WO2026028182A1 - Extracorporeal human brain perfusion apparatus with neural communication capability - Google Patents

Abstract

Provided herein is a bioreactor system for neural tissue, including a bioreactor configured to hold a neural tissue sample therein; a media reservoir in fluid communication with the bioreactor and configured to hold a fluid media; one or more fluid conduits fluidly connecting the bioreactor to the media reservoir; a cannula in fluid communication with the media reservoir and configured to couple to a blood vessel of the neural tissue sample; one or more sensors configured to measure one or more parameters of the fluid media and/or the neural tissue sample; and one or more pumps configured to cause media to flow from the media reservoir through the cannula.

Description

EXTRACORPOREAL HUMAN BRAIN PERFUSION APPARATUS WITH NEURAL COMMUNICATION CAPABILITY

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/678,723, filed August 2, 2024, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] Provided herein are systems and methods for isolating and maintaining brain tissue, useful for, for example, evaluation of potential therapeutic compositions for various neural conditions, disease, and/or disorders.

Description of Related Art

[0003] There are over 100,000 cases of brain cancer expected to be diagnosed each year in United States over the coming years. It is incredibly difficult to isolate and maintain viability of neural tissue, due to the complicated interconnectivity and significant number of variables necessary for proper functional of such tissue. Thus, it has been to this point impossible to utilize neural tissue for evaluation of interventions, such as therapeutic compounds.

SUMMARY OF THE INVENTION

[0004] Provided herein is a bioreactor system for neural tissue, including a bioreactor configured to hold a neural tissue sample therein; a media reservoir in fluid communication with the bioreactor and configured to hold a fluid media; one or more fluid conduits fluidly connecting the bioreactor to the media reservoir; a cannula in fluid communication with the media reservoir and configured to couple to a blood vessel of the neural tissue sample; one or more sensors configured to measure one or more parameters of the fluid media and/or the neural tissue sample; and one or more pumps configured to cause media to flow from the media reservoir through the cannula.

[0005] Also provided herein is a bioreactor system for neural tissue, including: a bioreactor; a neural tissue sample received within the bioreactor, the neural tissue sample including a plurality of cortical cells and at least one blood vessel; a media reservoir in fluid communication with the bioreactor and holding an artificial blood composition therein; one or more fluid conduits fluidly connecting the bioreactor to the media reservoir and defining a closed system including the media reservoir and the bioreactor; a cannula in fluid communication with the one or more fluid conduits and coupled to the at least one blood vessel of the neural tissue sample; one or more sensors configured to measure one or more parameters of the artificial blood composition and/or the neural tissue sample, the one or more sensors including a pH sensor, a flow rate sensor, a pressure sensor, a temperature sensor, and an oxygen sensor; and one or more pumps configured to cause the artificial blood composition to flow from the media reservoir to the at least one blood vessel through the cannula.

[0006] Further non-limiting embodiments are set forth in the following numbered clauses:

[0007] 1. A bioreactor system for neural tissue, comprising: a bioreactor configured to hold a neural tissue sample therein; a media reservoir in fluid communication with the bioreactor and configured to hold a fluid media; one or more fluid conduits fluidly connecting the bioreactor to the media reservoir; a cannula in fluid communication with the media reservoir and configured to couple to a blood vessel of the neural tissue sample; one or more sensors configured to measure one or more parameters of the fluid media and/or the neural tissue sample; and one or more pumps configured to cause media to flow from the media reservoir through the cannula.

[0008] 2. The system of clause 1 , wherein the bioreactor comprises a container having a top, bottom, a sidewall defining an interior configured to hold the neural tissue sample, and at least two openings configured to fluidly connect the bioreactor to the one or more fluid conduits.

[0009] 3. The system of clause 1 or clause 2, wherein the one or more pumps are arranged within the media reservoir.

[0010] 4. The system of any of clauses 1 -3, wherein the one or more pumps are arranged between the media reservoir and the bioreactor.

[0011 ] 5. The system of any of clauses 1 -, wherein one or more of the one or more pumps are a peristaltic pump and/or a roller pump.

[0012] 6. The system of any of clauses 1 -5, wherein the media reservoir is configured to hold an artificial blood composition.

[0013] 7. The system of any of clauses 1 -6, further comprising one or more additional reservoirs configured to hold one or more compositions, the one or more additional reservoirs in fluid communication with the one or more fluid conduits. [0014] 8. The system of any of clauses 1 -7, wherein the one or more additional reservoirs are arranged between the media reservoir and the bioreactor.

[0015] 9. The system of any of clauses 1 -8, wherein at least one of the one or more additional reservoirs is configured to hold heparin.

[0016] 10. The system of any of clauses 1 -9, wherein at least one of the one or more additional reservoirs is configured to hold a therapeutic composition.

[0017] 1 1 . The system of any of clauses 1 -10, further comprising an oxygenator in fluid communication with the one or more fluid conduits.

[0018] 12. The system of any of clauses 1 -1 1 , wherein the oxygenator is arranged between the media reservoir and the bioreactor.

[0019] 13. The system of any of clauses 1 -12, further comprising one or more filters arranged between the media reservoir and the bioreactor.

[0020] 14. The system of any of clauses 1 -13, wherein the one or more filters comprises a filtration membrane.

[0021] 15. The system of any of clauses 1 -14, wherein the one or more sensors comprise a pressure sensor, an oxygen sensor, a temperature sensor, a flowrate sensor, and/or a pH sensor.

[0022] 16. The system of any of clauses 1 -15, wherein the oxygen sensor is arranged in the bioreactor.

[0023] 17. The system of any of clauses 1 -16, further comprising a one or more temperature controllers.

[0024] 18. The system of any of clauses 1 -17, wherein one or more of the one or more temperature controllers is arranged in the bioreactor.

[0025] 19. The system of any of clauses 1 -18, wherein one or more of the one or more temperature controllers is arranged in the media reservoir and/or between the media reservoir and the bioreactor.

[0026] 20. The system of any of clauses 1 -19, further comprising an electrocorticography (ECoG) device.

[0027] 21 . The system of any of clauses 1 -20, wherein the ECoG device comprises at least one electrode lead arranged in the bioreactor and configured to detect an electrical signal from the neural tissue and at least one computing device in electrical communication with the at least one electrode lead and configured to acquire, amplify, filter, and/or analyze the electrical signal received from the at least one electrode lead. [0028] 22. The system of any of clauses 1 -21 , wherein the at least one electrode lead is configured to deliver electrical stimulation to the neural tissue and the at least one computing device comprises a pulse generator.

[0029] 23. The system of any of clauses 1 -22, further comprising a Faraday cage arranged on and/or in the bioreactor.

[0030] 24. The system of any of clauses 1 -23, wherein the system is a closed system.

[0031] 25. The system of any of clauses 1 -24, wherein the system is an open system.

[0032] 26. The system of any of clauses 1 -25, further comprising at least one computing device in communication with the one or more pumps and one or more sensors, and configured to modulate one or more parameters of the media based on data received from the one or more sensors.

[0033] 27. A bioreactor system for neural tissue, comprising: a bioreactor; a neural tissue sample received within the bioreactor, the neural tissue sample comprising a plurality of cortical cells and at least one blood vessel; a media reservoir in fluid communication with the bioreactor and holding an artificial blood composition therein; one or more fluid conduits fluidly connecting the bioreactor to the media reservoir and defining a closed system comprising the media reservoir and the bioreactor; a cannula in fluid communication with the one or more fluid conduits and coupled to the at least one blood vessel of the neural tissue sample; one or more sensors configured to measure one or more parameters of the artificial blood composition and/or the neural tissue sample, the one or more sensors comprising a pH sensor, a flow rate sensor, a pressure sensor, a temperature sensor, and an oxygen sensor; and one or more pumps configured to cause the artificial blood composition to flow from the media reservoir to the at least one blood vessel through the cannula.

[0034] 28. The system of clause 27, further comprising one or more additional reservoirs holding a therapeutic composition.

[0035] 29. The system of clause 27 or clause 28, wherein the neural tissue sample is a sample comprising one or more tumor cells and the therapeutic composition is a potential therapy for the tumor.

