Methods and systems for generating vascularized neurospheres

Abstract

The present invention introduces an approach to neural tissue engineering by developing vascularized neurospheres (VNSs) through novel methods, systems, and kits. These VNSs are generated using a unique blend of neural, vascular, and supportive cells, cultured in specialized hydrogel-containing media. The VNSs feature an integrated vascular network, serving as a physiologically relevant in vitro model. The invention also offers a grafting technique for therapeutic applications in neural disorders and injuries, as well as facilitating brain-computer interfacing. Beyond therapeutics, the VNSs have broad applications in drug development, synthetic biology, disease modeling, and more. The invention also includes kits for VNS generation and methods for interfacing VNSs with probes for applications of brain-computer interfacing. Overall, this patent represents a significant leap in the field, bridging the gap between in vitro and in vivo systems and opening new avenues in research, therapeutics, and bioengineering.

Description

[0001] METHODS AND SYSTEMS FOR GENERATING VASCULARIZED NEUROSPHERES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application 63 / 702,881, filed 3 October 2024. The entirety of the above-referenced application is incorporated herein by reference.

[0004] FIELD

[0005]

[0002] The present disclosure relates to methods and systems for generating vascularized neurospheres and the methods to manufacture the same, and three- dimensional (3D) brain cell culture models obtained by these methods.

[0006] BACKGROUND

[0007]

[0003] Research on stem cell derived human 3D brain organoids, also known as minibrains, has received attention in the last decade, due to the organoids’ potential in modeling brain-related developmental disorders. One of the significant challenges faced in the cultivation of these mini-brains is their vascularization. The absence of a vascular system in brain organoids limits their size and complexity due to the lack of nutrient and oxygen supply to inner cells. This results in necrosis in the core and hinders the accurate representation of in vivo brain physiology. Furthermore, the lack of vascularization impedes the study of neurovascular interactions, which are crucial in understanding various neurological conditions. Addressing the vascularization challenge is pivotal for the advancement of brain 3D cell culture technology, ensuring their utility in biomedical research and therapeutic applications.

[0008]

[0004] Previous development in vascularization of organoids include co-culturing of cortical organoid with endothelial cells (Yingchao Shi et al., Vascularized human cortical organoids (vOrganoids) model cortical development in vivo, 18 PLoS Biology (2020)). Further, via fusion of cortical organoid with a vascular spheroid (Myung Geun Kook et al., Generation of Cortical Brain Organoid with Vascularization by Assembling with Vascular Spheroid, 15 International Journal of Stem Cells 85-94 (2022); Liqing Song et al., Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues, 9 Scientific Reports (2019); Xin Yao Sun et al., Generation of Vascularized Brain Organoids to Study Neurovascular Interactions, 11 eLife (2022)) and using genetic engineering (Bilal Cakir et al., Engineering of human brain organoids with a functional vascular-like system, 16 Nature Methods 1169-1175 (2019), https: / / doi.org / 10.1038 / s41592-019-0586-5). Also, embedding of cortical organoid in with encapsulated endothelial cells in Matrigel (Missy T. Pham et al., Generation of human vascularized brain organoids, 29 NeuroReport 588-593 (2018)) was used to induce vascularization in organoids. Other approaches include microfluidics-based methods (Jungwook Paek et al., Microphysiological Engineering of Self-Assembled and Perfusable Microvascular Beds for the Production of Vascularized Three-Dimensional Human Microtissues, 13 ACS Nano 7627-7643 (2019); Idris Salmon et al., Engineering neurovascular organoids with 3D printed microfluidic chips, 22 Lab on a Chip 1615-1629 (2022)) and organoid engraftment into animal hosts (Abed Alfatah Mansour et al., An in vivo model of functional and vascularized human brain organoids, 36 Nature Biotechnology 432-441 (2018)).

[0009]

[0005] However, although several groups have been working on strategies to optimize vascularization on brain organoids and spheroids, it is still extremely difficult to obtain a full and reproducible vascularization.

[0010]

[0006] There is therefore a need for a method that allows the generation of vascularized neurospheres (VNSs).

[0011] SUMMARY

[0012]

[0007] The present disclosure relates to methods, systems, and kits for generating and utilizing vascularized neurospheres, providing an advancement in three-dimensional (3D) brain cell culture models. Developed using a distinct combination of neural cells, vascular cells, and supportive cells, the resulting VNSs feature an integrated vascular network, offering a physiologically relevant in vitro model system.

[0013]

[0008] A significant innovation of this disclosure lies in the methodological approach to the generation of these VNSs. By culturing a specific combination of cells in a specialized hydrogel-containing “VNS initiation” and later “differentiation medium”, the conditions are optimized to promote three-dimensional growth and concurrent vascular network formation within the evolving neurosphere.

[0014]

[0009] Furthermore, the present invention provides a groundbreaking therapeutic application through the grafting of the VNSs onto a patient's brain tissue. This grafting technique not only facilitates brain-computer interfacing but also holds immense potential in the realm of regenerative medicine, offering therapeutic solutions for a range of neural disorders and injuries.

[0010] Beyond their therapeutic implications, the VNSs developed through this invention find applications in a myriad of sectors including, but not limited to, drug development, drug screening, synthetic biology, biodefense studies, disease modeling, and developmental studies. The VNSs versatility, combined with their ability to be derived from both human and animal cells, further underscores their broad applicability. [Oi l] In addition to the methods, this disclosure provides kits that contain the requisite components for the generation of VNSs, simplifying the process for researchers and practitioners. Accompanied by comprehensive instructions, these kits are designed to be user-friendly, ensuring the consistent generation of high-quality VNSs.

[0015]

[0012] Moreover, this disclosure introduces methods to interface the VNSs with probes, enabling advanced applications in neuromorphic computing modules and the development of sophisticated brain-computer interfaces.

[0016]

[0013] In essence, this patent presents a multi-faceted advancement in the field of neural tissue engineering, bridging the gap between in vitro models and in vivo functionality, and unlocking a plethora of applications in research, therapeutics, and bioengineering.

[0017]

[0014] It is thus one object of the present invention to provide an approach to overcome the above mentioned lack of vascularization in neural tissue.

