RELATED APPLICATIONS

[0001] This application claims priority to US Patent Application No. 17/952,355, filed September 26, 2022. For the USA, this application is a continuation in part of US Patent Application No. 17/952,355, filed September 26, 2022. US Patent Application No. 17/952,355, filed September 26, 2022, is incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to sorbent compositions, and to cell culture bioreactors and the methods of cell culture, for example in vitro cultures of mammalian cells.

BACKGROUND OF THE INVENTION

[0003] Mammalian cell culture is one of the most common methods for the commercial production of recombinant proteins and for the isolation of endogenous proteins from those cells. However, higher cell densities in these cultures are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH.

[0004] The culture of mammalian cells in vitro changes their metabolism compared to in vivo conditions and results in higher glycolysis and glutaminolysis rates, which correspondingly results in higher lactate and ammonia production. The increased lactate and ammonia concentrations, in both stationary and stirred cultures, which sometimes also occurs in other cells culture, such as bacteria, reduces the cells’ growth and protein production. Increases in lactate and ammonia also increase cellspecific glucose and glutamine consumption rates, while also reducing the oxygen consumption rate of the cells. On the other hand, increasing the concentration of only one or the other of lactate or ammonia increases the production rate of the other one by the cells. The increase in concentration of lactic acid and ammonia results in decreasing the intracellular pH and acidification, which reduces, and even inhibits, cells’ growth, metabolism, and their specific productivity. The increase in lactic acid and ammonia can eventually result in apoptosis.

[0005] One of the most commonly used methods to inhibit excessive accumulation of lactate and ammonia in cell culture systems is to replace the spent culture medium (i.e., the medium that has high levels of lactate acid and ammonia) with fresh medium frequently. However, this replenishment tends to be very costly because the inhibitory concentrations of these factors are achieved in culture quickly, while the concentrations of other beneficial elements in the medium are still high and are replaced without being used by the cells. Alternative methods for selectively removing undesired elements from the medium and culture broth have been suggested. Three of these major techniques for removing these undesired factors include membrane techniques, extractive processes, and ion exchange resins. Membranes have very high capital cost and short lifetimes, extractive processes use solvents that are toxic to the microorganisms, and ion exchange resins are very costly.

BRIEF SUMMARY OF THE INVENTION

[0006] Activated carbon (AC), as a highly porous material that has a high surface area to volume ratio, has high affinity for organic materials and physically adsorbs polar materials such as alcohols and acids. However, putting culture broth or medium directly in contact with AC can result in uncontrolled adsorption of different components of the culture medium to the particles, with uncontrolled rates. Further, AC particles dispersed in a medium are difficult to separate from the medium. As a result, the treated culture medium might not be suitable for reusing with the cells.

[0007] This specification describes a sorption unit, and methods of making the sorption unit. The sorption unit has a sorbent material, such as activated carbon or charcoal, in a porous matrix. The porous matrix holds the sorbent material and thereby enables contacting the sorbent material with a culture medium without the sorbent material becoming dispersed in the medium. The porous matrix may also reduce the rate at which one or more components of the culture medium are removed by the sorbent material. Optionally, the sorption unit may be coupled with a semi-permeable membrane to inhibit one or more selected components of a culture medium from being removed by the sorption unit. In some examples, the semi-permeable membrane has a molecular weight cut off in the range of 0.1 to 1000 kDa. Optionally, removal of one or more selected components of a culture medium is inhibited by pre-loading the sorption unit with the selected components. Optionally, the sorption unit is pre-loaded with a compound so that the compound may be desorbed into a culture medium.

[0008] This specification also describes a cell culture bioreactor. The bioreactor has a cell culture vessel for containing cells and a liquid medium. The bioreactor also has a sorption unit in fluid communication with the cell culture vessel and/or the liquid medium. The sorption unit has a porous matrix and a sorbent material. Optionally, the bioreactor may have a porous barrier, for example a semi- permeable membrane, separating the cells from the sorption unit. In some examples, the porous barrier also separated large molecules or particles from the sorption unit.

[0009] This specification also describes a method of growing cells. The method includes growing cells in a cell culture medium and exposing the cell culture medium to a sorption unit. The sorption unit has a porous matrix and a sorbent material. Growing the cells may include expanding the cell population, differentiating the cells, remodeling the cells’ microenvironment, or growing the cells to form a 2D or 3D structure, a cell sheet, a tissue or an organ.

[0010] In some examples, the cell culture medium is exposed to the sorption unit while cells are growing in the cell culture medium. The cell culture medium may be contained in a vessel and the sorption unit may be placed in the same vessel. In this case, the cells are optionally separated from the sorption unit, for example by being spaced apart from the sorption unit or by being separated from the porous matrix by a porous barrier. In other examples, the cell culture medium is separated from the cells (e.g. removed from the vessel), exposed to the sorption unit, and replaced in contact with the cells. Optionally, whether the sorption unit is exposed to a culture medium with or without cells, the cell culture medium is not forced through the pores of the porous matrix from one side of the porous matrix to another side of the porous matrix, for example by way of a material pressure differential applied to opposing sides of the porous matrix.

[0011] In some examples, a sorption unit may remove one or more inhibitory compounds, such as ammonia or lactic acid, from a cell culture medium in fluid communication with cells. Optionally, the sorption unit may be loaded with one or more solutes or other components of the cell culture medium before exposing the cell culture medium to the sorption unit. Optionally, the sorption unit may be loaded with a nutrient such as glucose before exposing the cell culture to the sorption unit.

[0012] This specification also describes a composition having a porous matrix and particles of a sorbent dispersed in the porous matrix. In some examples, a) the porous polymer matrix is at least 1 mm thick, b) the polymer matrix has pores having a cross section in the range of 200 to 13,000 square micrometers, c) the porosity of the porous matrix is in the range of 50-90 percent, and/or d) the composition also has a semi-permeable membrane. In some examples, a semi-permeable membrane may be a dialysis or ultrafiltration membrane, a hollow fiber or tubular membrane, or a membrane having a molecular weight cut off in the range of 0.1 to 1000 kDa. Optionally, the sorbent may be charcoal or activated carbon. Optionally, the porous matrix may be made of a polymer, for example a hydrophilic polymer, for example a cellulosic polymer. A normally water-soluble polymer may be crosslinked, for example with citric acid. In some examples, the porous matrix includes polymer fibers. In other examples, the porous matrix includes a phase separated polymer. [0013] This specification also describes a method of producing a sorbent unit. The method includes mixing a water-soluble polymer, a crosslinker, a sorbent material and water to create a mixture, optionally placing the mixture into a form, and drying the mixture. The drying may include lyophilizing the mixture. The method may also include activating the crosslinker, for example by heating it. In some examples, the polymer is a cellulosic polymer such as methyl cellulose and the crosslinker is citric acid.

[0014] This specification also describes a method of removing a molecule from a solution. The method includes placing a sorption unit in fluid communication with the solution and allowing the sorption unit to remove, for example absorb or adsorb, the molecule. The sorption unit has a porous matrix that contains a sorbent material, for examples particles or a powder of the sorbent material may be dispersed in the matrix. In some examples, the sorption unit is partially or entirely immersed in the liquid. Optionally, the method may also include removing the sorption unit from the solution and recovering the molecule from the sorption unit. In some examples, the sorbent material is activated carbon or charcoal. In some examples, the porous matrix is made of a hydrophilic polymer, for example a cellulosic polymer or nylon. The porous matrix may be made of a water-soluble polymer that has been crosslinked, for example with citric acid. In some examples, the molecule removed is lactic acid or ammonia, or an ion associated with either of them. In some examples, the solution is a cell culture medium, which may contain growing cells. In some examples, the sorption unit is placed in fluid communication with the solution through a semi-permeable membrane.