[0036] 30. The system of any of clauses 27-29, wherein the neural tissue sample is a sample from a patient with epilepsy and the therapeutic composition is a potential therapy for epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGS. 1 A-1 B are a schematic of a system and processes that can be utilized with a system according to non-limiting embodiments described herein.

[0038] FIGS. 2A-2E show (A) Schematic of the tumor-infiltrated cortex resection and application of fluorescein dye for vascular visualization. The image on the right shows the resected specimen with the green arrow indicating the perfused area. (B) Sequential images showing the administration and spread of fluorescein dye through the cannulated artery in the perfused brain specimen, with black arrows indicating the dye's progression, confirming vascular patency. (C) Detailed views of fluorescein blush in perfused cortical tissue. Yellow dashed circles highlight areas with successful dye penetration. Magnified images show extensive dye distribution, indicating robust perfusion. (D) Comparative analysis between perfused and non-perfused specimens. The left schematic shows the non-perfused cortex with biopsy sites, while the right schematic illustrates the perfused cortex with a clear fluorescein blush, highlighting effective perfusion. (E) Comparative DAPI and Dextran staining between t = 0 and 1 hour after perfusion.

[0039] FIGS. 3A-3C show (A) Nissl staining of brain tissue sections at 0, 1 , and 4 hours. Perfused specimens exhibit well-preserved neuronal architecture (purple arrows), whereas non-perfused controls show significant neuronal degradation. Quantitative analysis shows higher Nissl signal, and neuron counts per ROI in perfused specimens at 1 hour (n=9, p=0.02) and 4 hours (n=9, p=0.01 ) (B) Caspase-

3 (CASP3) immunofluorescence staining at 0, 1 , and 4 hours. Perfused specimens display fewer Caspase-3 positive cells (red arrows) compared to non-perfused controls. Statistical analysis indicates significantly lower CASP3 signal intensity and fewer CASP3-positive ROIs in perfused specimens at 1 hour and 4 hours (n=9, p<0.001 ). (C) Hematoxylin and Eosin (H&E) staining showing cell density at 0, 1 , and

4 hours. Perfused specimens maintain higher cell density, with significantly elevated values at 0 hours (n=5, p<0.001 ), 1 hour (n=4, p<0.001 ), and 4 hours (n=4, p<0.001 ). Non-perfused specimens exhibit progressive tissue necrosis, while perfused specimens preserve tissue integrity.

[0040] FIGS. 4A-4F show (A-C) Schematic representation showing the brain tissue perfused with a controlled circulatory system. Evans Blue dye is introduced into the perfusion to assess BBB integrity, with intact BBB indicated by dye retention within the vascular compartment. (D) Representative images of brain tissue post-perfusion: (i) intact BBB shows no dye infiltration into the parenchyma, while (ii) a compromised BBB exhibits diffuse blue staining in the tissue. (E-F) Comparative drug permeability analysis for different compounds, highlighting variations in BBB penetration.

[0041] FIGS. 5A-5F show GFAP immunofluorescence staining at 1 and 4 hours post-LPS injection, showing increased GFAP signal in perfused specimens (purple arrows) compared to minimal GFAP expression in non-perfused controls. Quantitative analysis confirms higher GFAP signal in perfused specimens at 1 hour and 4 hours (n=8, p<0.01 ). IBA1 immunofluorescence staining at 1 and 4 hours post-LPS injection, highlighting pronounced microglial activation in perfused specimens (white arrows) with robust IBA1 expression, whereas non-perfused controls show lower IBA1 signal. Statistical analysis indicates significantly elevated IBA1 signal in perfused specimens at both time points (n=8, p<0.01 ). Regional GFAP and IBA1 staining across frontal, temporal, and parietal cortex. Perfused specimens show consistent glial activation across all regions, with significantly higher GFAP and IBA1 signals compared to sparse and uneven glial response in non-perfused controls. Quantitative comparisons reveal significant differences in GFAP and IBA1 signals between perfused and nonperfused specimens at both 1 hour and 4 hours across all regions analyzed (n=8, p<0.01 ).

[0042] FIG. 6 is a schematic diagram of example components of one or more devices of a system suitable for implementing methods as described herein according to non-limiting embodiments described herein.

DESCRIPTION OF THE INVENTION

[0043] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. [0044] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used.

[0045] Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of "1 to 10" is intended to include all sub-ranges between and including the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

[0046] As used herein "a" and "an" refer to one or more.

[0047] As used herein, the term "comprising" is open-ended and may be synonymous with "including", "containing", or "characterized by".

[0048] As used herein, the term "patient" or "subject" refers to members of the animal kingdom including but not limited to human beings, and "mammal" refers to all mammals, including, but not limited to human beings.

[0049] For purposes of the description hereinafter, the terms "end," "upper," "lower," "right," "left," "vertical," "horizontal," "top," "bottom," "lateral," "longitudinal," and derivatives thereof shall relate to the embodiments as they are oriented in the drawing figures. However, it is to be understood that the present disclosure may assume various alternative variations and step sequences, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes illustrated in the attached drawings, and described in the following specification, are simply exemplary and non-limiting embodiments or aspects of the disclosed subject matter. Hence, specific dimensions and other physical characteristics related to the embodiments or aspects disclosed herein are not to be considered as limiting.

[0050] Some non-limiting embodiments or aspects are described herein in connection with thresholds. As used herein, satisfying a threshold may refer to a value being greater than the threshold, more than the threshold, higher than the threshold, greater than or equal to the threshold, less than the threshold, fewer than the threshold, lower than the threshold, less than or equal to the threshold, equal to the threshold, etc. [0051] No aspect, component, element, structure, act, step, function, instruction, and/or the like used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles "a" and "an" are intended to include one or more items and may be used interchangeably with "one or more" and "at least one." Furthermore, as used herein, the term "set" is intended to include one or more items (e.g., related items, unrelated items, a combination of related and unrelated items, and/or the like) and may be used interchangeably with "one or more" or "at least one." Where only one item is intended, the term "one" or similar language is used. Also, as used herein, the terms "has," "have," "having," or the like are intended to be open-ended terms. Further, the phrase "based on" is intended to mean "based at least partially on" unless explicitly stated otherwise. In addition, reference to an action being "based on" a condition may refer to the action being "in response to" the condition. For example, the phrases "based on" and "in response to" may, in some non-limiting embodiments or aspects, refer to a condition for automatically triggering an action (e.g., a specific operation of an electronic device, such as a computing device, a processor, and/or the like).

[0052] As used herein, the term "communication" may refer to the reception, receipt, transmission, transfer, provision, and/or the like of data (e.g., information, signals, messages, instructions, commands, and/or the like). For one unit (e.g., a device, a system, a component of a device or system, combinations thereof, and/or the like) to be in communication with another unit means that the one unit is able to directly or indirectly receive information from and/or transmit information to the other unit. This may refer to a direct or indirect connection (e.g., a direct communication connection, an indirect communication connection, and/or the like) that is wired and/or wireless in nature. Additionally, two units may be in communication with each other even though the information transmitted may be modified, processed, relayed, and/or routed between the first and second unit. For example, a first unit may be in communication with a second unit even though the first unit passively receives information and does not actively transmit information to the second unit. As another example, a first unit may be in communication with a second unit if at least one intermediary unit processes information received from the first unit and communicates the processed information to the second unit. In some non-limiting embodiments or aspects, a message may refer to a network packet (e.g., a data packet and/or the like) that includes data. It will be appreciated that numerous other arrangements are possible. Communication may include one or more wired and/or wireless networks. For example, communication may include a cellular network (e.g., a long-term evolution (LTE) network, a third-generation (3G) network, a fourth-generation (4G) network, a fifth-generation (5G) network, a code division multiple access (CDMA) network, etc.), a public land mobile network (PLMN), a local area network (LAN), a wide area network (WAN), a metropolitan area network (MAN), a telephone network (e.g., the public switched telephone network (PSTN) and/or the like), a private network, an ad hoc network, an intranet, the Internet, a fiber optic-based network, a cloud computing network, and/or the like, and/or a combination of some or all of these or other types of networks.

