WO2025019395A2 - Methods for cryopreserving cells for modeling a human brain or a human blood-brain barrier - Google Patents

Abstract

This application relates to methods of cryopreserving cells for modeling a human brain, a human blood-brain barrier, or a human microvasculature. Also provided are assays based on an induced blood-brain barrier (iBBB), a multicellular integrated human brain tissue (miBrain), "just add neurons" (JANs), or an induced minimal microvascular cell combo (miVasC).

Description

METHODS FOR CRYOPRESERVING CELLS FOR MODELING A HUMAN BRAIN OR A HUMAN BLOOD-BRAIN BARRIER

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63/514,203, filed July 18, 2023, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This disclosure relates generally to methods of cry opreserving cells for modeling a human brain, a human blood-brain barrier, or a human microvasculature.

BACKGROUND OF THE INVENTION

Animal models and conventional 2D cell culture systems have been used to study neurodegenerative diseases, but they have not successfully translated their findings to human disease. It has been shown that 3D in vitro systems, such as organoids, are more efficient than 2D systems at recapitulating neurodegenerative phenotypes. However, most organoids lack physiological components such as vascular structures and neuro-myelin interactions, making them ill-equipped to study the complex multi-cellular interactions that are emerging as critical drivers of neurodegeneration. Cerebral organoids are also plagued with i) variability, ii) contain immature and embryonic cells that may create technical artifacts when modeling aging disorders, and iii) lack a high degree of tractability that enables dissection of complex genetic, molecular, and cellular interactions. In view of the foregoing, there remains a need to develop human cell-based platforms that are suitable for neurodegenerative disease modeling and drug screening.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic overview of an exemplary method according to various embodiments of this disclosure.

FIG. 2 is a schematic of the generation of induced blood-brain barrier (iBBB) and multicellular integrated human brain tissue (miBrain).

FIG. 3 is a schematic of an example of a cry opreservation workflow and thawing viability of the cells. FIG. 4 depicts the viability of the indicated thawed tissues using Trypan Blue. The graph shows the cell count on an automated cell counter.

SUMMARY OF THE INVENTION

The present application relates to methods of cry opreserving cells for modeling a human brain, a human blood-brain barrier, or a human microvasculature. In one aspect, provided is a method of cry opreserving cells for modeling a human brain or a human bloodbrain barrier (BBB), the method comprising: a) combining cells comprising mammalian primary, induced pluripotent stem cells (iPSC)- or embryonic stem cell-derived neurons, glia, and vascular cells at a predetermined cell ratio to obtain a cell mixture; b) resuspending the cell mixture in a freezing medium; c) transferring the resuspended cell mixture into a single container; d) pre-chilling the container at a first temperature followed by storing the container at a second temperature for a predetermined period of time, wherein the second temperature is lower than the first temperature and causes the cell mixture to freeze; and e) storing the container at a third temperature.

In one embodiment, the cell mixture comprises iPSC-derived cells, embryonic stem cell-derived cells or cells derived from primary cells. In one embodiment, the method further comprises prior to combining the cells, differentiating iPSC or embry onic stem cells into neurons, glia, and vascular cells. In one embodiment, the method further comprises differentiating the neurons, glia, and vascular cells into astrocytes, oligodendrocyte progenitor cells, brain endothelial cells, microglia, and pericytes.

In one embodiment, the cell mixture comprises brain endothelial cells, astrocytes, and pericytes. In one embodiment, the cell mixture comprises human neurons, astrocytes, oligodendrocyte progenitor cells, endothelial cells, and pericytes. In one embodiment, the cell mixture comprises brain endothelial cells and pericy tes. In one embodiment, the first temperature is about 4 °C. In one embodiment, the second temperature is about -80 °C. In one embodiment, the third temperature is from about - 80 °C to about -196 °C.

In one embodiment, the predetermined period time is from about 12 hours to about 36 hours. In one embodiment, the predetermined period of time is about 24 hours.