[0015] In some examples, the sorption unit delays the removal of a molecule from the solution relative to direct contact (i.e. contact without the porous matrix) of the same amount of the sorbent material with the solution. Optionally, the removal of a beneficial molecule from the solution may be reduced by pre-loading the sorption unit with one or more of those molecules. Optionally, the removal of a beneficial molecule from the solution may be reduced by placing a semi-permeable membrane between the solution and the sorption unit. The semi-permeable membrane may have a molecular weight cut-off below the molecular weight of the beneficial molecule.

[0016] This specification also describes a process for fabricating units containing sorbents such as carbon-based materials, for example activated carbon and charcoal powders. The powders are embedded within a polymeric porous network, for example methylcellulose, that is optionally crosslinked for example with citric acid, to create the units. Optionally a unit can be fabricated from natural materials. In some examples, by controlling the properties of the polymeric network, such as the chemical properties of the network, the pore sizes and porosity, and the degree of crosslinking, the absorption and/or release rates of one or more molecules can be adjusted. In order to selectively control the transport of small molecules with certain sizes, the units can be enclosed in, or associated with, membranes with different molecular weight cut-offs to limit the size of molecules that can be removed by the unit based on their molecular weight. Embedding sorbent powder in the polymeric network may stabilize the powder and prevents the powder from contaminating a liquid.

[0017] In some examples, a unit can be used to remove one or more molecules from a solution. A unit can also be used to remove a certain type of molecule from one solution and then release the molecule when the unit is contacted with another solution. In some examples, one or more captured molecules are extracted from a unit, for example by treating the unit with a solvent or other liquid solution such as sodium hydroxide. The extracted molecules are optionally used for another application. A treated unit may optionally be reused to capture more molecules.

[0018] In some examples, a unit can be used to increase the sustainability or lower the cost of cell culture, for example in bioreactors and fermenters or for different cell types, such as mammalian cells. In some examples, the units are used to keep the concentration of lactate and/or ammonia at low amounts for a period of time, for example lower than their toxic thresholds, to improve the performance of a cell culture method. The units can also be used in other situations where organic components need to be separated from a solution. The units can be used to remove undesired elements from a solution for example by putting the final product (e.g a sorption unit or sorption unit associated with a semi-permeable membrane) in direct contact with the solution or by perfusing the solution through a unit using a hollow fiber or tubular membrane, optionally with a defined molecular weight cut-off. Optionally, the removal and release of different molecules from a unit can be performed simultaneously.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0019] Fig. 1 shows an embodiment of the process of the present invention for making polymeric units with carbon-based particles embedded in a network.

[0020] Fig. 2 shows a plurality of embodiments of how the carbon particles can adsorb the target molecules. [0021 ] Fig. 3 shows hollow fiber tubes that have been inserted into the filter units of the present invention to limit the molecules that can be absorbed by the units based on their molecular weight.

[0022] Fig. 4 shows the absorption and desorption of molecules in the absence and presence of a molecular weight cut-off dialysis membrane.

[0023] Fig. 5 shows various carriers that can be used in conjunction with the filter units of the present invention.

[0024] Fig. 6 shows a process by which desorption and isolation of a molecule of interest may occur.

[0025] Fig. 7 shows a process by which different molecules can be selectively absorbed and/or desorbed to and/or from the filter unit by the selective use of different molecular weight cut-off dialysis membranes.

[0026] Fig. 8 shows the results of experiments with a methyl cellulosic polymeric matrix comprising activated carbon demonstrating that the filter units of the present invention can be used to both absorb/adsorb and desorb targeted molecules.

[0027] Fig. 9A is a scanning electron microscope (SEM) photograph of a methyl cellulose-based filter unit cut in a vertical plane.

[0028] Fig. 9B is a scanning electron microscope (SEM) photograph of the methyl cellulose-based filter unit of Fig. A cut in a horizontal plane.

[0029] Fig. 9C is a histogram of the area of 50 pores selected from the SEM photograph of Fig. 9B.

[0030] Fig. 10 is an exploded side view of a bioreactor with a filter unit.

[0031] Fig. 11 is an isometric exploded view of the bioreactor of Fig. 10 viewed from above.

[0032] Fig. 12 is an isometric exploded view of the bioreactor of Fig. 10 viewed from below. [0033] Fig. 13 is a partially exploded side view of the bioreactor of Fig. 10 with the filter unit assembled.

[0034] Fig. 14 is an isometric partially exploded side view of the bioreactor of Fig. 10 with the filter unit assembled viewed from above.

[0035] Fig. 15 is a cross-sectional side view of the assembled bioreactor of Fig. 10.

[0036] Fig. 16 is an isometric cross-sectional of the assembled bioreactor of Fig. 10.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Functionally, the term “filter unit” and similar terms are used loosely to describe a device useful for removing one or more selected solutes or particles from a liquid without the liquid necessarily passing through the filter unit. In some examples, the filter unit is merely placed in communication with the liquid or the liquid may flow past or around the filter unit. The pores or other cavities of the filter unit are typically wetted or substantially filled with liquid. In some examples, solutes or particles may move, for example by diffusion or Brownian motion, into pores or other cavities of the filter unit from a liquid surrounding the filter unit without a material flow of liquid passing through the filter unit. In other examples, the liquid may pass through the filter unit.

[0038] Structurally, the term “filter unit” and similar terms are typically used to refer to a combination including a porous matrix and a sorbent material. Optionally, the filter unit may also include, or be associated with or separated from a liquid by, a semi-porous membrane such as an ultrafiltration membrane or a dialysis membrane. The porous matrix may be made, for example, by the formation of pores in an ordinarily bulk material (for example by phase separation or other methods) or by aggregation of fibers or other media, for example by sintering or textile manufacturing methods.

[0039] Optionally, a composition including a sorbent material and a porous matrix but not including a semi-porous membrane may be called a filter block, sorption block or sorption unit. The terms may be used to refer specifically to that part of a filter unit that also contains a semi-porous membrane. However, in other cases these terms may be used to refer to a filter unit that does not include a semi-porous membrane or to an intermediate product. The word “block” is not intended to imply any particular size or shape of the composition.

[0040] The terms “absorption” and “adsorption”, and similar terms, may be used imprecisely or interchangeably herein to mean either absorption and/or adsorption. The terms “sorption” or “sorbent” or similar terms may be used to indicate absorption and/or adsorption and/or ion exchange but optionally ion exchange is excluded. Reference to a sorption media typically refers to particles dispersed or embedded into a matrix material. In some examples the matrix material may also perform sorption, for example by way of a swellable polymer matrix that absorbs a liquid. However, such swelling is primarily useful for wetting the pores of the polymer matrix and is not typically material in reducing the concentration of one or more selected solutes or particles from a liquid. A variety of terms such as adsorption, absorption, sorption, uptake or removal, may all be used to refer to the reducing the concentration of one or more selected solutes or particles from a liquid.

[0041] Sorbent materials used in the filter unit may include, for example, one or more of activated carbon, charcoal, zeolites, ion exchange resins, metals, metal oxides, metal-organic frameworks, silica, polymers and ceramics. Optionally, the sorbent material is carbon-based. Optionally, the sorbent material is not an ion exchange resin.