[0053] As used herein, the term "computing device" may refer to one or more electronic devices configured to process data. A computing device may, in some examples, include the necessary components to receive, process, and output data, such as a processor, a display, a memory, an input device, a network interface, and/or the like. A computing device may be a mobile device. As an example, a mobile device may include a cellular phone (e.g., a smartphone or standard cellular phone), a portable computer, a wearable device (e.g., watches, glasses, lenses, clothing, and/or the like), a personal digital assistant (PDA), and/or other like devices. A computing device may also be a desktop computer or other form of non-mobile computer.

[0054] As used herein, the term "server" may refer to or include one or more computing devices that are operated by or facilitate communication and processing for multiple parties in a network environment, such as the Internet, although it will be appreciated that communication may be facilitated over one or more public or private network environments and that various other arrangements are possible. Further, multiple computing devices (e.g., servers, mobile devices, etc.) directly or indirectly communicating in the network environment may constitute a "system."

[0055] As used herein, the term "system" may refer to one or more computing devices or combinations of computing devices (e.g., processors, servers, client devices, software applications, components of such, and/or the like). Reference to "a device," "a server," "a processor," and/or the like, as used herein, may refer to a previously-recited device, server, or processor that is recited as performing a previous step or function, a different device, server, or processor, and/or a combination of devices, servers, and/or processors. For example, as used in the specification and the claims, a first device, a first server, or a first processor that is recited as performing a first step or a first function may refer to the same or different device, server, or processor recited as performing a second step or a second function.

[0056] While this disclosure is focused on human cells and tissue, those of skill in the art will appreciate that the same principles can be applied to other species including rodent (e.g., mice and rats), ferret, guinea pig, dog, cat, and non-human primate species. In such cases the source of cells/tissue would be from the respective animals.

[0057] Provided herein is a system, and methods of using the same, for maintenance of neural tissue. Systems, and methods described herein, provide previously unavailable models for monitoring neural tissue in an in vitro setting and for assessing therapies for neuro-specific conditions, particularly in an individualized manner as the neural tissue may, in some non-limiting embodiments, be from a particular patient with a condition, and therapies for that specific patient and that patient’s specific condition may then be tested in an in vitro setting to provide guidance for clinical implementation.

[0058] Accordingly, in non-limiting embodiments a system 1000 may include a bioreactor 1 10. Bioreactors are known to those of skill in the art and may take the form of a commercially-available bioreactor or one that may be custom-built. In nonlimiting embodiments the bioreactor 110 includes a housing, which may have a top, bottom, and one or more sidewalls defining an interior configured to hold a tissue sample 1 12, such as a neural tissue sample, therein. In non-limiting embodiments the bioreactor 1 10 may further include a buffer therein in which the tissue sample 1 12 may be at least partially immersed. Suitable buffers for maintaining tissue viability are known and include saline, buffered salines (such as phosphate-buffered saline (PBS) and/or tris-buffered saline (TBS)), balanced salt solutions (such as Gibco Balanced Salt Solution) HEPES (2-[4-(2-hydroxyethyl)piperazin-1 -yl]ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (C6H15NO5S), BES (2-(bis(2- hydroxyethyl)amino)ethane sulfonic acid), MOPSO (2-Hydroxy-3- morpholinopropanesulfonic acid), ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid), TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), BICINE (CeHi2NNaO4), and/or Tricine (A/-[Tris(hydroxymethyl)methyl]glycine).

[0059] In non-limiting embodiments the bioreactor 1 10 may include one or more fluid inlets and/or outlets, to allow for the interior of the bioreactor to be placed into fluid communication with a fluid source. Accordingly, in non-limiting embodiments system 1000 may further include a reservoir 120 in fluid communication with the bioreactor and configured to hold a fluid media. In non-limiting embodiment the fluid may be a cell culture media. In non-limiting embodiments, the fluid may be another type of fluid useful for maintaining viability of tissue sample 1 12. In non-limiting embodiments, the fluid may be an artificial blood. A useful artificial blood composition may include an energy source for the tissue 1 12 (e.g., glucose), one or more buffers, a hemoglobin-based carrier (or other chaperone/carrier for oxygen), one or more perfluorocarbons, and/or one or more platelets. Artificial blood compositions are also available commercially, for example from HbO2 Therapeutics (under the tradename Hemopure) and Yale University (under the tradename BrainEx).

[0060] In non-limiting embodiments, system 1000 may further include one or more fluid conduits 190 fluidly connecting the bioreactor 1 10 to the media reservoir 120. Fluid conduits may be formed of any biocompatible material and may include any number of turns, bends, curves, and/or side branches (e.g., to allow for connection of one or more additional reservoirs to bioreactor system 1000 as described below). In non-limiting embodiments, system 1000 may include a cannula in fluid communication with the media reservoir 120 (for example, through one or more fluid conduits 190). A cannula may be any cannula and/or needle that is biocompatible, and such devices are commercially available and are known to those of skill in the art. A useful cannula may be configured to couple to a blood vessel of the tissue sample 1 12.

[0061] In non-limiting embodiments, one or more additional reservoirs 130 may be included in the system. These reservoirs may hold one or more compositions that may be of use in system 1000. For example, as described below tissue sample 1 12 may be a tissue sample from an individual with a condition and/or disease, and one or more additional reservoirs 130 may include one or more potential therapeutics, so that system 1000 may be useful for testing efficacy and/or toxicity in an in vitro setting prior to a clinical use. Useful compositions can include anticoagulants (such as, for example heparin), anti-cancer compounds, anti-epileptic compounds, and/or any potential therapeutic for a neuro-specific condition and/or disease. One or more additional reservoirs 130 may be arranged anywhere within system 1000 (e.g., the arrangement in FIG. 1 is not limiting). In non-limiting embodiments, one or more additional reservoirs 130 may be arranged between the media reservoir 120 and the bioreactor 1 10. [0062] The term “fluid circuit” is used herein to refer to a fluid connection that may include a circuit including one or more fluid conduits 190 and their connection to bioreactor 1 10, media reservoir 120, and/or one or more additional reservoirs 130. In non-limiting embodiments, such a fluid circuit (and thus, system 1000), may be a closed system (e.g., fluid pumped from media reservoir 120 is eventually returned to media reservoir 120), or an open system (e.g., media pumped from media reservoir 120 is pumped into a waste container (not shown) after flowing through bioreactor 1 10).

[0063] In non-limiting embodiments, system 1000 may further include one or more sensors 170. Sensor(s) 170 may be configured to measure one or more parameters of the bioreactor 110, the media within the bioreactor, the tissue 112, and/or the fluid media (e.g., the artificial blood composition). Useful sensor may include a pressure sensor, an oxygen sensor, a temperature sensor, a flowrate sensor, and/or a pH sensor. Sensor(s) 170 may be arranged in any useful configuration (e.g., the arrangement in FIG. 1 is not limiting), including within bioreactor 110, within tissue sample 1 12, within the fluid circuit defined by bioreactor 1 10, media reservoir 120, and one or more fluid conduits 190, and/or within a reservoir (e.g., 120 or 130). As will be described in greater detail below, sensors may be in communication with a computing device 180 (e.g., as shown in FIG. 6), to allow for modulation of one or more parameters of system 1000 (for example, by oxygenator 140, a temperature controller (182), a pump 150, and/or a fluid reservoir (e.g., 120 and/or 130). To this end, in nonlimiting embodiments system 1000 may include one or more temperature controllers 182, which may be arranged in bioreactor 110 and/or any useful location in the fluid circuit, for example in media reservoir 120.

[0064] In non-limiting embodiments, system 1000 may further included one or more pumps 150 configured to cause media to flow from the media reservoir 120 and any additional reservoirs 130 through one or more fluid conduit 190, for example to a cannula and thus to a blood vessel of the tissue sample 1 12. Any suitable pump may be used in system 1000, including, without limitation, peristaltic pumps, roller pumps, and/or rotary pumps. While FIG. 1 shows a certain placement of pumps 150 within system 1000, those of skill will appreciate that any arrangement of pumps 150 is possible, including, for example, in different places within the fluid circuit defined by bioreactor 1 10, media reservoir 120, and one or more fluid conduits 190, and/or within a specific structure (e.g., within bioreactor 1 10, media reservoir 120, and/or additional reservoir(s) 130).