[0018]

[0015] It is another object of the present disclosure to provide an approach suitable for 3D tissue culture which helps to promote vessel-like structure formation.

[0019]

[0016] It is another object of the present disclosure to provide an approach suitable for 3D tissue culture which helps to improve the functionality, viability and stability of 3D tissues.

[0020]

[0017] Provided herein are methods of generating VNSs, from human or animal-derived cells, including a heterogeneous population of neural cells, glial cells, endothelial cells, and pericytes.

[0021]

[0018] Methods of the present disclosure relates to methods of producing a plurality of homogeneous VNSs, the methods including: using progenitor cells; culturing the progenitor cells in hydrogel-containing “VNS initiation medium”; seeding the progenitor cells into a cell culture device comprising at least one compartment comprising a plurality of microwells; culturing the progenitor cells in the cell culture device under conditions that allow the cells to form vascularized neurospheres; and transferring the formed vascularized neurospheres into 3D culture dishes for long-term culture.

[0019] Methods of the present disclosure relate to producing a plurality of homogeneous VNSs, the methods including: using one or more of progenitor cells, primary cells, and cell lines; culturing one or more of progenitor cells, primary cells, and cell lines in hydrogel-containing “VNS initiation medium”; seeding the one or more of progenitor cells, primary cells, and cell lines into a cell culture device comprising at least one compartment comprising a plurality of microwells; culturing the one or more of progenitor cells, primary cells, and cell lines in the cell culture device under conditions that allow the cells to form VNSs; and optionally treating the one or more of progenitor cells, primary cells, and cell lines with various “VNSs differentiation media”, and / or transferring the formed vascularized neurospheres into 3D culture dishes for long-term culture.

[0022]

[0020] In some embodiments, the VNSs exhibit electrical activity.

[0023]

[0021] In another aspect, the disclosure relates to VNSs and other 3D cell culture models comprising or consisting of neural cells, glial cells, pericytes, and endothelial cells and optionally having a maximum size of about 5 mm, e.g., 1, 2, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0 mm.

[0024]

[0022] In another aspect, the disclosure pertains to methods for producing human brain proteins, wherein the human brain proteins are produced by VNSs according to the disclosure.

[0025]

[0023] In another aspect, the disclosure relates to methods for drug screening using VNSs generated by the methods of the disclosure.

[0026]

[0024] Neurospheres are herein defined as free-floating 3D cell clusters comprising neural precursor cells, while organoids are more complex structures that are frequently derived from stem cells. Neurospheres can be used to develop and generate brain spheres, which are herein defined as self-assembled 3D cell aggregates with cell types and a cytoarchitecture resembling human brain tissue. Put differently, neurospheres can, under the right culture conditions, mature into brain organoids.

[0027]

[0025] The term “vascularized neurosphere (VNS)” as used herein, refers to an organized mass of specific cell types generated in vitro that mimics at least to some degree the structure, marker expression, cellular composition, or function of a naturally occurring vascularized neural tissue. VNSs may be derived from stem cells (embryonic stem cells, induced pluripotent stem cells, patient-derived stem cells), neural progenitor cell lines (e.g. ReNcells) or commercially available cell lines. VNS may be used for singular and plural.

[0028]

[0026] Beyond the scope of biomedical research and therapeutic applications, the development of vascularized neurospheres (VNSs) holds significant promise for advancements in brain-computer interfaces (BCIs). BCIs aim to establish direct communication between the brain and external devices, which can aid in restoring sensory and motor functions or even enhance cognitive abilities. The fusion of BCIs with VNSs can pave the way for innovative treatments, interventions, and enhancements for a wide range of applications.

[0029]

[0027] Unless otherwise defined, 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0028] In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0030]

[0029] Other features and advantages of the invention will be apparent from the following detailed description, the figures, and from the claims. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the disclosure.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032]

[0030] Figure 1 is a series of representations of microscope images showing staining of vascularized neurospheres. VNS (day 28) are positive for stem cell, neuronal, glial, and vascular markers: Neuronal stem cell marker (SOX2), neuronal markers (TUJ 1 , NEUN), glial marker (GFAP) and vascular markers (SMA, VE-CADHERIN, VWF). Scale bar = 200pm: VWF scale bar = 100pm.

[0033]

[0031] Figures 2A and 2B are images of different shapes and sizes of neurodishes and their respective intermediate molds. Figure 2A shows different shapes and sizes of neurodish silicone intermediate molds. Figures 2B shows different shapes and sizes of neurodishes placed in a 6-well plate.

[0032] Figures 3A, 3B, and 3C are schematic illustrations of a micromanipulator as described herein and an example of a jig used to immobilize an electrode over a neurodish once it has been precisely positioned (Figure 3A, Figure 3B, Figure 3C).

[0034] DETAILED DESCRIPTION

[0035]

[0033] As summarized above, provided herein are methods of generating VNSs from human-derived cells, including a heterogeneous population of neural, glial, endothelial cells and pericytes.

[0036] Methods of Generatins Vascularized Neurospheres

[0037]

[0034] Methods of the present disclosure include culturing human progenitor and primary cells in a hydrogel-containing “VNS initiation medium” that includes basic fibroblast growth factor, epidermal growth factor, and endothelial growth supplement. A “VNS differentiation media” may be used to induce differentiation by withdrawal of basic fibroblast growth factor and epidermal growth factor in a manner sufficient to induce Tuj 1 and GFAP expressing cells.

[0038]

[0035] The VNS initiation media is any suitable media that promotes induction of progenitor and primary cells into mature neurons, glial cells, endothelial cells and pericytes. The VNS initiation media can include a base medium with one or more supplements. Suitable base media include, ReNcell™ NSC Maintenance Medium (Millipore), Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F12, and MEM. Other suitable media include Neurobasal™ medium and NSC™ (Life Technologies), Neural Stem Cell basal medium (Millipore) and Stemdiff™ (StemCell Technologies). Suitable supplements can include, N2 supplement, L-glutamine, heparin, non-essential amino acids, antibiotics, ascorbic acid, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF) and, basic fibroblast growth factor, epidermal growth factor, endothelial growth supplement (ScienCell) and pericyte growth supplement (ScienCell). It can also include a maximum of 5% fetal bovine serum (FCS).