[0042] Materials used to form the matrix may include, for example, one or more of polymers, metals and ceramics. A polymer may be a bio-based polymer or a synthetic polymer. Inherently water soluble polymers may be functionalized or crosslinked to make them less soluble or insoluble. In some examples, the matrix is formed of a hydrophilic polymer. The matrix may be made porous, for example, by one or more of phase separation (for example temperature induced phase separation or nonsolvent induced phase separation), sintering, or filament-based techniques such as filament deposition (e.g. by way of spinning or electrospinning), textile manufacturing techniques or depth filter manufacturing techniques. [0043] In some examples, a porous matrix is made by mixing water, a water- soluble polymer, sorbent particles and a cross linker for the polymer. The crosslinker may be active in the aqueous solution. Alternatively, the cross linker may be a latent crosslinker. In the case of a latent crosslinker, the mixture may be lyophilized to preserve its porous structure during drying. After drying, the latent crosslinker is activated, for example by heating.

[0044] The terms “lactic acid” and “lactate” are used without specificity. For example, removal of lactic acid or lactate may include removal of the other compound or both compounds. Similarly, the terms “ammonia” and “ammonium” are used without specificity. For example, removal of ammonia or ammonium may include removal of the other compound or both compounds.

[0045] In some examples, filter units contain sorbent particles or powders embedded within a porous matrix or network. The sorbent materials are optionally carbon-based materials, including but not limited to activated carbon and charcoal powders. The porous network is optionally a polymeric porous network, including but not limited to methylcellulose that is crosslinked with citric acid. The sorbent material and the matrix material create a functioning unit. These units can be used to remove one or more selected molecules from a solution. The units can also be allowed to absorb certain molecules and then release these molecules when the units are contacted with another solution. The absorption and release of different molecules can be performed simultaneously. By controlling the properties of the polymeric network, such as the chemical properties of the network, the pore sizes and porosity, the degree of crosslinking, and the amount of the sorbent particles, the sorption and release rates of different molecules can be adjusted. In order to selectively control the transport of particles or molecules with certain sizes, the units can be enclosed in dialysis membranes. The molecular weight cut-off of the dialysis membrane may be selected to limit the size of molecules that can be removed by the unit based on their molecular weight. Embedding sorbent powder in the polymeric network stabilizes the powder and prevents them from contaminating the liquid. Embedding the powder also adjusts the rate at which sorption and desorption is performed. It is also possible to extract the sorbed molecules from the unit and reuse the molecules and/or the unit. Molecules may be released by treating the units with proper solvents or other liquid solutions, such as sodium hydroxide with the proper molarity. The units can then be reused without adversely affecting their performance.

[0046] The units can be fabricated from natural materials. The units can be used to increase the sustainability of a cell culture process or lower the cost of cell culture in bioreactors and fermenters for different cell types, including mammalian cells and other microorganisms. Alternatively, the units can be used in other situations where one or more organic components need to be separated from a solution. The units can also be used to continuously remove undesired elements from the solution by directly putting the unit in contact with the solution directly or through a dialysis membrane. Optionally, a solution may be perfused through a unit using hollow fiber or tubular membranes, which may have defined molecular weight cut-offs. The units may be used to keep the concentration of lactate and/or ammonia at low amounts, preferably lower than their toxic thresholds, to improve performance of a cell culture method. Use of the units optionally involves both the trapping (sorption) of compounds and the release (desorption) of compounds that are removed from solutions.

[0047] 1. Fabrication of exemplary filter units:

[0048] In an embodiment, the active part of the units includes carbon-based particles or powders, for example activated carbon or activated charcoal. Activated charcoal is charcoal that has been treated with oxygen at very high temperatures to make it porous, thereby dramatically increasing the surface area of the charcoal, so that it can readily trap or adsorb impurities (e.g., small molecules). Activated carbons (AC) are carbonaceous materials with large specific surface area, superior porosity, high physicochemical-stability, and excellent surface reactivity, extensively used for adsorption of several environmental contaminants, gas separation, heterogeneous catalysis, gas storage, and gas masks, among other uses. Activated charcoal often has a negative charge associated with it due to the presence of oxygenated groups that allows it to bind compounds that are positively charged, or by allowing binding in a donor-acceptor type of relationship with small molecules (such as benzene derivatives like phenols wherein the activated charcoal is the donor and the phenol is the acceptor).

[0049] Examples of activated carbon or activated charcoal particles that can be used in this process include: activated carbon that has been modified with bis(2- ethylhexyl)phosphate (Sigma-Aldrich, St. Louis Missouri) or alternatively unmodified activated carbon (Sigma-Aldrich, St. Louis Missouri), either of which are suited for animal cell culture and/or plant cell culture.

[0050] In an alternative embodiment, sorption units are made using lump charcoal pieces that can be used by smashing them into small particles and using sieves for separating particles with known and uniform particle sizes. The uniform charcoal size can then be embedded in the matrix. By using different sieve sizes, different small particle charcoal can be used. The adsorption rate is lower if these particles are used instead of activated carbon. The lump charcoal that can be used is charcoal that one might typically use for barbequing (that does not have a flame inducing solvent accompanying it).

[0051] In an embodiment, the units are made by physically trapping carbonbased particles, optionally of different sizes and surface area ratios, in a polymeric network. Any polymer capable of forming stable networks with controlled properties can be used. Methyl cellulose (MC) is one example of a polymer that can be used. It is readily isolatable from bark, wood or leaves of plants (such as cotton). In a variation, MC can be used for making these units due to its ability to form a stable porous network when crosslinked with citric acid.

[0052] In an embodiment, the process of making a unit of the present invention is shown in Figure 1 . First, a solution of the polymer is made with a predefined weight percentage, in this case MC is dissolved in deionized water. Methyl cellulose can be purified from plant cell walls, or alternatively it can be prepared by reacting methyl chloride with alkali cellulose (for example by treating the cellulose with a base like sodium hydroxide) to generate the methyl ether of cellulose. [0053] In an embodiment, higher weight percentages of the MC result in slower mass transfer and slower sorption/release rates. Similarly, lower weight percentages of the MC result in faster mass transfer and faster sorption/release rates. Subsequently, a desired amount of carbon-based particles can be mixed with this solution. In the case of MC, citric acid dissolved in deionized water is also added to the mix at a pre-defined amount. Once a homogenous solution is made, defined volumes can be added to molds with predefined shapes and sizes wherein the defined volume is sufficient to completely or partially fill the mold of the predefined size and shape. In an embodiment, the solution is then flash-frozen in liquid nitrogen (so that the mixture goes to the solid state quickly) so as to avoid precipitation or settling of the particles. The samples are then freeze-dried (lyophilized) to form the solid units of the present invention. After freeze-drying, the filter units are heated at defined temperatures for a defined amount of time to initiate the crosslinking reaction with citric acid. In an embodiment, the heating step is specific to MC and might not be needed if other polymeric materials are used. For MC specifically, in one embodiment, the mixture may be heated at 190°C for 4 hours. The heating temperature and the timing affect the degree of crosslinking, and consequently molecular weight of the matrix and ultimately, may affect sorption rate. The amount of crosslinking affects the sorption and release rates. It should be understood that the amount of reactant time and temperature can be modified to get different amounts of crosslinking and different polymeric molecular weights. Thus, it should be recognized that different MCs with different molecular weights can be used to achieve different sorption/release rates.