[0065] In non-limiting embodiments, system 1000 may further include oxygenator 140. Oxygenator 140 may be in fluid communication with the one or more fluid conduits 190, media reservoir 120, and/or bioreactor 1 10. Suitable oxygenators can be those that provide dissolved oxygen to the fluid media (e.g., artificial blood composition), and are known to those of skill in the art, for example as commercially available from BioProcess International. Oxygenator 140 may be arranged anywhere within system 1000 (e.g., the arrangement in FIG. 1 is not limiting). In non-limiting embodiments, oxygenator 140 may be arranged between the media reservoir 120 and the bioreactor 1 10.

[0066] In non-limiting embodiments, system 1000 may include one or more filters 160. One or more filters 160 may be arranged anywhere within system 1000 (e.g., the arrangement in FIG. 1 is not limiting). In non-limiting embodiments, one or more filters 160 may be arranged between the media reservoir 120 and the bioreactor 1 10, for example downstream of oxygenator 140 and/or one or more of pumps 150. Suitable filters are known to those of skill in the art and may include filtration membranes and/or may be purchased commercially from, for example, Sigma-Aldrich, Sartorius, Wasteless Bio, and/or BioProcess International.

[0067] In non-limiting embodiments, system 1000 may further include an electrocorticography (ECoG) device. Such a device may include one or more electrode leads 184 and a computing device (e.g., computing device 180). In nonlimiting embodiments, the at least one electrode lead 184 may be arranged in the bioreactor 110, for example in tissue sample 1 12 (though electrodes for detecting field potentials without entering tissue are available), and may be configured to detect an electrical signal from the tissue sample 1 12. In non-limiting embodiments, at least one computing device 180 may be in electrical communication with the at least one electrode lead 184 and may be configured to acquire, amplify, filter, and/or analyze the electrical signal received from the at least one electrode lead 184. As used herein, "electrical communication," for example in the context of transmitting electrical pulses from a pulse generator to an electrode lead 184, refers to sending an electrical pulse produced by a pulse generator to the lead for providing electrical stimulation as described herein, typically through an electrically conductive lead, such as a wire. Stimulation may be delivered to mimic a condition and/or disease, and/or as a potential therapy for a condition and/or disease.

[0068] In non-limiting embodiments, system 1000 may further include a device to reduce and/or eliminate interference in electrical signal detection 1 14, such as a Farraday cage arranged on and/or in bioreactor 1 10.

[0069] In non-limiting embodiments the at least one electrode lead 184 may be configured to deliver electrical stimulation to the tissue sample 1 12. In non-limiting embodiments, the at least one computing device 180 may include or be in communication with a pulse generator capable of producing electrical pulses that may be delivered to tissue sample 112 through electrode lead 184.

[0070] As discussed above, in non-limiting embodiments, system 1000 may include at least one computing device 180. At least one computing device 180 may be in communication with the one or more pumps 150, one or more sensors 170, one or more temperature controllers 182, and/or one or more electrode leads 184. Computing device 180 may be configured to modulate one or more parameters of the media, the bioreactor 110, and/or the tissue sample 1 12 based on data received from the one or more sensors 170.

[0071] A device (e.g., computing device 180) for inclusion in system 1000 and/or implementing methods described herein may correspond to any element of a system 1000. In some non-limiting embodiments, such systems 1000 or devices 180 may include, with reference to FIG. 6, at least one device 200 and/or at least one component of device 200. The number and arrangement of components shown are provided as an example. In some non-limiting embodiments, device 200 may include additional components, fewer components, different components, or differently arranged components than those shown. Additionally, or alternatively, a set of components (e.g., one or more components) of device 200 may perform one or more functions described as being performed by another set of components of device 200. [0072] Device 200 may include a bus 202, a processor 204, memory 206, a storage component 208, an input component 210, an output component 212, and a communication interface 214. Bus 202 may include a component that permits communication among the components of device 200. In some non-limiting embodiments, processor 204 may be implemented in hardware, firmware, or a combination of hardware and software. For example, processor 204 may include a processor (e.g., a central processing unit (CPU), a graphics processing unit (GPU), an accelerated processing unit (APU), etc.), a microprocessor, a digital signal processor (DSP), and/or any processing component (e.g., a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), etc.) that can be programmed to perform a function. Memory 206 may include random access memory (RAM), read only memory (ROM), and/or another type of dynamic or static storage device (e.g., flash memory, magnetic memory, optical memory, etc.) that stores information and/or instructions for use by processor 204.

[0073] Storage component 208 may store information and/or software related to the operation and use of device 200. For example, storage component 208 may include a hard disk (e.g., a magnetic disk, an optical disk, a magneto-optic disk, a solid-state disk, etc.) and/or another type of computer-readable medium. Input component 210 may include a component that permits device 200 to receive information, such as via user input (e.g., a touch screen display, a keyboard, a keypad, a mouse, a button, a switch, a microphone, etc.). Additionally, or alternatively, input component 210 may include a sensor for sensing information (e.g., a global positioning system (GPS) component, an accelerometer, a gyroscope, an actuator, etc.). Sensors useful here may include biochemical sensors, electrochemical sensors, sensors for detecting autonomic tone, sensors for detecting sympathetic tone, and/or the like. Output component 212 may include a component that provides output information from device 200 (e.g., a display, a speaker, one or more light-emitting diodes (LEDs), etc.). Communication interface 214 may include a transceiver-like component (e.g., a transceiver, a separate receiver and transmitter, etc.) that enables device 200 to communicate with other devices, such as via a wired connection, a wireless connection, or a combination of wired and wireless connections. Communication interface 214 may permit device 200 to receive information from another device and/or provide information to another device. For example, communication interface 214 may include an Ethernet interface, an optical interface, a coaxial interface, an infrared interface, a radio frequency (RF) interface, a universal serial bus (USB) interface, a Wi-Fi® interface, a cellular network interface, and/or the like.

[0074] Device 200 may perform one or more processes described herein. Device 200 may perform these processes based on processor 204 executing software instructions stored by a computer-readable medium, such as memory 206 and/or storage component 208. A computer-readable medium may include any non-transitory memory device. A memory device includes memory space located inside of a single physical storage device or memory space spread across multiple physical storage devices. Software instructions may be read into memory 206 and/or storage component 208 from another computer-readable medium or from another device via communication interface 214. When executed, software instructions stored in memory 206 and/or storage component 208 may cause processor 204 to perform one or more processes described herein. Additionally, or alternatively, hardwired circuitry may be used in place of or in combination with software instructions to perform one or more processes described herein. Thus, embodiments described herein are not limited to any specific combination of hardware circuitry and software. The term "configured to," as used herein, may refer to an arrangement of software, device(s), and/or hardware for performing and/or enabling one or more functions (e.g., actions, processes, steps of a process, and/or the like). For example, "a processor configured to" may refer to a processor that executes software instructions (e.g., program code) that cause the processor to perform one or more functions.

[0075] In non-limiting embodiments, a system 1000 may include a bioreactor 1 10, a neural tissue sample 1 12 received within the bioreactor 1 10. In non-limiting embodiments, the neural tissue sample 1 12 may include a plurality of cortical cells and at least one blood vessel. As used here, the term “cortical cell” may refer to a neuron (or any part thereof such as an axon and/or a dendrite), a glial cell, any cell or portion thereof that forms the blood-brain barrier, and/or a neural stem cell. In nonlimiting embodiments system 1000 may include a media reservoir 120 in fluid communication with the bioreactor 1 10 and holding an artificial blood composition therein. In non-limiting embodiments, system 1000 may include one or more fluid conduits 190 fluidly connecting the bioreactor 1 10 to the media reservoir 120 and defining a closed system including the media reservoir 120 and the bioreactor 130. In non-limiting embodiments, one or more fluid conduits 190 may include a cannula coupled to the at least one blood vessel of the neural tissue sample 1 12. In nonlimiting embodiments, system 1000 may include one or more sensors 170 configured to measure one or more parameters of the artificial blood composition and/or the neural tissue sample 112. In non-limiting embodiments the one or more sensors 170 may include a pH sensor, a flow rate sensor, a pressure sensor, a temperature sensor, and/or an oxygen sensor. In non-limiting embodiments, system 1000 may include one or more pumps 150 configured to cause the artificial blood composition to flow from the media reservoir 120 to the at least one blood vessel through the cannula. [0076] In non-limiting embodiments, system 1000 may further include one or more additional reservoirs 130 holding a therapeutic composition. In non-limiting embodiments, the neural tissue sample 112 is a sample comprising one or more tumor cells and the therapeutic composition is a potential therapy for the tumor. In nonlimiting embodiments, the neural tissue sample 1 12 is a sample from a patient with epilepsy and the therapeutic composition is a potential therapy for epilepsy.