[0039]

[0036] The amount of the basic fibroblast growth factor and epidermal growth factor present in the VNS initiation media may be an amount suitable to induce maturation of cells and vessel-like structure formation. In some cases, basic fibroblast growth factor and epidermal growth factor are present in the “VNS initiation medium” at a concentration of about 10 ng / mL or more, e.g., 10 ng / mL to 50 ng / mL, e.g., 15 ng / mL, 20 ng / mL, 25 ng / mL or 30 ng / mL. In some embodiments, the concentration of basic fibroblast growth factor and epidermal growth factor in the “VNS initiation medium” may be different at different times during the culturing.

[0040]

[0037] The amount of hydrogel present in the hydrogel-containing VNS initiation media may be an amount suitable to induce maturation of cells and vessel-like structure formation. In some cases, Matrigel® (Corning) is present in the “VNS initiation medium” at a concentration of about 1% or more e.g. 1.5%, 2%, 2.5%, 3%, 4%, 5%. In some embodiments, the concentration of Matrigel® in the VNS induction medium may be different at different times during the culturing e.g. 0.5%, 1.5%, 2%, 2.5%, 3%. In some cases, other hydrogels including but not limited to Cultrex™, Geltrex™ or Okamatrix may be applied at different concentrations at different times during culturing. In some cases, other extracellular matrix-associated components e.g. laminin, collagen or fibronectin may be applied at different concentrations at different times during culturing.

[0041]

[0038] The amount of endothelial cell growth supplement (ECGS) (supplied as 50X solution by ScienCell) present in the VNS initiation media may be an amount suitable to induce maturation of endothelial cells and vessel-like structure formation. In some cases, ECGS is present in the “VNS initiation medium” at a concentration of about IX or more. In some embodiments, the concentration of ECGS in the “VNS initiation medium” may be different at different times during the culturing and range from 0.5X- 2X.

[0042]

[0039] The amount of pericyte growth supplement (PGS) (ScienCell) present in the VNS initiation media may be an amount suitable to induce maturation of pericytes and vessel-like structure formation. In some cases, PGS is present in the “VNS initiation medium” at a concentration of about IX or more. In some embodiments, the concentration of PGS in the “VNS initiation medium” may be different at different times during the culturing and range from 0.5X-2X.

[0043]

[0040] The culturing of the VNSs in the “VNS initiation medium” (e.g., all of the one or more initiation media) may take any suitable number of days to start differentiation into mature neurons, glial cells, endothelial cells and pericytes, according to methods disclosed herein. In some cases, the VNSs are cultured in the “VNS initiation medium” for about 10 days or more, about 5 days or more and in some cases 14 days or less. In some embodiments, the methods include culturing the VNSs in an differentiation medium that includes 5% FCS.

[0041] The “VNS differentiation media” may be any suitable media that promotes function and viability of neural cells, glial cells, endothelial cells and pericytes. The “VNS differentiation media” may include a base medium with one or more supplements. Suitable base media include, ReNcell™ NSC Maintenance Medium (Millipore), Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F12, and MEM. Other suitable media include Neurobasal™ medium and NSC™ (Life Technologies), Neural Stem Cell basal medium (Millipore), BrainPhys™ (StemCell Technologies), and Stemdiff™ (StemCell Technologies). Suitable supplements include endothelial growth supplement (ScienCell) and pericyte growth supplement (ScienCell). It may also include a maximum of 5% fetal bovine serum (FCS). Suitable supplements include, N2 supplement, L- glutamine, heparin, non-essential amino acids, antibiotics, ascorbic acid, brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF) and brain derived neurotrophic factor.

[0044]

[0042] VNSs can be derived from any animal, including mammals, such as, but not limited to human, cephalopod, mouse, and rat. In some embodiments the VNS cells are of human origin including, but not limited to, ReNcell™ CX Human Neural Progenitor Cell Line (Millipore), Human Brain Vascular Pericytes (HBVP) (ScienCell and Human Brain Endothelial Cells 5i (HBEC) (ATCC®).

[0045]

[0043] The cells used in this disclosure may be commercial cells such as Human Brain Vascular Pericytes (31200, ScienCell), ReNcell VM or CX Neural Progenitor Cell Line (SCC008 / SCC007, Millipore Sigma) and Human Brain Microvascular Endothelial Cells (1000, ScienCell). In one embodiment, patient-derived cells or cells known to carry specific genetic mutations or alterations may be used in the methods of the disclosures. In another embodiment, microglial cells and / or oligodendrocytes may be included in the methods of the disclosure.

[0046]

[0044] Maturity of the VNSs may be shown by the presence of maturity markers such as NeuN and GFAP. They also comprise mature vasculature expressing markers such as VE-Cadherin SAM, and vWF (Fig.l).

[0047]

[0045] In another embodiment, the VNSs may be shown by detection of electrophysiological activity and the presence of maturity markers such as NeuN and GFAP. They also comprise vasculature expressing markers as VE-Cadherin, SMA, and vWF (Fig.l).

[0046] In another embodiment, the vascularized neurospheres express at least one neural stem cell marker, one differentiated early neuronal marker and one late neuronal marker. Vasculature is confirmed with a minimum of expression of VE-Cadherin and vWF.

[0048]

[0047] The preferred cell ratio of neuronal progenitor cells, endothelial cells and pericytes used for initiation of VNS may be, but not limited to, 4: 1 : 1. In some embodiments the cell ratios may be 6:2: 1, 6: 1 : 1, 9: 1 : 1, 4:2: 1 and 9:2: 1.

[0049]

[0048] In another embodiment, the cells used in this disclosure can be derived from induced pluripotent stem cells (iPSCs), transdifferentiated cells, cell lines, and primary cells.

[0050]

[0049] In the methods according to the disclosure, the cells are preferably cultured at 37°C and 5% CO2.

[0051]

[0050] Culturing the VNSs in the differentiation medium may be continued for any suitable amount of time. In some cases, the VNSs are cultured for about 80 days. The minimum amount of time required to generate a VNS is about 30 days.