[0054] Various embodiments may use different molecular weight MC polymer matrices. Higher molecular weight methyl cellulose matrices generally tend to have higher viscosities. Optionally, a MC polymer matrix may be used that is anywhere between 15 and 4000 centipose. Methyl cellulose can be procured from commercially available sources at viscosities from about 15 to about 4000 centipose (Sigma Aldrich, St. Louis, Missouri). In an embodiment, hydroxypropyl cellulose can be obtained at levels between 50-50,000 centipose (Dupont, Wilmington, DE). It should be understood that any of a plurality of different viscosity solutions can be produced. [0055] In other embodiments, other polymer matrices can be used. Alternative polymers include other modified polymers or cellulose ethers, such as ethylcellulose (EC), hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC) and carboxymethylcellulose (CMC). Cellulose is the most abundant polysaccharide found in nature; it is a regular and linear polymer composed of (1— >4) linked [3-d- glucopyranosyl units. This particular [3-(1 — >4) configuration together with intramolecular hydrogen bonds gives a rigid structure. Aggregates or crystalline forms are a result of inter-molecular hydrogen bonds occurring between hydroxyl groups, and this structure makes these polymers particularly useful in the sorption units.

[0056] In a variation, it is contemplated that the filters can be made with other natural or synthetic polymers, such as alginate with calcium chloride as crosslinker, chitosan with aldehydes, such as formaldehyde or glutaraldehyde, as crosslinker, or even different nylons, such as nylon-6, 6. Non-water soluble polymers such as nylon do not require the crosslinking step as they are not readily soluble in aqueous solutions used in culture medium or broth. In the case of using non-water-soluble polymers, it should be understood that other solvents such as formic acid can be used to dissolve the polymer and, after the porous network is formed and solvents are removed. Units with non-water-soluble polymers can also be used directly, similar to the use of water soluble polymers (like methyl cellulose).

[0057] In an embodiment, other premade products where carbon-based particles are trapped within a stable network can be used for the same purpose (sorption and release) but with limited control over adsorption/release profiles. Unfortunately, the premade products may also possess limited applications due to difficulties surrounding their sterilization. However, if the premade product may be able to survive being autoclaved, the premade product may have use in a context where they need to be sterilized. Examples of such premade products include carbon sponge filters, which may be procured from Amazon, (Amazon, Seattle, Washington) or application specific filter, activated carbon loaded paper, Grade 72 (Sigma-Aldrich, St. Louis, Missouri). Because these do not have the capture/release profiles of the filters that are made by the process of the instant invention, they may have more limited uses for removing lactic acid and ammonia from in vitro cell cultures of mammalian cells.

[0058] In one embodiment, figure 1 shows the process of making polymeric units with carbon-based particles embedded in the network matrix. The left most figure shows a polymer solution 1 to which cross-linker 2 (e.g., citric acid) and the carbonbased powder 3 (as described above) are added. A mechanical means of stirring may be added (such as a stir bar 4) so as to facilitate the dissolution of the mix. The resulting mix 6 is added to molds 5 of a certain size and shape and rapidly frozen as shown in the middle panel. The rapid freezing may occur by adding the mold to liquid nitrogen (or liquid helium). In the right most panel, the freeze-dried unit 7 (which has the solvent removed) is shown that is ready for use after heating is performed to complete the crosslinking reaction.

[0059] 2. Absorption/release applications of the units:

[0060] Carbon particles could be added directly to the media or broth in the absence of the polymer matrix. However, if the carbon particles are added directly, then they will show a high-speed adsorption of most if not all of the elements of the media without discrimination. When recycling the media and reusing it is the goal, this method is not applicable. The activated carbon particles will remain suspended in the media and their removal may prove to be difficult. In any event, even if one were able to remove the activated charcoal from the media, the one or more removal step(s) that would be necessary would make the re-use of the media not cost-effective.

[0061] In an embodiment, the units where carbon-based powders are trapped within the polymeric network can be used directly in contact with a liquid such as a cell growth medium. In this case, the unit may remove all of the components of the media, but at a slower rate. Depending on the weight percentage of the polymer solution used in first step of fabrication, the degree of crosslinking, as well as the amount of activated carbon, this rate can be adjusted. Chemical and physical properties of the polymeric network can also affect this rate. For example, the rates can be higher if a hydrophilic polymer, such as MC, is used, as compared to when the network is hydrophobic. A certain degree of control can be applied to the molecules absorbed by the units by controlling the molecular weight of the molecules that can be absorbed. This can be achieved by separating the unit from the solution with a membrane with a defined molecular weight cut-off, for example in the range of 0.1 to 1000 kDa. Membranes with smaller cut-offs allow more strict control over the molecules absorbed to the unit. A 0.1 to 1 kDa cut-off, for example a 500 Da cut-off, can be used where the goal is to remove lactate and/or ammonia (see Figure 2). The membrane 14 is shown in figures 2C and 4C.

[0062] Figure 2A show activated carbon, which has been added directly to the media. The arrows show that molecules present in the media are adsorbing to the activated carbon (the circles). The embodiment in figure 2B shows a unit (the hatched box) which contains the polymer matrix containing activated carbon. Molecules in the media are being removed by the unit as indicated by the arrows. In this embodiment, the media is directly in contact with the unit. Figure 2C shows the unit which is inside a semi-permeable membrane, for example a dialysis membrane. Only the molecules in the media that are smaller than the molecular weight cut-off of the membrane are able to pass through the membrane to be removed by the unit (which contains the polymer matrix and the activated carbon). In this embodiment, the media is indirectly in contact with the unit.

[0063] One alternative to the dialysis membrane that is shown in figure 2C is an embodiment that comprises hollow-fiber or tubular membranes. Similar to dialysis membranes in other forms, hollow-fiber and tubular membranes have defined molecular weight cut-offs and/or pore sizes. They can be embedded within the units (i.e. , within the polymer matrix) in order to allow the passage of a solution through them either actively by perfusion or passively, for example by gravitational force. Molecules that are bigger than the hollow-fiber or tubular membrane cut-off are not able to pass. It is also possible to embed the units inside the fibers or tubes and allow the solution to perfuse around the fibers or tubes (Figure 3).

[0064] Figure 3 shows two embodiments of the present invention. In embodiment 3A, the unit 7 contains hollow-fiber or tubular membranes 8 that allow the passage of solution through the hollow-fiber or tubular membranes 8 in the position and direction of arrows 9. The hollow-fiber or tubular membranes are similar to a flat sheet dialysis membrane because only some molecules that are present in the solution (media) moving through lumens of the membranes (in a vertical direction as shown in Figure 3A) can pass through pores of the membranes to adsorb to the activated carbon in the unit 7 (unit 7 comprises a polymer matrix and the activated carbon inside the polymer matrix). Accordingly, the tubes (hollow fiber membrane or tubular membrane) act as a filter so that the media that enters the tubes perfuses through the tubes. The media enters the tube with a higher concentration of the molecules (for example, lactic acid and ammonia) than are present when the media exits the tubes because as the media traverses the tubes, the molecules that are below the molecular weight cut-off perfuse through the tube to be captured by the unit 7.

[0065] Similarly, embodiment 3B shows tubes 8 (hollow fiber membrane or tubular membrane) wherein the medium traverses through the tubes that are situated in the unit 7. In this embodiment, the medium traverses in the direction of arrows 9 through the tubes by gravitational means. As the medium traverses through the hollowfiber tubes, the small molecules that are smaller than the molecular weight cut-off of the hollow-fiber tubes 8 allows these molecules to perfuse through the hollow-fiber tubes 8 and adsorb to the activated carbon, which is present in unit 7.

[0066] Although one use of these units 7 is removing smaller molecules, the units can also be used to release/desorb other molecules with a controlled rate. In some examples the units may create a constant concentration of those released factors in the solution (e.g., media). In an embodiment, this may be beneficial in the case of molecules that are used by the cells at higher rates, such as glucose, in order to keep their concentration constant (or at least replace some of the glucose that is consumed by cells) and improve the efficiency of the culture system. In order to release the molecules, the units can be placed in contact with a first solution having the target molecules to capture the target molecules. If this unit is put in contact with either a new solution that doesn’t contain the target molecule or only contains lower concentrations, then, either directly or indirectly through a dialysis membrane or tube, assuming the cut-off is larger than the molecule’s size, the unit will release the molecule to the solution at a defined rate (see Figure 4).