[0077] Also provided herein are methods of using a bioreactor system, such as system 1000, to maintain a tissue sample (e.g., a neural tissue sample) and/or screen one or more potential therapies for a condition or disease (e.g., a neural condition or disease). In non-limiting embodiments, a method may include harvesting or otherwise obtaining a tissue sample. In non-limiting embodiments the sample may include tissue and vasculature. In non-limiting embodiments the sample may be a neural tissue sample, for example a cortical tissue sample. In non-limiting embodiments, the bloodbrain barrier of the neural sample is intact. In non-limiting embodiments, the tissue sample may be placed into a bioreactor, for example, as described herein, and the vasculature may be cannulated. In non-limiting embodiments the vasculature is first flushed with heparin or another anticoagulant, for example to clean and/or dilate the vasculature, thereby making cannulation more likely to be successful. In non-limiting embodiments, the cannulated tissue is then maintained in the bioreactor, and the fluid media (e.g., artificial blood composition) is circulated through the fluid circuit to the tissue sample through the one or more fluid conduits and the cannula. In non-limiting embodiments, a computing device as described herein may receive data from one or more sensors as described herein and, based on such data, adjust one or more parameters of the bioreactor, pump(s), temperature controller(s), ECoG device, additional reservoirs, and/or fluid media (e.g., artificial blood composition). In nonlimiting embodiments such adjustments may be made automatically and may be tissue-specific and/or condition/disease specific.

[0078] In non-limiting embodiments, tissue in the bioreactor may be maintained or various therapies for a condition or disease may be assessed, for example by stimulating tissue with electrical stimulation (as described above) and/or introduction of one or more potential therapeutics to fluid media. As disclosed herein, the tissue sample may be a neural tissue sample, and may include tissue with an intact bloodbrain barrier, thus allowing for assessment of potential therapeutics’ ability to affect, interact with, and/or cross that barrier. Example

Methods

[0079] Extracorporeal Perfusion:

[0080] Upon surgical resection of brain tumors from consenting patients, the harvested cortical specimens were expeditiously divided into two experimental groups: perfused and non-perfused control. In the operating room, the resected cortex is transferred to a separate draped table. The sulci are split to expose the intrasulcal arteries. A 25G cannula is primed with heparin and papaverine to prevent air emboli. Once the artery is cannulated the specimen is flushed with heparin and papaverine and transferred in cold saline to the lab. The perfusion apparatus consisted of a peristaltic pump system (Reglo Digital Multichannel Peristaltic Pump (SKU: MF- 78018-10) designed for controlled and pulsatile perfusion. The tubing from the peristaltic pump (2-Stop Tygon E-LFL Pump Tubing SKU: MF-96449-10) was connected to an 25G arterial cannula (MEDIine Angiocath B-D381 112Z) , which was delicately introduced into the vasculature of the excised brain cortex specimen. The perfusion solution employed was a custom-designed human plasma-based media, comprising electrolytes, nutrients, and oxygen carriers to simulate the physiological environment. Throughout the perfusion process, critical parameters including tissue oxygen saturation (02), temperature, and pH were continuously monitored using inline sensors. Temperature sensors within the perfusion circuit maintained a normothermic environment, critical for preserving enzymatic and cellular functions. pH sensors ensured the solution remained within the physiological range, adjusting buffers as necessary to mimic the natural conditions of brain interstitial fluid. These meticulous controls were essential for maintaining the integrity and functionality of the brain tissue, providing a stable environment for subsequent analyses.

[0081] Perfusion Procedure:

[0082] Before initiating the extracorporeal perfusion, we injected surgical-grade fluorescein dye (AK-FLUOR® Fluorescein Sodium 10%) intraarterially to verify the vascular patency with brain specimens was injected intravascularly at a standardized concentration. The circulation of the dye throughout the vascular network of the brain specimens was observed with the naked eye. The presence of a uniform distribution of fluorescein within the vasculature indicated unobstructed blood flow, confirming the integrity and patency of the vessels. This step was critical for ensuring that the perfusion system could effectively deliver perfusate to all regions of the brain tissue, thereby facilitating consistent and reliable experimental conditions. Each specimen's vascular integrity was documented photographically as part of the experimental record, providing visual evidence of successful dye penetration and vessel functionality before the commencement of perfusion procedures.

[0083] The perfusion procedure was initiated immediately post-resection. The peristaltic pump was meticulously calibrated and primed to replicate physiological blood flow conditions. The human plasma-like media (Gibco™ Human Plasma-Like Medium (HPLM) Catalog number: A4899101 ), maintained at physiological temperature and pH, was perfused through the vasculature of the tumor at a controlled flow rate. The perfusion process is aimed at preserving the tissue's microenvironment, including oxygenation, nutrient supply, and waste removal, thereby maintaining the physiological conditions of the tumor as closely as possible. Simultaneously, the nonperfused control group underwent parallel processing without the introduction of the perfusion solution. These samples were handled under identical conditions, ensuring any observed differences could be attributed to the perfusion procedure.

[0084] Biopsies Extraction:

[0085] To assess the molecular and cellular responses at multiple time points, we employed a sterile punch biopsy technique, which is renowned for its precision and minimal tissue disruption. A 3 mm biopsy punch was used to extract tissue samples from predefined regions of interest within the brain specimens. These regions were selected based on their known susceptibility to ischemic damage. Biopsies were performed at the onset of perfusion and subsequently at one-hour intervals, culminating in a final sample collection at four hours post-mortem. This schedule was strictly adhered to for perfused and non-perfused specimens, facilitating a time-course analysis of ongoing biological processes within the brain tissue. Immediately following extraction, each biopsy sample was either fixed in 4% PFA (Thermo Scientific, Catalog number: A1 1313.22) Chemicals for histological processing and placed into a cryogenic vial, snap-frozen in liquid nitrogen, and stored at -80 °C to prevent degradation and to maintain the viability of RNA, proteins, and metabolites for subsequent analyses. To ensure reproducibility and statistical robustness, multiple biopsies were taken from symmetric regions across different specimens in each group. Additionally, all biopsies and subsequent analyses were conducted by personnel blinded to the group allocations to mitigate bias.

[0086] Hematoxylin and Eosin (H&E) Staining: [0087] Sample Collection and Perfusion Procedure:

[0088] Upon surgical resection of brain tumors from consenting patients, the excised tumor specimens were expeditiously divided into two groups: perfused and non-perfused. The perfusion procedure was initiated immediately, involving the establishment of physiological blood flow in the perfused group through cannulation. A specialized perfusion solution, mimicking the constituents of human plasma, was circulated through the vasculature of the tumor, with meticulous attention to preserving the natural perfusion environment.

[0089] Histological Processing:

[0090] Post-collection, the tumor specimens were promptly fixed in 4 % neutral buffered formalin for a duration ensuring optimal tissue fixation. Subsequently, the fixed tissues were meticulously embedded in paraffin (Thermo Scientific, Catalog number: 416770020), and 5-micron sections were meticulously obtained utilizing a microtome. To discern cellular architecture and overall tissue morphology, H&E staining, a gold standard histological technique, was meticulously employed. The paraffin-embedded sections underwent a sequential process of deparaffinization and rehydration before being subjected to hematoxylin staining (ABCAM, ab245880) for nuclear visualization and eosin staining for the delineation of cytoplasmic components. The stained sections were then systematically dehydrated and meticulously coverslipped.

[0091] Morphological Assessment:

[0092] A comprehensive morphological evaluation was undertaken by a blinded observer utilizing a light microscope. Random fields of view were systematically selected from both the perfused and non-perfused tumor samples for unbiased analysis. Cellular attributes such as nuclear morphology, cytoplasmic density, and overall tissue architecture were subjected to meticulous scrutiny. Advanced image analysis software (Nikon, NIS-Elements) facilitated the quantitative assessment of parameters such as cell density and nuclear-to-cytoplasmic ratio. Quantitative data, expressed as mean ± standard error of the mean (SEM), underwent rigorous statistical scrutiny. The divergence between perfused and non-perfused groups was statistically evaluated using appropriate tests using the software GraphPad prism, with a predefined significance level set at p < 0.05.