[0052]

[0051] The total time the VNSs are cultured in vitro may vary, depending on the time the VNSs are cultured in each medium. In some embodiments, the total time the VNSs are cultured in vitro is about 60 days or more, e.g., about 80 days or more.

[0053]

[0052] Culturing the VNSs, according to any of the methods of the present disclosure, may include seeding the cells at an initial population of 20,000 cells / well (96-well Clear Round Bottom Ultra-Low Attachment (Coming®)) or more, e.g., 100,000 cells / well.

[0054]

[0053] The maturation process may also include culturing the VNS in a free-floating culture from about day 10-14 of culture in 96-well Clear Round Bottom Ultra-Low Attachment wells.

[0055]

[0054] In another aspect, the VNSs are initiated in a custom-made cell culture device, referred to as a “neurodish,” that allows the cells to form vascularized neurospheres, with or without centrifugation. The neurodishes have a curved bottom and are made by first designing a mold that is to have the same dimensions as the neurodish itself. The mold will be used to cast an intermediate mold, which will finally be used to cast the neurodish itself. The mold is 3D printed, e.g., with a Form 3B SLA Printer (Formlabs), using a resin, such as Clear V4 Resin (Formlabs), and cured, e.g., in Form Cure (Formlabs), under curing conditions, e.g., at about 50 to 70°C, e.g., at 60°C, for a sufficient time, e.g., 10-20 minutes, e.g., 15 minutes. The silicone intermediate mold is cast, e.g., using Ecoflex 00-20 (Smooth On, Inc.) (Figure 2A). The neurodishes can be made of 2% Agarose in DMEM / F12 with a hemispherical base of radius of about 2 mm, (e.g., 1.50 mm, 2.00 mm, 2.50 mm) followed by a section with tapered walls with a taper angle of about 25° (e.g., 20°, 22.5° 25°, 30°). This is followed by a cylindrical section of diameter 10mm, which extends such that the total depth of the dish is 13mm. The outer dimensions may be made such that the dish fits within a 6 well plate. Neurodish- cultured spheres do not need to be transferred to a free-floating culture, but can be put on a rocker after 10-14 days.

[0056]

[0055] In another aspect, the VNSs are initiated in neurodishes coated with 1% Pluronic® F-127 solution to prevent adherence of cells with or without centrifugation (e.g. 60g, 70g, 80g, 90g, 100g, 110g, 120g, for e.g. 30 sec, 60 sec, 100 sec, 120 sec). Immediately, after seeding of cells into the neurodishes an electrode can be lowered into the well to allow vascularized sphere formation around the probe to record electrical activity after maturation of spheres. A custom-made micromanipulator can be used for positioning of the electrode.

[0057]

[0056] In another aspect, the VNSs are initiated in neurodishes that include built-in electrode(s) at the bottoms of the respective wells. In practice, VNSs form around the electrode and this growth enables the electrodes to be used for the recording of electrical activity after maturation of VNSs.

[0058]

[0057] In another aspect, the VNSs are initiated in neurodishes coated with 1% Pluronic® F-127 solution to prevent adherence of cells with or without centrifugation (e.g. 60g, 70g, 80g, 90g, 100g, 110g, 120g, for e.g. 30 sec, 60 sec, 100 sec, 120 sec). Immediately, after seeding of cells into the neurodishes, one or more perfusable tubes, composed of a biocompatible material, such as stainless steel, silicone, polystyrene, polydimethylsiloxane, polyethylene terephthalate, poly(methyl methacrylate), polycarbonate, teflon, or glass for example, may be lowered into the well to allow vascularized sphere formation around the tube to allow induction of perfusion of the VNSs. A micromanipulator can be used for positioning of the electrodes.

[0059]

[0058] In another aspect, the VNSs are initiated in neurodishes or (96-well Clear Round Bottom Ultra-Low Attachment (Corning®)) coated with 1% Pluronic® F-127 solution to prevent adherence of cells with or without centrifugation (e.g. 60g, 70g, 80g, 90g, 100g, 110g, 120g, for e.g. 30 sec, 60 sec, 100 sec, 120 sec). Immediately, after seeding of cells into the neurodi shes / wells, an inlet tube and an outlet tube may be lowered into the well to allow vascularized sphere formation around the tubes to allow perfusion through the VNS after maturation.

[0060]

[0059] In another aspect, the VNSs are initiated in neurodishes or (96-well Clear Round Bottom Ultra-Low Attachment (Corning®)) coated with 1% Pluronic® F-127 solution to prevent adherence of cells with or without centrifugation (e.g. 60g, 70g, 80g, 90g, 100g, 110g, 120g, for e.g. 30 sec, 60 sec, 100 sec, 120 sec). After a minimum of 2 days, the VNSs may be fused with other spheres / organoids / 3D cell culture constructs from other tissues.

[0061]

[0060] In another aspect, the VNSs can be used for 3D bioprinting by placing the VNSs in a hydrogel bath and connecting the VNSs to bioprinted vessel-like structures.

[0062]

[0061] In another aspect, the VNSs may be initiated using 3D bioprinting. VNS cell suspension could be deposited using a bioprinter or by hand pipetting into a hydrogel bath (container filled with a special hydrogel). This promotes self-aggregation of the cells into a VNS while at the same time providing a matrix for them to pattern and migrate into. This embodiment is related to the other embodiments where a singular coherent spherical structure is formed but is supplemented by an extracellular matrix to form organotypic structures.

[0063]

[0062] In another aspect, the VNSs can be initiated using casting of VNS cell suspension instead of 3D printing. Voids in an initial hydrogel can be created placing sacrificial materials meant to be removed after the hydrogel cures. The curing of the action can be thermal -based curing, photo-based curing, or other types of curing obvious to one skilled in the area of curable gels. The sacrificial material could be sacrificed depending on the material and could be thermal based sacrifice (melting away for example), or by dissolving, or other methods obvious to one skilled in the area of material science. The materials could be cast pluronic, cast gelatin, 3D printed PEG, or many other materials, made by various techniques, depending on the compatibility with the casting strategy. Once the sacrificial material is sacrificed, voids are left in the hydrogel and the cell suspension can be poured or cast into the voids. The hydrogel can then be placed in an incubator on a rotating device or shaker to help keep the cells distributed so as not to fall by gravity to the bottom of the voids within the mold.

