FIELD OF THE DISCLOSURE

[0002] This disclosure general relates to systems and methods of monitoring cell cultures in bioreactor systems. In particular, the present disclosure relates to apparatuses and methods for in-situ biomass monitoring of a cell culture within a fixed bed bioreactor system using electrical and / or optical sensing within the bioreactor.BACKGROUND

[0003] In the bioprocessing industry, large-scale cultivation of cells is performed for purposes of the production of hormones, enzymes, antibodies, vaccines, therapeutic proteins, and cell therapies. Cell and gene therapy markets are growing rapidly, with promising treatments moving into clinical trials and quickly toward commercialization. However, one cell therapy dose can require billions of cells or trillions of viruses. As such, being able to provide a large quantity of cell products in a short amount of time is critical for clinical success.

[0004] A significant portion of the cells used in bioprocessing are anchorage dependent, meaning the cells need a surface to adhere to for growth and functioning. Traditionally, the culturing of adherent cells is performed on two-dimensional (2D) cell-adherent surfaces incorporated in one of a number of vessel formats, such as T-flasks, petri dishes, cell factories, cell stack vessels, roller bottles, and other multilayered vessels (e.g., the HYPERStack® from Coming Inc.). These approaches can have significant drawbacks, including the difficulty in achieving cellular density high enough to make it feasible for large scale production of therapies or cells.Attorney Docket No.: SP24-268

[0005] Alternative methods have been suggested to increase volumetric density of cultured cells. These include microcamer cultures performed in stir tanks; hollow fiber bioreactors, in which cells may form large three-dimensional aggregates as they proliferate in the interspatial fiber space; and packed-bed bioreactors. In packed-bed or fixed-bed bioreactors, a cell substrate is packed or fixed within a bioreactor and used to provide a surface for the attachment of adherent cells. Medium can be perfused along the surface or through the semi-porous substrate to provide nutrients and oxygen needed for the cell growth,

[0006] One of the significant issues with traditional fixed bed bioreactors is the nonuniformity of cell distribution inside the bed. For example, the packed bed can function as a depth filter with cells predominantly trapped at the inlet regions or other regions of relatively low flow and / or high substrate density, resulting in a gradient of cell distribution during the inoculation step. In addition, due to random fiber packaging, flow resistance and cell trapping efficiency of cross sections of the packed bed are not uniform. For example, medium flows fast though the regions with low cell packing density and flows slowly through the regions where resistance is higher due to higher number of entrapped cells. This creates a channeling effect where nutrients and oxygen are delivered more efficiently to regions with lower volumetric cells densities and regions with higher cell densities are being maintained in suboptimal culture conditions. In addition, because of the random arrangement of fibers in the traditional packed or fixed bed substrates, it can be difficult for bioreactor users to predict cell culture performance, since the substrate arrangement and / or packing varies between cultures. Monitoring of the cell culture health or progress is also difficult. For example, the presence of the fixed bed itself limits options for effectively monitoring the health of the culture and the biomass production.

[0007] Regardless of the platform used, it is desirable for users to have information on cell behavior, virus production, and / or culture progress during the cell culture process. However, the process of seeding, proliferating and transfecting or harvesting cells can require several days to multiple w eeks. One of the identified draw backs of adherent-based bioreactors is the difficulty in taking substrate samples to directly assess the state of the cells and the overall cell culture progress. Taking substrate samples risk contaminating the entire culture or, in the case of non-uniform platforms, providing misleading, inaccurate, or non-representative data. To maintain process control and consistency and determine process endpoints or transitions, it is desirable to monitor biomass or cell density inside the reactor during the cell culture run. ThisAttorney Docket No.: SP24-268 is often completed using commercial capacitance probes in suspension reactors today. In a conventional suspension cell reactor, cells are suspended in media and well mixed. Sampling of a small volume of the media is considered generally representative of the cell density of the entire reactor. For this reason, commercial capacitance probes that monitor a region having a diameter of less than 20 mm have been adopted industrially to provide a direct measure of cells in the media. The measurement is sensitive to the environment and requires calibration, but has been generally adopted industrially for suspension technology,

[0008] In fixed-bed bioreactors, it can be difficult to directly monitor cells on the surface of the substrate due to the optical density of the substrate. Further, because cells are not mobile or mixed, some spatial variation in the cell density is possible. Also, bubbles or suboptimal processes can contribute to spatial cell density variation in the reactor. These limitations in monitoring of fixed beds, in addition to the other challenges of fixed beds discussed above, have led to a dearth of adequate cell culture monitoring for fixed-bed bioreactors.

[0009] Thus, there is a need for bioreactor systems and methods that enable real-time and aseptic monitoring of cell culture progress, including cell proliferation and density. There is also a need for such monitoring to be conducted in-line or in-situ and in a representative sample of the cell culture, or in a way that provides biomass spatial information, without disrupting the cell culture.SUMMARY

[0010] According to embodiments of this disclosure, a cell culture bioreactor is provided that is capable of biomass sensing within a fixed bed substrate for culturing cells using at least one biomass sensor disposed within a cell culture vessel of the bioreactor and having at least one conductor configured to generate an electric field passing through at least a portion of the fixed bed. The following is a description of various aspects of implementations of the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter, The implementations are intended to illustrate a few aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible implementations.

[0011] According to embodiments of this disclosure, a cell culture bioreactor for in-situ biomass sensing is provided that includes a cell culture vessel with at least one inlet, at least one outlet, and an interior reservoir disposed between the at least one inlet and the at least one outlet. The bioreactor also includes a fixed bed having a substrate for culturing cells adheredAttorney Docket No.: SP24-268 to a surface thereof the fixed bed being disposed in the interior reservoir. The bioreactor also includes at least one biomass sensor including a pair of electrode structures each having a plurality of interdigitated electrode branches, the pair of electrode structures being disposed within the fixed bed; and an impedance analyzer electrically coupled to the pair of electrode structures, wherein the at least one biomass sensor can detect a change in impedance between the pair of electrode structures.

[0012] It is to be understood that the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this description. The draw ings illustrate the various embodiments described herein, and together w ith the description serve to explain the principles and operations of the claimed subject matter.BRIEF DESCRIPTION OF THE DR WINGS

[0013] Figure 1 is a schematic representation of a cell culture bioreactor, according to one or more embodiments,

[0014] Figure 2 is a schematic representation of cell culture bioreactor, according to one or more embodiments.

[0015] Figure 3A is a schematic view of a fixed-bed bioreactor w ith electrodes positioned within a central guide rod and around a periphery of the cell culture vessel, according to embodiments of this disclosure.

[0016] Figure 3B is a view of a fixed-bed bioreactor with an electrode positioned w ithin a central guide rod and around a periphery’ of the cell culture vessel, accordi ng to embodiments of this disclosure.

[0017] Figure 4 is an illustration of a cell culture bioreactor using alternating pairs of electrodes in a central guide rod, according to embodiments of this disclosure.

[0018] Figure 5 is a cross-sectional view of a bioreactor w ith two guide rods having embedded electrodes, according to embodiments of this disclosure.

[0019] Figure 6A is a cross-sectional view' of a bioreactor with two guide rods having embedded induction coils, according to embodiments of this disclosure.Attorney Docket No.: SP24-268

[0020] Figure 6B is a cross-sectional view of a bioreactor w ith a guide rod having two embedded induction coils, according to embodiments of this disclosure.

[0021] Figure 7 is a schematic view of redundant electrode pairings, according to embodiments of this disclosure,

[0022] Figure 8A is an illustration of an unrolled mesh cell culture substrate w ith metallized sections as electrodes, according to embodiments of this disclosure.

[0023] Figure 8B is a plan view7of the mesh substrate like the one in Figure 8A in a rolled cylindrical configuration, according to embodiments of this disclosure.

[0024] Figure 9 A is a plan view' of a mesh substrate in a cylindrical roll w ith a continuous electrode in the rolled mesh and a counter electrode at the central guide rod, according to embodiments of this disclosure.

[0025] Figure 9B is a cross-sectional elevation view' of the bioreactor of Figure 9A where the central counter electrode is segmented, according to embodiments of this disclosure.

[0026] Figure 10 is a cross-sectional view of a bioreactor with an unrolled substrate showing segmented electrodes in the substrate, according to embodiments of this disclosure.

[0027] Figure 11A is an exploded perspective view of an induction coil in between tw o rolled substrate fixed beds, according to embodiments of this disclosure.

[0028] Figure 1 IB is an exploded perspective view of a core for a fixed bed with two, vertically spaced induction coils, according to embodiments of this disclosure.

[0029] Figure 11C is an exploded perspective view of a fixed bed w ith tw o, concentrically spaced induction coils, according to embodiments of this disclosure.

[0030] Figure 12A is a perspective view of a cylindrical bioreactor vessel using the top and bottom surfaces as the sensing interface of a biomass sensor, with annular electrodes, according to embodiments of this disclosure.

[0031] Figure 12B is a perspective view of a cylindrical bioreactor vessel using the top and bottom surfaces as the sensing interface of a biomass sensor, w ith split annular electrodes, according to embodiments of this disclosure.

[0032] Figure 12C is a cross-section view of the bioreactor of Figures 12A or 12B, according to embodiments of this disclosure.

[0033] Figure 13 is a perspective view' of a bioreactor using an array of electrodes of a top side and a ground electrode on the bottom side, according to embodiments of this disclosure.Attorney Docket No.: SP24-268

[0034] Figure 14 is a diagram of a four-electrode biomass sensor, according to embodiments of this disclosure.

[0035] Figure 15A is a cross-section view in elevation of a rolled fixed bed using the four-electrode based design with multiple working electrodes and additional reference electrodes, according to embodiments of this disclosure.

[0036] Figure 15B is a plan view7of the rolled fixed bed of Figure 15A, according to embodiments.

[0037] Figure 16A is an exploded view7of a stacked fixed bed with a four-electrode sensing array, according to embodiments of this disclosure.

[0038] Figure 16B is an exploded view of a stacked fixed bed with a four-electrode sensing array, according to embodiments of this disclosure.

[0039] Figure 17 is a schematic of a modeled electric circuit, according to embodiments.

[0040] Figure 18 is a plan view7of interdigitated electrodes for biomass sensing, according to embodiments.

[0041] Figure 19 is an elevation view7of the interdigitated electrodes for biomass sensing of Figure 18, according to embodiments.

[0042] Figure 20 is a representation of a simplified computational domain of interdigitated electrodes used for modeling, according to embodiments.

[0043] Figure 21 is a graph of the potential distribution of interdigitated electrodes based on modeling, according to embodiments.

[0044] Figure 22 is a graph showing the modeled decay in potential along a vertical distance, y, of interdigitated electrodes, according to embodiments.

[0045] Figure 23 is a graph show'ing the vertical distance at w hich the value of potential drops less than 95% of the applied potential and its relation to the interdigitated electrodes line width, according to embodiments.

[0046] Figure 24 is a graph of the electric field generated between branches of the interdigitated electrodes, according to embodiments.

[0047] Figure 25 is a graph showing the decay in electric field along a line, y, of the interdigitated electrodes of different line widths, according to embodiments.

[0048] Figure 26 is a graph comparing the potential decay along the line, y, as electrode spacing of interdigitated electrodes is increased, according to embodiments.Attorney Docket No.: SP24-268

[0049] Figure 27 is a graph showing 95% voltage drop distance versus electrode spacing of the interdigitated electrodes, according to embodiments.

[0050] Figure 28 is a graph of the variation in length scale associated with interdigitated electrodes as the Debye length of the solution is changed, according to embodiments.

[0051] Figure 29 is a perspective view of a partially unrolled fixed bed showing interdigitated electrodes of a biomass sensor placed betw een layers of the fixed bed, according to embodiments,

[0052] Figure 30 is a side view' of interdigitated electrodes between layers of cell culture substrate, according to embodiments.

[0053] Figure 31 is a schematic of an electrochemical model for interdigitated electrodes used in a fixed-bed bioreactor biomass sensor, according to embodiments.

[0054] Figure 32 is a photograph of line-type interdigitated electrodes, according to embodiments.

[0055] Figure 33 is a photograph of circular-type interdigitated electrodes, according to embodiments.

[0056] Figure 34 is a graph of impedance data over a frequency range from interdigitated electrodes used during cell culture in a fixed-bed bioreactor, according to embodiments.

[0057] Figure 35 is a plot of biomass capacitance over time based on impedance data from interdigitated electrodes, according to embodiments.

[0058] Figure 36A is a graph of cyclic voltammetry data for planar and nanoporous interdigitated electrodes, according to embodiments.

[0059] Figure 36B is a graph compared the current generated for the nanoporous and planar interdigitated electrodes of Figure 36A, according to embodiments.

[0060] Figure 37 are electrochemical impedance spectroscopy plots for planar, nanotextured printed, and microfabricated nanoporous electrodes, according to embodiments.DETAILED DESCRIPTION

[0061] Various embodiments of the disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.Attorney Docket No.: SP24-268

[0062] Due to some of the inherent limitations of adherent-based bioreactors discussed above, sensors, bioreactor systems, and methods for accurately measuring biomass or cell density are needed. Embodiments of this disclosure achieve this goal in multiple ways, including enabling sensing and measuring of biomass or cell density over larger portions of the bioreactor to ensure representative values. In embodiments, measuring multiple and / or discrete locations of the bioreactor is possible, which can provide the additional benefit of assessing reactor variability. Aspects of embodiments include advanced configurations that offer ease of implementation, improvements in sensitivity, and the possibility of measuring an array of locations to determine variability over larger areas of the bioreactor. Aspects of embodiments include compatibility with fixed beds of different configurations, such as cylindrically rolled substrate beds and stacked substrate beds, as discussed below.

[0063] Embodiments of this disclosure include biomass sensors employing arrays of conductive elements (e.g., electrodes and / or inductors) within a bioreactor to interrogate multiple regions of a substrate fixed bed within the bioreactor. The electrodes or inductors can detect within the fixed bed capacitance or impedance changes, which can be calibrated to a cell density level. Biomass sensors using a plurality of conductors (e.g., 2 or more electrodes or more than 1 inductor coil) can enable inferring spatial differences in the reactor providing the user with more information and enabling optimization of the uniformity of process. Optimized uniformity can lead to better overall yield and cell health. Where there are no specific controls for uniformity in the process, a process endpoint can be chosen based on maximizing the overall reactor viable cell density, instead of being based on only on location (or few locations) within the rector.

[0064] Biomass monitoring is important for cell culturing platforms of all scales, but becomes especially important or valuable as the size of the reactor increases, as opening and sampling reactors grows increasingly impractical and risky in large-scale or manufacturinglevel environments due to, among other challenges, threats to sterility of the cell culture. In addition, as the size of the reactor increases, the possibility of variations in cell density within reactor can also increase. On the one hand, an indication of the total cell density is desirable, primarily; however, if not only total cell density but also spatial variation within the reactor can be determined, the process can be optimized to maximize uniformity and cell density. For this reason, it is desirable to have biomass sensors capable of interrogating multiple (potentially overlapping) regions of the reactor. Multiple regions of the fixed bed of the bioreactor may beAttorney Docket No.: SP24-268 interrogated by having components (e.g., electrodes) of the biomass sensor in various regions of the bioreactor or by relative physical motion of the sensors and the reactor so that a single component can effectively move to different regions within the bioreactor.

[0065] Embodiments of this disclosure include systems and methods for monitoring, measuring, and / or predicting the density and / or proliferation of adherent cells inside a fixed bed bioreactor. Embodiments do not require access to samples of the bioreactor cell substrate or fixed bed, nor do they require opening the bioreactor or perfusion loop to the exterior environment (and thus risking contamination of the culture). Rather, embodiments disclosed herein enable, real-time, in-situ, and aseptic monitoring of a cell culture within a fixed-bed bioreactor. In addition, embodiments of this disclosure enable monitoring the cell culture in different regions or zones of the fixed bed. The embodiments described herein are scalable across all ranges of bioreactor systems, and can be used from process development to production or manufacturing scale systems. According to embodiments, fixed-bed bioreactor systems, as well as fixed bed cell substrates themselves, are provided with one or more biomass sensors built into the bioreactor vessel and / or the substrate fixed bed. These sensors can be integral components of an aseptic bioreactor system.

[0066] Embodiments of this disclosure also include methods of monitoring biomass (e.g., cell density and / or proliferation) within a fixed bed reactor using one or more sensors in one or more zones within a fixed bed. By distributing multiple sensors in multiple zones of the fixed bed or using an array of sensor components capable of gathering spatial information, it is possible to monitor cell distribution and proliferation uniformity through the fixed bed. The advantages of embodiments disclosed include the ability to actively monitor the bioreactor or cell culture in real time without the need to perform physical sampling of the fixed bed material for offline analysis. Continuous monitoring of the cell culture allows end users to actively adjust the bioprocess steps that are dependent on the progression of culture processes inside the fixed bed bioreactor, and to identify the timing of positive or negative changes in the state of the cell culture to help pinpoint process parameters in need of changing and optimization. The ability to characterize and log the progression of a cell culture also allows end users to monitor and record batch-to-batch consistency of the process. This type of tracking of progression and consistency among cell culture runs can be incredibly advantageous.