[0093] Immunofluorescence Staining: [0094] The tissue sections were permeabilized with 0.3% Triton X-100 (Thermo Fischer, HFH10) in PBS (Thermo Fischer, 10010023) for 10 minutes and blocked with 10% normal goat serum (Invitrogen™, R37624) in PBS for 1 hour at room temperature to prevent non-specific binding. Primary antibodies were diluted in 1 % NGS in PBS and incubated with the tissue sections overnight at 4°C. The primary antibodies used were anti-GFAP (glial fibrillary acidic protein) (Cell Signaling, 12389T) to label astrocytes, anti-IBA1 (ionized calcium-binding adapter molecule 1 ) (Cell Signaling, 17198T) to label microglia, and anti-Caspase-3 to detect apoptotic cells (Cell Signaling, 9664T). Following primary antibody incubation, the sections were washed three times with PBS and incubated with species-specific secondary antibodies conjugated to fluorescent dyes (e.g., Alexa Fluor 488, Alexa Fluor 594) (Cell signaling, 4412S, 8889S) for 1 hour at room temperature in the dark. The sections were then washed in PBS and counterstained with DAPI (4',6-diamidino-2-phenylindole) to label cell nuclei (Invitrogen™, R37606). Negative controls, omitting the primary antibody, were included to verify the specificity of the staining. Additionally, isotype-matched control antibodies were used to ensure the accuracy of the immunofluorescence signals.

[0095] This immunofluorescence protocol allowed for the precise localization and quantification of astrocytic and microglial activation, as well as the assessment of apoptosis in both perfused and non-perfused brain tissues.

[0096] Nissl Staining:

[0097] Sample Preparation:

[0098] Human brain specimens were collected post-mortem and divided into two groups: one subjected to our extracorporeal brain perfusion system and the other left non-perfused as a control. Both sets of specimens were sectioned coronally at 5 mm intervals to ensure consistency in analysis.

[0099] Fixation and Processing:

[00100] All sections were fixed in 4% paraformaldehyde (Thermo Fischer, Catalog number: J61899.AP) for 24 hours at 4°C to preserve cellular architecture. After fixation, sections were washed in phosphate-buffered saline (PBS) (Thermo Fischer, Catalog number: J61 196.AP) three times to remove residual fixative, followed by a dehydration sequence through graded ethanol (70%, 95%, 100%) and subsequent clearing in xylene. Sections were stained using 0.1 % Cresyl violet, a dye (ABCAM, ab246817) that selectively binds to Nissl substance in neuronal cytoplasm, facilitating the visualization of neuronal structure and morphology. Staining was performed for 15 minutes to ensure adequate penetration and staining intensity. After staining, excess dye was removed by differentiation in 95% ethanol, a step critical for enhancing the visual contrast of the stained Nissl bodies. Sections were then rehydrated briefly, cleared again in xylene, and mounted onto slides with a xylene-based mounting medium.

[00101] Microscopic Examination and Image Analysis:

[00102] Stained sections were examined under a Nikon Elipse Ti2 microscope equipped with digital imaging capabilities. High-resolution images were captured from consistent anatomical locations across all specimens. Neuronal morphology, particularly the integrity of Nissl substance, was assessed quantitatively by measuring the density and clarity of Nissl bodies within neurons. The structural integrity of neurons in perfused versus non-perfused specimens was compared using image analysis software. Parameters such as Nissl body density, neuronal soma size, and overall neuronal architecture were quantified and statistically analyzed. This comparative assessment allowed us to determine the efficacy of the perfusion system in preserving neuronal structure.

[00103] Electrophysiological Signal Acquisition and Recording:

[00104] To evaluate the electrical activity of the brain specimens, we employed a custom-built probe specifically designed for high-resolution electrophysiological recordings. This probe, developed in collaboration with an engineering team, features multiple microelectrodes capable of detecting subtle electrical signals from the cortical surface of the brain tissue. The probe was carefully positioned on the surface of the perfused brain specimens to ensure optimal contact and signal acquisition.

[00105] Concurrently, we perfused the brain specimens with a hemoglobin-based media, obtained from the Sestan group. This media was specially formulated to mimic the oxygen-carrying capacity and nutrient profile of human blood, thereby supporting the metabolic demands of the brain tissue. The media was delivered through our extracorporeal perfusion system via a peristaltic pump, which provided a controlled and pulsatile flow, closely replicating natural cerebral perfusion. This setup ensured that the brain tissue received a consistent supply of oxygen and nutrients, maintaining its viability and functionality during the ex vivo experiments.

[00106] Metabolomic Analysis Sample Collection and Preparation [00107] Perfused tumor specimens and non-perfused control tissue samples were collected from our experimental model. Perfusion was performed using a carefully optimized protocol to mimic physiological conditions and enhance the preservation of tissue metabolites. Non-perfused controls were collected directly after excision without undergoing perfusion. Both groups were snap-frozen immediately after collection to minimize metabolic degradation and stored at -80 °C until analysis. Samples were homogenized using an ice-cold solvent mixture (methanol: water: chloroform, 2:2:1 ), facilitating the separation of polar and non-polar fractions through centrifugation. The polar fraction, containing metabolites critical to central carbon metabolism, was extracted for metabolomic analysis.

[00108] Targeted Metabolomic Analysis

[00109] Targeted metabolomic profiling focused on quantifying metabolites involved in essential pathways, such as energy metabolism (TCA cycle and glycolysis) 26-30, redox homeostasis 31 -34 (glutathione metabolism), amino acid biosynthesis, and nucleotide metabolism. UHPLC-MS/MS was conducted with analyte-specific parameters, including optimized collision energies and precursor/product ion transitions. Calibration curves were generated using authentic standards for each targeted metabolite to ensure high specificity and quantification accuracy. Quality control samples were interspersed throughout the analysis to assess instrument stability and reproducibility.

[00110] Untargeted Metabolomic Analysis

[00111 ] The untargeted metabolomic analysis aimed to capture a broader spectrum of metabolites to identify novel metabolic alterations in perfused versus non-perfused samples 35, 36. LC-MS/MS was performed using high-resolution Orbitrap mass spectrometry in positive and negative ionization modes. Data-dependent acquisition (DDA) enabled the simultaneous collection of high-resolution MS1 data and fragmentation spectra (MS2). Data preprocessing, including peak alignment, feature extraction, and deconvolution, was performed using Compound Discoverer software. Compound identification relied on exact mass, isotope patterns, and fragmentation spectra matched against in-house and publicly available libraries.

[00112] Statistical and Pathway Enrichment Analysis

[00113] Metabolite intensities were normalized to tissue weight, log-transformed, and subjected to statistical analysis. Differential metabolites between perfused and non-perfused samples were identified using two-tailed t-tests with p-values adjusted for multiple comparisons via the Benjamini-Hochberg method. Fold changes (Iog2- transformed) were calculated to assess relative metabolite abundance. Pathway enrichment analysis was conducted through MetaboAnalyst, integrating Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations. Bubble plots were used to visualize pathway-level changes, with enrichment ratios derived from metabolite set overlap, and adjusted p-values indicating pathway significance.

