Fluidized bed extracorporeal treatment device

Abstract

The disclosure provides an extracorporeal devices, systems and methods for removing a pathogen or a toxin from a biological fluid of a subject comprising contacting a biological fluid from the subject with a loosely packed bed of adsorption media, such as a fluidized bed, wherein the adsorption media comprises heparin, to remove the pathogen or the toxin to form a treated biological fluid; and infusing the treated fluid into the subject.

Description

FLUIDIZED BED EXTRACORPOREAL TREATMENT DEVICECROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 603,913, filed November 29, 2023, which application is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Devices can be used to filter or separate various constituents from fluids. Filters can contain adsorbents and media materials that separate based on size, affinities, and other functionalities of the constituents of the fluids.

[0003] Despite the advances in the field, there is a need in the art for media beds that are efficient separators. The present disclosure satisfies this need and offers other advantages as well.BRIEF SUMMARY

[0004] The present disclosure relates to devices and methods for separating and removing pathogens, toxins, bacteria, cancer cells, cancer mediators and viruses from biological fluids. The disclosure has particular advantages in connection with separating and removing pathogens and toxins from whole blood and blood components, such as plasma and serum.

[0005] As such, in one embodiment, the present disclosure provides an extracorporeal method for removing a pathogen or a toxin from a biological fluid of a subject, the method comprising: contacting a biological fluid from the subject with a loosely packed bed of adsorption media, wherein the adsorption media comprises heparin, to remove the pathogen or the toxin to form a treated biological fluid; and infusing the treated fluid into the subject.

[0006] The method provides a loosely packed adsorption bed, which in certain aspects is a fluidized bed.

[0007] In another embodiment, the present disclosure provides a device for removing a pathogen, a cancer cell or cancer mediator, or a toxin from a biological fluid of a subject, the device comprising: a container having a loosely packed bed of adsorption media disposed therein, wherein the adsorption media comprises heparin; and wherein the container has a first screen or endplate and a second screen or endplate to retain the loosely packed bed of absorption media.

[0008] The device provides a loosely packed adsorption bed, which becomes a fluidized bed during use.

[0009] These and other aspects, objects and advantages will become more apparent when read with the figures and detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 illustrates a system embodiment of the present disclosure.

[0011] FIG. 2 illustrates a device embodiment of the present disclosure.

[0012] FIG. 3 illustrates a system for removing a pathogen or a toxin or a cancer cell from a biological fluid of a subject.DETAILED DESCRIPTION

[0013] The present disclosure relates in part to methods and devices for treating biological fluids such as blood to remove toxins and pathogens. The methods and devices use a bed of adsorption media that becomes a fluidized bed in use. The adsorption bed treats the sample such as blood to cleanse it. The treated or “cleansed” sample can then be continuously or intermittently reinfused into the subject. Advantageously, the current devices and methods remove toxins, pathogens and / or cancer cells or mediators, and thereby cleanse the biological fluid in a therapeutic, prophylactic or diagnostic procedure.

[0014] In certain aspects, the containers having adsorption media disposed therein do not have an adsorption bed compression in use. Instead, the bed is loosely packed such that the adsorption media is not pressed together as typically the use of compression results in flow constrictions or dead-end flow spaces. In certain aspects, by using adsorption media which isnon-rigid, gel-like or a deformable media with heparin bound, or affixed thereto, the bed of adsorption media becomes fluidized in use.I. Definitions

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein the following terms have the following meanings.

[0016] As used herein, the terms “about” and “approximately equal” are used herein to modify a numerical value and indicate a defined range around that value. If “X” is the value, “about X” or “approximately equal to X” generally indicates a value from 0.90X to 1.10X. Any reference to “about X” indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. Thus, “about X” is intended to disclose, e.g., “0.98X.” When “about” is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 6 to 8.5” is equivalent to “from about 6 to about 8.5.” When “about” is applied to the first value of a set of values, it applies to all values in that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%, about 9%, or about 11%.”

[0017] As used herein, the terms “comprising” or “comprises” are intended to mean that the compositions, devices, and methods include the recited elements, but do not exclude others. “Consisting essentially of’ refers to those elements required for a given embodiment. The phrase permits the presence of additional elements that do not materially affect the basic and novel or functional character! stic(s) of the given embodiment (e.g., compositions, devices, and methods). “Consisting of’ refers to compositions, devices, methods, and respective components thereof, as described herein, which are exclusive of any element not recited in that description of the embodiment. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0018] As used herein, the term “adsorption media” refers to a material having a surface that is modified, functionalized, coated, and the like, with a composition (i.e., adsorbent) which comprises heparin, heparan sulfate, and mixtures thereof. Optionally the adsorption media may have the inherent surface properties needed to bind the desired adsorbates without the need for a coating. In certain aspects, the adsorbent is a glycosaminoglycan, such as heparin, heparan sulfate, and mixtures thereof, and, optionally one or more additional polymers or carbohydrates or glycosaminoglycans, such as, for example, polyethyleneimine,chondroitin sulfate, dermatan sulfate, keratan sulfate, sialic acid / sialylated glycans, and / or hyaluronic acid.