[0067] In conventional large-scale cell culture bioreactors, different types of packed-bed bioreactors have been used. Usually, these cell culture beds contain porous matrices to retainAttorney Docket No.: SP24-268 adherent or suspension cells, and to support growth and proliferation. Packed-bed matrices provide high surface area to volume ratios, so cell density can be higher than in the other systems. However, the packed bed often functions as a depth filter, where cells are physically trapped or entangled in fibers of the matrix. Thus, because of linear flow of the cell inoculum through the packed bed, cells are subject to heterogeneous distribution inside the packed bed, leading to variations in cell density through the depth or width of the packed bed. For example, cell density may be higher at the inlet region of a bioreactor and significantly lower nearer to the outlet of the bioreactor. In another example, non-uniformities in the packed bed create a channeling effect in which cell culture media preferentially flows in certain areas of the bed while be restricted from reaching other areas of the bed, again leading to non-uniform cell distribution and nonuniform or inconsistent medium or nutrient distribution. This non-uniform distribution of the cells inside of the packed bed significantly hinders scalability and predictability of such bioreactors in bioprocess manufacturing, and can even lead to reduced efficiency in terms of grow I h of cells or viral vector production per unit surface area or volume of the packed bed.|0068] Another problem encountered in packed bed bioreactors disclosed in prior art is the channeling effect, described above. Due to random nature of packed substrate material and / or random arrangements of nonwoven fibers, the local fiber density’ at any given cross section of the packed bed is not uniform. Medium flow’s quickly in the regions with low fiber density (high bed permeability) and much slower in the regions of high fiber density (low er bed permeability). The resulting non-uniform media perfusion across the packed bed creates the channeling effect, which manifests itself as significant nutrient and metabolite gradients that negatively impact overall cell culture and bioreactor performance. Cells located in the regions of low7media perfusion will starve and very’ often die from the lack of nutrients or metabolite poisoning. Cell harvesting is yet another problem encountered w’hen bioreactors packed with non-woven fibrous scaffolds are used. Due to packed-bed functions as depth filter, cells that are released at tire end of cell culture process are entrapped inside the packed bed, and cell recovery’ is very low’. This significantly limits utilization of such bioreactors in bioprocesses where live cells are the products. Thus, the non-uniformity leads to areas with different exposure to flow’ and shear, effectively reducing the usable cell culture area, causing non- uniform culture, and interfering with transfection efficiency and cell release.Attorney Docket No.: SP24-268

[0069] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide bioreactor systems, fixed bed cell substrates, and methods of using such bioreactor systems and substrates that enable efficient and high-yield cell culturing for anchorage-dependent cells and production of cell products (e.g,, proteins, antibodies, viral particles). Embodiments include a porous cell-culture matrix made from an ordered and regular array of porous substrate material that enables uniform cell seeding and media / nutrient perfusion, as well as efficient cell harvesting. Embodiments also enable scalable cell-culture solutions with substrates and bioreactors capable of seeding and growing cells and / or harvesting cell products from a process development scale to a full production size scale, without sacrificing the uniform performance of the embodiments. For example, in some embodiments, a bioreactor can be easily scaled from process development scale to product scale with comparable viral genome per unit surface area of substrate (VG / cm2) across the production scale. The harvestability and scalability of the embodiments herein enable their use in efficient seed trains for growing cell populations at multiple scales on the same cell substrate. In addition, the embodiments herein provide a cell culture matrix having a high surface area that, in combination with the other features described, enables a high yield cell culture solution. In some embodiments, for example, the cell culture substrate and / or bioreactors discussed herein can produce 1016to 1018viral genomes (VG) per batch.

[0070] In addition, the embodiments disclosed herein enable not only cell attachment and growth to a cell culture substrate, but also the viable harvest of cultured cells. The inability to harvest viable cells is a significant drawback in current platforms, and it leads to difficulty in building and sustaining a sufficient number of cells for production capacity. According to an aspect of embodiments of this disclosure, it is possible to harvest viable cells from the cell culture substrate, including between 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the cells that are harvested, at least 80% are viable, at least 85% are viable, at least 90% are viable, at least 91 % are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95 % are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable. Cells may be released from the cell culture substrate using, for example, trypsin, TrypLE or Accutase.

[0071] According to one or more embodiments, a cell culture bioreactor can include a cell substrate within the bioreactor vessel. The substrate can be deployed in a packed bed or fixed bed bioreactor configuration, or in other configurations within a three-dimensional cultureAttorney Docket No.: SP24-268 chamber of the bioreactor vessel. Due to contamination concerns, the vessel can be a single¬ use vessel that can be disposed of after use.

[0072] A cell culture bioreactor system is provided, according to one or more embodiments, in which the cell culture substrate is used within an interior reservoir or culture chamber of a bioreactor vessel. Figure 1 shows an example of a cell culture bioreactor 100 that includes a bioreactor vessel 102 having an interior re servo ir 104 in the interior of the bioreactor vessel 102. Within the interior reservoir 104 is a fixed bed cell culture substrate 106 that is made from a stack of cell substrate layers 108. The substrate layers 108 are stacked with the first or second major side of a substrate layer facing a first or second major side of an adjacent substrate layer. The bioreactor vessel 100 has an inlet 110 at one end for the input of media, cells, and / or nutrients into the interior reservoir 104, and an outlet 112 at the opposite end for removing media, cells, or cell products from the interior reservoir 104. By allowing stacking of substrate layers in this way, the system can be easily scaled up without negative impacts on cell attachment and proliferation, due to the defined structure and efficient fluid flow through the cell substrate material. While the vessel 100 may generally be described as having an inlet 110 and an outlet 112, some embodiments may use one or both of the inlet 110 and outlet 112 for flowing media, cells, or other contents both into and out of the interior reservoir 104. For example, inlet 110 may be used for flowing media or cells into the culture chamber 104 during cell seeding, perfusion, or culturing phases, but may also be used for removing one or more of media, cells, or cell products through the inlet 110 in a harvesting phase. Thus, the terms ‘-inlet” and “outlet” are not intended to restrict the function of those openings.

[0073] In Figure 1, the bulk flow direction is in a direction from the inlet 110 to the outlet 112, and, in this example, the first and second major sides of the substrate layers 108 are perpendicular to the bulk flow direction. However, embodiments include a fixed bed with a stack of substrates that have first and second sides that are parallel to a bulk flow direction, or a fixed bed having a cylindrical roll of one or more substrate layers. Thus, the substrate of embodiments of this disclosure can be employed in multiple configurations. The fixed bed can be sized and shaped to fill the interior reservoir defined by the culture chamber so that the interior reservoir is filled with cell growth surfaces to maximize efficiency in terms of cells per unit volume.Attorney Docket No.: SP24-268

[0074] Figure 2 shows an example of a cell culture bioreactor 100' similar to the one in Figure 1, except that the fixed bed 116 is arranged as a cylindrical rolled substrate, resulting in a number of layers of substrate 118 in the cylindrical roll. The longitudinal axis of the fixed bed 116 is substantially parallel to the longitudinal axis 115 of the bioreactor vessel. In the example shown in Figure 2, the cylindrical roll 116 is arranged in the bioreactor such that the layers of substrate 118 are parallel to a general bulk media flow direction, which is from the inlet 130 to the outlet 132. Optionally, the fixed bed 116 can be placed around a center guide rod 120, which may function to help hold the roll’s shape or to align the roll within the reactor. In addition, although not shown in Figure 1, a fixed bed of stacked layers of substrate can also use a guide rod that extends through some kind of opening in the layers of substrate. In the case of stacked substrate layers, the guide rod can be used to hold the layers in place and can even be keyed to the layers such that individual layers are held in a predetermined rotational alignment with other layers. In embodiments, it is contemplated that multiple guide rods can be used in a single fixed bed.

[0075] According to embodiments of this disclosure, the fixed bed cell substrate includes a structurally defined surface area for adherent cells to attach and proliferate that has good mechanical strength and forms a highly uniform multiplicity of interconnected fluidic networks when assembled in a packed bed or other bioreactor. In particular embodiments, a mechanically stable, non-degradable woven mesh can be used as the substrate to support adherent cell production. The cell culture matrix disclosed herein supports attachment and proliferation of anchorage dependent cells in a high volumetric density format. Uniform cell seeding of such a matrix is achievable, as well as efficient harvesting of cells or other products of the bioreactor. In addition, the embodiments of this disclosure support cell culturing to provide uniform cell distribution during the inoculation step and achieve a confluent monolayer or multilayer of adherent cells on the disclosed substrate, and can avoid formation of large and / or uncontrollable 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. Thus, the matrix eliminates diffusional limitations during operation of the bioreactor. In addition, the matrix enables easy and efficient cell harvest from the bioreactor. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor.Attorney Docket No.: SP24-268

[0076] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., non-woven substrates of randomly ordered fibers), embodiments of this disclosure include a cell culture substrate having a defined and ordered structure. The defined and order structure allows for consistent and predictable cell culture results. In addition, the substrate has an open porous structure that prevents cell entrapment and enables uniform flow through the packed bed. This construction enables improved cell seeding, nutrient delivery, cell growth, and cell harvesting. According to one or more particular embodiments, the matrix is formed with a substrate material having a thin, sheet-like construction having first and second sides separated by a relatively small thickness, such that the thickness of the sheet is small relative to the width and / or length of the first and second sides of the substrate. In addition, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometry that allows cells to adhere to the surface of the substrate material as if it w ere approximately a two-dimensional (2D) surface, while also allowing adequate fluid flow around the substrate material and through the openings. In some embodiments, the substrate is a polymer-based material, and can be formed as a molded polymer sheet; a polymer sheet with openings punched through the thickness; a number of filaments that are fused into a mesh-like layer; a 3D-printed substrate; or a plurality of filaments that are woven into a mesh layer. The physical structure of the matrix has a high surface-to-volume ratio for culturing anchorage dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor in certain ways discussed here for uniform cell seeding and growth, uniform media perfusion, and efficient cell harvest.

[0077] In one or more embodiments, a fiber may have a diameter in a range of about 10 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm; or about 150 pm to about 300 pm. On a microscale level, due to the scale of the fiber compared to the cells (e.g., the fiber diameters being larger than the cells), the surface of monofilament fiber is presented as an approximation of a 2D surface for adherent cells to attach and proliferate. Fibers can be woven into a mesh with openings ranging from about 100 pm x 100 pm to about 1000 pm x 1000 pm. In some embodiments, the opening may have a diameter of about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; or about 200 pm to about 300 pm. These ranges of the filament diameters and opening diameters are examples ofAttorney Docket No.: SP24-268 some embodiments, but are not intended to limit the possible feature sizes of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is chosen to provide efficient and uniform fluid flow through the substrate when, for example, the cell culture matrix comprises a number of adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0078] Factors such as the fiber diameter, opening diameter, and weave type / pattem will determine the surface area available for cell attachment and growth. In addition, when the cell culture matrix includes a stack, roll, or other arrangement of overlapping substrate, the packing density of the cell culture matrix will impact the surface area of the packed bed matrix. Packing density can vary with the packing thickness of the substrate material (e.g., the space needed for a layer of the substrate). For example, if a stack of cell culture matrix has a certain height, each layer of the stack can be said to have a packing thickness determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness will vary based on fiber diameter and weave, but can also vary based the alignment of adjacent layers in the stack. For instance, due to the three-dimensional nature of a woven layer, there is a certain amount of interlocking or overlapping that adjacent layers can accommodate based on their alignment with one another. In a first alignment, the adjacent layers can be tightly nestled together, but in a second alignment, the adjacent layers can have zero overlap, such as when the lower-most point of the upper layer is in direct contact with the upper-most point of the lower layer. It may be desirable for certain applications to provide a cell culture matrix with a lower density packing of layers (e.g., when higher permeability is a priority) or a higher density of packing (e.g., when maximizing substrate surface area is a priority). According to one or more embodiments, the packing thickness can be from about 20 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm.

[0079] The above structural factors can determine the surface area of a cell culture matrix, whether of a single layer of cell culture substrate or of a cell culture matrix having multiple layers of substrate). For example, in a particular embodiment, a single layer of woven mesh substrate having a circular shape and diameter of 6 cm can have an effective surface area of about 68 cm2. The ‘"effective surface area,” as used herein, is the total surface area of fibers inAttorney Docket No.: SP24-268 a portion of substrate material that is available for cell attachment and growth. Unless stated otherwise, references to “surface area” refer to this effective surface area.

[0080] The substrate mesh can be fabricated from monofilament or multifilament fibers of polymeric materials compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide. Mesh substrates may have a different patterns or weaves, including, for example knitted, warp-knitted, or woven (e.g., plain weave, twilled weave, dutch weave, five needle weave).

[0081] The surface chemistry of the mesh filaments may need to be modified to provide desired cell adhesion properties. Such modifications can be made through the chemical treatment of the polymer material of the mesh or by grafting cell adhesion molecules to the filament surface. Alternatively, meshes can be coated with thin layer of biocompatible hydrogels that demonstrate cell adherence properties, including, for example, collagen or Matrigel®. Alternatively, surfaces of filament fibers of the mesh can be rendered with cell adhesive properties through the treatment processes with various types of plasmas, process gases, and / or chemicals known in the industry. In one or more embodiments, however, the mesh is capable of providing an efficient cell growth surface without surface treatment.

[0082] In some embodiments, at least a portion of an internal or interior surfaces of a bioreactor or cell culture device may be more particularly adapted for cell growth. For example, a cell culture surface of the bioreactor or cell culture device may be treated w ith a coating to encourage or discourage cells to stick to the cell culture surface. The bioreactor or cell culture device may comprise a cell adherent coating on one or more interior surfaces. Any suitable cell adherent coating may be used, such as tire nonlimiting examples of CORNING CELLBIND® (Coming Incorporated, Coming, NY), CORNING PRIMARIA™ (Coming Incorporated, Coming, NY), and CORNING PURECOAT™ amine and carboxyl (Coming Incorporated, Coming, NY) surfaces. The bioreactor or cell culture vessel may comprise a cell non-adherent coating on one or more interior surfaces. Any suitable cell non¬ adherent coating may be used as the coating, such as a CORNING Ultra-Low Attachment (Coming Incorporated, Coming, NY) surface. Nonlimiting examples of ultra-low binding materials for coating include one or more of perfluorinated polymers, olefins, agarose, non¬ ionic hydrogels such as polyacrylamides, polyethers such as polyethyleneoxide, polyols such as polyvinylalcohol or mixtures thereof.Attorney Docket No.: SP24-268

[0083] By using a structurally defined culture matrix of sufficient rigidity, high-flow-resistance uniformity across the matrix or packed bed is achieved. According to various embodiments, the substrate fixed bed can be deployed in monolayer or multilayer formats. This flexibility eliminates diffusional limitations and provides uniform delivery of nutrients and oxygen to cells attached to the matrix. In addition, the open matrix lacks any cell entrapment regions in the packed bed configuration, allowing for complete cell harvest with high viability at the end of culturing. The matrix also delivers packaging uniformity for the fixed bed, and enables direct scalability from process development units to large-scale industrial bioprocessing unit. The ability to directly harvest cells from the packed bed eliminates the need of resuspending a matrix in a stirred or mechanically shaken vessel, which would add complexity and can inflict harmful shear stresses on the cells. Further, the high packing density of the cell culture matrix yields high bioprocess productivity in volumes manageable at the industrial scale. Examples of bioreactor contemplated in embodiments of this disclosure include ones having diameters ranging from a few centimeters, or 6 centimeters, to over 85 centimeters, and / or bioreactors containing fixed beds with total surface areas ranging from.25 m2to 1000 m2or greater. The “surface area” of the fixed bed refers to the total surface of the substrate that is available for adherent ceil attachment.

[0084] According to embodiments of this disclosure, a cell culture bioreactor is provided that includes one or more biomass sensors capable of in-situ biomass sensing during a live cell culture. The cell culture bioreactor includes a cell culture vessel having a vessel wall defining an interior space for housing a cell culture substrate. The vessel wall has an interiorfacing surface surrounding the interior space, as well as an exterior facing surface. The substrate is arranged as a fixed bed within the interior space of the cell culture vessel and is surrounded by the vessel wall. The substrate is designed for adhering cells thereto for cell culture. That is, cells can be seeded into the bioreactor vessel such that they attached or adhere to the substrate where, during the cell culture, the cell population may grow, spread, and / or otherwise be acted upon (e.g., transfected, harvested, etc.). The bioreactor also includes at least one biomass sensor within the cell culture vessel. The biomass sensor includes at least one conductor configured to generate an electric field passing through at least a portion of the fixed bed in which the biomass is desired to be monitored during the cell culture. The at least one biomass sensor is configured to measure at least one of capacitance and impedance.Attorney Docket No.: SP24-268

[0085] In aspects of embodiments, the at least one biomass sensor includes a plurality of biomass sensors. The plurality of biomass sensors can be arranged or designed to interrogate multiple regions (whether discrete or overlapping) of the fixed bed. For example, at least two biomass sensors of the plurality of biomass sensors can measure at least one of capacitance and impedance in at least two different regions of the fixed bed. As used herein, reference to “■different regions” being sensed or interrogated can refer to regions that overlap (i.e., regions that may share some portion of the fixed bed in common), but which are not identical; or it can refer to independent, non-overlapping regions of the fixed bed.

[0086] According to embodiments, the cell culture bioreactor can also include a guide rod disposed within the cell culture vessel. The guide rod may extend in a direction parallel to a longitudinal axis of the cell culture vessel. The guide rod may be described as having a height extending in that same direction. The guide rod extends through the fixed bed such that the guide rod is at least partially surrounded by the substrate over at least a portion of the height of the guide rod. For example, some portion of the substrate can be physically attached to the guide rod to hold the substrate in place. In some embodiments, the guide rod has a shape that is keyed to a cutout or hole formed in the substrate, with the shape designed to hold the substrate in a desired orientation. For example, with woven fiber substrates, the rotational alignment of various substrates in a layered fixed bed can be controlled to influence fluid flow through the bioreactor, increase or decrease how densely packed the substrate layers are, or both.

[0087] The guide rod or alignment rod can be positioned in a center of the cell culture vessel or the fixed bed. Alternatively, the guide rod can be placed asymmetrically within the fixed bed. For example, if the guide rod is place off-center of the vessel or fixed bed, the location of the matching cutout in the substrate material can be placed predetermined location of substrate layers of a stacked fixed bed so that, when the cutouts in the various layers are aligned to fit the guide rod, the orientations of the fibers in the substrate layers have a predetermined alignment. For example, if it desired for some substrate layers to be rotated 45 degrees with respect to some other layers in the stack, the cutout can be placed at an angle of 45 degrees with respect to the cutout in other layers. To align the cutouts, the layers must be rotated 45 degrees, thus ensuring the fibers are in the predetermined 45-degree orientation with respect to oilier layers.Attorney Docket No.: SP24-268

[0088] When using the guide rod to house an electrode, tire electrode can be placed within the guide rod, or on the guide rod itself. The guide rod electrode then forms one half of the circuit required for improved capacitance measurements. The electrode forming the other half of the circuit can be located at the edge of the reactor (either inside the outer wall or outside the outer wall) in order to measure biomass in a bulk of the reactor, in addition, multiple electrodes can be located both in the guide rod and outside the mesh area to enable spatial resolution within different regions of the bioreactor. The electrodes can be wired such that any two pairs can be used for interrogation enabling advanced positional resolution. As used herein, “interrogation” or “interrogate” refers to the act of sensing an electrical property in a portion of the bioreactor to detect or measure biomass.