Results

[00114] Extracorporeal Perfusion Maintains Brain Function:

[00115] We designed an extracorporeal human brain perfusion system to sustain and monitor resected brain lobectomy specimens from neurosurgical procedures as depicted in (FIGS. 1A-1 B). The brain cortex was excised from a brain tumor patient and prepared for perfusion. The arterial cannula was connected to the resected brain tissue within a temperature-controlled chamber, ensuring continuous circulation of a hemoglobin-based perfusate, delivered using roller and peristaltic pumps to achieve pulsatile flow (FIG. 1A). Key physiological parameters, including temperature, pH, pressure, flow rate, and tissue oxygen levels, were continuously monitored through integrated sensors, with real-time data acquisition for electroencephalographic (ECoG) activity. Vital signs of the brain tissue over the four-hour perfusion period indicated rigorous temperature control, stable perfusate delivery as monitored by flow rate and pressure, effective pH buffering, and adequate tissue oxygenation, supporting cellular metabolism (FIG. 1 B). Hourly punch biopsies allowed for subsequent analyses of apoptosis, immune response, neural cellular integrity, and metabolic activity. Nissl staining and caspase-3 immunofluorescence on these biopsies revealed significant preservation of neuronal architecture and reduced apoptosis in perfused specimens compared to non-perfused controls. (FIGS. 3A-3C)

[00116] Fluorescein Dye Confirms Effective Perfusion

[00117] To visualize vascular patency and assess the efficacy of our perfusion system, fluorescein dye was utilized in resected brain cortex specimens (FIGS. 2A- 2E) The resected cortex was highlighted by the application of fluorescein dye to visualize vascular structures (FIG. 2A). The administration of fluorescein dye through the cannulated artery showed the progressive spread of the dye within the vasculature, confirming effective perfusion and vascular integrity (FIG. 2B). Detailed views of the fluorescein blush, with regions where the dye successfully permeated the cortical tissue marked by yellow dashed circles, further demonstrate the extensive dye distribution indicative of robust perfusion (FIG. 2C). These findings confirm the vascular patency and functional capacity of our perfusion system to deliver perfusate efficiently across the resected brain tissue. The consistent distribution of fluorescein and the observed blush in perfused specimens underscore the potential of our system to maintain adequate vascular flow and tissue viability ex vivo.

[00118] Perfusion Preserves Neuronal Integrity and Reduces Apoptosis:

[00119] To evaluate the efficacy of our perfusion system, we conducted a detailed analysis of neuronal integrity, apoptosis, and cell density in perfused and non-perfused brain specimens over a four-hour period (FIGS. 3A-3C). Nissl staining 20, 21 demonstrated that perfused specimens exhibited well-preserved neuronal architecture, with prominent Nissl bodies, while non-perfused controls showed significant neuronal degradation (FIG. 3A) Quantitative analysis revealed a significantly higher Nissl signal and a greater number of neurons per region of interest (ROI) in perfused specimens at both 1 hour (n=9, p=0.02) and 4 hours (n=9, p=0.01 ) compared to controls (FIG. 3A). Apoptosis was assessed using Caspase-3 (CASP3) immunofluorescence staining, which identified fewer Caspase-3 positive cells in perfused specimens at all time points (FIG. 3B). The perfused group showed significantly lower CASP3 signal intensity and fewer CASP3-positive ROIs at 1 hour (n=9, p<0.001 ) and 4 hours (n=9, p<0.001 ), indicating reduced apoptosis (FIG. 3B). Cell density was evaluated using Hematoxylin and Eosin (H&E) staining, which showed that perfused specimens maintained higher cell density over time (FIG. 3C). Perfused specimens had significantly higher cell density at 0 hours (n=9, p<0.001 ), 1 hour (n=9, p<0.001 ), and 4 hours (n=9, p<0.001 ) compared to non-perfused controls. Moreover, non-perfused specimens exhibited progressive tissue necrosis, whereas perfused specimens preserved tissue integrity, as indicated by cell density measurements (FIG. 3C). These findings collectively demonstrate that our perfusion system effectively preserves neuronal structure, reduces apoptosis, and maintains cell viability in resected human brain tissue, underscoring its potential as a robust ex vivo model for studying brain physiology and pathology.

[00120] Perfused Brain Specimens Show Regional Glial Activation:

[00121] To assess the impact of our perfusion system on glial activation and inflammatory responses, we injected brain tissue specimens with 2.5ul of lipopolysaccharide (LPS) and analyzed them using immunofluorescence staining. (FIGS 5A-5E) presents the results of these analyses, comparing perfused and non- perfused specimens over a four-hour-period. Immunofluorescence staining for glial fibrillary acidic protein (GFAP), a marker of astrocytic activation 22, showed a substantial increase in GFAP signal intensity in perfused specimens at both 1 hour and 4 hours post-LPS injection compared to non-perfused controls, which showed minimal GFAP expression (FIGS. 5A-5F). Quantitative analysis confirmed significantly higher GFAP signal in perfused specimens at both time points (n=8, p<0.01 ) (FIGS. 5A-5F). Ionized calcium-binding adapter molecule 1 (IBA1 ) staining, a marker of microglial activation 23, revealed pronounced microglial activation in perfused specimens with robust IBA1 expression at 1 hour and 4 hours post-LPS injection, while non-perfused controls showed markedly lower IBA1 signal (FIGS. 5A-5F). Statistical analysis indicated significantly elevated IBA1 signal in perfused specimens at both time points (n=8, p<0.01 ) (FIGS. 5A-5F). Regional GFAP and IBA1 staining across different brain regions (frontal, temporal, and parietal cortex) demonstrated consistent glial activation in perfused specimens, with significantly higher GFAP and IBA1 signals compared to non-perfused controls, which exhibited sparse and uneven glial response (FIGS. 5A-5F). Quantitative comparisons revealed significant differences in GFAP and IBA1 signals between perfused and non-perfused specimens at both 1 hour and 4 hours across all regions analyzed (n=8, p<0.01 ) (FIGS. 5A-5F). These results demonstrate that our perfusion system effectively maintains glial and inflammatory responses in resected brain tissue, suggesting a preserved capability for immune reactivity. The significant activation of astrocytes and microglia in perfused specimens underscores the system's potential for studying neuroinflammatory processes ex vivo. [00122] Sustained Electrocorticographic Activity in Perfused Brain Specimens: [00123] To evaluate the preservation of neural activity in perfused brain specimens, we continuously monitored potential electrocorticographic (ECoG) signals. Our perfusion system demonstrated the ability to maintain an ECoG activity over the experimental period. Perfused specimens exhibited consistent and well-defined ECoG waveforms, indicating sustained neural viability and function. Compared to nonperfused controls, which showed a rapid decline in ECoG activity, perfused specimens retained stable electrical signals throughout the first hour of the four-hour monitoring period. These findings underscore the efficacy of our perfusion system in preserving electrophysiological function for a certain amount of time, confirming its potential as a reliable ex vivo model for studying brain activity and pathophysiology. The sustained ECoG activity in perfused specimens highlights the system's capability to maintain the intricate electrical signaling of the brain, crucial for investigating neurophysiological processes and therapeutic interventions.

[00124] Enhanced Metabolic Profile in Perfused Brain Specimens

[00125] To assess the metabolic integrity of perfused brain specimens, we conducted comprehensive metabolomic analyses comparing perfused and nonperfused controls. Our results demonstrated a significantly more robust metabolic profile in the perfused specimens.

[00126] Targeted Analysis Highlights Metabolic Differences Between Perfused and Non-Perfused Samples

[00127] Targeted metabolomic analysis provided critical insights into the metabolic stabilization conferred by perfusion. Perfused samples exhibited a pronounced upregulation in amino acid metabolism pathways, with arginine biosynthesis showing a 3.4-fold increase compared to non-perfused controls (p = 0.002). This pathway is crucial for maintaining nitric oxide production and polyamine synthesis, both essential for tumor growth and signaling. Similarly, valine, leucine, and isoleucine biosynthesis demonstrated significant upregulation (2.9-fold, p = 0.004), reflecting the preservation of branched-chain amino acid (BCAA) metabolism under perfused conditions. TCA cycle intermediates, including citrate (+3.6-fold), succinate (+4.1 -fold), and malate (+2.8-fold), were markedly elevated in perfused samples (p < 0.01 ). This suggests enhanced preservation of central carbon metabolism pathways, enabling more accurate profiling of energy metabolism. Additionally, glutathione metabolism showed a notable increase in reduced glutathione (GSH) levels (+4.2-fold, p < 0.001 ), indicating a reduction in oxidative degradation in perfused specimens.