[0019] As used herein, the term “adsorbate” refers to a pathogen, cancer cell, cancer mediator, or toxin that has an affinity for the adsorbent. In the context of the present disclosure, a sample obtained from a subject will contain one or more adsorbates such as pathogenic bacteria or a cancer cell. When contacted with the adsorption media of the present disclosure, the adsorbates bind to the surface of the adsorption media and are thereby removed from the sample. Examples of pathogens that can be removed from the blood using a heparinized substrate according to the disclosure include viruses such as adenovirus, coronavirus, dengue virus, Hepatitis B, Hepatitis C, HIV, HPV, Cytomegalovirus, and others. Bacteria such as Bacillus anthracis, Chlamydia pneumoniaem, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, MRS A, Streptococcus pyrogenes, Yersinia enterocolitica, and others. Parasites such as Giardia lambitia, plasmodium spp. and others.

[0020] As used herein, the term “cancer cell” includes circulating tumor cells (CTCs) and cancer stem cells (CSCs) that shed from a tumor, organ or tissue. Cancer cells are cells that divide continually, forming solid tumors or flooding the blood or lymph with abnormal cells. Cancer cells are created when the genes responsible for regulating cell division are damaged.

[0021] As used herein, the term “cancer mediator” includes a circulating cell or protein that contributes to the pathogenesis or metastases of tumors. Examples of such mediators include, but are not limited to, circulating tumor cells responsible for metastases, circulating growth factors that contribute to angiogenesis of tumors, circulating cytokines that contribute to angiogenesis, circulating heparanases that degrade heparan sulfate segments on endothelial cell walls that can lead to tumor cell invasion, and circulating fibrin or thrombin that leads to venous thromboembolism. An “adsorbate” includes cancer mediators.

[0022] As used herein, the term “fluidized bed” refers to a solid adsorption media dispersed in a fluid and acting as a fluid. A fluidized bed refers to a mixture state of two different phases, solid and fluid. The fluidized bed behaves as a fluid material. A fluidized bed is a physical phenomenon that occurs when solid such as an adsorption media such as heparinized media, disposed within a container or cartridge under the right conditions behaves like a fluid including a gaseous fluid. For example, the adsorption media being loosely packed without intentional compression within the container, or an inert gas being pumped into the container, or the velocity of the fluid being pumped into the container is high enough to create afluidized bed, or when pressurized fluid enters the adsorption media are all illustrative ways to achieve fluidization. In one aspect, the fluidized bed comprises solid particles (such as ridged beads) kept in motion by a downward or an upward flow of a fluid, such as whole blood. In certain aspects, fluidized beds operate by balancing the downward gravity forces of the weight of the adsorption particles with the upward forces created by high gas and / or fluid flow. Fluidization or suspension can be carried out on a large range of material densities and particle sizes.

[0023] As used herein, the term “sample” refers to any biological sample that could contain an analyte / adsorbate obtained from a subject infected with a pathogen or toxin. Non-limiting examples of samples include whole blood, serum, and plasma.II. Embodiments

[0024] In one embodiment, the present disclosure provides an extracorporeal method for removing a pathogen, cancer cell or mediator, or a toxin from a biological fluid of a subject, the method comprising: contacting a biological fluid from the subject with a loosely packed bed of adsorption media, wherein the adsorption media comprises heparin, to remove the pathogen or the toxin to form a treated biological fluid; and infusing the treated fluid into the subject.

[0025] The method can be a continuous or a batch process.

[0026] In certain aspects, the loosely packed bed of adsorption media is a fluidized bed. In certain aspects, the loosely packed bed of adsorption media allows the media to act as a fluid. The solid media can act as a fluid due to contact with the biological fluid. In certain instances, the biological fluid is blood. In certain aspects, the media comprises heparin.

[0027] In certain aspects, the heparin polymers are “full-length” having a mean molecular weight within the range of from about 6 kDa to about 40 kDa, such as, for example, about 11 kDa, or about 6, 8, 10, 12, 14, 15, 16, 18, 20, 21, 22, 24, 26, 28, 30, 32, 34, 36, 38 or about 40kDa or about 12 kDa to about 15 kDa.

[0028] In certain aspects, the adsorption media is Sepharose. Sepharose is a bead-formed gel prepared from agarose by a purification process that removes the charged polysaccharides and gives a gel with only a small number of residual charged groups. Sepharose is stable inwater and salt solutions over the pH range of 4 to 9 and in the absence of oxidizing agents. Cross-linked Sepharose is called Sepharose CL.

[0029] In certain aspects, the adsorption media is Sephadex. Sephadex is a bead-formed gel prepared by cross-linking dextran with epichlorohydrin. The gel is hydrophilic due to the large number of hydroxyl groups, and it therefore swells readily in water and electrolyte solutions. The G-types of Sephadex differ in their degree of cross-linking and hence in their degree of swelling upon immersion in aqueous fluids, and their fractionation range.

[0030] In certain aspects, the adsorption media is Sephacryl. Sephacryl is prepared by covalently cross-linking allyl dextran with N,N-methylene bisacrylamide and gives a rigid gel with a carefully controlled range of pore sizes.

[0031] In certain aspects, the adsorption media is a hollow bead such as hollow spherical cellulose beads or hollow microspheres. The hollow beads can be made of a carbohydrate, synthetic or natural polymer, dextran, agarose, or other deformable materials.

[0032] In certain aspects, the adsorption media is a foamed polymer bead. The polymer can be a thermoplastic polymer.