[0067] Figure 4A shows an embodiment where the unit 7 is undergoing loading by molecules that are adsorbing to the unit 7. Arrows 11 indicate the general direction of the concentration gradient of the molecules. That is, more molecules are adsorbing to the unit 7 than are being desorbed meaning that the concentration of molecules that absorb to the unit in the medium 10 is decreasing. Optionally, a saturation point of molecules absorbed to the unit 7 is reached. The loaded unit 7 can be used, for example, as in figure 4B, wherein more molecules are desorbed than are being absorbed. If a concentration gradient is introduced to the loaded unit wherein the medium does not have the molecule that is loaded in the unit (for example, if a new medium is introduced that does not contain the absorbed molecule), a concentration gradient is created by the introduction of the newly introduced medium 13. The consequence of the newly introduced medium 13 is to have the unit 7 desorb the molecules such that the net concentration of molecules is moving in the direction of arrows 12. That is, the medium 13 is having the loaded molecule on the unit 7 desorb from the unit 7, so that molecules are being added to the newly introduced medium 13. Similarly, figure 4C undergoes the same net movement (arrows 12) of molecules from the unit 7 into the newly introduced medium 13 even if a dialysis membrane 14 is introduced.

[0068] Alternatively, in another embodiment, the molecules intended to be released from the unit can be used with a carrier and then embedded in the polymeric network containing activated carbon. These carriers could be in the form of polymeric reservoirs and liposomes (Figure 5A), matrix systems (Figure 5B), dendrimers (Figure 5C), or other means that provide controlled release profile and can be inserted into the polymer matrix of the present invention (Figure 5D). The use of these controlled delivery systems provides another level of control over the release (desorption) of the small molecules. [0069] 3. Desorption of the adsorbed/absorbed elements:

[0070] In an embodiment, the molecules removed by a unit can be released by from the unit using a solvent with higher affinity to the target molecules. In one embodiment, acetone can be used for both lactate and ammonia. Alternatively, a 0.1 N NaOH solution (or a NaOH solution of other concentration) can be used with lactate as the capacity of the units to hold lactate decreases with increasing pH, thereby allowing lactate to be desorbed from the unit. Other solvents such as C1-6 alcohols can be used as well. In one variation, isopropanol can be used. If the solvent is chosen with care such that it doesn’t damage the polymeric network, the filter can be reused, optionally indefinitely. The solvent can then be allowed to evaporate, and the desorbed molecules can be collected for other applications. (Figure 6). Figure 6 shows a two step process wherein different molecules that are captured by the unit can be selectively desorbed. First, the filter unit 7 is introduced to a particular solvent 15 that allows the desorption 16 of the molecule of interest. In essence, the particular solvent 15 is extracting the desired molecule into the solvent. In the second step of the process, the solvent 17 is evaporated to leave the molecule of interest 18. The process may be facilitated by using a methodology similar to the process that a rotary evaporator uses. By using reduced pressure, the solvent can be evaporated more rapidly at a lower temperature than would be necessary in the absence of the reduced pressure. In a subsequent step, the process may be repeated in a separate solvent to extract other molecules that may be absorbed to the unit.

[0071] In one embodiment, a classical distillation procedure can be implemented to separate the components based on their molecular weights (using the boiling points) to collect the solvent at the end to be reused in the process. If different components are adsorbed to the units, and those components require different solvents for desorption, a step-wise treatment with these solvents can be performed instead of the distillation process. Thus, the polymeric network can provide a unique process whereby desorption of different absorbed molecules can occur as needed.

[0072] In another embodiment, it is also possible to put solvents in contact with the filters indirectly through dialysis membranes with an incremental increase in their molecular weight cut-offs (Figure 7). This multi-step process will allow different molecules to be adsorbed to different filters based on their molecular weights and simplify the separation of the molecules in the downstream processes. Accordingly, the earlier used filter that is used in conjunction with a lower molecular weight cut-off dialysis membrane will have molecules that are of lower molecular weight than the filters that are used subsequently that are used with higher molecular weight cut-off dialysis membranes. Figure 7 shows a three-step process that illustrates the overall process. In figure 7A, the unit 7a has a molecular weight cut-off dialysis membrane 20 that allows molecules that are smaller than the cut-off to be absorbed by the filter unit 7a. The relative sizes of arrows 19, 21 , and 23 are indicative of the relative molecule sizes by molecular weight that are being absorbed by the filter unit 7. In figure 7B, a molecular weight cut-off dialysis membrane 22 is used that is greater than the molecular weight cut-off dialysis membrane 20 used in 7A. Accordingly, molecules are adsorbed by filter unit 7b that are of a molecular weight that is between the molecular weight cut-off dialysis membrane 20 used in figure 7A and the molecular weight cutoff dialysis membrane 22 used in figure 7B. In figure 7C, no molecular weight cut-off dialysis membrane is used and molecules that are of any size are adsorbed by the filter unit 7c. This allows different filter units 7a-c to have different molecules absorbed by them.

[0073] It should be understood that this process can be used in conjunction with different solvents and with concentration gradients as discussed above so that the process can be made even more selective in isolating molecules that are adsorbed/desorbed by/from the filter unit.

[0074] One advantage to the use of the filter units of the present invention relative to using activated carbon without a polymeric matrix is the facility with which the filter units can be removed. Because the filter units are self contained with the activated carbon in them, they can simply be removed by lifting them out of solution. In contrast, activated carbon needs to be filtered, which not only requires additional time but sometimes leads to the inability to remove all of the activated carbon from the solution (media). [0075] Regarding the desorption rate, the present invention contemplates that different solvents can be used to adjust the rate of desorption with the possibility that mixes of solvents may be used to give the ideal desorption rate. Moreover, the use of dialysis membranes may be used to also adjust the rate and the types of compounds that are desorbed from the units. By varying the number of units used, the polymeric composition of the units, different molecular weight cut-off dialysis membranes, and the types of solvents used, one can achieve an almost infinite number of possible desorption rates.

[0076] In an embodiment, the present invention relates to compositions and methods of creating and using filter units that comprise a polymeric matrix into which activated carbon has been inserted. In a variation, the present invention relates to a filter unit that comprises a) a polymer matrix and b) activated carbon. In a variation, the polymer matrix comprises a cellulosic polymer matrix. In a variation, the polymer matrix comprises methyl cellulose.

[0077] In an embodiment, the activated carbon is activated charcoal and the activated charcoal is present in the polymer matrix. In a variation, the filter unit is substantially free of solvent. In a variation, the filter unit is substantially free of water because any solvent that was present has been removed by evaporation or by freeze drying.

[0078] In an embodiment, the filter unit further comprises a semi-permeable membrane, for example one or more of a dialysis membrane or a hollow-fiber or tubular membrane. The semi-permeable membranes may have a molecular weight cut-off of 0.1 -5000 Da, or alternatively, 0.1 -4000 Da, or alternatively, 0.1 -3000 Da, or alternatively, 0.1-2000 Da, or alternatively 0.1-1000 Da, or alternatively, 0.1 -500 Da.

[0079] In an embodiment, the filter unit may be further used with carriers that may be in the form of polymeric reservoirs and liposomes that are often used as drug delivery vehicles, in other matrix systems, with dendrimers, or with other carriers that provide a controlled release profile and can be inserted into the polymer matrix. [0080] In an embodiment, the polymeric matrix is methyl cellulose. Alternatively, the polymeric matrix is a different cellulosic polymer, an alginate polymer, a chitosan polymer, or a nylon polymer.