In one embodiment, the predetermined ratio is about 5 : 1 : 1 by cell density of brain endothelial cells, astrocytes, and pericytes. In one embodiment, the cell mixture comprises about 1 x 10⁶ to about 10 x 10⁶ brain endothelial cells/mL, about 1 x 10⁶ to about 2 x 10⁶ astrocytes/mL, and about 1 x 10⁶ to about 2 x 10⁶ pericytes/mL. In one embodiment, the cell mixture comprises about 5 x 10⁶ brain endothelial cells/mL, about 1 x 10⁶ astrocytes/mL, and about 1 x 10⁶ pericytes/mL. In one embodiment, the cell mixture comprises about 1 x 10⁶ to about 10 x 10⁶ neurons/mL, about 1 x 10⁶ to about 10 x 10⁶ brain endothelial cells/mL. about 0.1 x 10⁶ to about 2 x 10⁶ astrocytes, about 0.1 x 10⁶ to about 2 x 10⁶ oligodendrocyte progenitor cells (OPCs)ZmL, and about 0.1 x 10⁶ to about 2 x 10⁶ pericytes/mL. In one embodiment, the predetermined ratio is about 5 : 1 : 1 : 1 : 1 by cell density of brain endothelial cells, astrocytes, OPCs, microglia, and pericytes. In one embodiment, the cell mixture comprises about 2.5 x 10⁶ neurons, about 2.5 x 10⁶ brain endothelial cells/mL, about 0.5 x 10⁶ astrocytes/mL, about 0.5 x 10⁶ OPCs/mL, about 0.5 x 10⁶ microglia/mL, and about 0.5 x 10⁶ pericytes/mL.

In one embodiment, the step of combining comprises combining the cells in a combination medium comprising:

(a) astrocyte media and 10 ng/mL VEGF-A (vascular endothelial growth factor A

(b) 500 mL human endothelial serum-free (hECSR) media, 5 mL penicillinstreptomycin, 5 mL NEAA (non-essential amino acid), 5 mL CD (chemically defined) lipids, 5 mL astrocyte grow th supplement, 10 mL serum-free supplement for neuronal cells, 500 pl insulin, 1 pM dibutyl-cAMP, 50 pg/mL ascorbic acid, 10 ng/mL NT3, 10 ng/mL IGF (insulin-like growth factor), 100 ng/mL biotin. 60 ng/mL T3, 10 ng/mL VEGF, and 1 pM SAG; or

(c) 500 mL hECSR media, 5 mL penicillin-streptomycin, 5 mL NEAA, 10 mL B27 supplement, 50 ng/mL VEGF, 20 ng/mL FGF, and 10 ng/mL FGF. In one embodiment, the combination medium of (c) further comprises 5 pg/ml doxycycline. In one embodiment, the freezing medium comprises 60% FBS-free serum replacement, 30% hECSR, 10% DMSO, 10 pM Y27632 ROCK Inhibitor, and 50 ng/mL VEGF-A. In one embodiment, the method comprises resuspending the cell mixture in 1 mL of the freezing medium.

In one embodiment, the cell mixture comprises 3 to 6 different cell types. In one embodiment, the cell mixture has about 1 x 10⁶ to about 10 x 10⁶ cells/mL. In one embodiment, the cell mixture has about 7 x 10⁶ cells/mL.

In one embodiment, provided is an assay that utilizes cells prepared by the methods described herein. In one embodiment, the assay is an assay based on induced blood-brain barrier (iBBB). just add neurons (JANs), a multicellular integrated human brain tissue (miBrain), or an induced microvascular cell combo (miVasC).

DETAILED DESCRIPTION OF THE INVENTION

This disclosure relates to a method of cryopreserving cells for modeling a human brain, a human microvasculature, or a human blood-brain barrier. Advantageously, the methods of cryopreservation described herein facilitate ready -to-use assays for modeling the human brain, the human blood-brain barrier, and the human microvasculature. Thus, the present methods facilitate the time required to complete such assays. Moreover, provided are human cell-based platforms that are suitable for neurodegenerative disease modeling and drug screening. Accordingly, the present invention addresses a long-felt need of facilitating neurodegenerative disease modeling and drug screening.