[0033] The size of the substrate can be selected according to the volume of the sample to be treated or other parameters. In some aspects, each bead of the plurality of hollow beads has an average outer diameter of about 1 pm to about 1 mm, e.g., 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 45 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, or 1 mm. In certain aspects, the hollow beads are about 100 pm to about 600 pm, or about 100 pm to about 500 pm, or about 100 pm to about 400 pm, or about 100 pm to about 300 pm.

[0034] In other aspects, each bead of the plurality of hollow beads has an average diameter of about 10 pm to about 400 pm, e.g., 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, 45 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 105 pm, 110 pm, 115 pm, 120 pm, 125 pm, 130 pm, 135 pm, 140 pm, 145 pm, 150 pm, 155 pm, 160 pm, 165 pm, 170 pm, 175 pm, 180 pm, 185 pm, 190 pm 195 pm, 200 pm or more. Generally, a particle size in the range of 20-400 pm, or 20-300 pm or 20-200 pm such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 pm is useful, butin high flow rate applications larger particles may be required. In certain instances, particles at sizes of 120 pm and below are used with plasma and serum.

[0035] The solid substrate may comprise one or more hollow or solid fiber pieces or particles. In an embodiment, wherein the solid substrate comprises hollow fibers, the hollow fibers can comprise a material selected from the group consisting of polysulfones, polyfluorocarbons, polyamides, polynitriles, polypropylenes, cross linked alginates, and cellulose. Other materials commonly used in hollow fibers for medical applications may also be employed. The hollow fiber may comprise a polysulfone.

[0036] In certain instances, the adsorption medium includes a group of porous polymeric particles, which are formed to selectively retain various adsorbates. The polymeric particles are predominantly mesoporous, with a pore size ranging from 2 to 70 nm or from 5 to 50 nm.

[0037] In certain instances, each polymer particle possesses a porous hydrophobic core. The surface of the hydrophobic particles can be modified to provide a hydrophilic coating, which imparts a high degree of biocompatibility with blood. The hydrophilic coating is desirably thin and permeable so as to allow penetration of various adsorbates to the hydrophobic porous core of the particles.

[0038] The hydrophobic cores can be composed, for example, of crosslinked polymeric materials prepared by polymerization or copolymerization of styrene, ethylstyrene, a- m ethyl styrene, divinylbenzene, diisopropenyl benzene, trivinylbenzene, alkyl methacrylate as methyl methacrylate, butyl methacrylate. The hydrophilic biocompatible coating of the particles can be composed for example of the following materials: polyvinylpyrrolidone, polyhydroxyethyl methacrylate, carboxymethylcellulose, polyurethane.

[0039] In other instances, the porous beads may be formed out of any of a variety of materials, including porous carbon, pyrolyzed polymer, zeolite, or any other porous nanostructure. Certain beads are mesoporous carbon beads formed of polymeric carbon. Beads of this type are able to absorb particular molecular species in a manner that is dependent on pore size. The size of the pores in the beads may vary widely depending on the target molecular species but will generally be between 0.2 and 50 nm in diameter.

[0040] The porous beads are generally between 50 pm and 500 pm in diameter, or between about 100 pm and 300 pm, or about 200 pm in diameter. It has been found that smaller diameter beads are more effective at removing target molecular species. However, smallerbeads are more difficult to produce and also may require non-woven fabrics for entrapment of the beads, resulting in a greater pressure drop across the filter medium, potentially leading to hemolysis and activation of white blood cells during use.

[0041] In operation, undesirable molecules or toxins having a molecular weight in the range of from about 100 Daltons to about 450,000 Daltons are sorbed.

[0042] The adsorbent can be contained in cartridge or a suitable container to hold blood and be in direct contact with blood and blood products but incapable of escaping from the container.

[0043] In certain instances, the beads are placed in a container where, due to a density similar to blood products, the beads remain naturally dispersed throughout the blood or blood product, allowing undesirable molecules in the blood to be sorbed by the beads.

[0044] In certain aspects, the adsorption media is disposed or contained within a container. The container can be a column, a chamber, or a cartridge. The container holds the loosely packed bed of adsorption media and allows the biological fluid to contact the adsorption media for efficient adsorption of toxins and pathogens and thereby remove the toxins and pathogens from the biological fluid through contact with the media. The container has a head space such that the media is not compressed.

[0045] In certain aspects, “loosely packed” media means that the container has a volume that is larger than the adsorption media disposed therein. The volume of the container is about 5% larger than the volume of the adsorption media contained therein. In certain aspects, there exists a headspace above the adsorbent media in the container. The volume of the container is larger than the media i.e., has a headspace by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and / or 95% larger than the volume of the adsorption media.

[0046] In certain aspects, the container comprises a first end plate or screen (e.g., top plate) and a second end plate or screen (e.g., bottom plate) to retain the adsorption media.

[0047] In certain aspects, the adsorption media has a density less than the biological fluid. In this manner, if the biological fluid is directed downward onto the adsorption media, the media will become fluidized by tending to float on the biological fluid.

[0048] In certain aspects, the biological fluid flows through an inlet of the container from the first end plate to the second end plate, in a top to a bottom direction.

[0049] In certain aspects, the inlet of the container or treatment module is configured to redirect fluid flow to improve contact of the biological fluid with the adsorption media. For example, in one aspect, the inlet in the container separates into a plurality of inlets or a manifold within the container with multiple jets. Alternatively, the inlet rotates within the container to direct the stream of biological fluid in more than one direction.