[0081] In an embodiment, the filter unit is capable of removing lactate and/or ammonia. In a variation, the filer unit removes both lactate and ammonia. In a variation, the lactate and/or ammonia can be desorbed from the filter unit. In some examples, or depending on the cells being cultured and the culture method, the filter unit may also remove other compounds such as reactive oxygen species, glutamate, urea, free radicals or peroxides.

[0082] In an embodiment, the filter unit further comprises a crosslinker. In a variation, the crosslinker allows the crosslinking of the methyl cellulose to generate a polymer matrix that is able to incorporate activated charcoal. In a variation, different and varying amounts of crosslinker and methyl cellulose can be added to change the physical properties of the polymer matrix.

[0083] In an embodiment, the crosslinker is citric acid, and the filter unit is made by dissolving the polymer matrix into a solvent, adding the activated carbon and crosslinker to form a solution, optionally heating the solution, pouring the solution into a form, freezing the form containing the solution, and freeze drying the solution to remove the solvent. In a variation, the filter unit is removed from the form and added to a solution that contains a molecule that is to be removed from the solution.

[0084] In an embodiment, the present invention relates to methods of producing a filter unit comprising: dissolving a polymer or a monomer into a solvent to generate a solution, adding a crosslinker and activated carbon to the solution to generate polymer matrix that comprises the activated carbon in a mixed solution, pouring the mixed solution into a form, rapidly freezing the mixed solution and the form, and lyophilizing the mixed solution to remove the solvent. In a variation, the monomer that is added to the solution polymerizes to form a polymer. In an embodiment, the polymer matrix may comprise a homopolymer, a co-polymer, or a terpolymer. In a variation, different monomers may be added to the solvent to generate a co-polymer or a terpolymer. In an embodiment, the monomer may be glucose or another sugar. In an embodiment, the sugar may be allose, altrose, glucose, mannose, gulose, idose, galactose, or talose.

[0085] In an embodiment, the method uses a polymer matrix that comprises one or more members selected from the group consisting of a cellulosic polymer, an alginate polymer, a chitosan polymer, and a nylon polymer. In a variation, the crosslinker may be citric acid, calcium chloride, formaldehyde or glutaraldehyde. In a variation, the polymer matrix comprises a nylon that is nylon-6, 6.

[0086] In an embodiment, the method uses a polymer matrix that comprises methyl cellulose that has been crosslinked with citric acid. In a variation, the method further comprises a heating step. In a variation, the heating step is used to crosslink the polymer and/or to allow the activated charcoal to incorporate into the polymer matrix.

[0087] In an embodiment, the present invention relates to a method of removing a molecule from a solution, the method comprising adding a filter unit to the solution, wherein the filter unit comprises a polymer matrix that contains activated carbon, allowing the filter unit to adsorb the molecule, and optionally removing the filter unit from the solution, thereby removing the molecule from the solution. In a variation, the activated carbon is activated charcoal, and the activated charcoal is present inside the polymer matrix, and wherein the polymer matrix is methyl cellulose. In a variation, the polymer matrix has been crosslinked with a crosslinker. In a variation, the crosslinker is citric acid. In a variation, the molecule is recovered by desorbing the molecule from the filter unit.

[0088] In an embodiment, the method of the present invention relates to removing lactic acid and/or ammonia from a cell culture media using the filter units as described above. In a variation, the lactic acid and/or ammonia can be recycled and used for other purposes. In a variation, the filter unit can be recycled and re-used to remove more lactic acid and/or ammonia. In a variation, the filter unit can be used in conjunction with a dialysis membrane. In a variation, the filter unit can be used with deionized water or another appropriate solvent. Other solvents include lower alkanols, acetone, or other solvents that may be used in biological systems. In a variation, the filter unit comprises a polymeric matrix that is made by combining methyl cellulose, activated carbon/charcoal, and citric acid. In a variation, the filter unit does not contain any solvent. In a variation, the filter unit is added to a bioreactor that is undergoing cell growth/expansion to remove lactic acid and/or ammonia as the cells expand/propagate. In a variation, a plurality of filter units can be used to more rapidly remove the lactic acid and/or ammonia. In a variation, the polymer matrix physical properties can be adjusted so as to adjust the rate of removal of the lactic acid and/or ammonia. In a variation, the physical properties can be adjusted by adjusting the viscosity of the polymer matrix, and/or the concentrations of the various components that are used to make the polymer matrix.

[0089] In a cell culture process or bioreactor, a sorption unit may be placed in communication with a cell growth medium (i.e. medium containing the cells or in contact with the cells) while the cells are growing. Cell growth may include one or more of cell population expansion, cell differentiation, or cell construct remodeling.

[0090] In some examples, the sorption unit is located within the same vessel that contains the cells or otherwise in direct contact with a volume of the medium containing the cells. In this case, no fluid flow devices, for example tubes or pumps, are needed to move the medium from a first vessel containing the cells to a second vessel containing the sorption unit. In this way, the need for equipment such as a second vessel, tubes or pumps is reduced. Further, since circulation of a medium typically involves an increase in the total volume of the medium required, the consumption of the medium may be reduced. However, in other examples the sorption unit may be located in a separate vessel. In these examples, the flow rate between the cell culture vessel and the vessel containing the sorption medium may be controlled to adjust the rate of sorption. In this case, the increase in total medium volume may be minimized by providing a small liquid volume in the second vessel or by flowing the media through a tube, which may be a hollow fiber or tubular membrane as shown in Fig. 3A or a more porous tube. [0091] In bioreactors where the medium is mixed, for example by way of an impeller or by movement of the bioreactor (for example as in a shaker flask, wave bioreactor or roller bottle) or by alternating suction and expulsion of the medium, mixing the medium may also enhance flow of the medium around the outside of sorption unit and/or renew the medium around the sorption unit. However, the medium is not forced through the sorption, i.e. into one side of the matrix, through pores of the matrix and out another side of the matrix, by way of a pressure or pressure differential. T urbulence or impulses resulting from movement of the medium tend to be random, localized and/or with minimal net force directed into the matrix such that the average pressure around the matrix is essentially the ambient pressure or static head pressure of the medium in the vessel. While the pores are not necessarily configured to exclude the bulk flow of liquid through the matrix, the pores provide channels of material length, for example at least 1 mm on average, where bulk liquid flow is restricted by the small cross-sectional area of the pores. The transport of small molecule compounds, such as lactate and ammonia, from the medium into the matrix and into contact with the sorption material may thereby be primarily driven by diffusion even when the medium is refreshed around the outside of the matrix. Refreshing the medium around the outside of the matrix is optional but may be useful, in particular where a large or uncontrolled concentration gradient might otherwise develop between an area where the cells are concentrated and the sorption unit.

[0092] In some examples, the sorption unit is separated from the cells such that cells do not contact the sorption unit directly. In the case of adherent cell or cells grown into 2D or 3D constructs, the separation may be provided by a distance or space between the sorption unit and the cells. For example, the sorption unit may be spaced apart from surfaces or structures (e.g. pillars or scaffolds) holding the cells. Optionally, the sorption unit may be removed from the bioreactor during cell seeding procedures and put into the bioreactor after cells have settled or adhered to a surface or structure of the bioreactor. Alternatively or additionally, the sorption unit may be separated from cells by a semi-permeable barrier, such as a mesh or membrane, with openings sized to exclude the cells, or the pores of the matrix may be sized to exclude cells. Optionally, the semi-permeable barrier may be a dialysis or ultrafiltration membrane sized to also exclude selected proteins or other large molecules, for example growth factors, or selected particles, for example viruses or exosomes, from the sorption unit. In the case of non-adherent cell culture, the cells may be permitted to intermittently contact the sorption or the cells may be separated from the matrix by a semi-permeable barrier.