[0089] According to embodiments, the cell culture vessel has an inlet and an outlet that are in fluid communication w ith the interior space of the cell culture vessel in which the substrate resides. The cell culture bioreactor is configured for fluid to flow through the cell culture vessel such that it flows into the interior space via the inlet, through the substrate fixed bed, and out via the outlet. This flow path (in through the inlet, through the fixed bed, and out through the outlet) can be in a direction substantially parallel to the longitudinal axis of the cell culture vessel.

[0090] In embodiments, the guide rod extends in a direction parallel to the longitudinal axis of the bioreactor. In addition, embodiments include bioreactors where fluid flow into the fixed bed from an inlet on one end of the bioreactor, then through the fixed bed, and out of the bioreactor through an outlet on the opposite side of the vessel from the inlet. In fixed bed reactors, there can be nonuniformities in cell seeding, growth, and overall concentrations along the direction of fluid flow. For example, it is possible that the fixed bed can act as a depth filter, with a higher concentration of cells near the inlet wfiere the fixed bed traps cells and has access to the freshest media and higher concentration of cell nutrients, but lower concentrations near the outlet, where some cells may never reach and the media is depleted by upstream cells. Therefore, being able to have interrogate the fixed bed at different points along the length of the fixed bed (i.e., in the media flow direction) can provide valuable insight into the uniformity of the cell culture,

[0091] According to embodiments, the at least one conductor can include a first interior electrode disposed within an outer perimeter of the fixed bed of the substrate. The outer perimeter of the fixed bed refers to the outermost extent of the fixed bed. In aspects ofAttorney Docket No.: SP24-268 embodiments, the first interior electrode is disposed inside a guide rod in tire fixed bed. In further aspects of embodiments, the guide rod is hollow. The at least one conductor can further include a second interior electrode disposed within an outer perimeter of the fixed bed of the substrate. The second interior electrode can be disposed mside the guide rod. While either of the first and second interior electrodes can be disposed inside the guide rod, embodiments are not limited to this arrangement, and one or more of the first and second interior electrodes can also be located elsewhere inside the perimeter of the fixed bed. In embodiments, the first interior electrode and the second interior electrode are arranged at different positions along the height of the guide rod. In aspects of embodiments, the first interior electrode and tire second interior electrode are configured to generate an electrical field from the first interior electrode to the second interior electrode that extends at least partially into the fixed bed.

[0092] In embodiments, the at least one conductor includes a first exterior electrode disposed on at least one of the interior-facing surface of the vessel wall, the exterior facing surface of the vessel wall, and within the vessel wall. The at least one conductor can also include a second exterior electrode disposed on at least one of the interior-facing surface of the vessel wall, the exterior facing surface of the vessel wall, and within the vessel wall. The second exterior electrode can be disposed on a different portion of the cell culture vessel than the first exterior electrode. For example, the first and second exterior electrodes can be positioned at different heights of the vessel wall, with the height of the vessel wall extending in a direction of the longitudinal axis of the bioreactor vessel. In aspects of embodiments, tire first exterior electrode and the second exterior electrode are configured to generate an electrical field from the first exterior electrode to the second exterior electrode that extends at least partially into the fixed bed. In embodiments, the at least one conductor includes a cylindrical capacitor plate electrode. The cylindrical capacitor plate electrode can be attached to the guide rod or the vessel wall.

[0093] In aspects of embodiments, the first interior electrode and the first exterior electrode are configured to generate an electrical field from the first interior electrode to the first exterior electrode that extends at least partially into the fixed bed. In aspects, the at least one conductor includes a plurality of electrodes to generate an electric field from any one individual electrode of tlie plurality of electrodes to any one of two or more of the other of the plurality of electrodes.Attorney Docket No.: SP24-268

[0094] In embodiments, the cell culture bioreactor includes a plurality of guide rods disposed within the cell culture vessel, and each of the plurality of guide rods extends in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod, Each of the plurality of guide rods extends through the fixed bed such that each of the plurality of guide rods is at least partially surrounded by the substrate over at least a portion of the height. Multiple guide rods of the plurality of guide rods include a conductor of the at least one conductor,

[0095] In additional aspects of embodiments, the at least one conductor can include one or more central electrodes running vertically through the fixed bed and parallel to the longitudinal axis of the cell culture vessel, and disposed within an interior of the fixed bed such that the one or more central electrodes are separated from the vessel wall by at least a portion of the substrate. The at least one conductor can further include a plurality of peripheral electrodes disposed on at least one of the interior-facing surface of the vessel wall, the exterior facing surface of the vessel wall, and within the vessel wall. The plurality of peripheral electrodes are spaced apart from each other in a direction parallel to the longitudinal axis of the cell culture vessel.

[0096] According to embodiments, the least one conductor includes at least one inductor configured to measure inductance in at least a portion of the fixed bed in which biomass is desired to be sensed. In aspects of embodiments, the at least one inductor includes a plurality of inductors to measure inductance in at least two different regions of the fixed bed.

[0097] The cell culture bioreactor can further include a guide rod within the cell culture vessel. The guide rod extends in a direction parallel to a longitudinal axis of the cell culture vessel — in a direction of a height of the guide rod. The guide rod is designed to extend through the fixed bed such that the guide rod is at least partially surrounded by the substrate over at least a portion of the height. The guide rod can function as a support for the fixed bed and, in some embodiments, can hold the substrate material of the fixed bed in specified orientations. For example, some portion of the substrate can be physically attached to the guide rod to hold the substrate in place. In some embodiments, the guide rod has a shape that is keyed to a cutout or hole formed in the substrate, with the shape designed to hold the substrate in a desired orientation. For example, with woven fiber substrates, the rotational alignment of various substrates in a layered fixed bed can be controlled to influence fluidAttorney Docket No.: SP24-268 flow through the bioreactor, increase or decrease how densely packed the substrate layers are, or both.

[0098] In embodiments, the at least one conductor comprises a first interior inductor disposed within an outer perimeter of the fixed bed of the substrate. In other words, the first interior inductor is positioned somewhere within the circumference, perimeter, or outer bounds of the fixed bed. By this arrangement, a field produced by the first interior inductor passes through the bed. In aspects, the first interior inductor is disposed inside the guide rod or around an exterior of the guide rod. To facilitate this, the guide rod can have a hollow interior. In embodiments, the at least one conductor also includes a second interior inductor disposed within an outer perimeter of the fixed bed of the substrate. The second interior inductor can also be disposed inside the guide rod, for example. In some embodiments, the first interior inductor and the second interior inductor are arranged at different positions along the height of the guide rod. As such, the first and second interior inductors can be used to interrogate the fixed bed at different positions or heights of the fixed bed.

[0099] In aspects of embodiments, a plurality of guide rods can be used within the cell culture vessel. In such cases, each of the plurality of guide rods can extend in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod, and each of the plurality of guide rods extends through the fixed bed such that each of the plurality of guide rods is at least partially surrounded by the substrate over at least a portion of the height. In some cases, multiple guide rods of the plurality of guide rods can include an inductor of the at least one inductor. In embodiments, the at least one inductor is positioned on at least one of the interior-facing surface of the vessel wall, the exterior facing surface of tire vessel wall, and within the vessel wall. The at least one inductor can include a coil inductor.

[0100] In embodiments, the at least one conductor includes a moveable conductor able to move relative to tire fixed bed while the moveable conductor is generating the electric field. The moveable conductor can be moved to different locations in order to interrogate different regions of the fixed bed. In aspects, the moveable conductor moves in a direction parallel to the longitudinal axis of the cell culture vessel. In examples of embodiments, the moveable conductor moves inside a hollow interior of the guide rod. The movement of the conductor can be automated or manually operated by a user of the bioreactor. The guide rode can have a hollow interior that opens to an exterior of the bioreactor where a user or machine can moveAttorney Docket No.: SP24-268 the conductor along the height of the guide rod. In addition, the at least one conductor can include one or more stationary conductors that are not designed to move relative to the fixed bed while the one or more stationary conductors are generating the electric field.

[0101] Aspects of embodiments include further include an analyzer operatively connected to the at least one conductor such that the analyzer can process signals from the at least one conductor. The analyzer can measure at least one of a capacitance, inductance, and reactance based on signals from a pair of electrodes of the at least one conductor. The analyzer can generate information about the state of the cell culture at a given moment in time, the change or rate of change in biomass in the fixed bed, and spatial information about cell growth in the fixed bed based on at least one of electrical properties indicated by the signals and physical position of the at least one conductor. The analyzer can interpret signals from the biomass sensor to understand the state of cells, cell by-products, analytes, nutrients, and more in the bioreactor. The electrical properties include at least one of capacitance, inductance, reactance, resonant frequencies, amplitude resonance across a range of frequencies, and an interaction of near and far field space, as a function of frequency, between electrodes.

[0102] In embodiments of the cell culture bioreactor, the cell culture vessel has a top wall and a bottom wall, the fixed bed is disposed between the top wall and the bottom wall, and the at least one conductor includes one or more annular electrodes disposed on at least one of the top wall and the bottom wall. The one or more annular electrodes are disposed on at least one of an exterior surface, an interior surface, or within the top wall or the bottom wall. The one or more annular electrodes, in embodiments, include a plurality of annular electrodes arranged concentrically with each other. As used herein, ‘"annular” means, unless stated otherwise, having a shape like an annulus, and can include a full a full annulus (e.g., the region between two full, concentric circles) or a partial annular (e.g., the region between a portion of two concentric circles, such as two semicircles or two arcs of circles). Thus, the one or more annular electrodes can include at least one of an annular electrode and an annular sector electrode. The plurality of annular electrodes is arranged about a longitudinal axis of the packed bed, so that the longitudinal axis is in approximately the center of the centric circles of the annular shape. In embodiments, the one or more annular electrodes include a plurality of annular sector electrodes. The plurality of annular sector electrodes can include one or more half-annulus electrodes. Aspects of embodiments include the at least one conductor further including a central electrode disposed concentrically inside the one or moreAttorney Docket No.: SP24-268 annular electrodes. In embodiments, the one or more annular electrodes include a plurality of annular electrodes arranged to have radial symmetry.

[0103] In embodiments of the cell culture bioreactor, the cell culture vessel includes a top wall and a bottom wall, the fixed bed being disposed between the top wall and the bottom wall, and the at least one conductor including a plurality of electrodes disposed on at least one of the top wall and the bottom wall. The plurality of electrodes are disposed on at least one of an exterior surface, an interior surface, or within the top wall or the bottom wall. The plurality of electrodes include a ground electrode disposed on one of the top wall and the bottom wall, and a remainder of the plurality electrodes disposed on the other of the top wall and the bottom wall. The remainder of the plurality of electrodes includes two or more electrodes,

[0104] In embodiments, the substrate is arranged as at least one of a cylindrical roll of substrate material and a plurality of substrate layers in a stacked configuration. In aspects of embodiments, the substrate is made of a plurality of woven fibers forming a plurality of openings in the substrate through which fluid and cells can flow.

[0105] In some embodiments, at least a portion of the plurality of woven fibers can include a metallic conductor, wherein the at least one conductor comprises the metallic conductor of the portion of the plurality of woven fibers. The metallic conductor can include a conductive coating on the portion of the plurality of woven fibers. The metallic conductor can include a metallic fiber incorporated into the plurality of woven fibers. In yet other aspects, the metallic conductor includes a thin sheet of conductive material on the portion of the plurality of woven fibers,

[0106] In embodiments where tire substrate includes the cylindrical roll of substrate material, the cylindrical roll of substrate material can include a sheet of substrate material that has a plurality of sections of the portion of the plurality of woven fibers including the metallic conductor. The plurality of sections are separated from each other by another portion of the plurality of woven fibers that do not include the metallic conductor. Each of the plurality of sections can extend over a height of the fixed bed and is separated from each other by the another portion of the plurality of woven fibers in a circumferential direction of the cylindrical roll. Each of the plurality of sections is separated from each other by the another portion of the plurality of woven fibers in a direction parallel to the longitudinal axis of the fixed bed. In an aspect of embodiments, each of the plurality of sections is wired forAttorney Docket No.: SP24-268 independent electrical control, meaning that the charge, current, or voltage of individual sections can be controlled independently of at least some of the other sections. In this way, individual electrode sections can be selectively employed by the biomass sensor to gather biomass information in different portions of the fixed bed. Each of the plurality of sections can include a partial, single, or multiple revolution about the cylindrical roll.

[0107] In embodiments, the substrate can include multiple cylindrical rolls of substrate material, where a first cylindrical roll of the multiple cylindrical rolls is nested concentrically within a second cylindrical roll of the multiple cylindrical rolls. The at least one conductor can include a first induction coil disposed between the first cylindrical roll and the second cylindrical roll. The at least one conductor can further include a second induction coil disposed between the first cylindrical roll and the second cylindrical roll, where the first cylindrical roll and the second cylindrical roll are disposed at different heights within the fixed bed.

[0108] In embodiments where the substrate includes die plurality of substrate layers in a stacked configuration, the metallic conductor can be integrated into at least a portion of the plurality of substrate layers.

[0109] In further aspects of embodiments, the at least one conductor comprises at least four electrodes that include at least two current-carrying electrodes and at least two voltage-sensing electrodes. The at least one biomass sensor is designed to pass current between the at least two current-carrying electrodes while voltage drop is measured between two of the at least two voltage-sensing electrodes. The at least two voltage-sensing electrodes are disposed between the at least two current-carrying electrodes. At least a portion of the substrate is disposed between two of the at least two voltage-sensing electrodes. The at least two current-carrying electrodes includes at least one working electrode and a counter electrode, and the at least two voltage-sensing electrodes includes at least two reference electrodes. Tire at least one working electrode can include a plurality of working electrodes, and the at least two reference electrodes can include more than two reference electrodes. The counter electrode and at least one reference electrode are disposed on a first side of at least a portion of the substrate, and at least one working electrode and at least one other reference electrode are disposed on a second side of the at least a portion of the substrate that is an opposite side of the portion of the substrate from the first side. The substrate includes at least one of a cylindrical roll of substrate material and a plurality of substrate layers in a stackedAttorney Docket No.: SP24-268 configuration. When the substrate includes a cylindrical roll of substrate material, the counter electrode can be disposed in the center of the cylindrical roll along a longitudinal axis of the cylindrical roll. In these embodiments, the cell culture bioreactor can include a guide rod disposed within the cell culture vessel, where the guide rod extends in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod. The guide rod extends through the fixed bed such that the guide rod is at least partially surrounded by the substrate over at least a portion of the height. The counter electrode can be disposed inside or on the guide rod. In embodiments, one or more layers of the cylindrical roll are disposed between any pair of reference electrodes of the at least two reference electrodes. In further aspects of embodiments, one or more layers of the cylindrical roll are disposed between the counter electrode and any one working electrode of the at least one working electrode. In embodiments where the substrate includes a plurality of substrate layers in a stacked configuration, one or more layers of the plurality of substrate layers are disposed between any pair of reference electrodes of the at least two reference electrodes. Further, one or more layers of the plurality of substrate layers can be disposed between the counter electrode and any one working electrode of the at least one working electrode.

[0110] As described herein, the disclosed biomass sensors can be employed in bioreactors having various fixed bed configurations, including rolled beds or stacked beds. The uniformity in structure and fluid flow performance has numerous advantages in cell culture performance. However, embodiments are not limited to these examples, and can include bioreactors with packed beds, fluidized beds, hollow fiber beds, suspension cultures, or microcarriers or beads in suspension. In addition, embodiments described herein are applicable to bioreactors scales or sizes, from product development scale to pilot scale to production or manufacturing scale.

[0111] Figures 3 A shows a schematic view of a fixed-bed bioreactor 200, according to embodiments of this disclosure, with a fixed-bed substrate 201 surrounding a central guide rod 202, and interior electrodes 204 positioned within the guide rod 202. Around a periphery of the fixed bed or the cell culture vessel, there are exterior electrodes 206. The exterior electrodes 206 around the wall of the vessel could be positioned on or next to an interior or exterior of the vessel wall, or otherwise embedded in the wall i tself. The relative orientation of electrodes (e.g., wires) could be concentric, as in Figure 3A. Alternatively, the electrodesAttorney Docket No.: SP24-268 can be arranged to create a 3D x-y-z matrix to further improve the spatial sensitivity of the biomass sensor system.

[0112] An additional Electro Magnetic Interference (EMI) shield can be positioned around the bioreactor vessel to reduce other environmental (e.g., room or system related electronics) interference with the biomass sensor detection. Such shielding could be tailored in shape and or electrophysical properties to focus or otherwise reinforce the signals used for biomass detection. Additionally, the system can be driven using specific frequencies, and utilize frequency filters, to avoid common environmental noises, such as those occurring in the 50- 60Hz range.

[0113] While Figure 3A shows three interior electrodes 204 and three exterior electrode 206, this is only an example of embodiments contemplated by this disclosure. While the exterior electrodes in Figure 3 A are depicted as each surrounding a circumference of the fixed bed, the exterior electrodes 206 can be divided into any number of electrodes along the circumference direction, allowing independent interrogation of sections of the fixed bed for better spatial information. Figure 3B shows an alternative arrangement with only one interior electrode 214, and five exterior electrodes 216. A switch 218 is used to choose the exterior electrode 216 to pair with the interior electrode 214 at any given time.

[0114] Embodiments of this disclosure include cell culture bioreactors with biomass sensors wired to switch certain electrodes on or off in the signal path, or otherwise process signals from individual electrodes in a biomass sensing operation.

[0115] Figure 4 is a schematic illustration of a cell culture bioreactor 220 using alternating pairs of electrodes 224a-224f in a guide rod 222, according to embodiments of this disclosure. This example configuration shows a center guide rod 222 with alternating pairs of electrodes w ith the field 225 partially projecting into the area of interest in the fixed bed 221. For clarity and simplicity, the field 225 is only depicted on one side of the guide rod 222. This method does not require external electrode pairs, like exterior electrodes 206 in Figure 3A, which might sacrifice the ability to interrogate the peripheral regions of the fixed bed, in some cases. Figure 5 shows a configuration of a cell culture bioreactor 230 in which multiple guide rods 232a, 232b are included; each having one or more individual electrodes 234. With multiple guide rods 232a, 232b, the distance and sensing area can be individually optimized. Further, each guide rod can include discrete electrodes 234 to enable spatial sensitivity.Attorney Docket No.: SP24-268 Reactors with more than two guide rods containing electrodes are also contemplated as embodiments of this disclosure.