[0064]

[0063] In a similar aspect, the VNS cell suspension can be poured or cast into the voids of a 3D printed hydrogel. Alternatively, the 3D printed hydrogel can be soaked in a cell suspension bath. In yet another alternative, the 3D printed hydrogel can be fixed in a bioreactor that has the VNS cell suspension flowing throughout.

[0065]

[0064] In another aspect, the VNSs can be fused to a tissue-engineered blood vessel to enable perfusion. The tissue-engineered blood vessel may be produced in a variety of ways including, but not limited to, seeding of cells onto a natural or synthetic polymer scaffold, seeding of cells onto decellularized natural tissue, bioprinting of cells or cells and scaffold material, or self-assembly of cells. The tissue-engineered blood vessel will include vascular endothelial cells alone, or in combination with vascular smooth muscle cells, fibroblasts or pericytes. The VNSs and the tissue-engineered blood vessel may be placed in close proximity to each other to allow the vasculature of the VNS to connect with the tissue-engineered blood vessel, by self-assembly, enabling perfusion of the VNSs from the flow within the vessel.

[0066]

[0065] In another aspect, the VNSs according to the disclosure shows similar cell diversity. In particular, they comprise neural progenitor cells, neurons, astrocytes, pericytes and endothelial cells (Fig.1).

[0067]

[0066] In certain embodiments, the VNSs can be used to screen drugs and brain protein production.

[0068]

[0067] In one embodiment, the VNSs can be used for toxicity studies and drugresistance screening.

[0069]

[0068] In one embodiment, the VNSs can be used for disease-modeling. Diseases in a model of the VNSs can be induced through genetic manipulation of the VNSs, chemically induced, or physically induced.

[0070]

[0069] In one embodiment, the VNSs can be used for personalized drug testing, e.g., induced pluripotent stem cell (iPSC)-patient-derived VNSs.

[0071]

[0070] In one embodiment, the VNSs can be used for cell-replacement therapy, e.g., to replace diseased or damaged tissue.

[0072]

[0071] In one embodiment, the VNSs can be used clinically for e.g., implantation, therapeutics, or regeneration approaches.

[0073]

[0072] In one embodiment, the VNSs can be used for drug-delivery purposes, e.g., cells that are genetically modified to release drugs.

[0074]

[0073] In one embodiment, the VNSs can be used for personalized medicine to generate patient specific VNS.

[0074] In one embodiment, the VNSs can be used to generate a humanized animal model, e.g., implantation of a VNS into a rodent to generate data more closely to humans.

[0075]

[0075] In one embodiment, a central nervous system (CNS)-VNS and a peripheral nervous system (PNS)-VNS may be fused together. In another embodiment a CNS-PNS VNS and a muscle sphere may be fused together.

[0076]

[0076] In another embodiment, the VNSs are formed onto a probe that allows the sensing of neural activity, or the stimulation of neural activity, or both. The sensing of neural activity can be referred to as “reading” of neural activity. The stimulation of neural activity can be referred to as “writing” neural activity. In one example, the VNSs are grown onto a single electrode or onto a 3D electrode array. Examples of electrodes that can be used in this example include, but are not limited to: the Utah Array, the Michigan Probe, tungsten micro-electrodes, micro-electrode arrays, nanowire arrays, tetrode arrays, and electrode threads. Reading from neurons in this case would be accomplished by measuring voltage or current changes detected by the electrodes. Writing to neurons in this case would be accomplished by applying a voltage or current to one or more electrodes.

[0077]

[0077] In another embodiment, the placement of a probe is performed using a micromanipulator (FIG. 3). Referring to FIG. 3A, a removable micro-manipulator 1 holds the probe, allowing the user to position the probe stably and with high accuracy in three dimensions. The probe is mounted on a two-part probe holder 18, which comprises two sections, the section permanently attached to the probe 16 and the detachable section 14. The probe holder 18 is designed to be easily separable, such that the detachable section 14 can be removed with the removable micro-manipulator 1 while permanently attached section 16 remains attached to the probe. The probe holder 18 is mounted to a series of stages that provide fine positional control in x (controlled by the x stage 10), y (controlled by the y stage 4) and z (controlled by the z stage 8), and also allows for zeroing of the height of the probe using stage 12. Each stage uses a knob 6 for control. These stages are mounted to a removable base 2. The removable base is secured to a plate holder 26, which in turn holds the well plate containing a neurodish 22. The well plate is secured with retaining clips 24. A ground rod holder 20 allows for the ground rod to be held in position at a fixed distance from the probe tip as the probe is being positioned and after it is immobilized. The ground rod holder 20 can be moved before securing it down, to adjust the position of the ground rod relative to the probe tip.

[0078]

[0078] Referring to FIG. 3B, once the probe is in the desired position, a gantry system 38 is used to immobilize the probe relative to the well plate with neurodish. The position of the gantry is adjusted to match the position of the probe using slots. Slots 28 provides adjustment in the y axis, slots 30 provides adjustment in the z axis and slots 36 provides adjustment in the x axis. Once the gantry is in the correct position to line up with the probe, the permanently attached section of the probe holder 16 is secured to the gantry by tightening the bolts in the slots 28, 30 and 36, to fix the position of the probe. Then, the detachable section of the probe holder 14 and the permanently attached section 16 are separated and the removable base 2 and the plate holder 26 are separated, allowing the removable micro-manipulator assembly 1 to slide out.

[0079]

[0079] Referring to FIG. 3C, the probe remains fixed in place relative to the well plate 22, by being attached to the probe holder mount 34, which is in turn attached to the gantry base 32 which is finally attached to the plate holder 26, that also holds the well plate 22.

[0080]

[0080] In another example, the VNSs are grown onto an optical fiber or optical fiber array. The sensing of neural activity is performed through any established optical method of sensing neural activity. Examples of “reading” methods include, but are not limited to: optogenetics-based methods, calcium imaging, 2-photon calcium imaging, and the use of voltage-sensitive dyes. Stimulation of the neurons can be achieved using any established or experimental optical method such as optogenetics-based methods, 2- photon optogenetics (and similar multi-photon methods), photothermal, magnetothermal, the use of optically-caged compounds, or plasmonic nanotransducers.