Methods for cryopreserving cells

In one aspect, the method comprises: (a) combining cells comprising mammalian primary, induced pluripotent stem cell (iPSC)- or embryonic stem cell-derived neurons, glia, and vascular cells at a predetermined cell ratio to obtain a cell mixture; (b) resuspending the cell mixture in a freezing medium; (c) transferring the resuspended cell mixture into a single container; (d) pre-chilling the container at a first temperature followed by storing the container at a second temperature for a predetermined period of time, wherein the second temperature is lower than the first temperature and causes the cell mixture to freeze; and (e) storing the container at a third temperature. As used herein, the term “container’" refers to any container, for example, a vial, designed to store materials at temperatures ranging from about 4 °C to about -196 °C. An example of a container is a cryovial.

As used herein, the term “prechilling” or “prechilled” refers to reducing the temperature to below room temperature.

As used herein, the term “cry opreserving” or “cry opreservation” refers to the longterm conservation of storing tissues or living cells at ultralow temperatures (such as about -196 °C). Cry opreservation usually involves a controlled rate freezing process, wherein the temperature of the cellular material is reduced to about -80 °C at a rate 1 °C/min, followed by further reducing the temperature by placing the material into liquid nitrogen. In some cases, the cellular material is prechilled at 4 °C in a Mr. Frosty™ Freezing Container prior to being transferred to -80 °C. Cryopreservation allows for high cell recovery with over 90% cell viability post-thaw.

As used herein, the term "‘freezing medium” refers to any suitable medium for cry opreservation of living cells or tissues. The freezing medium can contain a cryoprotective agent such as DMSO or glycerol. Cryoprotective agents can permeate the cell membrane or can be impermeable substances (U.S. Patent No. 10,472,606). In a particular example, the freezing medium comprises 60% FBS-free serum replacement (such as KnockOut™ Serum Replacement (KSR)), 30% hECSR (Human endothelial serum-free medium), 10% DMSO, 10 pM Y27632 ROCK Inhibitor, and 50 ng/mL VEGF-A.

In one embodiment, the brain or BBB is mammalian such as a human. In other embodiments, the brain or BBB is non-mammalian such as a reptile, chicken, or amphibian.

In one embodiment, the freezing medium comprises 60% FBS-free serum replacement, 30% hECSR, 10% DMSO, 10 pM Y27632 ROCK Inhibitor, and 50 ng/mL VEGF-A. In one embodiment, the method comprises resuspending the cell mixture in 0.5 mL to 5 mL of the freezing medium such as about 0.5 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL. or any amount in-between. In one embodiment, the method comprises resuspending the cell mixture in 1 mL of the freezing medium.

In one embodiment, the single container is a vial designed to store materials at temperatures ranging from about 4 °C to about -196 °C. The vial can be a cry ovial. In one embodiment, the first temperature is about 4 °C such as about 3.9 °C, about 4.0 °C. or about 4. 1 °C. In one embodiment, the second temperature is about -80 °C such as about -70 °C, about -80 °C, or about -81 °C. In one embodiment, the third temperature is from about -80 °C to about -250 °C such as about -80 °C, about -85 °C, about -90 °C, about - 95 °C, about -100 °C, about -110 °C, about -120 °C, about -130 °C, about -140 °C, about - 150 °C, about -160 °C. about -170 °C. about -180 °C, about -190 °C, about -200 °C, about - 210 °C, about -220 °C. about -230 °C. about -240 °C. about -250 °C. or any temperature inbetween. In one embodiment, the third temperature is from about -80 °C to about -196 °C. In one embodiment, the third temperature is from about -80 °C to about -130 °C.

In one embodiment, the predetermined period time is from about 2 hours to about 72 hours such as about 2 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 25 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 71 hours, about 72 hours, or any period of time in-between. In one embodiment, the predetermined period time is from about 12 hours to about 36 hours such as about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, or any period of time in-between. In one embodiment, the predetermined period of time is about 24 hours.