[0116] Figures 6A and 6B depict embodiments of cell culture reactors 300, 310 using multiple induction coils within one or more guide rods. The embedded coils are used to sense impedance in the field external to the coil. In Figure 6A, two guide rods 302, 303 house two induction coils 304, 305 (one in each guide rod). The fields 307 produced by the induction coils 304, 305 reach into the surrounding fixed bed. The regions interrogated by the induction coils 304, 305 may overlap in part, as shown, or not. In Figure 6B, multiple induction coils 314, 315 are placed inside a single guide rod. Embodiments include single or multiple induction coils per guide rod whether using a single or multiple guide rod. Additionally, when using multiple induction coils, a guide rod can include more than two induction coils.

[0117] By including an array of electrodes, embodiments include using advanced algorithms to effectively map out the capacitance and subsequent cell density in a bioreactor in high resolution. Additionally, the electrodes in the array can be organized such that loss of one pair (or multiple pairs, or an individual electrode) does not significantly impede the measurement mapping (i.e., the design can have built-in redundancy). Alternatively, the electrodes can be arranged such that if one electrode in a pair loses connectivity, the remaining electrode can potentially be paired with another active electrode to continue monitoring the region of interest For example, Figure 7 shows a diagram where four electrodes operate in two pairs: electrodes 402 and 404 in one pair, and electrodes 406 and 408 in another pair. During normal, uninterrupted operation, electrodes 402 and 404 are paired (symbolized by connection 410), and electrodes 406 and 408 are paired (symbolized by connection 412). However, if any one electrode is inoperable, it can be paired with another, as shown by the alternative connections 414. Correction factors can be added to account for the change in distance between electrodes, as needed.

[0118] As discussed herein, embodiments of this disclosure include bioreactors housing a cylindrical rolled substrate. In such embodiments, electrodes and / or inductor coils can be embedded in different orientations. Counter electrodes can still be placed in a guide rod or an external wall. Interior electrodes can be rolled into the mesh during manufacturing process. Various electrode lengths can enable electrodes to span a partial circumference, full circumference, or even have multiple revolutions. The equations relating capacitance, electrode sizes and permitivity for a cylindrical capacitor are well known. Specifically, theAttorney Docket No.: SP24-268 capacitance for cylindrical or spherical conductors can be obtained by evaluating the voltage difference between the conductors for a given charge on each. By applying Gauss’s law to an infinite cylinder in a vacuum, the electric field outside a charged cylinder is found to be:E — — - — Equation (1)The voltage between the cylinders can be found by integrating the electric field along a radial line:&. V = fh~dr = — In- Equation (2)r 2ne0aFrom the definition of capacitance and including the case where the volume is filled by a dielectric of dielectric constant k, the capacitance per unit length is defined as:— = — = Equation (3)a

[0119] In this case, the electrodes can be designed to maximize sensitivity in the region of interest. The electrode length and gaps between the electrodes can be balanced to achie ve the desired signal-to-noise (SNR) or sensitivity. Variations in the permitivity can be measured as cell growth accumulates for an indication of biomass. The above equations related to a “static” or “DC” magnitude of the capacitance for the environment. While a large area would increase the capacitance, the spacing between the electrodes would decrease the capacitance. Measuring impedance across a range of frequencies can reveal resonances within the overall system that can be much more sensitive than a DC capacitance measurement. A multitude of wavelength and electrode related attributes all taken in consideration of the physical relationship of the electrodes, would provide information about the reactor volume to map cell growth. Shifts in the resonant frequencies, amplitude response across a range of frequencies, and the interaction near / far field space (also a function of frequency) between electrodes.

[0120] Figure 8A shows an unrolled mesh substrate with metallized portions to act as electrodes, according to embodiments. As discussed herein, embodiments of this disclosure include cell culture substrate made of fibers (e.g., interwoven fibers). These fibers can be used as integrated electrodes, rather than using separate electrodes, for the biomass sensor. For examples, the fibers can be coating with a conductive material capable of acting as an electrode in embodiments discussed herein. In some examples, the fibers can be formed of, or can be intertwined w ith, a metallic conductor. In other examples, these sections of the substrate can have a metallic electrode (e.g., a metal wire or sheet) attached to the surface ofAttorney Docket No.: SP24-268 the substrate. These metallized substrate sections are then electrically connected to a remainder of the biomass sensor so that they are capable of acting as the one or more conductor of the biomass sensor. In embodiments, as shown in Figure 8A, the substrate 450 includes regular substrate sections 452 that do not incorporate a metallic conductor, and metallized sections 454. These substrate sections 452 and metallized sections 454 can be arranged in alternating fashion, as shown in an unrolled substate 450 in Figure 8A. There can be any number of substrate sections 452 and metallized sections 454 depending on the dimensions of the reactor and number of electrodes needed for the application. The width w of the metallized sections can be the same for each section, or can vary. For instance, if it is desired for the metallized section to make one-quarter, one-half, or one or more revolutions around a rolled cylindrical fixed bed, the width w of the metallized sections near the center longitudinal axis of the fixed bed may be shorter than required in sections near the periphery of the cylindrical roll.

[0121] Figure 8B shows a plan view of a mesh substrate like the one in Figure 8A in a rolled cylindrical configuration 460. As shown, the metallized sections 464 alternate with regular substrate sections 462, and a width of the metallized sections 464 increases as they get farther from the center of the roll so that each metallized section 464 can make approximately one revolution. It will also be appreciated from the view in Figure 8B how portions of the regular substrate sections 462 are disposed between metallized sections 464. As such, the metallized sections 464 on opposite sides of a regular substrate section 462 can be used as a pair of electrodes generating a field for biomass sensing of that substrate section 462.

[0122] In embodiments, rather than the fibers of the substrates being made conductive, solid electrode sheets are interspersed and rolled into the rolled substrate between layers of the substrate. Various combinations of pairs of electrodes can be interrogated to achieve spatial sensitivity of the capacitance. The electrode can be composed of either a metal mesh or a thin solid material. Because of the orientation of the electrode is parallel to the flow (with the flow direction being into or out of the page in Figure 8B), distribution of oxygen and nutrients would not be greatly if at all affected by the presence of these electrodes, if using solid electrodes.

[0123] As an alternative to the segmentation approach shown in Figures 8A and 8B, Figures 9 A and 9B show an embodiment that uses a rolled substrate 470 containing a singleAttorney Docket No.: SP24-268 continuous electrode 472 in combination with a segmented guide counter electrode 474 in the guide rod 476 to enable interrogation of specific locations. If capacitance measurements near the guide rod location provide enough information to quantify cell growth, this implementation could be simple and economical. Segmentation of the electrodes 474 embedded in the guide rod allow s for data collection for separate height levels within the fixed bed. Further segmentation of any of the electrodes in the vertical or axial direction (or radial, or combinations thereof) with discrete electrode strips that are shorted the length of the reactor are also contemplated. For example, Figure 10 show s an embodiment of a bioreactor 480 where the rolled fixed bed (shown in Figure 10 as partially unrolled, for clarity) contains metallized sections 484 of mesh substrate separated from each other by regular substrate sections 482 in the vertical direction (i.e., spaced in a direction parallel to the longitudinal axis of the cylindrical roll). This creates essentially parallel plate capacitors when rolled up and installed in the bioreactor vessel. Capacitance can be evaluated with a single guide rod electrode as a counter electrode or between each of these discrete metallized sections 484 to achieve spatial sensitivity. The guide rod 486 can contain electrodes with exposed contacts 488 to contact the metallized sections 484. The capacitance between electrodes in Figure 10 could be evaluated by using transformer coupling from outside tire vessel wall, in some embodiments. The rolled electrodes 484 can be partial, single or multiple revolutions.

[0124] Figure 11A show's an embodiment w here the at least one conductor includes an induction coil 500 positioned between two concentric rolled substrate fixed beds 502, 504. Hie inductor-style electrode is thus embedded within the rolled fixed bed of the bioreactor. Uris also has the advantage of being easy to assembly. As shown in Figure 1 IB, embodiments include discrete coil inductor sensors. Multiple coils 510, 512 can be wound along the same core 14 to achieve vertical sensitivity. In embodiments, multiple inductor coils 520, 522 can be placed concentrically, with substrate 524 disposed between the coils, as well as inside the innermost coil 520 and outside the outermost coil 522, to enable radial sensitivity, as shown in Figure 11C. These configurations enable detection of spatial variability within the fixed bed.

[0125] In embodiments, the conductors (e.g., electrodes or inductors) can be coupled to components (e.g., wiring, leads, controller, processor, etc.) outside of the bioreactor vessel via inductive coupling, capacitive coupling, or both. This can eliminate the need to penetrate the vessel w'alls or seals with conductors or wiring.Attorney Docket No.: SP24-268

[0126] The system can use impedance spectroscopy (i.e., impedance as a function of frequency) to provide additional spatial data as the near / far field sensitivity will vary as a function of frequency, particularly in the complex chemical environment within the bioreactor,

[0127] Checking and verification of the biomass measurement system can continue through the bioreactor process with intentional variation in flow7chemistry7or vessel contents (including bubbles of appropriate gasses). In-process referencing can be a valuable part of ensuring the integrity of an otherwise slow7ly vary ing process during the cell culture.

[0128] Given the relatively slow7growth, there is not necessarily the need for fast (e.g., millisecond scale) measurement response. Response time in seconds or minutes would suffice in most applications, with a precision overall volumetric response that might be in the tens of minutes. This will permit the use of faster detection schemes with substantial averaging to significantly reduce measurement noise by an order of magnitude or more. It could also permit multiplexing of precision circuitry among multiple probing connections or locations.

[0129] In embodiments, the surfaces of the bioreactor vessel can be used as the sensing interfaces of the biomass sensor, including the side walls of the reactor, as discussed above. Figures 12A-12C show7embodiments where the top and bottom of reactor are used as the sensing interfaces, as opposed to the side walls of the reactor. As shown in Figure 12A, the conductors 530 are arranged are a series of concentric annular and / or circular electrodes on the top and bottom of the reactor with the fixed bed 532 betw een tire top side 534 and the bottom side 536 of the reactor. The central electrode can be a circle, as shown, or an annulus like the other electrodes. The electrodes 530 are effectively plates with some finite area. While the electrodes are shown touching each other, ideally they w'ould be electrically isolated relative to one another. They are wired to a complex impedance machine where the reactance of pairs of the electrodes 530 can be measured. The reactance is a function of the cell density and therefore can be used to calculate the volumetric density of cells. Because the radial symmetry in the embodiment of Figure 12A, the calculations of the complex reactance can be greatly reduced. This makes it much easier to calculate the volumetric density. One of the drawbacks of the radially symmetric design above is that when the radius gets larger and larger, the amount of volume being sampled becomes larger and larger. And, if there are inlets and outlets, this spatial information may be impaired or lost. To combat that, the radial pieces could be divided, in some embodiments, as show a in Figure 12B.Attorney Docket No.: SP24-268

[0130] Figure 12B shows a variation in which the annular electrodes are partial- or semiannuluses 540. This means that as the radius gets larger and larger, the number of electrodes could be more than 1 per anulus. For example, the circular electrode could be divided into 3, 5, 10 or more multiple electrodes. This would provide more spatial information. However, using this configuration at some level the cellular density as a function of angle around would be limited.

[0131] As shown in Figure 12C, due to the independent wiring of each electrode 530, 540, any one electrode on a top or bottom of the reactor can be paired with anyone other electrode on the opposite end of the reactor to create a sensing field 542 therebetw een.

[0132] One potential drawback of the radially symmetric design in Figures 12A-12C is tire possible loss of information about specific angles. To combat this, smaller electrodes confined to a more localized area could be placed on the top side, bottom side, or both top and bottom sides of the bioreactor. An example of this is show n in Figure 13, w here the top side 550 of the bioreactor has multiple electrodes 52. The bottom side 551 of the bioreactor is simply a ground plane 554, with the fixed bed 556 disposed between the top side 550 and the bottom side 551. The complex reactance of each electrode can be measured to sense the field below7that electrode, represented by the nominal sampling volume 558 in Figure 13. This provides high spatial information. The size, spacing, density, and / or pattern / ) ay out of the electrodes shown in Figure 13 are an example only, and these could be further defined and customized by the application.

[0133] Some of the above-discussed embodiments involve different and multiple electrode configurations, wherein, essentially, the electrical property (e.g., capacitance) is measured with tw7o active electrodes. Due to the complicated kinetics of the charge transfer reaction at the surface of the electrodes it is not possible to accurately measure the electrical properties when the change in capacitance / resistance / impedance are of low magnitudes. As a result, the two electrode characterization methods may be unable to measure those changes in some cases. This problem can be further aggravated when the biomass and the cell culture matrix / solution within the reactor forms electrical double layer on the electrodes. To address these challenges, embodiments of this disclosure include a four-electrode sensing design and method based on an electrical impedance measuring technique that uses separate pairs of current-carrying and voltage-sensing electrodes to make more accurate measurements than the simpler and more usual two-electrode sensing. The four-electrode design avoids effects ofAttorney Docket No.: SP24-268 electrode polarization. Separation of current and voltage electrodes eliminates the lead and contact resistance from the measurement. This is an advantage for precise measurement of low impedance values.

[0134] The substrate in the reactor can be considered as a membrane / barrier. As the cell biomass grows on the mesh it changes the electrical properties (e.g., capacitance) of the mesh. In the four-electrode mode, the potentials for any electrochemical reactions that are occurring at the working (and counter) electrode(s) are not being measured. Instead, what is measured is the effect of an applied current on the solution itself or some barrier in that solution.

[0135] Figure 14 shows the basic electrode layout for the four-electrode arrangement 600, according to embodiments. Tire four-electrodes include a working electrode 602, a counter electrode 604, and two reference electrodes 606a, 606b. Between the two reference electrodes 606a, 606b is a membrane or barrier 608 (e.g., cell substrate and biomass). Current is passed between the working electrode 602 and the counter electrode 604 while voltage drop is measured between the reference electrodes 606a, 606b. The working electrode 602 and counter electrode 604 can be of similar construction or different constructions.

[0136] Figure 15A is a cross-section view in elevation of a rolled fixed bed 620 using the four-electrode based design with multiple working electrodes and additional reference electrodes, according to embodiments of this disclosure. The counter electrode 622 is positioned in the center of the rolled fixed bed 620 with a first reference electrode 626a. One the other side of one or more layers of substrate are a first working electrode 62.4a and second reference electrode 626b. This pattern repeats with a second working electrode 624b and third reference electrode 626c, and a third working electrode 624c and fourth reference electrode 626d. Figure 15B is apian view of the rolled fixed bed of Figure 15 A, according to embodiments. Although Figures 15A and 15B show seemingly a single layer of substrate between pairs of reference electrodes, embodiments include examples with multiple layers of substrate separating the reference electrodes. To measure biomass, current is passed through counter electrode 622 and, for example, the working electrode 624a. The voltage drop is then measured between the first reference electrode 626a and the second reference electrode 626b. This would be used to calculate the capacitance. Of course, the current can also be passed through one of the other w orking electrodes 624b or 624c, and the voltage drop measured between any t7o of the reference electrodes 626a-626d.Attorney Docket No.: SP24-268

[0137] In addition, while current can be passed between the counter electrode 622 and one of the working electrodes 624a-c, in some embodiments, c urrent can be passed between tw o working electrodes (e.g., 624a and 624b), with voltage drop between reference electrodes (e.g., 626d and 626c) being measured. If the total growth in a number of rolls needs to be measured, then the two outer most current carrying electrodes as well as the voltage measuring electrodes may be selected accordingly.

[0138] This configuration can create an annular space between two parts of the roll, as shown in Figure 15B; however, Figure 15B is not necessarily drawn to scale and the electrode can be thin enough to be slipped between closely spaced layers of substrate. In embodiments, the fixed bed can be continuous rolled fixed bed (as in Figure 15B), or the fixed bed can be made from a series of concentric annular beds, each annular bed separated from another by an annular space housing working and references electrodes. Also, while only one half of rolled fixed bed 620 is shown having an electrode array, there can be additional electrodes on the opposite side. Additionally, the electrodes can be moved around the rolled fixed bed to map the properties at a particular location. In some embodiments, each electrode can be formed as a continuous part to fully occupy the annular space, or at least make a partial revolution within or around the rolled fixed bed. The working electrode 622 can be placed inside a central guide rod, as discussed in embodiments herein,

[0139] The four-electrode sensing method can also be employed in a stacked substrate fixed bed, as shown in Figures 16A and 16B. In Figure 16A, a portion of a stacked bed is shown with a four-electrode array interleaved in the layers. In this example, three layers of substrate 640 are showm disposed between two reference electrodes 642a, 642b, all of which is disposed between a working electrode 644 and a counter electrode 646. Figure 16B is similar, but with the layers of substrate 650 being separated by an additional reference electrode 652c, as well as being disposed between reference electrodes 652a and 652b, all of which are disposed between working electrode 654 and counter electrode 656. Having additional reference electrodes, as in Figure 16B, can help achieve more localized assessment of electrical properties in the fixed bed that relate to biomass growth. Reference electrodes can be placed, for example, after every’ consecutive layer of substrate, or after a predetermined number of consecutive layers, according to the system needs.

[0140] The space between the electrode interface and the substate can be modeled using an equivalent electrochemical circuit. The fluid matrix of the biomass culture media exists as aAttorney Docket No.: SP24-268 solution resistance. The immediate interface of the electrode has the charge transfer resistance and the electrical double layer capacitance. By conducting an impedance spectroscopy scan, the resulting impedance versus frequency spectrum can be used to fit a model constructed from the description above. This will enable the discrimination of the capacitance solely due to the growth of the biomass layer. A generalized version of this model is shown in Figure 17, w here RE — 1 and RE-2 represent any pair of electrodes, =oi is the solution resistance, Rsystem is resistance in the biomass as well as in the electrode interface, and Cbiomass is the capacitance of interest. This can be used to determine the capacitance change specific to biomass.

[0141] Embodiments of this disclosure also include bioreactors that include interdigitated electrodes for sensing cell proliferation in-situ in a fixed-bed bioreactor, and related methods for sensing cell proliferation using interdigitated electrodes in a fixed-bed bioreactor. In aspects of these embodiments, bioreactors and methods allow monitoring cell growth in different locations inside the fixed bed. These embodiments are scalable across bioreactors of various sizes and scales, from so-called process development scale to production scale bioreactors.