[0050] In certain aspects, the inlet is configured to reverse fluid flow within the container to improve contact or mixing of the biological fluid with the adsorption media. In another aspect, the container comprises a recirculator that allows multiple passes through the container before leaving the container.

[0051] In certain aspects, the container is placed in series or in parallel with a blood circuit for extracorporeal membrane oxygenation (ECMO) or a blood circuit for cardiopulmonary bypass, which circuit includes a pump.

[0052] In certain aspects, the biological fluid is blood, whole blood or plasma.

[0053] In one embodiment, the present disclosure provides a device for removing a pathogen or a toxin from a biological fluid of a subject, the device comprising: a container having a loosely packed bed of adsorption media disposed therein, wherein the adsorption media comprises heparin; and wherein the container has a first endplate and a second endplate to retain the bed of absorption media.

[0054] In certain aspects, the loosely packed bed of adsorption media is a fluidized bed.

[0055] In certain aspects, the adsorption media is a non-rigid or a deformable media with heparin bound thereto.

[0056] In certain aspects, the adsorption media is a hollow bead or a foamed polymer bead.

[0057] In certain aspects, the adsorption media has a density less than the biological fluid.

[0058] In certain aspects, the biological fluid flows through an inlet of the container from the first end plate to the second end plate, in a top to a bottom direction.

[0059] In certain aspects, the inlet is configured to redirect fluid flow to improve contact of the biological fluid with the adsorption media.

[0060] In certain aspects, the inlet is configured to reverse fluid flow within the container to improve contact of the biological fluid with the adsorption media.

[0061] In certain aspects, the container is placed in series with a blood circuit for extracorporeal membrane oxygenation (ECMO) or a blood circuit for cardiopulmonary bypass, which circuit includes a pump.

[0062] In certain aspects, the biological fluid is whole blood, serum, or plasma.

[0063] In therapeutic indications, the cleansed sample may be continuously or intermittently returned by reinfusion into the subject. The cleansed sample can be infused into the subject immediately after it is formed. The cleansed sample can be held for any period of time prior to infusion into the subject. One or more components can be added to the cleansed sample subsequent to its formation and prior to the infusion. In some embodiments, liquid is added to the cleansed sample to adjust its volume subsequent to its formation and prior to the infusion. In some aspects, an anticoagulant is added to the sample prior to or subsequent to its formation and prior to the infusion. The infusion can be in the form of a discrete volume of sample cleansed with the method and device. The infusion can be in the form a continuous or semi-continuous stream. The amount of cleansed sample that can be infused in the claimed methods is not intended to be limited. It can range from less than 1 mL to above 1 L, up to and including the entire blood volume of the patient or subject when the sample comprises whole blood and when continuous recirculation back to the subject is employed.

[0064] In certain aspects, one or more ‘passes’ through the fluidized adsorption bed through the circuit or recirculator may be used if needed. In some aspects, the sample or biological fluid is withdrawn from the subject and the cleansed sample is infused into the subject at a rate of about 5 mL / min, about 10 mL / min, about 15 mL / min, about 20 mL / min, about 25 mL / min, about 30 mL / min, about 35 mL / min, about 40 mL / min, about 45 mL / min, about 50 mL / min, about 60 mL / min, about 70 mL / min, about 80 mL / min, about 90 mL / min, about 100 mL / min, about 150 mL / min, about 200 mL / min, about 250 mL / min, about 300 mL / min, about 350 mL / min, about 400 mL / min, about 450 mL / min, about 500 mL / min, about 550 mL / min, about 600 mL / min, about 700 mL / min, about 800 mL / min, about 900 mL / min, or about 1000 mL / min. Included in the main circuit of an ECMO orcardiopulmonary bypass device treating an adult human patient, blood flow may exceed 1000 mL / min and reach about 2000 mL / min, about 3000 mL / min, about 4000 mL / min or about 5000 mL / min or more.

[0065] During operation, the biological fluid contacts the adsorption media in a fluidized bed for efficient adsorption of toxins and pathogens. This allows higher flow rates and lower pressure drops (compared to packed beds) through adsorption beds of practical size in order to achieve adequate separation or purification of the treated biological fluid.

[0066] Covalent attachment of the polysaccharide, such as heparin, to a solid substrate provides better control of parameters such as surface density and molecular orientation of the immobilized molecules as compared to non-covalent attachment. These parameters can be advantageous in order to provide optimal cytokine, toxin or pathogen binding to the immobilized carbohydrate molecules. The surface concentration of heparin on the substrate can be in the range of 1-10 pg / cm2. Covalent end-point attachment means that the polysaccharide, such as heparin is covalently attached to the solid substrate via the terminal chemical group of the heparin molecule. Heparin can also be bound at multiple points by covalent or ionic bonds.