[00128] Untargeted Analysis Reveals Extensive Metabolic Perturbations

[00129] The untargeted metabolomic analysis identified 375 unique metabolites, with 43 showing statistically significant differences between the two groups (adjusted p < 0.05). Key metabolites included elevated levels of glutathione, ornithine, and S- adenosylmethionine in perfused specimens, reflecting preserved balance, urea cycle activity, and methylation potential, respectively. Conversely, non-perfused samples exhibited higher levels of nucleotide breakdown products such as inosine and hypoxanthine, consistent with post-excision degradation of nucleotides. Volcano plot analysis further highlighted the significant upregulation of glutathione (Iog2 fold change: +4.2, p < 0.001 ) and the downregulation of adenosine monophosphate (Iog2 fold change: -3.8, p = 0.005) in perfused samples. These findings underscore the protective effect of perfusion in stabilizing redox homeostasis and nucleotide turnover. [00130] Pathway Enrichment Emphasizes Metabolic Stabilization via Perfusion [00131] Pathway enrichment analysis revealed profound differences in metabolic pathways between perfused and non-perfused specimens. Among the most significantly enriched pathways were amino acid metabolism, the TCA cycle, and pyrimidine metabolism. The arginine biosynthesis pathway demonstrated an enrichment ratio of 14.7 (p < 0.001 ), highlighting its central role in perfused tissue metabolism. Similarly, the TCA cycle showed an enrichment ratio of 1 1 .2 (p < 0.001 ), reflecting the preservation of energy-generating pathways under perfused conditions. [00132] Enrichment analysis of pyrimidine metabolism revealed a 10.5-fold increase in pathway activity in perfused samples (p < 0.001 ), suggesting improved stabilization of nucleotide synthesis and turnover. This finding aligns with reduced levels of nucleotide degradation products in perfused specimens, further supporting the hypothesis that perfusion mitigates post-excision metabolic degradation. Additionally, pathways associated with redox homeostasis, such as glutathione metabolism, were significantly enriched, demonstrating the protective effects of perfusion on oxidative stress. Pathway bubble plots emphasize the most significantly altered pathways, with bubble size indicating enrichment ratio and color gradient representing p-value significance. Volcano plots highlight key metabolites driving the observed metabolic differences, underscoring the stabilization achieved in perfused specimens. Discussion

[00133] Our proposed extracorporeal brain perfusion system demonstrates significant potential in maintaining the viability, functionality, and metabolic integrity of resected human brain tissue ex vivo. By ensuring a continuous supply of oxygenated, nutrient-rich perfusate, our system effectively preserves the physiological conditions necessary for sustaining brain tissue health and activity, addressing substantial limitations inherent in traditional in vitro models. When compared to the landmark study Vrselja et al 19, which successfully restored and maintained cellular functions in the intact pig brain under ex vivo conditions, our findings extend these capabilities to human brain specimens, thus bridging a critical gap between animal models and human applications. Our study revealed sustained electrocorticographic (ECoG) activity in perfused specimens, indicating that neural circuits remained functional over extended periods ex vivo, paralleling the preservation of neuronal functions observed in the pig brain study. Additionally, our metabolomic analysis showed that perfused brain specimens maintained high levels of key metabolites, such as ATP, glutamate, and N-acetylaspartate, with reduced markers of metabolic stress. Furthermore, robust glial activation and immune responsiveness were observed in our perfused specimens following LPS injection, as evidenced by increased GFAP and IBA1 signals, demonstrating the preservation of immune responses, similar to the glial and vascular responses reported in the pig brain model. These findings substantiate the capability of our perfusion system to sustain human brain tissue ex vivo, offering a promising platform for further research into brain physiology and the development of novel therapeutic strategies. This study highlights the potential of extracorporeal perfusion systems for advancing neuroscience research and clinical applications, providing new insights into brain function and pathology.

[00134] While the present invention has been described in terms of the above detailed description, those of ordinary skill in the art will understand that alterations may be made within the spirit of the invention. Accordingly, the above should not be considered limiting, and the scope of the invention is defined by the appended claims.

Claims

THE INVENTION CLAIMED IS

1 . A bioreactor system for neural tissue, comprising: a bioreactor configured to hold a neural tissue sample therein; a media reservoir in fluid communication with the bioreactor and configured to hold a fluid media; one or more fluid conduits fluidly connecting the bioreactor to the media reservoir; a cannula in fluid communication with the media reservoir and configured to couple to a blood vessel of the neural tissue sample; one or more sensors configured to measure one or more parameters of the fluid media and/or the neural tissue sample; and one or more pumps configured to cause media to flow from the media reservoir through the cannula.

2. The system of claim 1 , wherein the bioreactor comprises a container having a top, bottom, a sidewall defining an interior configured to hold the neural tissue sample, and at least two openings configured to fluidly connect the bioreactor to the one or more fluid conduits.

3. The system of claim 1 , wherein the one or more pumps are arranged within the media reservoir.

4. The system of claim 1 , wherein the one or more pumps are arranged between the media reservoir and the bioreactor.

5. The system of claim 4, wherein one or more of the one or more pumps are a peristaltic pump and/or a roller pump.

6. The system of claim 1 , wherein the media reservoir is configured to hold an artificial blood composition.

7. The system of claim 1 , further comprising one or more additional reservoirs configured to hold one or more compositions, the one or more additional reservoirs in fluid communication with the one or more fluid conduits.

8. The system of claim 7, wherein the one or more additional reservoirs are arranged between the media reservoir and the bioreactor.

9. The system of claim 8, wherein at least one of the one or more additional reservoirs is configured to hold heparin.

10. The system of claim 8, wherein at least one of the one or more additional reservoirs is configured to hold a therapeutic composition.

1 1 . The system of claim 1 , further comprising an oxygenator in fluid communication with the one or more fluid conduits.

12. The system of claim 1 1 , wherein the oxygenator is arranged between the media reservoir and the bioreactor.

13. The system of claim 1 , further comprising one or more filters arranged between the media reservoir and the bioreactor.

14. The system of claim 13, wherein the one or more filters comprises a filtration membrane.

15. The system of claim 1 , wherein the one or more sensors comprise a pressure sensor, an oxygen sensor, a temperature sensor, a flowrate sensor, and/or a pH sensor.

16. The system of claim 15, wherein the oxygen sensor is arranged in the bioreactor.

17. The system of claim 1 , further comprising a one or more temperature controllers.

18. The system of claim 17, wherein one or more of the one or more temperature controllers is arranged in the bioreactor.

19. The system of claim 17, wherein one or more of the one or more temperature controllers is arranged in the media reservoir and/or between the media reservoir and the bioreactor.

20. The system of claim 1 , further comprising an electrocorticography (ECoG) device.

21 . The system of claim 20, wherein the ECoG device comprises at least one electrode lead arranged in the bioreactor and configured to detect an electrical signal from the neural tissue and at least one computing device in electrical communication with the at least one electrode lead and configured to acquire, amplify, filter, and/or analyze the electrical signal received from the at least one electrode lead.

22. The system of claim 21 , wherein the at least one electrode lead is configured to deliver electrical stimulation to the neural tissue and the at least one computing device comprises a pulse generator.

23. The system of claim 20, further comprising a Faraday cage arranged on and/or in the bioreactor.

24. The system of claim 1 , wherein the system is a closed system.

25. The system of claim 1 , wherein the system is an open system.

26. The system of claim 1 , further comprising at least one computing device in communication with the one or more pumps and one or more sensors, and configured to modulate one or more parameters of the media based on data received from the one or more sensors.

27. A bioreactor system for neural tissue, comprising: a bioreactor; a neural tissue sample received within the bioreactor, the neural tissue sample comprising a plurality of cortical cells and at least one blood vessel; a media reservoir in fluid communication with the bioreactor and holding an artificial blood composition therein; one or more fluid conduits fluidly connecting the bioreactor to the media reservoir and defining a closed system comprising the media reservoir and the bioreactor; a cannula in fluid communication with the one or more fluid conduits and coupled to the at least one blood vessel of the neural tissue sample; one or more sensors configured to measure one or more parameters of the artificial blood composition and/or the neural tissue sample, the one or more sensors comprising a pH sensor, a flow rate sensor, a pressure sensor, a temperature sensor, and an oxygen sensor; and one or more pumps configured to cause the artificial blood composition to flow from the media reservoir to the at least one blood vessel through the cannula.

28. The system of claim 27, further comprising one or more additional reservoirs holding a therapeutic composition.

29. The system of claim 28, wherein the neural tissue sample is a sample comprising one or more tumor cells and the therapeutic composition is a potential therapy for the tumor.

30. The system of claim 28, wherein the neural tissue sample is a sample from a patient with epilepsy and the therapeutic composition is a potential therapy for epilepsy.