[0142] As discussed herein, some solutions for monitoring cell cultures in bioreactors rely on inline sensors (e.g., glucose and / or lactate sensors) whose sensing is based on enzymatic reactions and capacitance-based sensors that sense capacitive build up across live cell membranes in an electric field. Some previous impedance-based biomass sensor systems rely on measuring cells attachment on the electrode itself that results in a measurable change in impedance. Based on the mechanism of detection, by placing the cells w ithin an electric field, viable cells with an intact cell membrane will get polarized, becoming a tiny capacitor. As the volume of membrane-bound ions increases within the electric field (corresponding to the biomass), so does the capacitance measurement. The type of sensor has been applied widely used for non-adherent cell culture system and for adherent cell bioreactors. How ever, such sensors can be bulky and in a form that is not conveniently inserted inside fixed-bed bioreactors.

[0143] In contrast to the monitoring techniques in the preceding paragraph, according to embodiments described herein, in-situ impedance-based sensors can monitorAttorney Docket No.: SP24-268 microenvironment metabolite change due to cell proliferation in the area adjacent to the sensor's electrode. This impedance-based sensor can sense a decrease of impedance instead of an increase in impedance. Additionally, embodiments include methods of increasing signal -to-noise ratio or signal intensity. Aspects of embodiments include electrodes with high surface area and methods of fabricating such electrodes.

[0144] In embodiments, compact interdigitated electrodes (IDEs) that are thin with microscale electrodes can easily be placed in tight spaces and in multiple locations within a fixed-bed bioreactor without majorly disrupting the cell culture process in the fixed bed. According to embodiments, as an electric field is applied to the electrodes, the microenvironment changes near the electrodes can be measured using, for example an impedance analyzer, which correlates to the total metabolites produced by the cells during cell growth period. A change in impedance can then be used to extract capacitance changes on the nearby cells using the electrochemical model of this disclosure.

[0145] Embodiments of this disclosure include the application of interdigitated electrodes (IDEs) inside fixed-bed bioreactors (FBRs). The fixed-bed bioreactors, in embodiments, can be a bioreactor containing a fixed bed containing a substrate as described in this disclosure. For example, the substrate can be a PET material. The substrate can have a uniform structure of fibers and openings formed through the substrate material. Tire fixed bed can be formed by rolling one or more sheets of substrate material into a cylindrical rolled fixed bed, or by stacking multiple layers of the substrate material to form a stacked fixed bed. Thus, the fixed bed can be considered a multi-layered bed in either the rolled or stacked configuration. Hie sensor can be placed inside the bioreactor in multiple locations with an electrical connection to the impedance analyzer. Unlike the typical impedance-based cells biomass sensor, embodiments of this disclosure can rely on the mechanism of local environmental changes close to the exposed electrodes, which are sandwiched tightly inside layers of the bed. Instead of relying on the mechanism of cells covering the electrode or the immobilization of receptors on the electrodes which leads to increase in impedance, embodiments of this disclosure use the bare two electrodes to sense the metabolite change in the local environment close to the area where cell growth is occurring. Tire change in the electrical field across the electrodes are caused by the metabolite’s composition change during cell growth. By measuring the change in electrical field, which is translated to the decrease in the localAttorney Docket No.: SP24-268 impedance, an electrochemical model of this disclosure can be used to extract the value of cells capacitance in the vicinity.

[0146] Embodiments of the IDE sensor implementation and methods have many advantages. The use of thin and compact IDEs in layers of a rolled or stacked fixed-bed substrate allows for minimal disruption in perfusion flow through the fixed bed. In addition, the IDEs and methods of this disclosure can provide in-situ measurement of cell proliferation inside the fixed-bed bioreactor without the need to have access opening into the internal environment of the bioreactor, which should remain sterile. Embodiments also allow microenvironment sensing at different locations inside the bioreactor, potentially providing uniformity information across the bioreactor or fixed bed. Also, an array of IDEs can be created along a desired direction or preferential location to create a spatial map of cell growth inside the fixed bed. The ability to sense an increase and a decrease in impedance due to overall metabolite composition allow s detailed monitoring of the health of the cells inside the bioreactor. In addition, tire electrochemical models proposed herein can be used to extract information that is specific to the cells inside the bioreactor.

[0147] Instead of relying on cells covering the electrode, as in some prior art biomass sensors, or the immobilization of receptors on the electrodes leading to an increase in impedance, embodiments of this disclosure use two bare electrodes to sense local environmental changes close to the exposed electrodes which is sandwiched tightly inside the bed. The basic mechanism is to sense the metabolite change in the local environment close to the area where cells growth occurring, therefore the change in the electrical field across the electrodes are caused by the metabolite's composition change during cells growth. While measuring the change in electrical field, which is translated to the decrease in the local impedance, an electrochemical model is used to extract the value of cells capacitance in the vicinity of the electrodes.

[0148] Figure 18 shows an example of interdigitated electrodes (IDE or IDEs) 700 according to embodiments of this disclosure. The IDEs 700 include alternating positive and negative electrodes. In embodiments, as shown in Figure 18, the positive electrode 702 and the negative electrode 704 each include a plurality of electrode branches 703 and 705, respectively, or “fingers.” The term “interdigitated” refers to the act of two or more tilings interlocking, like fingers of tw o clasped hands, an image evoked by the design in Figure 18. In embodiments, interdigitated electrodes of this disclosure can include a pair of electrodeAttorney Docket No.: SP24-268 structures each having a plurality of electrode branches that are interdigitated with the plurality of electrode branches of the other electrode structure. In embodiments, each electrode structure is individually addressable, and may be connected to its own connection pad. According to embodiments, the length and width of the electrodes are maintained uniform throughout the geometry. The electrodes are placed equidistant with a spacing of d. Once a positive and negative potential,and Vnis applied to the electrodes 702, 703, respectively, an electric field 708 is generated by the positive and negative electrodes,as shown in Figure 19. The electric field 708 formed between the positive electrode 702 and negative electrode 703 decays as the distance from the electrodes 702, 703 increases. Thus, there is an inherent length scale involved based on the geometrical parameters of the interdigitated electrodes. The operating principle of a biosensor is based on the fact that the presence of a cell culture interferes with the electric field, w hich changes the impedance of the sensor, and that change in impedance is then used to measure the activity of cell culture.

[0149] Since the electric field is one that determines the sensing capability of the IDE 700, the effect of the spacing and geometry of the electrodes on the electric field is examined herein. Based on these effects, embodiments of this disclosure can tune the geometry of the electrodes as needed. The Maxwell equations of electrostatics can be stated as:E = -V<»(4)V D = ^(5)wA=»a< (6)where E is the electric field, is the potential, Dis the electric field density, is the free charge density, J is the conduction current. In absence of free charge, equations (4) and (5) can be combined asV = 0 (7) For applied,and F, the above equation can be solved by using numerical techniques, such as FEM to obtain the space varying potential. Once the potential solution is obtained, the electric field can be obtained using equation (4) by performing the operations numerically.For numerical simulation of electric field generated in an IDE, the following set of assumptions are considered: (1 ) the thickness of the electrodes has no effect on the electricAttorney Docket No.: SP24-268 field formed; and (2) the electric field between a positive and negative electrode is representative of the whole IDE. These assumptions allow to simplify the domain for analysis to a 2D computational field as shown in Figure 20 from the actual 3D domain. The total width of the computational domain is IF = 2We. + Wp. Where, FE is the half width of the electrodes, We, ~ We / 2. L is the height considered above the surface of the electrode. For FE domain for analysis is created in CUBIT meshing software using QUAD elements. MOOSE, tensor mechanics module is used to achieve the FE solution. A constant potential of Vp- 10 V and F = -10 V is assumed to be applied on the positive and the negative electrodes, respectively. For other surfaces the boundary conditions are applied as ^ = 0 V -JF!2 <y < -W = dysp^ = 0 Vj’ = -IF 12-W, and y = W!2 + W, dx $ = 0V V z =L

[0150] The effect of the width of the electrodes on the electric field is examined by considering an input potential of Vp= 10 V and Vn= -10 V on positive and the negative electrodes, respectively. For example purposes, four configurations are examined, as shown in Table 1. For the all the configurations, the size of electrode width and spacing is maintained same, lire potential solution for the electrode width of We= 100 pm andWsp- 100 pm is shown in Figure 21. The potential decays as one moves farther away from the surface.Configuration We( pm) ft^( pm)! 100 1009 50 5020 204 10 10Table 1. Electrode width considered for analysis.

[0151] Figure 22 shows the comparison of decay in potential, or the length scale involved along the line y = ~W. - y / 2 for all the configurations in Table 1. It can be seen that with increase in width of electrode, the length scale associated with the decay of also increases. Next, the vertical distance at which the value of potential drops less than 95 percent of theAttorney Docket No.: SP24-268 applied potential and its relation to electrode width is shown in Figure 23. It is observed the length scale of potential decay bears a linear relation with the electrode width.

[0152] Similarly, the electric field developed between electrodes is shown in Figure 24. The larger the size of the arrows inidicates high magnitude of the electric field. Similar to potential field, the electric field also decrease as we away from the surface of the electrodes. This is further established through the Figure 25, which shows the decay in electric field along the line, y = ~We, ~ Wsp / 2.

[0153] Next, by considering the width of the electrodes to be constant, We= 100 pm, the effect of spacing between the electrodes on the potential field can be examined. Four spacing distances = 25 pin, 50 pm 75 pm, and 100 pm between the electrodes are considered, for example purposes. Figure 26 shows the comparison of potential decay along the liney - ~We’ / 2 as spacing is increased. The variation indicates that spacing between the electrodes positively influences the potential field. The relation of associated length with spacing is shown in Figure 27.

[0154] The above analysis of the geometry and field of the IDE is applicable only for interdigitated electrodes in a vacuum.. However, the presence of, e.g., salt solution or cell culture media will form an electrostatic double layer above the surface of the electrodes. Accordingly, the effect of salt solution on the length scale of the IDE system is now considered. In the presence of a salt solution, the above Laplace equation mentioned in Equation 4 is no longer applicable as upon application of potential free charges are formed above the surface of electrodes and the amount of free charge is dependent on the magnitude of the potential applied. To solve this system, the linearized Poisson-Boltzmann equation given as:d20 d2d> j,fl'x(ii) / c1is referred to as Debye length and its value is dependent on the solution and can be explicitly expressed as:Attorney Docket No.: SP24-268

[0155] The derivation of this equation can be found in standard texts on electrodynamics. The Debye length should be determined experimentally. Since the Debye length of our solution is not unknown, the effect of Debye length on the electric field should be considered. Figure 28 shows the variation of length scale associated with the electric field decay with respect to the Debye length of the solution. A lower value of Debye length severely affects the electric field formed in the solution. The variation of length scale is linear with respect Debye length till Debye length of the solution is equal to length of electrode after which point there is no effect of Debye length on the electric field.

[0156] Attention is now turned to implementations of the IDE in bioreactors and methods of using the IDE in the bioreactor setting, according to embodiments. Figure 29 shows an example of an embodiment in which an IDE 720 is disposed between substrate layers of a rolled substrate 722 for a fixed-bed bioreactor. Figure 30 shows how cells 730 growing on the substrate 732 will be near the IDE 736. The cells 730 in the vicinity of the IDE 736 w ill change the local environment due to changes in metabolite composition around the location where the cells are growing, thus changing the electrical field 734 across the electrodes. An electrochemical model of the electrode, substrate, cells, and cell culture media is depicted in Figure 31. The model is used to extract overall impedance to resistance into the media resistance (containing metabolites released by cells), electrode capacitance, electrode interfacial resistance, and biomass (cells) capacitance. In Figure 31, Ru represents the solution resistance, which is the resistance given by the cell culture media; CPE-1 is the electrode capacitance; CPE-2 is the biomass capacitance; Rp is the electrode interfacial resistance; Wd is the electrode diffusion; and R7 is the biomass resistance.

[0157] Due to long-term exposure of the electrodes and / or contacts to the complex mixture of media and cells, the interconnect on the electrode must be reliable and insulated from conducting buffer in the media. According to embodiments, interconnects made from a thermal cure silver epoxy are sealed with bio-grade silicone. IDEs placed on substrates of PET, polyimide, or polyetherimide (PEI) were tested inside fixed bed bioreactors, as examples of embodiments of IDEs, and have been shown to be effective in sensing the microenvironment change during cells culture. Non-limiting examples of suitable substrate materials for the IDE include polymer surfaces or membranes, glass, plastic sheets, ceramics, insulator-on-semiconductor, and fiberglass. In these example, line-based interdigitated electrodes 750 mounted on a substrate 752 of PE T or PEI were used with silver epoxyAttorney Docket No.: SP24-268 interconnects 754 insulated with bio-grade silicone, as shown in Figure 32 (similar to the one in Figure 18) with an electrode width of 100 pm and separation of 100 gm is demonstrated, how ever other design of IDE such as circular electrode 760, as shown in Figure 33, could also w7ork. These PET- or PEI-based electrodes w7ere used to test the ability of the electrode to sense the microenvironment change, as examples of embodiments. Cells w ere grow n inside a fixed-bed bioreactor for 4 days and the impedance change was monitored over the culture period, A 100-mV bias was applied and the impedance over a frequency sweep (100 Hz- 1 MHz) was measured. The substrate of the rolled fixed bed was made of w'oven PET fibers w ith diameters of 200 pm. The results are shown in Figure 34. In the beginning of the culture and up to 65 hours later, a decrease in impedance was observed. However, after 65 hours and due to stoppage of the perfusion pump for the bioreactor, the cells were not supplied appropriate nutrients, resulting in cells death or declining growth and this resulted in the increase in impedance.

[0158] Using the developed electrochemical model, the electrode capacitance (CPE-1) and the biomass (cell) capacitance (CPE-2) could be extracted from the decreasing impedance measurement, as shown in Figure 34. As the impedance decreases, the capacitance of nearby cells increases. In addition, increase in impedance measurement corresponded to the decrease in the biomass capacitance. Figure 35 is a plot of biomass / cells capacitance extracted from the impedance data using the electrochemical model. Up to around 80 hours, cell population increases from the beginning of the culture and this is reflected by an increase in biomass capacitance. At around 100 hours, the cell population dropped due to stoppage of the perfusion pump for the bioreactor, which resulted in decreasing capacitance. Between 100 and 200 hours, the cell population recovered after perfusion w as restored, resulting in increase of biomass capacitance. These results show the effective response from the embedded IDE sensors.

[0159] According to embodiments, the biomass sensor includes interdigitated electrodes (IDEs) with a linewidth of tlie electrodes being from about 10 pm to about 400 gm, or in some preferred embodiments from about 50 gm to about 200 gm, and line spacing of from about 50 gm to about 200 gm. The electrodes can be nanoporous or nanotextured electrodes. As used herein, “nanoporous” refers to a material having pores that are 100 nanometers or smaller in diameter. According to embodiments, the nanoporous electrodes are fabricated by co-deposition of Ag and Au to create the electrode pattern, and then HNCh is used for etchingAttorney Docket No.: SP24-268 to create nanopores. In other embodiments, the electrodes are printed, using an Ag seed layer on a flexible substrate and Au plating to create nanotexture. The top layer of the electrodes can be metallic gold with nanopores or nanotexturing. However, embodiments are not limited to these materials, and any suitable biocompatible conductor can be used, as would be understood by a person of ordinary skill in the art.

[0160] One challenge with electrodes used for cell culture biomass sensing is signal strength. Conventional electrodes, including planar IDEs, can suffer from low signal strength. Tire signa generated by the change in ions that occurs and that causes a change in capacitance of the IDEs can be increased by increasing the IDE line s urface area. According to embodiments, the surface area of the electrode lines can be increased (assuming line width and length are held constant) by creating nanopores or nanotexturing in the electrodes. This increases the signal strength even if the electric field is confined to the immediate surface of the electrode.

[0161] Cyclic voltammetry was performed to test planar and nanoporous gold electrodes. Tire results are shown in Figure 36A and Figure 36B. Figure 36A shows the increased micro Amps for the nanoporous Au (“npAu”) interdigitated electrode compared to the Au electrode with a planar surface. Figure 36B also shows this, where the nanoporous Au electrode shows an approximately 10-times increase in micro Amps compared to the planar electrode. The interdigitated electrodes had line widths of 100 pm. More current produced by the nanoporous IDEs suggests the increase in surface area. The electrolyte used was 5 mM KsFe(CN)6 in 1 X Phosphate Buffered Saline (PBS) solution. This proves that the nanoporous electrodes have more surface area, due to which they generate greater current. Electrodes having more surface area can have (1 ) more capacitance and (2) more faradaic reactions which involve electron exchange.

[0162] The inkjet-printed electrodes can be readily integrated inside the fixed bed bioreactor due to their mechanical flexibility. Inkjet printing can also have cost advantages compared to conventional microfabrication. The silver seed layer used in aspects of embodiments forms a template for producing the nanotextured gold layer. This avoids etching which involves corrosive materials. Also, a cyanide free gold plating process is used.

[0163] Printed electrodes can be prepared by creating a pattern using computer software. The electrode substrate (e.g., polyimide) can be plasma cleaned, and the surface may or may not be coated with silica, according to aspects of embodiments. To promote ink adhesion, theAttorney Docket No.: SP24-268 surface can also be modified with APTES or ethylenediamine, depending on how much ink spread is tolerated in a given application. Then the patterns can be printed using a metal organic decomposition ink. Nanoparticle silver can be used as a base layer to create the electrode template. Also to control ink spreading, the platen temperature can be controlled to, e.g., 50-70 degrees C, which improves print fidelity. In examples, the print resolution used was between 847-2540 dpi; and there were 2-5 pixels per line of the IDE. The printed pattern can be baked (e.g., 80 degrees C for 1 hour) or otherwise treated for solvent removal.Furthermore, the printed pattern can be annealed. In an example, the printed pattern was annealed at 95 degrees C for 1 hour, and then ramped to 180 degrees C at 2 degree C per minutes, and then held for 1 hour at 180 degrees C, followed by a ramp down at 5 degrees C per minute to room temperature. For electroplating, a three-electrode scheme was used with a potentiostat: Au plating scheme with sample as working electrode; titanium foil chosen as counter electrode (large surface area); 1.6 mm diameter Ag / AgCl reference electrode; 1 mM H[AuC14] electrolyte for 20-30 minutes at 1.5 V of constant potential, lire electrical circuit was closed only when plating is started, otherwise the Ag ink would disintegrate. About 0.5 nm of gold was plated, and then the electrode was annealed again. Controlling the annealing step can influence the porosity, as desired.