In one embodiment, the step of combining comprises combining the cells in a combination medium comprising:

(a) astrocyte media and 10 ng/mL VEGF-A;

(b) 500 mL human endothelial serum-free (hECSR) media, 5 mL penicillinstreptomycin, 5 mL NEAA, 5 mL CD lipids, 5 mL astrocyte growth supplement, 10 mL serum-free supplement for neuronal cells, 500 pl insulin, 1 pM dibutyl-cAMP, 50 pg/mL ascorbic acid, 10 ng/mL NT3, 10 ng/mL IGF, 100 ng/mL biotin, 60 ng/mL T3, 10 ng/mL VEGF, and 1 pM SAG; or

(c) 500 mL hECSR media, 5 mL penicillin-streptomycin, 5 mL NEAA. 10 mL B27 supplement, 50 ng/mL VEGF, 20 ng/mL FGF, and 10 ng/mL FGF. In one embodiment, the combination medium of (c) further comprises 5 pg/ml doxycycline.

In some embodiments, the method comprises prior to combining the cells, differentiating iPSC or embryonic stem cells into desired cell t pes, such as neurons, glia, and vascular cells. In some embodiments, the cell mixture comprises iPSC-derived cells, embry onic stem cell-derived cells or cells derived from primary cells. In some embodiments, the method comprises differentiating the neurons, glia, and vascular cells into neurons, astrocytes, oligodendrocyte progenitor cells (OPCs), brain endothelial cells, microglia, and pericytes.

Cells

In one embodiment, the cell mixture of the cryopreserved cells described herein comprises iPSC-derived cells, embry onic stem cell-derived cells or cells derived from primary cells. Primary cells are cells that are isolated or harvested directly from living tissues or organs. Embryonic stem cell-derived cells are derived from embryos that are approximately three to five days old. In one embodiment, the iPSC-derived cells exhibit inducible expression of ETV2 (Ets variant 2).

In one embodiment, the cell mixture comprises mammalian cells from any mammalian species, including but not limited to humans, dogs, sheep, cows, monkeys, goats, mice, and pigs. In one embodiment, the cell mixture comprises non-mammalian cells such as cells from chickens, amphibians, or reptiles.

In one embodiment, the iPSC-derived cells or embry onic stem cell-derived cells are differentiated into neurons, glia, and vascular cells. In one embodiment, the neurons, glia, and vascular cells are differentiated into astrocytes, oligodendrocyte progenitor cells, brain endothelial cells, microglia, and pericytes. Astrocytes can be identified based on their expression of SI 00b (SI 00 calcium binding protein B). Endothelial cells can be identified based on their expression of PECAM1 (platelet and endothelial cell adhesion molecule 1). Neurons can be identified based on their expression of NFH (neurofilament). Vascular cells can be identified based on their expression of VE-CAD (vascular endothelial-cadherin). Glial cells can be identified based on their expression of GFAP (glial fibrillary acidic protein). Microglia can be identified based on their expression of TMEM-119 (transmembrane protein 119). Pericytes can be identified based on their expression of PDGFRp (Platelet-derived growth factor receptor beta). As used herein, the term “differentiation” is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a blood cell, an immune cell, or a bone cell. A differentiated cell refers to a cell that has achieved a specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.

As used herein, the term “tissue” refers to a group or layer of similarly specialized cells performing certain special functions together.

As used herein, the term “stem cells” refers to cells with the ability to both replace themselves and to differentiate into more specialized cells. Their self-renewal capacity generally endures for the lifespan of the organism. A pluripotent stem cell can give rise to all the various cell types of the body. A multipotent stem cell can give rise to a limited subset of cell types.

In one embodiment, the cell mixture of the cryopreserved cells described herein comprises brain endothelial cells, astrocytes, and pericytes. In another embodiment, the cell mixture comprises human neurons, astrocytes, oligodendrocyte progenitor cells, endothelial cells, and pericytes. In one embodiment, the cell mixture comprises brain endothelial cells and pericytes.