[0081] The neurons in VNSs can be genetically modified to aid in or implement any optical-based measurement or stimulation method. The neurons or other cells in the VNS may be genetically modified for therapeutics purposes. The genetic modification can occur before the VNS is formed, or after. The neurons in VNSs can be genetically modified for other characteristics such as enhanced axonal or dendritic outgrowth.

[0081]

[0082] In another example, the probe on to which the VNSs are grown is an optrode which combines the functionality of electrodes and fiber optics as described above.

[0083] In some embodiments, it is possible to “read from” and “write to” every neuron in the VNSs. As one example, a VNS whose neurons have been genetically-modified to enable optogenetic stimulation, is grown onto the end of a fiber optic cable that is used for both 2-photon optogenetics and 2-photon calcium imaging in order to enable the reading and writing to most or all neurons in the sphere. Conventional calcium imaging has low temporal resolution, however higher temporal resolution techniques can be used. Example of techniques or methods for high temporal and spatial resolution for the detection of action potentials includes: fast-scanning two-photon microscopy, random access multiphoton microscopy, genetically encoded calcium indicators (GECIs) with fast kinetics, acousto-optic deflector scanning, and parallelized imaging.

[0082]

[0084] In another embodiment, a brain-computer interface is created by first growing the VNSs onto a probe, e.g., one VNS per probe with an appropriate probe size, then secondly, implanting the VNS-probe into a brain, either by preferably grafting it onto the surface of the brain, or direct implantation to a resected or sliced part of the brain. The VNS, grown around the probe, can be implanted into a brain sulcus beneath the pia mater, without penetrating or damaging the patient’s natural brain tissue. The cells of the VNS can be patient-derived, using transdifferentiated cells or induced pluripotent stem cells, for example. This procedure can be done in a much less destructive manner compared to traditional penetrating electrode methods.

[0083]

[0085] With traditional penetrating electrodes, even flexible thread electrodes, the interface with the brain is created by puncturing the brain with the electrodes themselves. This inevitably results in the destruction of thousands or millions of axons, dendrites, microglia, neurons, microvasculature, and most importantly, the destruction of the blood-brain barrier. This damage results in scar tissue formation, eventually compromising the quality of the recorded signal and degrading the ability to stimulate the neurons.

[0084]

[0086] Furthermore, electrodes are far less specific in their stimulation of neurons compared to optogenetic methods, especially 2-photon optogenetics. To create the braincomputer interface, the VNSs are first grown onto the probe and then grafted onto the brain without puncturing the patient’s natural brain tissue. This is followed by a period of time to allow the neurons in the VNSs to start integrating with the patient’s natural brain tissue, and vice versa. The axons and dendrites from the natural tissue and the implanted VNS eventually create synapses bidirectionally, thus allowing the natural activity of the brain to cause action potentials within the VNS which are then detected by the probe.

[0085]

[0087] Additionally, stimulation of neurons within the VNSs by the probe will result in downstream stimulation of the native neurons in the patient’s brain. Both this stimulation and sensing of neural activity is without any destruction of the patient’s own brain tissue, without a significant risk of scar tissue formation and subsequent signal quality degradation. With some embodiments using high resolution techniques, stimulation and sensing of every neuron’s spiking activity within the VNS volume is possible, such as the use of both 2-photon optogenetics and fast 2-photon calcium imaging. This “tissue-engineered brain-computer interface” (TE-BCI) could be used for the many applications envisioned by experts in the field of brain-computer interfacing without requiring the destruction of native brain tissue as is the case with most traditional brain-computer interfaces.

[0086]

[0088] In another embodiment, the VNSs, coupled with a probe for stimulating and sensing of neural activity, function as a bio-hybrid computing module. A traditional computing module has inputs, outputs, and a processor. The processor takes the inputs, processes it, and the result is sent through the output. In this embodiment, the probe’s stimulation capabilities serve as the input, while the probe’s sensing capabilities serves as the output. The neural tissue serves as the processor. By stimulating neurons in the sphere with electrodes or optogenetic-based stimulation for example, a cascade of neural activity results. The stimulated neurons stimulate more neurons, depending on the neural network’s axonal and dendritic connections. Finally, that cascade of activity could be sensed by the sensing part of the probe and sent to electronics or a computer for further processing.

[0087]

[0089] In a further embodiment, the output’ s resulting actions are sensed by a computer, and sent back to the input in order to create a closed-feedback loop. The input can be encoded using existing techniques used in brain-machine interfacing. The closed feedback loop allows for the neural tissue to continuously sense the outside world, process what was sensed, and act (by stimulating the output), and then further sense the result of its actions. This high-level description of continuous closed-loop learning is similar to how a baby may learn. On its own, this particular computing module may be considered a “neural module,” which in the field has been called other terms such as “biohybrid computing device,” “bio-hybrid Al,” “neuromorphic computer,” “learning module,” or “biological computer,” for example.

[0088]

[0090] The module could be left to learn on its own through its own closed-loop feedback, or learning methods could be employed to accelerate learning or program the neural tissue to process information in desired ways. Learning methods that could be employed by using the stimulation and sensing abilities of the probe that the sphere is attached to include but are not limited to: directed plasticity, long-term potentiation, long-term depression, spike-timing-dependent plasticity (STDP), homeostatic plasticity, metaplasticity, Hebbian learning, reinforcement learning, and Bayesian learning. The modules can act on their own or be connected to other modules in series or in parallel. The modules don’t necessarily need to be physically connected - they can be connected virtually through the internet or other communication devices while being located physically separated by even large distances. The neural module could be in the form of a “body-on-a-chip” in which other human cells in various chambers on the chip exist to create nutrients and process waste in order to support the long-term survival of the brain.