[0093] Referring to Figures 10-16, a cell culture bioreactor 100 has a substrate 102 and a holder 104. The substrate 102 is in the form of a dish and functions as a vessel for holding cells and a liquid cell growth and/or differentiation medium. In this example, the substrate 102 is an elastomer such as silicone. The holder 104 supports and stabilizes the substrate 102. While cells are growing, the bioreactor 100 may be placed inside an incubator or biosafety cabinet to provide a suitable temperature and gaseous environment around the bioreactor 100. In other examples, a more complex bioreactor may have an enclosed vessel and integrated fluid handling and process control devices. Optionally, the bioreactor 100 may be used to grow a cell sheet on the bottom of the substrate 102 as described in US Patent No. 11 ,718,830 B1 , Silicone- based Membrane Surface Chemistry and Topography Control for Making Self- Assembled Cell Sheets with Cell Aligning and Positioning, which is incorporated herein in its entirety by this reference to it. As described therein, cells are seeded into the substrate 102 to partial or full confluence and grown (differentiated and optionally expanded) to form a layer of cells and cell produced extra-cellular materials and matrices. Liquid media is removed and refreshed or replaced with other media as required, for example by aspirating old media from the substrate 102 and pipetting in new media into the substrate 102. Optionally, additional cells may be added in the same manner to form additional layers over the first layer. After a period of time, a cell sheet may be scraped from the bottom of the substrate 102.

[0094] In this example, a filter unit 106 is added to the bioreactor by immersing part of the filter unit 106 in media held by the substrate 102. The filter unit 106 has a frame 108 that rests on the top of the substrate 102. The frame 108 may have one or more vents 110 to allow gasses to escape from the frame 108 if required to allow media to reach the filter unit 106. Optionally, the frame 108 does not completely cover the substrate 102 but instead leaves openings 112 for gas exchange and/or for cell seeding or media exchanges. Optionally, the filter unit 106 may be removed when cells are added and replaced after the cells have settled and/or adhered to the substrate 102.

[0095] A sorption unit 114 is provided in the frame 108. A portion of the frame 108 extends downwards through a free surface of a liquid medium held in the substrate 102 to immerse some or all the sorption unit 114 in the medium. The sorption unit 114 has particles of a sorbent material dispersed in a porous matrix. In this example, the filter unit 106 optionally or selectively includes a semi-permeable membrane 116. The semi-permeable membrane 116 is associated with the sorption unit 114 by covering part of the opening at the bottom of the frame 108. In this example, the semi- permeable membrane 116 covers an opening in a holder 118 selectively attached to the bottom of the frame 108. Optionally, the holder 118 may retain the sorption 114 in the frame 108 or the sorption 114 may be connected directly to the frame 108. The semi-permeable membrane 116 prevents selected compounds in the medium from contacting the sorption unit 114. For different applications, a holder 118 may be provided with a semi-permeable membrane 116 of a selected appropriate pore size. Optionally, a holder 118 may have a mesh or other open material in place of the semi- permeable membrane 116 or the holder 118 may be omitted.

[0096] In other examples, a filter unit 106 may be otherwise held in a vessel containing a liquid medium and cells, above or below an upper surface of the medium, which may be a free surface or a surface defined by the vessel.

[0097] As the cells grow, compounds such as lactate and/or ammonia diffuse into the matrix and are sorbed. These compounds are thereby prevented from inhibiting growth of the cells. In this way, the consumption of cell growth or differentiation media may be reduced. For example, the volume of a medium held by the bioreactor may be reduced and/or the frequency of full or partial media exchanges may be reduced. Optionally, to reduce the sorption of beneficial compounds in the media, the sorption unit may be pre-saturated in a medium, or pre-saturated in one or more selected components of a cell growth or differentiation medium, for example components that able to pass through the semi-permeable barrier if any, before being placed in the bioreactor. Optionally, the sorption unit may also be saturated in one or more beneficial compounds, for example glucose, before being placed in the bioreactor or otherwise in fluid communication with the cells. In this way, the sorption unit may also be used to supply one or more beneficial compounds to the bioreactor, medium or cells. In cases where the absence of these beneficial compounds would be inhibitory, the consumption of media may be reduced. For example, the volume of media held by the bioreactor may be reduced and/or the frequency of media full or partial media exchanges may be reduced.

[0098] Cell culture typically proceeds for a defined period of time, for example 1 to 30 days. The amount of one or more inhibitory compounds, for example lactate or ammonia, that will be produced by the cells, and the amount of the inhibitory compound that can be tolerated at the end of the cell culture period, may be calculated in advance. Sorption unit requirements can be calculated based on experimentally derived data involving the sorption unit and the estimated production of inhibitory compounds. One or more sorption units may be present in the bioreactor at any time. Optionally, an initial number of sorption units may be removed and/or one or more new sorption units may be added during a cell culture run.

[0099] While lactate (lactic acid) and ammonia (ammonium) are inhibitory in high concentrations, both of these substances may be useful in lower concentrations. For example, some cells metabolize ammonium or lactate at lower concentrations. Lactate is used by some cells as a signaling molecule and can be used to control cellular behavior. In some examples the interaction between ammonia and lactic acid can help buffer the cell culture environment. Sudden changes in ammonium or lactate can disturb pH or other process parameters. Further, measurements of ammonium or lactate concentration may be useful in monitoring cell growth or process control. Complete and/or rapid removal of ammonium or lactate may be undesirable in some cell culture processes. Further a material that removes lactate and ammonia at high rates is likely to also remove other small molecules at high rates which may be undesirable in some cell culture processes. Accordingly, in at least some examples, the delayed sorption provided by the sorption unit is desirable relative to directly exposing the media to a sorbent such as activated carbon. In cases where the sorption unit is used to deliver a nutrient, a delayed or sustained release of the nutrient may be preferred over a rapid release of the nutrient.

[00100] Lactic acid is widely used in different industries, including medicine, brewing, and in the manufacture of different foodstuffs. Lactic acid can also be used as an ingredient in the production of biodegradable plastics that are renewable. Ammonia is also used in many different applications, such as its use for green fuels in transportation, fertilizers in agriculture, and other synthetic and therapeutic applications. Lactic acid (or lactate) and ammonia (or ammonium) extracted from cell culture medium may be recovered from the sorption unit and used for various purposes, including the applications described above. For example, adsorbed materials can be desorbed by treating activated carbon with solvents such as acetone or sodium hydroxide solution for use in other applications.

[00101 ] The sorption unit may be in any shape such as a sphere, cylinder, cube, sheet, or rectangular prism. Since, it at least some examples, liquid is not forced through the matrix, the matrix does not need to be thin in any direction. In some example, a matrix that includes at least a material amount, for example 50% or more, of pores that are at least 1 mm long, delays the sorption of media component by the particles dispersed in the matrix. Optionally, the sorption unit is at least 1 mm, at least 3 mm, or at least 5 mm thick.

[00102] In some examples, the matrix is made of a polymer. Optionally, a polymer matrix may be swellable and absorb the liquid medium. In some examples, the matrix may be made of a non-polymer, such as a metal or ceramic. For example, a porous ceramic matrix may be made by self-assembly or sintering. In some examples, the matrix may be made of fibers, for example in the form or a fabric or yam. The sorbent particles may be attached to the fibers or physically trapped between fibers. [00103] Examples

[00104] Example 1 . Fabrication of methyl cellulose (MC) based filter units

[00105] Filter units were made using a 6 %wt/V solution of MC 15 centipoise (cp). After MC was dissolved, citric acid and activated carbon were added to achieve 1 %wt/V for both. Molds with 1 x1 cm cross-section were used with 1.5 mL of this solution. After the filter units were flash-frozen and freeze-dried, heating was performed at 190°C for 4 hrs to complete the crosslinking and stabilization of the units. The cross- sectional and top view of the units is shown in Figure 8D.