In one embodiment, the cell mixture comprises about 1 x 10² to about 10 x 10¹⁰ brain endothelial cells/mL, about 1 x IO² to about 2 x IO¹⁰ astrocytes/mL, and about 1 x 10² to about 2 x IO¹⁰ pericytes/mL. For example, the cell mixture comprises about 1 x 10², about 1 x 10^?, about 1 x 10⁴, about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, or any number in-between of brain endothelial cells/mL, astrocytes/mL, and pericytes/mL. In one embodiment, the cell mixture comprises about 1 x 10⁶ to about 10 x 10⁶ brain endothelial cells/mL, about 1 x 10⁶ to about 2 x 10⁶ astrocytes/mL, and about 1 x 10⁶ to about 2 x 10⁶ pericytes/mL.

In one embodiment, the cell mixture comprises about 5 x 10² to about 5 x IO¹⁰ brain endothelial cells/mL, about 1 x 10² to about 1 x IO¹⁰ astrocytes/mL, and about 1 x 10² to about 1 x IO¹⁰ pericytes/mL. For example, the cell mixture comprises about 5 x 10², about 5 x IO³, about 5 x 10⁴, about 5 x 10⁵, about 5 x 10⁶, about 5 x 10⁷, about 5 x 10⁸, about 5 x 10⁹, about 5 x IO¹⁰ or any number in-between of brain endothelial cells/mL. In another example, the cell mixture comprises about 1 x 10², about 1 x 10³, about 1 x 10⁴, about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, or any number in-between of brain endothelial cells/mL, astrocytes/mL, and pericytes/mL. In one embodiment, the cell mixture comprises about 5 x 10⁶ brain endothelial cells/mL, about 1 x 10⁶ astrocytes/mL, and about 1 x 10⁶ pericytes/mL.

In one embodiment, the cell mixture comprises about 1 x 10² to about 10 x IO¹⁰ neurons/mL, about 1 x 10² to about 10 x IO¹⁰ brain endothelial cells/mL, about 0.1 x 10²to about 2 x IO¹⁰ astrocytes/mL, about 0.1 x 10²to about 2 x IO¹⁰ OPCs/mL, and about 0. 1 x 10² to about 2 x 10¹⁰ pericytes/mL. For example, the cell mixture comprises about 1 x 10², about

1 x 10³, about 1 x 10⁴, about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x IO⁹, about 1 x 10¹⁰ or any number in-between of brain endothelial cells/mL. The cell mixture comprises about 0.1 x 10², about 0.1 x 10³, about 0.1 x 10⁴, about 0.1 x 10³, about 0.1 x 10⁶, about 0.1 x 10⁷, about 0.1 x 10⁸, about 0.1 x 10⁹, about 0.1 x 10¹⁰, about 1 x 10², about 1 x 10³, about 1 x 10⁴, about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, about 2 x 10², about 2 x 10³, about 2 x 10⁴, about 2 x 10⁵, about 2 x 10⁶, about

2 x 10⁷, about 2 x 10⁸, about 2 x 10⁹, about 2 x IO¹⁰, or any number in-between of astrocytes/mL, OPCs/mL, and pericytes/mL. In one embodiment, the cell mixture comprises about 1 x 10⁶ to about 10 x 10⁶ neurons/mL, about 1 x 10⁶ to about 10 x 10⁶ brain endothelial cells/mL, about 0. 1 x 10⁶ to about 2 x 10⁶ astrocytes/mL, about 0. 1 x 10⁶ to about 2 x 10⁶ OPCs/mL, and about 0.1 x 10⁶to about 2 x 10⁶ pericytes/mL.