[0091] In a further embodiment, the neural module described above could have its input, output, or both, in communication with the brain of one or more humans that uses a brain-machine interface. This setup is an implementation of what is sometimes referred to as a standalone or shared exo-cortex. The exo-cortex can be used for various applications previously envisioned in the field of brain-computer interfacing and science fiction, including, but not limited to: expanding the memory capacity of a human brain, improving memory, controlling an external humainoid robot, sensing things not normally sensed by humans (by connecting sensors like radiowave sensors, cameras, etc. to the exo-cortex), improving intelligence, accessing the internet, communicating with others by thinking, accelerating learning, and more.

[0089] EXAMPLES

[0090]

[0092] The disclosure is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0091] Example 1 - Vascularized Neurosphere Generation and Maintenance

[0092]

[0093] To initiate and maintain VNSs we used the following method: ReNcells™ CX (Millipore), Human Brain Endothelial Cells 5i (HBEC-5i) (ATCC®) and Human Brain Vascular Pericytes (HBVP) (ScienCell) were cultured in accordance with the data sheet. To generate vascularized neurospheres (VNSs), cells were mixed in a ratio of 4: 1 : 1 (ReNcells:HBEC-5i:HBVP) in “VNS initiation medium” (ReNcell TM NSC Maintenance Medium (Millipore) and Complete HBEC-5i Medium mixed 1 : 1 and supplemented with 20 ng / mL FGF-2, 20 ng / mL EGF (final concentrations) and 2% Matrigel (Corning). The cell suspension with a total volume of 200pL / well and a total cell number of 20,000 cells / well was pipetted into a 96-well Clear Round Bottom UltraLow Attachment Microplate (Coming, # 7007). Then the 96-well plate was centrifuged at 100g for 1 min and afterwards put into an incubator set to 37°C, 5% CO2. After 48h the medium was changed to VNS differentiation medium (ReNcell TM NSC Maintenance Medium (Millipore) and Complete HBEC-5i Medium mixed 1 : 1). The VNS were cultured for a total of 14 days in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (Corning) and the media was changed every other day.

[0093]

[0094] At day 14, VNSs were transferred into a free-floating culture using 10 cm dishes containing lOmL of VNS differentiation medium. Media was changed every 2-4 days and VNS were cultured up to 80 days.

[0094]

[0095] The results are shown in Figure 1, where we confirmed the presence of neurons, glia cells and vessel-like structures in the VNSs at day 28.

[0095] Example 2 - Medium Composition

[0096]

[0096] For initiation and long-term culture of VNSs we established the following media compositions: Complete HBEC-5i Medium consisted of DMEM:F12 (ATCC®) with Endothelial growth supplement (ECGS) (Sciencell), 10% Fetal bovine serum (FBS; ATCC®) and 1% Antibiotic-Antimycotic Solution (100X) (Caisson Labs). “VNS initiation medium” consisted of ReNcell™ NSC Maintenance Medium and Complete HBEC-5i Medium in a ratio of 1 : 1, 2% Matrigel (Coming) and 20ng / mL FGF-2 and 20 ng / mL EGF (final concentrations). “VNS differentiation medium” consisted of ReNcell™ NSC Maintenance Medium and Complete HBEC-5i Medium in a ratio of 1 : 1.

[0097]

[0097] The results are shown in Figure 1, where we confirmed the presence of neurons, glia cells and vessel-like structures in the VNSs at day 28 using the above-mentioned media compositions.

[0098] Example 3 - Preparation of Neurodishes

[0099]

[0098] The neurodishes were made by designing a mold designed to have the same dimensions as the neurodish itself. The mold was used to cast an intermediate mold, which was used to cast the neurodish itself. The mold was designed using Fusion 360 software (Autodesk). The mold was 3D printed with a Form 3B SLA Printer (Formlabs) using Clear V4 Resin (Formlabs). Afterwards, the molds were washed in 100% Isopropyl alcohol for 10 minutes and cured in Form Cure (Formlabs) at 60°C for 15 min. The silicone intermediate mold was casted using Ecoflex 00-20 (Smooth On, Inc) (Figure 2A). A 2% agarose DMEM / F12 (Corning) solution was used to fill the silicone intermediate mold and was left to solidify at room temperature for about 1-2 hours (Figure 2B). The Neurodishes were coated using 1% Pluronic® F-127 in lx phosphate- buffered saline (PBS) solution and rinsed one time with lx PBS prior to usage.

[0100] Example 4 - Preparation of Micromanipulator for Electrode Placement

[0101]

[0099] The micromanipulator comprised an X, Y and Z stage to precisely position an electrode in the center of the Neurodish placed in a 6-well plate (Figure 3 A). In addition, it contained a jig to immobilize the electrode over the Neurodish once it had been precisely positioned (Figure 3B). Once the electrode had been immobilized, the micromanipulator could be detached, and the electrode on the jig could be placed in the incubator for culturing of the VNS.

[0102] Example 5 - Vascularized Neurosphere Fixation and Cryosectioning

[0103]

[0100] To characterize the said VNSs, they were collected from cell culture with a wide- bore tip. After washing with lx PBS, VNSs were fixed with 4% paraformaldehyde (PF A) at room temperature for 30 - 60 min on a rocker (Benchmark). PFA was discarded and VNS were washed 3 times with lx PBS. Next, the VNSs were incubated with 30% Sucrose solution overnight. Then the spheres were transferred to a Cryomold, embedded with OCT compound and frozen at -80°C or on dry ice. 15-20 pm thick sections were cut at the cryostat and mounted onto glass slides.

[0104] Example 6 - Immunofluorescent Staining

[0105]

[0101] To permeabilize and block the cryosectioned spheres, Perm -Block-Buffer (5% BSA, 0.3% Triton X-100 in lx PBS) was added to the cryosections and incubated for at least 1 h at room temperature. After blocking, a primary antibody diluted in Perm-Block- Buffer was added to the VNS and incubated overnight at 4°C. Then the cryosections were washed at least 3 times with lx PBS, in total for at least 15 min at room temperature. Next, a secondary antibody solution diluted in Perm-Block-Buffer was prepared and pipetted on the cryosections and incubated at room temperature for 1 h in the dark. Next, the cryosections were washed at least 3 times with lx PBS, in total for at least 15 min at room temperature. A cell nuclei counterstain solution (2pM DAPI) was added onto the cryosections and incubated for 3 min at room temperature in the dark Afterwards, the cryosections were washed at least 3 times with lx PBS and a cover slip was mounted onto the glass slide. The results are shown in Figure 1, where we confirmed the presence of neurons, glia cells and vessel-like structures in the VNS at day 28.