[0164] Figure 37 shows the Electrochemical Impedance Spectroscopy (EIS) plots for a nanoporous gold (Au) electrode, nanotextured Au electrode, and planar (i.e., standard, non-nanoporous, non-nanotextured) Au electrode. The nanotextured electrodes (microfabricated nanoporous Au IDEs designated as np Au and printed nanotextured Au IDEs designated as Printed) have more baseline impedance Z for the frequency range of 0.1 to 100000 Hz, in comparison to the conventional planar electrode. Higher baseline indicates more dynamic range and greater change in signal for next steps in cellular sensing.

[0165] Embodiments of this disclosure also include optical-based systems and methods for monitoring biomass and cell condition in and / or around fixed-bed bioreactors. Embodiments include those that provide specific, detailed information on biomass and cell condition, and are compatible with directly monitoring a fixed bed reactor, media flow, or both. Advantages of these optical-based systems and methods include being non-destructive, non-invasive, label-free, fast, quantitative, sensitive, specific, compatible with multiple designs and / or ways to implement, and scalable.hi TecnqueAttorney Docket No.: SP24-268

[0166] There are several optical methodologies that can be used to determine and monitor biomass, according to embodiments of this disclosure, such as (but not limited to) native fluorescence, Mie scattering, terahertz imaging / spectroscopy, magnetic resonance, optical i NDttonesrucve-coherence tomography, and radiography. Native fluorescence can be done using multiple wavelengths to target specific molecules within cells, and can either make use of ultraviolet (UV) or avoid it if there is coni NIonnvasvec- ern about cell damage over time from LTV. It may also be used to identify contaminates. Additionabl LFaereel-ly, Mie scattering is a relatively simple technique that is broadly applicable for any cell line since it is based on cell size. X-ray and terahertz radiation and radio waves can bypass the fixed bed Ft basioreactor materials (including the substrate) to selectively interrogate the cells without having to be integrated into the bioreactor; inii Qtttuanaveparticular, terahertz wavelengths and radio waves do not damage cells. Additionally,ii Stensvemagnetic resonance and optical coherence tomography can image cells within 3D matrices and provide positional information within a sampled volumifi Specce. Utilizing coalescence and “light stripping” with optical waveguides to perform cell growth measurements via transmission iddiil Pdtroves aonalosses can be inexpensive to implement and compatible with current ifitnormaon bioreactor vesseliblih Cttompae w designs. ii dil FBRtt monorngrecy

[0167] These techniques share several characteristics that make them particblihi Cduttompaie w mealarly useful for quantifying cells within a fixed bed reactor (FBR) system. Table 2 summarizes t fliihtow monornaeiblihlld Cttsompae w roee benefits. One advantage to highlight, in particular, is the ability to provide detailed, specific b fid bdtt susraexee information on the cells beyond just biomass. This specificity can help determine cell heallbl Scaaeth, growth phase, and provide feedback to tune the conditions to optimize cell development. These techniques can also be paired with non-optical techniques to provide more complete coverage of biomass within the fixed bed reactor (bo th spatially and temporally, depending on deployment / integration of each technique).Native fluorescence Partial Yes Yes Yes Yes Yes Yes Yes Partial Yes Partial YesMie scattering Yes Yes Yes Yes Yes Yes Partial Yes Partial Yes Partial YesAttorney Docket No.: SP24-268 Terahertz Yes Yes Yes Partial Yes Yes Yes Yes Yes Yes Yes Yes miaging / spectroscopyMagnetic resonance Yes Yes Partial Partial Yes Yes Yes Yes Yes Yes Yes Yes Optical coherence Yes Yes Yes Partial Yes Yes Partial Yes Yes Yes Yes Yes tomographyRadiography Partial Yes Yes Yes Yes Yes Partial No Yes No Yes YesOptical coalescence Yes Yes Yes Yes Yes Yes No No Yes No Yes Yes Table 2. Summary of optical technique advantages.

[0168] Native florescence can be derived from a number of sub-cellular components, e.g., proteins, coenzymes, micronutrients, DNA. One could target the appropriate molecule(s) of interest for selective excitation and emission, or use multiple wavelengths to probe different criteria for cell health and growth. Table 3 provides examples of major cellular fluorophores and their excitation and emission peaks, which range from the UV to the visible range. To take this a step further, this characteristic emission could be used to determine cell health as well, on a finer scale than the typical “alive” vs. “dead”. Chemometric analysis may enable further sensitivity to small perturbations.Wavelength, nmFluorophcres Excitation EmissionProteins and amino acidsTryptophan 287 348Collagen 360 405Elastin 290 340Porphyrin 440 630Enzymes and coenzymesIWH 325-350 440-482Riboflavin 450 520-535T able 3. Examples of major cellular fluorophores and their excitation and emission peaks.

[0169] Mie scattering is elastic light scattering from particles who have diameters similar to or larger than the wavelength of incident light, generally in the 0.2-10-micron range. The scattering signal is proportional to the square of the particle diameter, and can be used to effectively identify living cells by size as well as discriminate between live and dead cells. The scattered light can also provide information about shape, internal structures, and the refractive index of the cells. Uris method is flexible and has several benefits such as being non-destructive, fast, and relatively simple to perform, and has been used in R& D applications for cell interrogation. Mie scattering is also advantaged because the scattering isAttorney Docket No.: SP24-268 predominantly forwar d, meaning that the signal to noise is typically very' strong, and can be increased in further by using multispectral imaging combined with chopping / AC coupling the source.

[0170] Terahertz imaging / spectroscopy is particularly usefill for non-invasive cell detection as terahertz radiation is defined as 30-3000 microns in w avelength, thus existing between the microw ave and infrared regions of the electromagnetic spectrum. This implies that the radiation can bypass the fixed bed reactor materials to selectively interrogate the cells. It is also a label-free method so no invasive technologies are needed to prep the cells. Terahertz radiation is non-ionizing and unless higher power should have minimal impact on cells. Terahertz imaging by millimeter wave scanner is commonly used in airports to screen passengers for concealed weapons. Components for Terahertz imaging are becoming more '■■comodilx " with the increasing high frequency used for communications. It is also actively- being used for warehouse imaging box contents.

[0171] Magnetic resonance (i.e., MMR, MRI) has been used for decades to investigate cells and tissues, including mapping and monitoring biomass density in 3D matrices. Typically seen as a complex technique, one can utilize existing equipment for mapping by calibrating against known cell densities. Alternatively, one can also use commercially available RF microcoils to design a magnetic resonance cell to fit the dimensions of the bioreactor vessel or fixed bed area for selective interrogation. Open geometries enable tire sensor to sit against a bioreactor rather than the entire bioreactor needing to fit within a large magnet. Permanent magnets reduce complexity and operating cost and avoid cryogenic cooling.

[0172] Optical coherence tomography (OCT) makes use of refractive index variations to generate contrast within non-transparent heterogenous materials (e.g., cells within tissue scaffolds or cell culture substrates). Cells can be identified, and their position, distribution, and general morphology can be extracted from a 3D section of substrate. Polarization sensitive OCT (PS-OCT) detects birefringence characteristics. PS-OCT is being used for differentiating live cells from dead ones in such applications as skin burns and eye retina evaluations.

[0173] Radiography, utilizing 2D or 3D digital x-ray imaging, is used to non-destructively image materials based on differences in density or path length. It is widely used in both medical and industrial fields. This is a very simple technique that can be used to image theAttorney Docket No.: SP24-268 cells as they grow on the mesh substrate within the fixed bed bioreactor. The changes in density can be correlated with the relative amount of biomass within the reactor.

[0174] According to embodiments, some of the above optical observation methods are implemented using a large-scale source lamp for excitation and a large format detector for information on a large fraction of the fixed bed bioreactor. The benefits are that this is simple, relatively inexpensive, does not require sterility, can scan any dimensions as needed, and can conform to multiple bioreactor designs and materials. The wavelength is chosen such that the bioreactor vessel housing material is transparent at the excitation and detection wavelengths of interest. A small-scale source (e.g., point, cone) in combination with motorized axes may also be used. With a reactor housing of suitable transparency to the excitation wavelength, it is also possible to use waveguide excitation light around the entire reactor which will then be emitted into the reactor.

[0175] In an aspect of some embodiments, the bioreactor includes top and bottom fluid distribution plates between which the fixed bed is disposed, and these distribution plates are seeded with fiber optics or related technology that can transmit the excitation to and the emission from the cells in the fixed bed. Because the excitation and emission wavelengths are different, each optical source could also serve as the detector if special optics are used. The detector can be a camera (i.e,, for imaging the fixed bed reactor directly), a spectrometer, or another light gathering device like a photomultiplier tube coupled with additional optical components, as needed.

[0176] In some embodiments, information may only be able to be captured on the outermost layers of the mesh substrate, as the penetration depth of UV and visible light is low (on the scale of millimeters or less). While this is still useful information, an additional aspect of some embodiments is used to realize mapping of more of the fixed bed. Specifically, internal guide rod(s) used in the fixed bed, as described above, can embed multiple source locations and detector locations for mapping out the biomass as a function of location.Examples of ways to introduce and extract light are fiber optics, wave guides, and mirrored surfaces.

[0177] Some embodiments use optical interrogation for monitoring the media flow at the outlet, inlet, and / or media reservoir. Optical interrogation could be used to detect cells, cell fragments, and metabolites to indicate the state of the process (e.g., using multiple interrogation sites around the bioreactor can provide insight into consumption rates and wasteAttorney Docket No.: SP24-268 production). For example, online cell monitoring can be used to assess the effectiveness of the seeding process. Further, the presence of cells in the recirculation loop above a predetermined threshold could indicate a problem with the bioprocess or lack of available surface for further cell proliferation, perhaps signaling the end of the phase, A low-flow bypass can be used to reduce optical length or fluid density depending on the detector. A small quantity' of media can also be withdrawn continuously for analysis with the volume either replaced or insignificant in comparison to system volume. Or, conversely, a port in the fixed bed reactor can be used for periodic sampling (without removing from the system) in a similar manor. Optical methods can be visual, fluorescence, NIR, or similar to detect relevant material in the reactor. Fluorescence excitation emission matrices (EEMs), for instance, may directly assess fluorescent compounds in cell culture media as well as indirectly assess species or conditions that quench or alter the fluorescence spectrum.

[0178] In embodiments, micro- and nanofluidic devices (e.g., micro-electro-mechanical systems, lab-on-a-chip) can be used to isolate samples for analysis within the low flow region or other media flow areas. They are used for flow cytometry, cell manipulation, and cell analysis, in addition to other uses. Benefits include small analysis volumes, high throughput, multiplexing or parallel analyses, high degree of control, automation capabilities, and compact device sizes. Microfluidics devices are compatible with several of the optical techniques described in this document, as well as a plethora of other techniques for chemical and physical analyses. Examples of what can be done on a device include sample collection, filtration, reactions (e.g. lysing, fluorescent labeling, enzymatic reactions), analyses (optical, chemical, physical), and waste disposal.

[0179] In embodiments, optical coalescence is used to strip light out of a predefined light path or waveguide as cell growth accumulates on the waveguides surface. This methodology can be implemented in numerous manners such as polymer-coated optical fiber, polymer-coated glass rods, or even using modified polymer wafer(s). Tire polymer used in some embodiments can be any biocompatible polymer, including polystyrene, polyethylene terephthalate (PET), polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide, for example, but other polymers are also possible. In embodiments, the polymer used is one that has a large difference in refractive index between the chosen polymer and water (or the cell culture media). For example, when using PET substrates, the PET and processes specific to processing the PETAttorney Docket No.: SP24-268 wafers to make them viable surfaces for cell atachment can be also applied to these proposed waveguides. There is a considerably large change in refractive index between water (or nutrient media solution) and PEI' (—1,33 vs — 1.55 at 850 nm). Ibis large change in refractive index helps to contain the light within the PET waveguide layer; however, as cell growth accumulates on the w aveguide surface the cells themselves have an index of refraction (-1.39 @ 850 nm) between that of the PET waveguide and tire surrounding media. The accumulating cells reduce the critical angle at which light can escape the waveguide, thus transmission or back reflection losses will increase as cell growth accumulates. Waveguidebased detectors can be also integrated into the vessel wall or guide rod, providing an application with minimal interference to the current reactor vessel design and the critically important laminar flow characteristics of the nutrient media,

[0180] The PET wafers scatter light aggressively, but the light scatter is still detectable. Light scattered through tire reactor vessel may be measured over time and trends analyzed for correlation to cell growth, lire scattered light will be inhibited by the cells as they accumulate. This effect may be further maximized by selecting wavelengths at which the cells being grown have characteristically higher absorption, further improving the signal-to-noise characteristics. Light may be scattered from guide rod to guide rod, guide rod to vessel, or vice versa.

[0181] The cell culture matrix can be arranged in multiple configurations within tire culture chamber depending on the desired system. For example, in one or more embodiments, the system includes one or more layers of the substrate with a width extending across the width of a defined cell culture space in the culture chamber. Multiple layers of the substrate may be stacked in this way to a predetermined height. The substrate layers may be arranged such that the first and second sides of one or more layers are perpendicular to a bulk flow direction of culture media through the defined culture space within the culture chamber, or the first and second sides of one or more layers may be parallel to the bulk flow direction. In one or more embodiments, the cell culture matrix includes one or more substrate layers at a first orientation with respect to the bulk flow, and one or more other layers at a second orientation that is different from the first orientation. For example, various layers may have first and second sides that are parallel or perpendicular to the bulk flow direction, or at some angle in between.Attorney Docket No.: SP24-268

[0182] In one or more embodiments, the cell culture system includes a plurality of discrete pieces of the cell culture substrate in a packed bed configuration, where the length and or width of the pieces of substrate are small relative to tlie culture chamber. As used herein, the pieces of substrate are considered to have a length and / or width that is small relative to the culture chamber when the length and / or width of the piece of substrate is about 50% or less of the length and / or width of the culture space. Thus, tlie cell culture system may include a plurality of pieces of substrate packed into the culture space in a desired arrangement. The arrangement of substrate pieces may be random or semi-random, or may have a predetermined order or alignment, such as the pieces being oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° relative to the bulk flow direction).

[0183] The “defined culture space,” as used herein, refers to a space within the culture chamber occupied by the cell culture matrix and in which cell seeding and / or culturing is to occur. Tlie defined culture space can fill approximately the entirety of the culture chamber, or may occupy a portion of the space within the culture chamber. As used herein, the “bulk flow direction” is defined as a direction of bulk mass flow of fluid or cul ture media through or over tlie cell culture matrix during the culturing of cells, and / or during the inflow or outflow of culture media to the culture chamber.

[0184] In some embodiments of this disclosure, there is only a single bulk flow direction within the defined culture space, the packed bed, and / or the bioreactor vessel, such that the liquid or media flow proceeds in predominately one direction from the bioreactor inlet through the packed bed to the bioreactor outlet. The liquid or media flow is uninterrupted by any complicated flow paths within the packed bed space and proceeds through the packed bed in predominately one direction. This avoids complicating flow paths used in some conventional bioreactors where flow spacers, separators, or channels are used to help distribute cell culture media through a cell culture substrate, often because of the inherent non-uniformity of the bioreactor or cell culture substrate. However, in embodiments of the current disclosure, such complicated flow paths are not necessary, and the media flow can be maintained in a single direction from the inlet of the bioreactor to the outlet of the bioreactor. The foregoing is not intended to preclude the use of flow distributor plates at the inlet and outlets of the bioreactor plate, which can be used to distribute fluid across a width of the bioreactor vessel and / or control pressure differentials within the reactor, but do not otherwise affect tlie bulk flow direction through the packed bed and / or within the cell culture space within tlie bioreactor vessel interior.Attorney Docket No.: SP24-268

[0185] The packed bed cell culture matrix of one or more embodiments can include a substrate material constructed to have a uniform and ordered porous structure. The substrate may be referred to as a “structurally defined” su bstrate meaning that the su bstrate has a physical structure that is non-random, but instead is ordered according to defined parameters. In one or more embodiments, the structurally defined substrate includes a plurality of openings defining a porosity of the substrate, the plurality of openings being arrayed in a regular or uniform pattern in each substrate piece or layer. In one or more embodiments, the packed bed cell culture substrate may include a woven cell culture mesh substrate without any other form of cell culture substrate disposed in or interspersed with the cell culture matrix. That is, the woven cell culture mesh substrate of embodiments of this disclosure are effective cell culture substrates without requiring the type of irregular, non-woven substrates used in existing solution. This enables cell culture systems of simplified design and construction, while providing a high-density cell culture substrate with the other advantages discussed herein related to flow uniformity, harvcstability. etc.

[0186] In one or more embodiments, a matrix is provided with a structurally defined surface area for adherent cells to attach and proliferate that has good mechanical strength and forms a highly uniform multiplicity of interconnected fluidic networks when assembled in a packed bed or other bioreactor. In particular embodiments, a mechanically stable, non-degradable ■woven mesh can be used as the substrate to support adherent cell production. The cell culture matrix disclosed herein supports attachment and proliferation of anchorage dependent cells in a high volumetric density format. Uniform cell seeding of such a matrix is achievable, as well as efficient harvesting of cells or other products of the bioreactor. In addition, the embodiments of this disclosure support cell culturing to provide uniform cell distribution during the inoculation step and achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrix, and can avoid formation of large and / or uncontrollable 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. Thus, the matrix eliminates diffusional limitations during operation of the bioreactor. In addition, the matrix enables easy and efficient cell harvest from the bioreactor, lire structurally defined matrix of one or more embodiments enables complete cell recovery’ and consistent cell harvesting from the packed bed of the bioreactor.