[0067] In certain aspects, the device is shown being used in a system 100 disclosed in FIG. 1. System 100 conveys blood or a biological fluid to / from patient 110 and treats the blood within a treatment module 120. For example, the blood or biological fluid is removed from the patient 110 at point 111 and enters the treatment device 120. The treatment contains the fluidized bed. The biological fluid is conveyed along conduit 118 and the biological fluid enters the top of the treatment device or module 120 and descends to the bottom of the device in a clockwise manner. A pump 150 is used to withdraw a biological fluid from the subject and circulate the biological fluid through the system 100. The pump 150 can be a Harvard apparatus or syringe pump or withdraw / infuse pump. The cleansed biological fluid is conveyed within conduit 160 and is infused into the patient 110 at point 180. In some aspects, fluid may be introduced at positions into the treatment device or module 120, at particular flow rates, and the like, to aid in establishing the fluidized bed.

[0068] In certain aspects, the device 200 having a fluidized bed is shown in FIG. 2. The biological fluid such as blood enters an inlet 210 and exits an outlet 220. In certain aspects, the adsorption media disposed within the device has a density less than the biological fluid. In certain aspects, the biological fluid flows through an inlet 210 of the container from thefirst end plate near the inlet to the second end plate at the exit 220, in a top to a bottom direction. The device or cartridge has “loosely packed” media, wherein the device 200 has a volume that is larger than the volume of the adsorption media. FIG. 2 shows that that the volume of the container is at least 30% larger than the volume of the adsorption media contained therein. In certain other aspects, the volume of the container is larger than the media and has a headspace by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and / or 95% larger than the volume of the adsorption media.

[0069] One advantage of the fluidized bed of the present disclosure is the large surface-to- volume ratio. The large surface-to-volume ratio of the adsorption media in the fluidized bed increases the rate of removal of the absorbate (e.g. pathogen or the toxin) compared to a nonfluidized bed.

[0070] In one aspect, the adsorption media is loosely packed bed without compression and the solid particles are kept in motion by a downward flow of a fluid, such as whole blood. The term “fluidized bed” refers to a solid adsorption media acting as a fluid. A fluidized bed refers to a mixture state of two different phases, solid and fluid. The fluidized bed behaves as a fluid material. The efficiency of mixing and contact of the adsorption media with the biological fluid is enhanced in the fluidized bed compared to a fixed densely packed adsorption bed.

[0071] In certain aspects, the adsorption media are hollow, or foamed polymer beads especially for example, if the substrate polymer media has a higher density than the fluid being treated.

[0072] In certain aspects, an optional nozzle in attached on the inside of the container, which gently and continuously redirects blood flow, e.g., by rotating or alternating among multiple inlets, which improves turnover of the floating beads for more uniform contact of blood with the media.

[0073] In certain aspects, the fluidized bed allows higher flow rates at a reduced pressure drop through the cartridge and media. In certain instances, this is useful for inserting a treatment module in series with the main blood circuit during ECMO or cardiopulmonary bypass, which operates at a higher flow rate.III. Applications

[0074] The devices, methods and systems disclosed herein are useful in therapeutic and diagnostic applications. When a biological fluid such as whole blood is contacted with the adsorption media of the present disclosure, the pathogens, cancer cells, cancer mediators and / or toxins bind to the surface of the adsorption media and are thus removed. Examples of pathogens that can be removed from the blood using a heparinized substrate according to the disclosure include viruses such as coronaviruses, dengue virus, Hep B, and Hep C, and others. Bacteria such as Bacillus anthracis, Chlamydia pneumoniaem, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, MRSA and others.

[0075] One example of a disease to be treated according to the disclosure is sepsis. Sepsis is generally considered to be a systemic response to an infection which can lead to organ failure and often death. The condition can be triggered by a bacterial, viral, parasitic or fungal infection. The condition is known to be particularly dangerous in hospitals where patients may already be immunocompromised. During sepsis, the patient experiences a so- called cytokine storm and the body’s immune system attacks healthy tissue that leads to multiple organ failure in highly perfused organs. Reducing TNF-a and other inflammatory molecules modulates the immune response and acts as an organ preservation strategy. Additionally, any heparin-binding pathogens in the blood can be removed which helps reduce further colonization and reduces the amount of antibiotics needed to treat an infection. This improves patient safety by reducing side effect risks associated with antibiotic therapy.

[0076] In certain aspects, the devices and methods of the present disclosure can be combined with other methods to filter or treat mammalian blood. For example, a cartridge or column with a fluidized bed as disclosed can be used in series with conventional extracorporeal circuits such as CPB, hemodialysis, and oxygenation. For example, the device or container is placed in series with a blood circuit for extracorporeal membrane oxygenation (ECMO) or a blood circuit for cardiopulmonary bypass, which circuit includes a pump.

[0077] In other aspects, the devices and methods can be used for a number of diagnostic applications including oncology, virology and infectious diseases. Diagnostic applications include isolation of any cell type and the option of additional cell characterization on column or post column. One application is the identification of pathogens such as viruses, bacteria, fungi and protozoa from a patient sample. Another application is the isolation and characterization of cancer cells, such as circulating tumor cells (CTCs) and cancer stem cells (CSCs). Isolation of CTCs is useful for early cancer detection, characterization of tumorcells, individualized cancer treatment, monitoring disease treatment, monitoring disease progression or remission.