[0106]

[0102] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, and the figures, the foregoing description and figures are intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A method of creating a vascularized neurosphere, the method comprising: obtaining a mixture of neural cells, glial cells, and vascular cells; culturing the cells in the mixture in a hydrogel-containing “VNS initiation medium” consisting of endothelial, pericyte and neuronal growth factors and “VNS differentiation medium” reduced in endothelial, pericyte and neuronal growth factors under conditions promoting three-dimensional growth and vascular network formation within developing neurospheres.

2. The method of claim 1, wherein the neural and glial cells are selected from the group consisting of: progenitor cells, primary cells, or cell lines.

3. The method of claim 1 or 2, wherein the vascular cells are selected from the group consisting of: progenitor cells, primary cells, or cell lines.

4. The method of any of claims 1-3, further comprising grafting the VNS onto a patient's brain tissue by creating an incision through the layers of tissue covering the brain, and positioning the VNS in a stable direct contact with the brain near the functional area of interest, after which the VNS and nearby brain tissue are covered in a stable hydrogel that is then cured.

5. The method of any of claims 1-3, wherein the “VNS initiation medium” comprises basic fibroblast growth factor, epidermal growth factor, and endothelial growth supplement.

6. The method of claim 5, further comprising seeding the mixture of cells into a cell culture device comprising at least one compartment with a plurality of microwells wherein the cell culture device is a Round Bottom Ultra-Low Attachment Microplate.

7. The method of claim 5, further comprising seeding the mixture of cells into a cell culture device comprising at least one compartment with a plurality of microwells wherein the cell culture device is a neurodish, a custom-made cell culture device that has a curved bottom and is made by using a 2% agarose DMEM / F12 solution.

8. The method of claim 5, further comprising seeding the mixture of cells into a cell culture device comprising at least one compartment with a plurality of microwells wherein the cells are centrifuged at 60g to 120g for 30 sec to 120 sec.

9. The method of claim 5, further comprising seeding the mixture of cells into a cell culture device comprising at least one compartment with a plurality of microwells wherein the cells are cultured in hydrogel-containing “VNS initiation medium” andwherein the VNS are cultured in the “VNS initiation medium” for a period ranging from 1-3 days.

10. The method of claim 5, further comprising seeding the mixture of cells into a cell culture device comprising at least one compartment with a plurality of microwells wherein the VNS are cultured in the “VNS differentiation medium” for a period ranging from 3-80 days.

11. The method of claim 5, further comprising seeding the mixture of cells into a cell culture device comprising at least one compartment with a plurality of microwells further comprising transferring the formed VNS into 3D culture dishes for long-term culture.

12. The method of claim 5, wherein the cells comprise neural progenitors, pericytes, and endothelial cells in a ratio selected from the group consisting of 4: 1 :1, 6:2:1, 6: 1 :1, 9: 1 : 1, 4:2: 1, and 9:2: 1.

13. The method of claim 5, further comprising: initiating VNSs using 3D bioprinting or casting; placing said VNSs in a hydrogel bath; and connecting said VNSs to bioprinted vessel-like structures to induce angiogenesis, and subsequently perfusing the VNSs through their vasculature using the bioprinted vessel-like structures.

14. The method of claim 5, further comprising connecting said VNSs to cast vessellike structures to induce angiogenesis and perfusing the VNSs through their vasculature via the cast vessel-like structures.

15. The method of claim 5, further comprising: culturing human neural progenitor cells, human brain microvascular cells, and human brain vascular pericytes; mixing said cells in a ratio of 4: 1 : 1 in “VNS initiation medium”; transferring the cell suspension into a 96-well Clear Round Bottom Ultra-Low Attachment Microplate; culturing for at least 3 days; changing the medium to “VNS differentiation medium”; and transferring the VNS into a free-floating culture for extended culture.

16. A vascularized neurosphere comprising an integrated vascular network, neural cells selected from the group consisting of progenitor cells, primary cells, and cell lines, vascular cells such as endothelial cells and pericytes, and supportive cells such as glial cells.

17. The method of any of Claims 1-17, further comprising first forming a VNS made from patient-derived cells onto a probe selected from the group consisting of electrodes, optical-based probes, and combinations thereof, wherein said probe is capable of both stimulating and sensing neural activity, and secondly, grafting the VNS-probe structure stably onto the patient’s brain without damaging the patient’s native neural or brain vasculature tissue, and thirdly allowing the VNS and patient tissue to integrate into each other neurally and vascularly, and fourthly using the setup for brain-computer interfacing applications.

20. A method of in vitro screening of a test substance, the method comprising: contacting a test substance to a vascularized neurosphere of any one of claims 1-19; and detecting an effect of the test substance on one or more of the cell types within the contacted VNS.

21. The method of claim 20, wherein the effect of the test substance affects the viability, gene expression, or morphology of the VNS or cells.

22. The method of claim 20, further comprising screening the effect of the test substance on a VNS derived from patient-specific cells.

23. The method of claim 20, comprising employing a VNS developed from cells carrying a disease-causing mutation, including but not limited to mutations in amyloid precursor protein (APP), presenilin 1 or 2 (PSEN 1 or 2), dardarin (LRRK2), alpha- synuclein (SNCA), Sodium channel protein type 1 subunit alpha (SCN1A), DEP domain-containing 5 (DEPDC5), and tuberous sclerosis complex 1 or 2 (TSC1 / 2).

24. A method of any one of claims 1-23, wherein the resulting VNS is utilized for applications selected from the group consisting of: drug discovery, personalized medicine, disease modeling, toxicity testing / screening, and other bioengineering applications.

25. A kit for obtaining a vascularized neurosphere (VNS), the kit comprising: a hydrogel facilitating three-dimensional growth; and a plurality of human cells comprising one or more of neural cells, vascular cells, and supportive cells.

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