In one embodiment, the cell mixture comprises about 1 x 10²to about 2.5 x IO¹⁰ neurons/mL, about 1 x 10²to about 2.5 x 10¹⁰ brain endothelial cells/mL, about 0.5 x 10²to about 1.0 x IO¹⁰ astrocytes/mL, about 0.5 x 10²to about 1.0 x IO¹⁰ OPCs/mL, about 0.5 x 10² to about 1.0 x 10¹⁰ microglia/mL, and about 0.5 x 10²to about 1.0 x 10¹⁰ pericytes/mL. For example, the cell mixture comprises about 1 x 10², about 1 x 10³, about 1 x 10⁴, about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, about 2 x 10², about 2 x 10', about 2 x 10⁴, about 2 x 10⁵, about 2 x 10⁶, about 2 x 10⁷, about 2 x 10⁸, about 2 x 10⁹, about 2 x IO¹⁰, about 2.5 x 10², about 2.5 x 10³. about 2.5 x 10⁴, about 2.5 x 10⁵. about 2.5 x 10⁶, about 2.5 x 10⁷, about 2.5 x 10⁸, about 2.5 x 10⁹, about 2.5 x IO¹⁰ or any number in-between of brain endothelial cells/mL. The cell mixture comprises about 0.5 x 10², about 0.5 x 10³, about 0.5 x 10⁴, about 0.5 x 10⁵, about 0.5 x 10⁶, about 0.5 x 10⁷, about 0.5 x IO⁸, about 0.5 x 10⁹, about 0.5 x IO¹⁰, about 1 x 10². about 1 x 10³, about 1 x 10⁴. about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x 10¹⁰ or any amount in-between of astrocytes/mL, OPCs/mL. microglia/mL, and pericytes/mL. In one embodiment, the cell mixture comprises about 2.5 x 10⁶ neurons/mL, about 2.5 x 10⁶ brain endothelial cells/mL, about 0.5 x 10⁶ astrocytes/mL, about 0.5 x 10⁶ OPCs/mL, about 0.5 x 10⁶ microglia/mL, and about 0.5 x 10⁶ pericytes/mL.

In one embodiment, the cell mixture comprises about 0.1 x 10²to about 2 x IO¹⁰ pericytes/mL and about 1 x 10² to about 2.5 x IO¹⁰ brain endothelial cells/mL. For example, the cell mixture comprises about 1 x 10², about 1 x 10³, about 1 x 10⁴, about 1 x 10³, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, about 2 x 10², about 2 x 10³, about 2 x 10⁴, about 2 x 10⁵, about 2 x 10⁶, about 2 x 10⁷, about 2 x 10⁸, about 2 x 10⁹, about 2 x IO¹⁰, about 2.5 x 10². about 2.5 x 10³, about 2.5 x 10⁴. about 2.5 x 10⁵, about 2.5 x 10⁶. about 2.5 x 10⁷, about 2.5 x 10⁸, about 2.5 x 10⁹, about 2.5 x IO¹⁰ or any number in-between of brain endothelial cells/mL. In another example, the cell mixture comprises about 0.1 x 10², about 0.1 x 10³, about 0.1 x 10⁴, about 0. 1 x 10⁵, about 0. 1 x 10⁶, about 0.1 x 10⁷, about 0.1 x 10⁸, about 0.1 x 10⁹, about 0. 1 x IO¹⁰, about 1 x 10², about 1 x 10³, about 1 x 10⁴. about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, about 2 x 10², about 2 x 10³, about 2 x 10⁴, about 2 x 10⁵, about 2 x 10⁶, about 2 x 10⁷, about 2 x 10⁸, about 2 x 10⁹, about 2 x IO¹⁰, or any number in-between of pericytes/mL.

In one embodiment, the predetermined cell ratio of the cryopreserved cells is about 2: 1 : 1; about 3: 1 : 1; about 4: 1: 1; about 5: 1 : 1; about 6:1:1; about 7: 1: 1; about 8: 1 : 1; or about 9: 1 : 1 by cell density of brain endothelial cells, astrocytes, and pericytes. In one embodiment, the predetermined cell ratio of the cryopreserved cells is about 5: 1: 1 by cell density of brain endothelial cells, astrocytes, and pericytes.

In one embodiment, the predetermined cell ratio of the cryopreserved cells is about 2: 1: 1: 1; about 3: 1: 1: 1; about 4: 1: 1: 1; about 5: 1: 1 : 1 : 1; about 6: 1 : 1: 1; about 7: 1 : 1: 1; about 8: 1 : 1 : 1 ; or about 9: 1: 1: 1 by cell density of brain endothelial cells, astrocytes, OPCs, microglia, and pericytes. In one embodiment, the predetermined cell ratio of the cryopreserved cells is about 5: 1 : 1: 1 : 1 by cell density⁷ of brain endothelial cells, astrocytes, OPCs, microglia, and pericytes. In one embodiment, the cell mixture described herein comprises 2 to 8 (e.g., 2, 3, 4, 5, 6, 7, or 8) different cell types. In one embodiment, the cell mixture comprises 3 to 6 different cell types.