[0078] The devices and methods of the present disclosure provide an in vitro method for concentrating a wide range of infectious pathogens and toxins present in a biological sample obtained from a subject who is suspected of being infected with the pathogens. The method comprises: (a) contacting the biological sample obtained from the subject with a broadspectrum adsorption media such as heparin in a fluidized bed under conditions that form an adhering complex comprising the adsorption media and the pathogens; (b) separating the adhering complex from components of the sample that are not included in the complex while maintaining the complex, e.g, by washing the adhering complex with a buffer solution; and (c) collecting pathogens of the adhering complex by applying an elution buffer to the complex, thereby concentrating the infectious pathogens in an eluent. In some embodiments, the method further comprises detecting the isolated infectious pathogens. In some instances, detecting the isolated infectious pathogens comprises a colorimetric assay, an immunoassay, an enzyme-linked immunosorbent assay (ELISA), a PCR-based assay, a pathogen growth assay with optional staining, or a combination thereof. In some instances, pathogens or cancer cells separated from the media or still bound to the media may be grown in culture to increase their number and used to identify the pathogen or cancer cell and / or test its susceptibility to drugs or other treatments.

[0079] Diagnostic applications of the disclosure can be used in a variety of settings. In certain embodiments, diagnostics are utilized in a research setting such as academia, biotechnology or A pharmaceutical company. In other embodiments, the columns and methods of the disclosure can be used in point of care settings including emergency rooms, intensive-care units, patients’ bedsides, physician's offices, pharmacies, and blood banks.

[0080] In one embodiment, the disclosure provides an extracorporeal method for removing a pathogen or a toxin from a biological fluid of a subject. As shown in FIG. 3, in step 301, the blood or biological fluid is contacted with fluidized device in order to remove CTCs, or other cellular targets or toxins. Typical flow rates are between 100 and 5000 mL / min. In step 312, cells are bound to adsorption media and the biological fluid is cleansed. In step 333, the blood or biological fluid is rinsed from the filter device. Using a side port, or the bottom port of the device, it is possible to circulate culture medium through the device to propagate captured cells. In one aspect, a secondary filtration vessel with a sub-micron porefilter is used to capture cells that release from adsorption media. In step 343, the secondary filtration vessel is removed so cells can be used for diagnostic or screening purposes. The cells can be used for identification, sensitization tests, or for screening patients for disease.

[0081] In certain aspects, the methods and devices described herein can be used in therapeutic and diagnostic applications with cancer cells such as circulating tumor cells (CTCs). Isolation of CTCs is useful for early cancer detection, therapeutic applications, characterization of tumor cells, individualized cancer treatment, monitoring disease treatment, monitoring disease progression or remission.

[0082] In certain aspects, the cancer cells or CTC can be cultured. Once isolated, for example, the CTCs are placed in cell culture conditions. By “cell culture” or “culture” is meant the maintenance of cells in an artificial, in vitro environment. The term “cell culture” also encompasses cultivating individual cells and tissues.

[0083] The cells being cultured according to the present disclosure, whether primary or not, can be cultured and plated according to the experimental conditions as needed by a person of skill in the art. Cells may or may not be plated onto the surface of culture vessels using attachment factors. If attachment factors are used, the culture vessels can be precoated with other natural, recombinant, or synthetic attachment factor or factors or peptide fragments thereof, such as but not limited to collagen, fibronectin and natural or synthetic fragments thereof.

[0084] The cell seeding densities for each experimental condition can be manipulated for the specific culture conditions needed. For routine culture in plastic culture vessels, an initial seeding density of from about 1 x 104to about 1-10 x 105cells per cm2is fairly typical, e.g., 1 x 106cells are often cultured in a 75 cm2culture flask. Using the methods of the present disclosure, however, even a single cell can be plated initially. Thus, the methods of the present disclosure can be performed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more cells for an initial cell seeding. Of course, higher cell seeding numbers can be used, such as but not limited to 1 x 103, 1 X 104, 1 X 105and so on.

[0085] Mammalian cells are typically cultivated in a cell incubator at about 37°C at normal atmospheric pressure. The incubator atmosphere is normally humidified and often contain about from about 3-10% carbon dioxide in air. Temperature, pressure and CO2 concentration can be altered as necessary, provided the cells are still viable. Culture medium pH can be in the range of about 7.1 to about 7.6, in particular from about 7.1 to about 7.4, and even moreparticular from about 7.1 to about 7.3. It may be necessary in some cases, like colon cancer cells, to use incubators with low oxygen (2%) rather than the normal 20%. The incubator atmosphere may thus be from about 0.5% to about 30% oxygen.

[0086] The cell culture media of the present disclosure can be any aqueous-based medium and can include any “classic” media such as, but not limited to DMEM (Dulbecco’s Modified Essential Medium), Ham’s F12 medium, Ham’s F-10 medium, RPMI 1640, Eagle’s Basal Medium (EBM), Eagle’s Minimum Essential Medium (MEM), HEPES, Medium 199 and the like. The culture medium can also be combinations of any of the classical medium, such as but not limited to, a combination of DMEM and F12 Media.

[0087] Additional ingredients can optionally be added to the culture medium used in the methods of the present disclosure. Such additional ingredients include but are not limited to, amino acids, vitamins, inorganic salts, adenine, ethanolamine, D-glucose, heparin, N-[2- hydroxy ethyl] piperazine-N’-[2-ethanesulfonic acid] (HEPES), hydrocortisone, insulin, lipoicacid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, triiodothyronine (T3), thymidine and transferrin. Alternatively, insulin and transferrin may be replaced by ferric citrate or ferrous sulfate chelates. Each of these additional ingredients is commercially available.