[00106] Example 2. Removal and release of toluidine blue

[00107] Toluidine blue (TB) was used as a target molecule to show the case of both sorption and release (desorption) of the small molecules from the units. TB was used because it can be visualized by the naked eye or measured by way of the absorbance of light. A calibration curve was prepared for TB by measuring the absorbance of its solution at different concentrations ranging from 0-50 pg/mL at 590 nm. The results can be seen in the graph shown in the left-hand graph of figure 8A. Using Beer’s law, the extinction coefficient was calculated.

[00108] To first show the ability of the filter units of example 1 in removing TB from its aqueous solution, three conditions were used with a 50 pg/mL TB solution and samples were taken every few hours to measure the absorbance of the TB solution. First, activated carbon powders were used without the MC polymeric network. Second, the filter units (comprising the polymeric network and the activated carbon) were used in direct contact with the solution. And third, the units were used indirectly through a dialysis membrane with a 2 kDa molecular weight cut-off. The activated carbon powders adsorbed the entire TB in less than 1 hour while it took up to 24 hours for the filter units to remove the majority of the TB. Although the molecular weight of TB was significantly smaller than the molecular weight cut-off of the dialysis membrane, the presence of the dialysis membrane slowed down the removal of the TB from the solution (see the right-hand graph of Figure 8A). [00109] In the next experiment, the units were exposed each to 10 mL of a 50 pg/mL solution of TB for 24 hours. An absorbance measurement revealed an almost complete removal of TB from the solution. These saturated filter units were then put in direct and indirect contact with 2 mL deionized water, either directly by placing the unit directly in the deionized water or indirectly through 2 kDa dialysis membranes, and the absorbance of the TB aqueous solution was measured every few hours over a 24-hour period. Both the direct and indirect contact showed the slow release of TB and, similar to before, the presence of the membrane resulted in a slower release rate. The release rate was also significantly slower than the removal rate (Figure 8B).

[00110] Loaded filter units were also treated with both acetone and isopropanol, and desorption of TB molecules started in both cases after only 5 minutes (Figure 8C).

[00111 ] These results show that the filter units, in this example comprising a polymeric matrix and activated carbon, are effective at removing a molecule such as TB from a solution. Although the sorption rate is not as fast for the filter units relative to activated carbon without the polymer matrix, it should be understood that this slower rate may be desirable in cases where one wants removal to be slower. It is expected that one can add additional filter units to speed the process so that the rate can be adjusted to the ideally desired rate. Moreover, the polymeric matrix can be manipulated as described herein to adjust the rate of uptake of one or more selected chemical species.

[00112] Example 3. Pore size and porosity measurements

[00113] Filter units made as described in Example 1 were cut from their horizontal and vertical planes and were observed under scanning electron microscope (SEM). Views of the vertical plane showed long fibers going from down to top (as shown in Fig. 9A) but pores of the filters were observed from the horizontal plane (as shown in Fig. 9B). The size ranges of these pores were measured using Imaged. 50 pores were manually selected, and their surface area was shown using a histogram (shown in Fig. 9C). These pores range from 200 to 13000 pm ² in area but the majority of them are smaller than 2000 pm ² . [00114] Porosity of the filter units was measured using ethanol displacement (liquid substitution) method. Filter units were submerged in a known volume of ethanol (Vi). Ethanol was used to avoid swelling or dissolution of the filter units. After 2 hours of treatment on ice, the total volume of the solution and filter was measured (V2). The filter unit was then removed, and the remaining volume of the ethanol was measured (V3). This was repeated four times and the porosity of the filter units (82.83±10.24%) was measured using Equation 1 .

[00115] Equation 1

[00116] Example 4. Removal of glucose

[00117] Performance of the filter units of Example 1 in removing glucose was tested by using glucose test paper strips. A 2wtA/% solution of glucose was prepared in deionized water and deionized water without any glucose was used as control. A filter unit with dimensions of 1 .5*1 ,5x0.5cm was used with 10mL of the glucose solution and after 15 minutes, measurements were performed. The concentration of the glucose solution had lowered from 2 to 0.1 % during this time.

[00118] Example 5. Fabrication of nylon-based filter unit

[00119] Nylon 6,6 is dissolved in formic acid inside a fume hood. Different concentrations of the polymer solution (2-20 wtA/%) can be used to create filters with different porosities to achieve different filtration rates. Higher polymeric solution concentrations result in lower filtration rates.

[00120] After a couple of hours of stirring and once a homogenous polymeric solution is achieved, the desired amount of activated carbon powder is added to the solution and stirring is continued to homogenize this blend as well. Activated carbon amount can be in the range of 1 -25 wtA/%. Higher amounts of the powder result in faster removal rates.

[00121 ] Once the solution is homogenized, the porous filter units are made with adding the polymer and carbon solution to deionized water as the antisolvent of the polymer. While a 200mL volume of deionized water is stirred at speed of 500rpm, 1 mL of the polymer and powder solution is added to it using a 1 mL pipette tip. The stirring is continued for 10 minutes until the entire formic acid is dissolved in the deionized water and the nylon 6,6 is solidified. At this point the filter unit that now has a fibrous or porous structure is transferred to another beaker containing 200m L of fresh deionized water and is stirred for another 30 min at a speed of 200rpm to make sure all of its solvent is removed.

[00122] After this, the filter units are dried at room temperature over night to remove any water residues.

[00123] Example 6. Fabrication of polycaprolactone-based filter unit

[00124] Polycaprolactone (PCL) with different molecular weights (either 45kDa or 90kDa) is dissolved in 1 :9 volume ratio of dichloromethane and ethanol. Different concentrations from 0.5 to 5 wtA/% can be used for making filter units depending on the molecular weight of the polymer used and the desired properties of the final filter. The desired amount of activated carbon can be added to the solution similar to before.

[00125] Once the solution is homogenized, the polymeric solution is used using electrospinning to create nonwoven electrospun mats. Electrospinning parameters can be within this range: Nozzle can have gauge 16-20, the distance between the tip of the nozzle and the collector can be 5-15cm, the DC voltage between the nozzle and collector can be 2-20kV.

[00126] Electrospinning parameters can be adjusted based on the polymeric solution concentration, nozzle gauge, distance and voltage between nozzle tip and collector surface to create mats with smaller fibers (smaller than 1 pm in diameter), larger fibers (larger than 10pm), or mats with mixed fiber sizes. Porosity and pore sizes of the mats can also be controlled by adjusting these parameters. These parameters can be adjusted to increase or decrease filtration rates.

[00127] PCL is a biodegradable polymer and depending on its molecular weight and fiber sizes in the mats, the biodegradation process can take from 6 months to more than 2 years. This can be beneficial from a sustainability perspective. [00128] It should be understood and it is contemplated to be within the scope of the present invention that any feature that is disclosed or used herein can be combined with any other feature even if they are not mentioned together as long as they are compatible with each other. When ranges are mentioned, it is within the scope of the invention that any endpoint that fits within that range is contemplated as an end-point for a sub-range, even if that sub-range and/or end point is not specifically recited. Moreover, modifications to the present invention can be made that do not deviate from the spirit and scope of the present invention. In any event, the present invention is defined by the below claims.