In one embodiment, the cell mixture has about 1 x 10² to about 10 x IO¹⁰ cells/mL such as about 1 x 10², about 1 x 10³, about 1 x 10⁴, about 1 x 10⁵, about 1 x 10⁶, about 1 x 10⁷, about I x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, or any number of cells/mL in-between. In one embodiment, the cell mixture has about 1 x 10⁶ to about 10 x 10⁶ cells/mL such as about 1 x 10⁶, about 1 x 10⁷, about 1 x 10⁸, about 1 x 10⁹, about 1 x IO¹⁰, about 2 x 10⁶, about 2 x 10⁷, about 2 x 10⁸, about 2 x 10⁹, about 2 x IO¹⁰, about 3 x 10⁶, about 3 x 10⁷, about 3 x 10⁸, about 3 x 10⁹, about 3 x IO¹⁰, about 4 x 10⁶, 4 x 10⁷, about 4 x 10⁸, about 4 x 10⁹, about 4 x IO¹⁰, about 5 x 10⁶, about 5 x 10⁷, about 5 x 10⁸, about 5 x 10⁹, about 5 x IO¹⁰, about 6 x 10⁶. about 6 x 10⁷, about 6 x 10⁸. about 6 x 10⁹, about 6 x IO¹⁰, about 7 x 10⁶. about 7 x 10⁷, about 7 x 10⁸, about 7 x 10⁹, about 7 x IO¹⁰, about 8 x 10⁶, about 8 x 10⁷, about 8 x 10⁸, about 8 x 10⁹, about 8 x IO¹⁰, about 9 x 10⁶, 9 x 10⁷, about 9 x 10⁸, about 9 x 10⁹, about 9 x IO¹⁰, about 10 x 10⁶, about 10 x 10⁷, about 10 x 10⁸, about 10 x 10⁹, about 10 x IO¹⁰ or any number of cells/mL in-between. In one embodiment, the cell mixture has about 7 x 10⁶ cells/mL.

Notably, the above-described cryopreserved cells maintain at least about 50% cell viability post-thaw such as at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. Cell viability can be determined using methods as known in the art. For example, cell viability can be determined by Trypan Blue cell counting. Other methods of determining cell viability include, but are not limited to, performing an MTT assay or an ATP cell viability luciferase assay.

Assays and methods of use thereof

As used herein, the term “assay” refers to a test or an experimental procedure that utilizes cells prepared from the method disclosed herein. In some embodiments, the assay may use cells, as depicted in FIG. 1, that model a brain or a blood brain barrier. In one embodiment, the assay is an assay based on induced blood-brain barrier (iBBB), just add neurons (JANs), a multicellular integrated human brain tissue (miBrain), or an induced microvascular cell combo (rmVasC).

An assay based on JANs comprises the addition of neurons to astrocytes, OPCs, microglia progenitors, endothelial cells, and/or pericytes. An assay based on miVasC comprises endothelial cells and pericytes to model the human microvasculature. In one embodiment. miVasC enables the addition of microvasculature to co-culture. 3D tissue, and/or organoids. An assay based on iBBB comprises astrocytes, endothelial cells, and pericytes. An assay based on miBrain comprises neurons, astrocytes, OPCs, microglia progenitors, endothelial cells, and/or pericytes to model brain tissue.

In some embodiments, the assays described herein are used to perform drug screening. For example, drugs can be screened for treating neurodegenerative diseases. In some embodiments, the neurodegenerative disease is selected from the group comprising Alzheimer’s disease-associated cerebral amyloid angiopathy, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, spinal muscular atrophy, motor neuron disease, and Amyotrophic lateral sclerosis (ALS).