[0187] By using a structurally defined culture matrix of sufficient rigidity, high-flow-resistance uniformity across the matrix or packed bed is achieved. According to variousAttorney Docket No.: SP24-268 embodiments, the matrix can be deployed in monolayer or multilayer formats. This flexibility eliminates diffusional limitations and provides uniform delivery of nutrients and oxygen to cells attached to the matrix. In addition, the open matrix lacks any cell entrapment regions in the packed bed configuration, allowing for complete cell harvest with high viability at the end of culturing. The matrix also delivers packaging uniformity for the packed bed, and enables direct scalability from process development units to large-scale industrial bioprocessing unit. Tire ability to directly harvest cells from the packed bed eliminates the need of resuspending a matrix in a stirred or mechanically shaken vessel, which would add complexity and can inflict harmful shear stresses on the cells. Further, the high packing density of the cell culture matrix yields high bioprocess productivity in volumes manageable at the industrial scale.

[0188] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., nonwoven substrates of randomly ordered fibers), embodiments of this disclosure include a cell culture substrate having a defined and ordered structure. The defined and order structure allows for consistent and predictable cell culture results. In addition, the substrate has an open porous structure that prevents cell entrapment and enables uniform flow through the packed bed. This construction enables improved cell seeding, nutrient delivery, cell growth, and cell harvesting. According to one or more particular embodiments, the matrix is formed with a substrate material having a thin, sheet-like construction having first and second sides separated by a relatively small thickness, such that the thickness of tire sheet is small relative to the width and / or length of the first and second sides of the substrate. In addition, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometry that allows cells to adhere to the surface of the substrate material as if it were approximately a two-dimensional (2D) surface, while also allowing adequate fluid flow around the substrate material and through die openings. In some embodiments, the substrate is a polymer-based material, and can be formed as a molded polymer sheet; a polymer sheet with openings punched through the thickness; a number of filaments that are fused into a mesh-like layer; a 3D-printed substrate; or a plurality of filaments that are woven into a mesh layer. The physical structure of the matrix has a high surface-to- volume ratio for culturing anchorage dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor in certain ways discussed here for uniform cell seeding and grow th, uniform media perfusion, and efficient cell harvest.Attorney Docket No.: SP24-268

[0189] According to some embodiments, a method of cell culturing is also provided using bioreactors with the matrix for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0190] Illustrative Implementations

[0191] The following is a description of various aspects of implementations of the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The implementations are intended to illustrate a few aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible implementations.

[0192] According to Aspect 1 as disclosed herein, a cell culture bioreactor is provided that may comprise a cell culture vessel comprising a vessel wall, the vessel -wall may have an interior-facing surface and an exterior facing surface; a substrate disposed in the cell culture vessel, arranged as a fixed bed within the cell culture vessel and surrounded by the vessel wall, the substrate being configured for adhering cells thereto for cell culture; at least one biomass sensor disposed within the cell culture vessel and comprising at least one conductor configured to generate an electric field passing through at least a portion of the fixed bed, wherein the at least one biomass sensor is configured to measure at least one of capacitance and impedance within the cell culture vessel.

[0193] Aspect 2 pertains to the cell culture bioreactor of Aspect I, wherein the at least one biomass sensor comprises a plurality of biomass sensors.

[0194] Aspect 3 pertains to the cell culture bioreactor of Aspect 2, wherein at least two biomass sensors of the plurality of biomass sensors are configured to measure at least one of capacitance and impedance in at least two different regions of the fixed bed.

[0195] Aspect 4 pertains to the cell culture bioreactor of any of Aspects 1 -3, further comprising a guide rod disposed within the cell culture vessel, wherein the guide rod extends in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod, wherein the guide rod extends through the fixed bed such that the guide rod is at least partially surrounded by the substrate over at least a portion of the height,

[0196] Aspect 5 pertains to the cell culture bioreactor of any of Aspect s 1 -4, wherein the cell culture vessel comprising an inlet and an outlet that are in fluid communication with an interior of the cell culture vessel in which the substrate is disposed.Attorney Docket No.: SP24-268

[0197] Aspect 6 pertains to the cell culture bioreactor of Aspect 5, wherein the cell culture bioreactor is configured for fluid to flow through the cell culture vessel such that it flows in via the inlet, through the substrate, and out via the outlet in a direction substantially parallel to the longitudinal axis of the cell culture vessel.

[0198] Aspect 7 pertains to the cell culture bioreactor of any of Aspects 1-6, wherein the at least one conductor comprises a first interior electrode disposed within an outer perimeter of the fixed bed of the substrate.

[0199] Aspect 8 pertains to the cell culture bioreactor of Aspect 7, wherein the first interior electrode is disposed inside the guide rod.

[0200] Aspect 9 pertains to the cell culture bioreactor of Aspect 8, wherein the guide rod comprises a hollow interior.

[0201] Aspect 10 pertains to the cell cul ture bioreactor of any of Aspects 7-9, wherein the at least one conductor comprises a second interior electrode disposed within an outer perimeter of the fixed bed of the substrate.

[0202] Aspect 11 pertains to tire cell culture bioreactor of Aspect 10, wherein tire second interior electrode is disposed inside the guide rod.

[0203] Aspect 12 pertains to the cell culture bioreactor of Aspect 10 or Aspect 11, wherein the first interior electrode and the second interior electrode are arranged at different positions along the height of the guide rod.

[0204] Aspect 13 pertains to the cell culture bioreactor of any of Aspects 10-12, wherein the first interior electrode and the second interior electrode are configured to generate an electrical field from the first interior electrode to the second interior electrode that extends at least partially into the fixed bed.

[0205] Aspect 14 pertains to the cell culture bioreactor of any of Aspects 1-13, wherein the at least one conductor comprises a first exterior electrode disposed on at least one of the interior-facing surface of the vessel wall, the exterior facing surface of the vessel wall, and within the vessel wall.

[0206] Aspect 15 pertains to the cell culture bioreactor of Aspect 14, wherein the at least one conductor comprises a second exterior electrode disposed on at least one of the interiorfacing surface of the vessel wall, the exterior facing surface of the vessel wall, and within the vessel wall, and wherein the second exterior electrode is disposed on a different portion of the cell culture vessel than the first exterior electrode.Attorney Docket No.: SP24-268

[0207] Aspect 16 pertains to the cell culture bioreactor of Aspect 15, wherein the first exterior electrode and the second exterior electrode are arranged at different positions along a height of the cell culture vessel, the height of the cell culture vessel extending in a direction of the longitudinal axis of the cell culture vessel.

[0208] Aspect 17 pertains to the cell culture bioreactor of Aspect 15 or Aspect 16, wherein the first exterior electrode and the second exterior electrode are configured to generate an electrical field from the first exterior electrode to the second exterior electrode that extends at least partially into the fixed bed.

[0209] Aspect 18 pertains to the cell culture bioreactor of any of Aspects 14-16, wherein the first interior electrode and the first exterior electrode are configured to generate an electrical field from the first interior electrode to the first exterior electrode that extends at least partially into the fixed bed.

[0210] Aspect 19 pertains to the cell culture bioreactor of any of Aspects 1-18, where the at least one conductor comprises a plurality of electrodes configured to generate an electric field from any one individual electrode of the plurality of electrodes to any one of two or more of the other of the plurality of electrodes.

[0211] Aspect 20 pertains to the cell culture bioreactor of any of Aspects 1-19, wherein the at least one conductor comprises a cylindrical capacitor plate electrode.

[0212] Aspect 21 pertains to the cell culture bioreactor of Aspect 20, wherein the cylindrical capacitor plate electrode is attached to the guide rod or the vessel wall.

[0213] Aspect 22 pertains to the cell culture bioreactor of Aspects 1-21, further comprising a plurality of guide rods disposed within the cell culture vessel, wherein each of the plurality of guide rods extends in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod, wherein each of the plurality of guide rods extends through the fixed bed such that each of the plurality’ of guide rods is at least partially surrounded by the substrate over at least a portion of the height.

[0214] Aspect 23 pertains to the cell culture bioreactor of Aspect 22, wherein multiple guide rods of the plurality of guide rods comprise a conductor of the at least one conductor.

[0215] Aspect 24 pertains to the cell culture bioreactor of any of Aspects 1-23, wherein the at least one conductor comprises: one or more central electrodes running vertically through the fixed bed parallel to the longitudinal axis of the cell culture vessel and disposed within an interior of the fixed bed such that the one or more central electrodes are separated from theAttorney Docket No.: SP24-268 vessel wall by at least a portion of the substrate, and a plurality of peripheral electrodes disposed on at least one of the interior-facing surface of the vessel wall, the exterior facing surface of the vessel wall, and within the vessel wall, the plurality7of peripheral electrodes being spaced apart from each other in a direction parallel to the l ongitudinal axis of the cell culture vessel.

[0216] Aspect 25 pertains to the cell culture bioreactor of any of Aspects 1-24, wherein the least one conductor comprises at least one inductor configured to measure inductance in at least a portion of the fixed bed.

[0217] Aspect 26 pertains to the cell culture bioreactor of Aspect 25, wherein the at least one inductor comprises a plurality of inductors configured to measure inductance in at least two different regions of the fixed bed.

[0218] Aspect 27 pertains to the cell culture bioreactor of Aspect 25 or Aspect 26, further comprising a guide rod disposed within the cell culture vessel, wherein the guide rod extends in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod, wherein the guide rod extends through the fixed bed such that the guide rod is at least partially surrounded by the substrate over at least a portion of the height.

[0219] Aspect 28 pertains to the cell culture bioreactor of any7of Aspects 25-27, wherein the at least one conductor comprises a first interior inductor disposed within an outer perimeter of the fixed bed of the substrate.

[0220] Aspect 29 pertains to the cell culture bioreactor of Aspect 28, wherein the first interior inductor is disposed inside the guide rod or around an exterior of tire guide rod.

[0221] Aspect 30 pertains to the cell culture bioreactor of Aspect 29, wherein the guide rod comprises a hollow interior.

[0222] Aspect 31 pertains to the cell culture bioreactor of an of Aspects 28-30, wherein the at least one conductor comprises a second interior inductor disposed within an outer perimeter of the fixed bed of the substrate.

[0223] Aspect 32 pertains to the cell culture bioreactor of Aspect 31, wherein the second interior inductor is disposed inside the guide rod.

[0224] Aspect 33 pertains to the cell culture bioreactor of Aspect 31 or Aspect 32, wherein the first interior inductor and the second interior inductor are arranged at different positions along the height of the guide rod.Attorney Docket No.: SP24-268

[0225] Aspect 34 pertains to the cell culture bioreactor of any of Aspects 27-33, further comprising a plurality of guide rods disposed within the cell culture vessel, wherein each of the plurality of guide rods extends in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod, wherein each of the plurality of guide rods extends through the fixed bed such that each of the plurality of guide rods is at least partially surrounded by the substrate over at least a portion of the height.

[0226] Aspect 35 pertains to the cell culture bioreactor of Aspect 34, wherein multiple guide rods of the plurality of guide rods comprise an inductor of the at least one inductor.

[0227] Aspect 36 pertains to tlie cell culture bioreactor of any of Aspects 25-35, wherein the at least one inductor is disposed on at least one of the interior-facing surface of the vessel wall, the exterior facing surface of the vessel wall, and within the vessel wall,

[0228] Aspect 37 pertains to the cell culture bioreactor of any of Aspects 25-36, wherein the at least one inductor comprises a coil inductor.

[0229] Aspect 38 pertains to the cell culture bioreactor of any of Aspects 1-37, wherein the at least one conductor comprises a moveable conductor configured to move relative to the fixed bed while the moveable conductor is generating tlie electric field.

[0230] Aspect 39 pertains to the cell culture bioreactor of Aspect 38, wherein moveable conductor is configured to move in a direction parallel to the longitudinal axis of the cell culture vessel.

[0231] Aspect 40 pertains to the cell culture bioreactor of Aspect 38 or Aspect 39, wherein the moveable conductor is configured to move inside tire guide rod having a hollow interior.

[0232] Aspect 41 pertains to the cell culture bioreactor of any of Aspects 38-40, wherein the at least one conductor comprises one or more stationary conductors that are not configured to move relative to the fixed bed while the one or more stationary conductors are generating the electric field.

[0233] Aspect 42 pertains to the cell culture bioreactor of any of Aspects 1-41, further comprising an analyzer operatively connected to the at least one conductor such that the analyzer is configured to process signals from the at least one conductor.

[0234] Aspect 43 pertains to the cell culture bioreactor of Aspect 42, wherein the analyzer is configured to measure at least one of a capacitance, inductance, and reactance based on signals from a pair of electrodes of the at least one conductor.Attorney Docket No.: SP24-268

[0235] Aspect 44 pertains to the cell culture bioreactor of Aspect 43, wherein the analyzer is configured to generate spatial information about cell growth in tlie fixed bed based on at least one of electrical properties indicated by the signals and physical position of the at least one conductor.

[0236] Aspect 45 pertains to the cell culture bioreactor of Aspect 44, wherein the electrical properties include at least one of capacitance, inductance, reactance, resonant frequencies, amplitude resonance across a range of frequencies, and an interaction of near and far field space, as a function of frequency, between electrodes.

[0237] Aspect 46 pertains to the cell culture bioreactor of any of Aspects 1-45, wherein the cell culture vessel comprises a top wall and a bottom wall, the fixed bed being disposed between the top wall and the bottom wall, and wherein the at least one conductor comprises one or more annular electrodes disposed on at least one of the top wall and the bottom wall.

[0238] Aspect 47 pertains to the cell culture bioreactor of Aspect 46, wherein the one or more annular electrodes are disposed on at least one of an exterior surface, an interior surface, or within the top wall or the bottom wall.

[0239] Aspect 48 pertains to the cell culture bioreactor of Aspect 46 or Aspect 47, wherein the one or more annular electrodes comprise a plurality of annular electrodes arranged concentrically 'ith each other.

[0240] Aspect 49 pertains to the cell culture bioreactor of Aspect 48, wherein the plurality annular electrodes are arranged about a longitudinal axis of the packed bed.

[0241] Aspect 50 pertains to the cell culture bioreactor of any of Aspects 46-49, wherein the one or more annular electrodes comprise at least one of an annular electrode and an annular sector electrode.

[0242] Aspect 51 pertains to the cell culture bioreactor of Aspect 50, wherein the one or more annular electrodes comprise a plurality of annular sector electrodes.

[0243] Aspect 52 pertains to the cell culture bioreactor of Aspect 51, wherein the plurality of annular sector electrodes comprises one or more half-annulus electrodes.

[0244] Aspect 53 pertains to the cell culture bioreactor of any of Aspects 46-52, wherein the at least one conductor further comprises a central electrode disposed concentrically inside the one or more annular electrodes.Attorney Docket No.: SP24-268

[0245] Aspect 54 pertains to the cell culture bioreactor of any of Aspects 46-53, further comprising an analyzer operatively connected to the at least one conductor such that the analyzer is configured to process signals from the at least one conductor.

[0246] Aspect 55 pertains to the cell culture bioreactor of Aspect 54, wherein the analyzer is configured to measure reactance based on signals from a pair of electrodes of the at least one conductor.

[0247] Aspect 56 pertains to the cell culture bioreactor of any of Aspects 46-55, wherein the one or more annular electrodes comprises a plurality of annular electrodes, the plurality of annular electrodes being arranged to have radial symmetry.

[0248] Aspect 57 pertains to the cell culture bioreactor of any of Aspects 1-45, wherein the cell culture vessel comprises a top wall and a bottom wall, the fixed bed being disposed between the top wall and the bottom wall, and wherein the at least one conductor comprises a plurality of electrodes disposed on at least one of the top wall and the bottom wall.

[0249] Aspect 58 pertains to the cell culture bioreactor of Aspect 57, wherein the plurality of electrodes is disposed on at least one of an exterior surface, an interior surface, or within the top wall or the bottom wall.

[0250] Aspect 59 pertains to the cell culture bioreactor of Aspect 57 or 58, wherein the plurality of electrodes comprise a ground electrode disposed on one of the top wall and the botom wall, and a remainder of the plurality electrodes disposed on the other of the top wall and the bottom wall.

[0251] Aspect 60 pertains to the cell culture bioreactor of Aspect 59, wherein the remainder of the plurality of electrodes comprises two or more electrodes.

[0252] Aspect 61 pertains to the cell culture bioreactor of any of Aspects 1-60, wherein the substrate comprises at least one of a cylindrical roll of substrate material and a plurality of substrate layers in a stacked configuration.

[0253] Aspect 62 pertains to the cell culture bioreactor of any of Aspects 1-61, wherein the substrate comprises a plurality of woven fibers forming a plurality of openings in the substrate through which fluid and cells can flow.

[0254] Aspect 63 pertains to the cell culture bioreactor of Aspect 62, wherein at least a portion of the plurality of woven fibers comprise a metallic conductor, wherein the at least one conductor comprises the metallic conductor of the portion of the plurality of woven fibers.Attorney Docket No.: SP24-268

[0255] Aspect 64 pertains to the cell culture bioreactor of Aspect 63, wherein the metallic conductor comprises a conductive coating on the portion of the plurality of woven fibers.

[0256] Aspect 65 pertains to the cell culture bioreactor of Aspect 63, wherein the metallic conductor comprises a metallic fiber incorporated into the plurality’ of woven fibers.

[0257] Aspect 66 pertains to the cell culture bioreactor of Aspect 63, wherein the metallic conductor comprises a thin sheet of conductive material on the portion of the plurality of woven fibers,

[0258] Aspect 67 pertains to tire cell culture bioreactor of any of Aspects 63-66, wherein the substrate comprises the cylindrical roll of substrate material, the cylindrical roll of substrate material comprising a sheet of substrate material that comprises a plurality of sections of the portion of the plurality of woven fibers comprising the metallic conductor, the plurality of sections being separated from each other by another portion of the plurality of woven fibers that do not comprise the metallic conductor.

[0259] Aspect 68 pertains to the cell culture bioreactor of Aspect 67, wherein each of the plurality of sections extends over a height of the fixed bed and is separated from each other by the another portion of the plurality of woven fibers in a circumferential direction of the cylindrical roll.

[0260] Aspect 69 pertains to the cell culture bioreactor of Aspect 67, wherein each of the plurality of sections is separated from each other by the another portion of the plurality of woven fibers in a direction parallel to the longitudinal axis of the fixed bed.

[0261] Aspect 70 pertains to the cell culture bioreactor of any of Aspects 67-69, wherein each of the plurality of sections is wired for independent electrical control.