[0088] Amino acid ingredients which may be included in the media of the present disclosure include but are not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L- glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L- lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L- tyrosine and L-valine.

[0089] Vitamin that may be added include but are not limited to biotin, choline chloride, D- Ca+2- pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine and vitamin Bl 2.

[0090] Inorganic salt ingredients which may be added include but are not limited to calcium salt (e.g., CaCh), CuSCh, FeSCh, KC1, a magnesium salt, e.g., MgCh, a manganese salt, e.g., MnCh, sodium acetate, NaCl, NaHCCh, Na2HPO4, Na2SO4 and ions of the trace elements selenium, silicon, molybdenum, vanadium, nickel, tin and zinc. These trace elements may be provided in a variety of forms, preferably in the form of salts such as Na2SeO3, Na2SiO3, (NH4)6Mo7O24, NH4VO3, NiSC , SnCl and ZnSO.

[0091] Additional ingredients include but are not limited to heparin, epidermal growth factor (EGF), at least one agent increasing intracellular cyclic adenosine monophosphate (cAMP) levels, and at least one fibroblast growth factor (FGF). Heparin, EGF, the cAMP- increasing agent(s) and FGF(s) may be added to the basal medium or they may be admixed in a solution of, for example, Dulbecco’s Phosphate Buffered Saline (DPBS) and stored frozen until being added to basal medium to formulate the medium to be used in the methods of the present disclosure.

[0092] The culture medium used in the methods of the present disclosure optionally comprises a calcium source. In one embodiment, the calcium source is serum or a serum replacement. In another embodiment, the calcium source is a calcium-containing salt that is added to the medium. If serum is used as a calcium source, the serum can be in a concentration (v / v) of from about 1% to about 35%. In select embodiments, the serum is at a concentration of from about 1% to about 20%, or from about 1% to about 15%, or from about 1% to about 10%, or from about 1% to about 5%. If a serum substitute or serum replacement is used as the calcium source, these can be added to the medium according to the manufacturer's suggested protocol. Examples of serum substitutes include but are not limited to commercially available substitutes such as Ultroser™ from Pall Corporation, milk or milk fractions such as but not limited to nonfat dry milk filtrate.

[0093] The methods of the present disclosure optionally comprise inhibiting rho associated coiled-coil protein kinase (ROCK) in the culture. In one embodiment, inhibiting ROCK is accomplished by addition of a ROCK inhibitor to the culture medium. Examples of ROCK inhibitors include but are not limited to Y-27632, HAI 100, HAI077, Thiazovivin and GSK429286. Another way of inhibiting ROCK kinase would be through the use of RNA interference (RNAi).

[0094] The present disclosure provides methods of stimulating growth of CTCs in vitro which may or may not include the use of feeder cells. In some embodiments, the methods of the present disclosure do not include the use of feeder cells. In some embodiments, the methods of the present disclosure include the use of feeder cells. The term “feeder cells” is used herein as it is in the art. Namely, feeder cells are cells that are cultured with the CTCs of the present disclosure. As used herein, “culturing with CTCs” (or “culturing with circulating tumor cells”) means that the feeder cells are cultured sharing the same medium and sharing the same vessel with the CTCs. Thus, the feeder cells need not be in directcontact with the CTCs and, for example, can be physically separated from the CTCs, e.g., by a porous filter, although both sets of cells are in the same vessel sharing the same medium.

[0095] Feeder cells can be from any mammal and the animal source of the feeder cells need not be the same animal source as the CTCs being cultured. For example, feeder cells may be, but are not limited to mouse, rat, canine, feline, bovine, equine, porcine, non-human and human primate feeder cells. The types of feeder cells used are typically spleenocytes, macrophages thymocytes and / or fibroblasts. One example of a feeder cell that may be used in the methods of the present disclosure is a population of J2 cells. The J2 cells are a subclone of mouse fibroblasts derived from the established Swiss 3T3 cell line. In one embodiment, the J2 cells are gamma irradiated. In another embodiment, the J2 cells are treated with mitomycin C.

[0096] In another embodiment, medium conditioned with feeder cells is used in place of culturing feeder cells with the CTCs. Preparing conditioned medium is routine in the art. As used herein, “conditioned medium” is any medium where all or a percentage of the medium has been previously used in culture. In yet another embodiment, feeder cell extract can be added to the medium in place of feeder cells themselves.

[0097] In certain instances, the loosely packed bed of adsorption media acting as a fluidzed bed augments impaired glycocalyx barrier function. In this regard, the fluidized bed as described herein can be used as described in WO 2020 / 231830, which is incorporated herein by reference. The adsorption media includes glycosaminoglycan structures and, optionally, proteoglycan core proteins, which are conducive to enhancing and / or restoring the impaired glycocalyx barrier function in a sample. The contacted sample is subsequently separated from the adsorption media, producing a treated sample that can be infused into the subject.

[0098] In certain instances, the glycocalyx-mimetic adsorption media aids in a member selected from the group consisting of vascular permeability, adhesion of leucocytes, adhesion of platelets, mediation of shear stress, and modulation of an inflammatory process.

[0099] In certain instances, the adsorption media acts as an endothelial surface layer to protect and / or maintain glycocalyx function.

[0100] In certain instances, the adsorption media reduces a member selected from the group consisting of capillary leak syndrome, edema formation, inflammation, platelet hyperaggregation, hypercoagulation, and loss of vascular responsiveness.