[0262] Aspect 71 pertains to tire cell culture bioreactor of any of Aspects 67-70, wherein each of the plurality of sections comprises a partial, single, or multiple revolution about the cylindrical roll.

[0263] Aspect 72 pertains to the cell culture bioreactor of Aspect 61 or Aspect 62, wherein the substrate comprises multiple cylindrical rolls of substrate material, and wherein a first cylindrical roll of the multiple cylindrical rolls is nested concentrically within a second cylindrical roll of the multiple cylindrical rolls.

[0264] Aspect 73 pertains to tire cell culture bioreactor of Aspect 72, wherein tire at least one conductor comprises a first induction coil disposed between the first cylindrical roll and the second cylindrical roll.Attorney Docket No.: SP24-268

[0265] Aspect 74 pertains to the cell culture bioreactor of Aspect 73, wherein the at least one conductor further comprises a second induction coil disposed between the first cylindrical roll and the second cylindrical roll, the first cylindrical roll and the second cylindrical roll being disposed at different heights within the fixed bed,

[0266] Aspect 75 pertains to the cell culture bioreactor of any of Aspects 61-66, wherein the substrate comprises the plurality of substrate layers in a stacked configuration, and wherein the metallic conductor is integrated into at least a portion of the plurality of substrate layers.

[0267] Aspect 76 pertains to the cell culture bioreactor of any of Aspects 1-75, wherein the at least one conductor comprises at least four electrodes, the at least four electrodes comprising at least two current-carrying electrodes and at least two voltage -sen sing electrodes.

[0268] Aspect 77 pertains to the cell culture bioreactor of Aspect 76, wherein the at least one biomass sensor is configured to pass current between the at least two current-carrying electrodes while voltage drop is measured between two of the at least two voltage-sensing electrodes.

[0269] Aspect 78 pertains to the cell culture bioreactor of Aspect 76 or Aspect 77, wherein the at least two voltage -sen sing electrodes are disposed between the at least two currentcarrying electrodes.

[0270] Aspect 79 pertains to the cell culture bioreactor of any of Aspects 76-78, wherein at least a portion of the substrate is disposed between two of the at least two voltage-sensing electrodes.

[0271] Aspect 80 pertains to tire cell culture bioreactor of any of Aspects 76-79, wherein the at least two current-carrying electrodes comprise at least one working electrode and a counter electrode, and the at least two voltage-sensing electrodes comprise at least two reference electrodes.

[0272] Aspect 81 pertains to the cell culture bioreactor of Aspect 80, wherein the at least one working electrode comprises a plurality of working electrodes.

[0273] Aspect 82 pertains to the cell culture bioreactor of Aspect 80 or Aspect 81, wherein the at least two reference electrodes comprise more than two reference electrodes.

[0274] Aspect 83 pertains to the cell culture bioreactor of any of Aspects 80-82, wherein the counter electrode and at least one reference electrode are disposed on a first side of atAttorney Docket No.: SP24-268 least a portion of the substrate, and at least one working electrode and at least one other reference electrode are disposed on a second side of the at least a portion of the substrate that is an opposite side of the portion of the substrate from the first side.

[0275] Aspect 84 pertains to the cell culture bioreactor of any of Aspects 80-83, wherein the substrate comprises at least one of a cylindrical roll of substrate material and a plurality of substrate layers in a stacked configuration.

[0276] Aspect 85 pertains to the cell culture bioreactor of any of Aspects 80-84, wherein the substrate comprises a cylindrical roll of substrate material.

[0277] Aspect 86 pertains to the cell culture bioreactor of Aspect 85, the counter electrode is disposed in the center of the cylindrical roll along a longitudinal axis of the cylindrical roll.

[0278] Aspect 87 pertains to the cell culture bioreactor of Aspect 85 or Aspect 86, further comprising a guide rod disposed within the cell culture vessel, wherein the guide rod extends in a direction parallel to a longitudinal axis of the cell culture vessel and in a direction of a height of the guide rod, wherein the guide rod extends through the fixed bed such that the guide rod is at least partially surrounded by the substrate over at least a portion of the height, and wherein the counter electrode is disposed inside or on the guide rod.

[0279] Aspect 88 pertains to the cell culture bioreactor of any of Aspects 85-87, wherein one or more layers of the cylindrical roll are disposed between any pair of reference electrodes of the at least two reference electrodes.

[0280] Aspect 89 pertains to the cell culture bioreactor of any of Aspects 85-88, wherein one or more lay ers of the cylindrical roll are disposed between the counter electrode and any one working electrode of the at least one working electrode,

[0281] Aspect 90 pertains to tire cell culture bioreactor of any of Aspects 80-84, wherein the substrate comprises a plurality of substrate layers in a stacked configuration.

[0282] Aspect 91 pertains to the cell culture bioreactor of Aspect 90, wherein one or more layers of the plurality of substrate layers are disposed between any pair of reference electrodes of the at least two reference electrodes.

[0283] Aspect 92 pertains to the cell culture bioreactor of Aspect 90 or Aspect 91, wherein one or more layers of the plurality of substrate layers are disposed between the counter electrode and any one working electrode of the at least one working electrode.

[0284] Aspect 93 pertains to the cell culture bioreactor of any of Aspects 1-92, wherein the cell substrate comprises a structurally defined multi-layered substrate.Attorney Docket No.: SP24-268

[0285] Aspect 94 pertains to the cell culture bioreactor of Aspect 93, wherein each layer of the multi-layered substrate comprises a physical structure and a porosity that are substantially regular and uniform.

[0286] Aspect 95 pertains to the cell culture bioreactor of Aspect 93 or Aspect 94, wherein the cell substrate comprises a substantially uniform porosity.

[0287] Aspect 96 pertains to the cell culture bioreactor of any of Aspects 93-95, wherein the structurally defined multi-layered substrate comprises at least one of a molded polymer lattice, a 3D-printed lattice, and a woven substrate.

[0288] Aspect 97 pertains to the cell culture bioreactor of Aspect 93, wherein the cell substrate comprises a plurality of substrate layers, at least a portion of the plurality of substrate layers are not separated by a spacer material or barrier, or are in physical contact ■with each other.

[0289] Aspect 98 pertains to the cell culture bioreactor of any of Aspects 1-96, wherein the cell culture vessel is configured for cell culture media to flow continuously from the inlet toward the outlet during a cell culture period.

[0290] Aspect 99 pertains to the cell culture bioreactor of any of Aspects 1-98, wherein the at least one biomass sensor is configured to measure a change in at least one of impedance and capacitance between the pair of electrodes over a period of time while culturing cells w ithin the reservoir.

[0291] Aspect 100 pertains to the cell culture bioreactor of Aspect 99, wherein the at least one biomass sensor is configured to monitor a proliferation of adherent cells in the cell substrate disposed at least between the pair of electrodes.

[0292] Aspect 101 pertains to the cell culture bioreactor of any of Aspects 1-100, further comprising a controller configured to receive signals from the at least one biomass sensor, the controller comprising a microprocessor for analyzing the signals measured by the sensor system.

[0293] Aspect 102 pertains to the cell culture bioreactor of Aspect 101, wherein the controller comprises a set of instructions stored in memory which, when executed by the microprocessor, causes the controller to predict the proliferation of adherent cells in the cell substrate based on the signals detected by the sensor system.

[0294] Aspect 103 pertains to a method of monitoring biomass during a cell culture of cells in the cell culture bioreactor of any one of Aspects 1-102, the method comprising: culturingAttorney Docket No.: SP24-268 the cells on the fixed bed using a cell culture medium perfused through the cell culture bioreactor; measuring at least one of impedance and capacitance across at least a portion of the fixed bed during the culturing of the cells.

[0295] Aspect 104 pertains to the method of monitoring biomass of Aspect 103, wherein the measuring occurs in real time and aseptically.

[0296] Aspect 105 pertains to the method of monitoring biomass of Aspect 103 or Aspect 104, further comprising determining at least one of a density and proliferation of cells in the fixed bed.

[0297] Aspect 106 pertains to the method of monitoring biomass of any of Aspects 103- 105, wherein the measuring comprises measuring the impedance or capacitance using a pair of electrodes that are spaced apart from each other with at least a portion of the fixed bed being disposed between the pair of electrodes.

[0298] Aspect 107 pertains to the method of monitoring biomass of any of Aspects 103- 106, wherein the measuring the at least one of impedance and capacitance comprises taking multiple or continuous measurements over a period of time during a cell culture.

[0299] Aspect 108 pertains to a cell culture bioreactor configured for in-situ biomass sensing, the bioreactor comprising: a cell culture vessel comprising at least one inlet, at least one outlet, and an interior reservoir disposed between the at least one inlet and the at least one outlet; a fixed bed comprising substrate configured for culturing cells adhered to a surface thereof, the fixed bed disposed in the interior reservoir; at least one biomass sensor comprising: a pair of electrode structures each comprising a plurality’ of interdigitated electrode branches, the pair of electrode structures being disposed within the fixed bed; and an impedance analyzer electrically coupled to the pair of electrode structures, wherein tire at least one biomass sensor is configured to detect a change in impedance between the pair of electrode structures.

[0300] Aspect 109 pertains to the bioreactor of Aspect 108, wherein the pair of electrode structures comprises a first electrode structure and a second electrode structure, the first electrode structure being connected to a first connection pad and the second electrode structure being connected to a second connection pad.

[0301] Aspect 110 pertains to the bioreactor of Aspect 108 or 109, wherein each electrode structure of the pair of electrode structures is individually addressed.Attorney Docket No.: SP24-268

[0302] Aspect 111 pertains to the bioreactor of any one of Aspects 108-110, wherein the at least one biomass sensor comprises a plurality of pairs of electrode structures each comprising a plurality of interdigitated electrode branches.

[0303] Aspect 112 pertains to the bioreactor of Aspect 111, wherein the plurality of pairs of electrodes are disposed in different regions of the fixed bed.

[0304] Aspect 113 pertains to the bioreactor of Aspect 111 or 112, wherein the biomass sensor further comprises one or more electrical switches arranged such that a voltage can be applied across a single pair of electrode structures at a given time.

[0305] Aspect 114 pertains to the bioreactor of any one of Aspects 108-113, wherein the fixed bed comprises multiple layers of the substrate.

[0306] Aspect 115 pertains to the bioreactor of Aspect 114, wherein the multiple layers of the substrate are arranged as a plurality of stacked layers of substrate or as a cylindrical roll of one or more pieces of substrate.

[0307] Aspect 116 pertains to the bioreactor of Aspect 114 or 115, wherein at least a portion of the multiple layers of the substrate are not separated from each other by any structure other than other substrate layers, cell culture media, cells being cultured, or a portion of the biomass sensor.

[0308] Aspect 117 pertains to the bioreactor of any one of Aspects 114-116, wherein the pair of electrode structures are disposed between adjacent layers of the substrate in the fixed bed.

[0309] Aspect 118 pertains to the bioreactor of any one of Aspects 108-117, wherein the plurality of interdigitated electrode branches of the pair of electrodes are not in physical contact with the substrate.

[0310] Aspect 119 pertains to the bioreactor of any one of Aspects 108-118, wherein the biomass sensor is configured to detect changes in impedance in a cell culture media in the interior reservoir, the changes in impedance being caused by changes in metabolites in the cell culture media.

[0311] Aspect 120 pertains to the bioreactor of any one of Aspect 108-119, wherein the impedance analyzer is configured to extract capacitance changes based on the detected changes in impedance.Attorney Docket No.: SP24-268

[0312] Aspect 121 pertains to the bioreactor of any one of Aspects 108-120, wherein the impedance analyzer is configured to correlate decreases in impedance with increased capacitance of cells and increased cell biomass.

[0313] Aspect 122 pertains to the bioreactor of Aspect 121, wherein the impedance analyzer is configured to correlate increases in impedance with decreased capacitance of cells and decreased cell biomass.

[0314] Aspect 123 pertains to the bioreactor of any one of Aspects 108-122, wherein the pair of electrode structures comprise a constant line width.

[0315] Aspect 124 pertains to the bioreactor of Aspect 123, wherein the line width of the pair of electrode structures is from about 10 μm to about 400 μm, or from about 50 μm to about 200 μm.

[0316] Aspect 125 pertains to the bioreactor of any one of Aspects 108-124, wherein each of the plurality of interdigitated electrode branches are spaced from another of the plurality of interdigitated electrode branches by a line spacing, wherein the line spacing is from about 10 μm to about 400 μm, or from about 50 μm to about 200 μm.

[0317] Aspect 126 pertains to the bioreactor of Aspect 125, wherein the line spacing is equal to the line width.

[0318] Aspect 127 pertains to the bioreactor of any one of Aspects 108-126, wherein the plurality of interdigitated electrode branches comprises electrode surfaces that are nanoporous or nanotextured.

[0319] Aspect 128 pertains to the bioreactor of Aspect 127, wherein the electrode surfaces of the interdigitated electrode branches are etched.Definitions

[0320] “Wholly synthetic’’ or “fully synthetic” refers to a cell culture article, such as a microcarrier or surface of a culture vessel, that is composed entirely of synthetic source materials and is devoid of any animal derived or animal sourced materials. The disclosed wholly synthetic cell culture article eliminates the risk of xenogeneic contamination.

[0321] “Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.

[0322] “Users” refers to those who use the systems, methods, articles, or kits disclosed herein, and include those who are culturing cells for harvesting of cells or cell products, orAttorney Docket No.: SP24-268 those who are using cells or cell products cultured and / or harvested according to embodiments herein.

[0323] “About” modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, viscosities, and like values, and ranges thereof, or a dimension of a component, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example: through typical measuring and handling procedures used for preparing materials, compositions, composites, concentrates, component parts, articles of manufacture, or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. Tire term “about” also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture.

[0324] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0325] The indefinite article “a” or “an” and its corresponding definite article “the” as used herein means at least one, or one or more, unless specified otherwise.

[0326] Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” for gram(s), “mL” for milliliters, and “rt” for room temperature, “nm” for nanometers, and like abbreviations).

[0327] Specific and preferred values disclosed for components, ingredients, additives, dimensions, conditions, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The systems, kits, and methods of the disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values described herein, including explicit or implicit intermediate values and ranges.

[0328] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is notAttorney Docket No.: SP24-268 otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that any particular order be inferred.

[0329] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the embodiments may occur to persons skilled in the art, the disclosed embodiments should be construed to include everything within the scope of the appended claims and their equivalents.

Claims

Attorney Docket No.: SP24-268 What is claimed:

1. A cell culture bioreactor configured for in-situ biomass sensing, the bioreactor comprising:a cell culture vessel comprising at least one inlet, at least one outlet, and an interior reservoir disposed between the at least one inlet and the at least one outlet;a fixed bed comprising substrate configured for culturing cells adhered to a surface thereof, the fixed bed disposed in the interior reservoir;at least one biomass sensor comprising:a pair of electrode structures each comprising a plurality of interdigitated electrode branches, the pair of electrode structures being disposed within the fixed bed; andan impedance analyzer electrically coupled to the pair of electrode structures, wherein the at least one biomass sensor is configured to detect a change in impedance between the pair of electrode structures.

2. The cell culture bioreactor of claim 1, wherein the pair of electrode structures comprises a first electrode structure and a second electrode structure, the first electrode structure being connected to a first connection pad and the second electrode structure being connected to a second connection pad.

3. The cell culture bioreactor of claim 1 or claim 2, wherein each electrode structure of the pair of electrode structures is individually addressed.

4. The cell culture bioreactor of any one of claims 1-3, wherein the at least one biomass sensor comprises a plurality of pairs of electrode structures each comprising a plurality of interdigitated electrode branches,5. The cell culture bioreactor of claim 4, wherein the plurality of pairs of electrodes are disposed in different regions of the fixed bed.

6. The cell culture bioreactor of claim 4 or claim 5, wherein the biomass sensor further comprises one or more electrical switches arranged such that a voltage can be applied across a single pair of electrode structures at a given time.Attorney Docket No.: SP24-2687. The cell culture bioreactor of any one of the preceding claims, wherein the fixed bed comprises multiple layers of the substrate.

8. The cell culture bioreactor of claim 7, wherein the multiple layers of the substrate are arranged as a plurality of stacked layers of substrate or as a cylindrical roll of one or more pieces of substrate.

9. The cell culture bioreactor of claim 7 or claim 8, wherein at least a portion of the multiple layers of the substrate are not separated from each other by any structure other than other substrate layers, cell culture media, cells being cultured, or a portion of the biomass sensor.

10. The cell culture bioreactor of any one of claims 7-9, wherein the pair of electrode structures are disposed between adjacent layers of the substrate in the fixed bed.

11. The cell culture bioreactor of any one of claims 1-10, wherein the plurality of interdigitated electrode branches of the pair of electrodes are not in physical contact with the substrate.

12. The cell culture bioreactor of any one of claims 1-11, wherein the biomass sensor is configured to detect changes in impedance in a cell culture media in the interior reservoir, the changes in impedance being caused by changes in metabolites in the cell culture media.

13. The cell culture bioreactor of any one of claims 1-12, wherein the impedance analyzer is configured to extract capacitance changes based on the detected changes in impedance.

14. The cell culture bioreactor of any one of claims 1-13, wherein the impedance analyzer is configured to correlate decreases in impedance with increased capacitance of cells and increased cell biomass.Attorney Docket No.: SP24-268 15. The cell culture bioreactor of claim 14. wherein the impedance analyzer is configured to correlate increases in impedance with decreased capacitance of cells and decreased cell biomass.

16. The cell culture bioreactor of any one of claims 1-15, wherein the pair of electrode structures comprise a constant line width.

17. The cell culture bioreactor of claim 16, wherein the line width of the pair of electrode structures is from about 10 μm to about 400 μm, or from about 50 μm to about 200 μm.

18. The cell culture bioreactor of any one of claims 1-17, wherein each of the plurality’ of interdigitated electrode branches are spaced from another of the plurality of interdigitated electrode branches by a line spacing, wherein the line spacing is from about 10 μm to about 400 μm, or from about 50 μm to about 200 μm.

19. The cell culture bioreactor of claim 18, wherein the line spacing is equal to the line width.

20. The cell culture bioreactor of any one of claims 1-19, wherein the plurality of interdigitated electrode branches comprises electrode surfaces that are nanoporous or nanotextured.

21. The cell culture bioreactor of claim 20, wherein the electrode surfaces of the interdigitated electrode branches are etched.

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