[0101] In certain instances, the adsorption media reduces shedding of the glycocalyx during reperfusion of a tissue.

[0102] In certain instances, the tissue is heart tissue during coronary by-pass surgery.

[0103] In certain instances, the tissue is perfused during an organ transplant.

[0104] In certain instances, the method treats acute respiratory distress syndrome (ARDS) in the subj ect.

[0105] In certain instances, the method improves oxygen saturation in the subject.

[0106] In certain instances, the method improves hemodynamic stability in the subject.

[0107] In certain instances, the adsorption media reduces shedding of the glycocalyx during sepsis.

[0108] In certain instances, the adsorption media removes tumor necrosis factor (TNF)- alpha and bacterial lipopolysaccharide (LPS).

[0109] In certain instances, the adsorption media reduces the risk of organ failure.

[0110] In certain instances, the adsorption media reduces shedding of the glycocalyx resulting from atherosclerosis or diabetes.[OHl] In certain instances, the adsorption media removes low-density lipoproteins (LDL).

[0112] In certain instances, the adsorption media binds heparin binding protein (HBP).

[0113] In certain instances, the treated sample has a HBP content that is reduced by about 10% to about 100% compared to the HBP content of the sample prior to treatment.

[0114] In certain instances, the adsorption media binds a member selected from the group consisting of exotoxins, endotoxins, ultra large von Willebrand factor (ULVWF), histones, exosomes, microvesicles and cytokines.IV. Examples

[0115] This example illustrates the device and system in combination with additional filtration / separation steps. In an embodiment of the treatment method, the withdrawal and infusion of blood can be performed in a continuous loop as in FIG. 1, which loop comprises a part of the bloodstream of the subject. In a further aspect, the treatment loop is combined with other methods to filter or treat mammalian blood. For example, a fluidized bed cartridgecan be used in series with conventional extracorporeal circuits such as CPB, hemodialysis, and oxygenation.

[0116] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirt of the disclosure.

Claims

WHAT IS CLAIMED IS:

1. An extracorporeal method for removing a pathogen or a toxin from a biological fluid of a subject, the method comprising: contacting a biological fluid from the subject with a loosely packed bed of adsorption media, wherein the adsorption media comprises heparin, to remove the pathogen or the toxin to form a treated biological fluid; and infusing the treated fluid into the subject.

2. The method of claim 1, wherein the loosely packed bed of adsorption media is a fluidized bed.

3. The method of claim 1 or 2, wherein the adsorption media is a non- rigid or a deformable media with heparin bound thereto.

4. The method of any one of claims 1-3, wherein the adsorption media is a hollow bead or a foamed polymer bead.

5. The method of any one of claims 1-4, wherein the adsorption media is contained within a container selected from a column, a chamber, or a cartridge.

6. The method of claim 5, wherein the container comprises a first end plate and a second end plate to retain the adsorption media.

7. The method of any one of claims 1-6, wherein the adsorption media has a density less than the biological fluid.

8. The method of claim 7, wherein the biological fluid flows through an inlet of the container from the first end plate to the second end plate, in a top to a bottom direction.

9. The method of claim 8, wherein the inlet is configured to redirect fluid flow to improve contact of the biological fluid with the adsorption media.

10. The method of claim 8, wherein the inlet is configured to reverse fluid flow within the container to improve contact of the biological fluid with the adsorption media.

11. The method of claim 5, wherein the container is placed in series or parallel with a blood circuit for extracorporeal membrane oxygenation (ECMO) or a blood circuit for cardiopulmonary bypass, which circuit includes a pump.

12. The method of any one of claims 1-11, wherein the biological fluid is blood or plasma.

13. The method of any one of claims 1-12, wherein the toxin is a cancer cell or a cancer mediator.

14. A device for removing a pathogen or a toxin from a biological fluid of a subject, the device comprising: a container having loosely packed bed of adsorption media disposed therein, wherein the adsorption media comprises heparin; and wherein the container has a first endplate and a second endplate to retain the bed of absorption media.

15. The device of claim 14, wherein the loosely packed bed of adsorption media is a fluidized bed.

16. The device of claim 14 or 15, wherein the adsorption media is a non- rigid or a deformable media with heparin bound thereto.

17. The device of any one of claims 14-16, wherein the adsorption media is a hollow bead or a foamed polymer bead.

18. The device of any one of claims 14-17, wherein the adsorption media has a density less than the biological fluid.

19. The device of any one of claims 14-18, wherein the biological fluid flows through an inlet of the container from the first end plate to the second end plate, in a top to a bottom direction.

20. The device of claim 19, wherein the inlet is configured to redirect fluid flow to improve contact of the biological fluid with the adsorption media.

21. The device of claim 19, wherein the inlet is configured to reverse fluid flow within the container to improve contact of the biological fluid with the adsorption media.

22. The device of any one of claims 14-21, wherein the container is placed in series or in parallel with a blood circuit for extracorporeal membrane oxygenation(ECMO) or a blood circuit for cardiopulmonary bypass, which circuit includes a pump.

23. The device of any one of claims 14-21, wherein the biological fluid is blood or plasma.

24. The device any one of claims 14-21, wherein the toxin is a cancer cell or a cancer mediator.

25. The device any one of claims 14-21, wherein the toxin is LDL.

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