In one embodiment, assays described herein are used to screen for drugs that treat vascular disorders. Examples of vascular disorders include, but are not limited to, carotid artery disease, deep vein thrombosis, mesenteric artery disease, peripheral artery disease, renal artery disease, or aneurysm.

In one embodiment, the above-described cells are used to treat neurodegenerative diseases in a subject in need thereof.

As used herein, a ‘"subject” or “individual” means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cows, horses, pigs, sheep, goats, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a human or a non-human mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders. The terms, “individual,” “patient,” and “subject” can be used interchangeably herein. A subject can be male or female. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein byreference in their entireties.

As used herein and in the appended claims, the singular forms "a." "‘and,’⁷ and "the" include plural references unless the context clearly dictates otherwise.

The term '‘about’’ refers to a range of values which would not be considered by a person of ordinary skill in the art as substantially different from the baseline values. For example, the term “about” may refer to a value that is within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%. 3%, 2%, 1%. 0.5%, 0.1%, 0.05%, or 0.01% of the stated value, as well as values intervening such stated values.

As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically- excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

The use of any and all examples, or exemplary language (e.g. “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherw ise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. As used herein, the phrases "in one embodiment,’' “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

As used herein, the terms “and/or” or

means any one of the items, any combination of the items, or all of the items with which this term is associated.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLES

Example 1. Development of an off-the-shelf cryopreserved human brain tissue.

1. Summary

A method was developed to cryopreserve pooled human brain cells (Example 3) into ready-to-use assays modeling the human brain and the human blood-brain barrier. The assays’ names are multicellular integrated human brain tissue (miBrain) and induced bloodbrain barrier (iBBB).

Differentiation

All cell types were generated from human induced pluripotent stem cells (iPSCs). The source of iPSC can be any donor, or they can be obtained from commercial entities. Neurons, glia, and vascular cells were generated from iPSCs using published protocols or a combination of published protocols (Abud, E.M., et al., iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron, 2017. 94(2): p. 278-293 e9; Blanchard, J.W., et al.. Reconstruction of the human blood-brain barrier in vitro reveals a pathogenic mechanism of APOE4 in pericytes. Nat Med, 2020. 26(6): p. 952-963; Chambers, S.M., et al., Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol, 2009. 27(3): p. 275-80; Douvaras, P., et al., Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Reports, 2014. 3(2): p. 250-9; Patsch, C., et al., Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat Cell Biol, 2015. 17(8): p. 994-1003; Qian, T., et al., Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells. Sci Adv, 2017. 3(11): p. el701679; Tew. J., et al., An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells. Stem Cell Reports, 2017. 9(2): p. 600-614; Wang, K., et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA. Sci Adv, 2020. 6(30): p. eaba7606.; Zhang, Y., et al., Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron, 2013. 78(5): p. 785-98). Details on each protocol can be found in Example 2. For all cell types, the shortest differentiation protocol takes 1 week, the longest protocol takes 75 days. This duration time is optimized because the cells generated from protocols taking more than 2 weeks are banked and expanded upon thaw.

Using iPSCs is advantageous because it enables deriving the final tissue from any donor and multiple genetic backgrounds. However, the cry opreservation protocol also works using cells derived from embryonic stem cell lines or primary cells from any mammalian species.

Brain tissue assembly and cryopreservation

After all cells were ready after differentiation or at least 1 week after thaw, the tissue was assembled and cryopreserved. Details on the methods are described in Example 3. The cells were pooled altogether with pre-established cell ratios before being cryopreserved. Each cryovial contained a pool of all cells at a conserved cell density. The cell density was 5 xlO⁶ neurons/ml, 5 x 10⁶ endothelial cells/ml, and 1 x 10⁶ cells/ml for each of the remaining cell types: pericytes, astroglia, oligodendroglia and microglia. Each cryovial contained 7 x 10⁶ pooled cells per 1 ml. This number was based on optimal cell freezing densities between 1 x 10⁶ to 10 x 10⁶ cells in 1 ml. In summary, the critical steps are: (1) the cells are pooled altogether wi th pre-established cell ratios before being cryopreserved, and (2) the freezing media contain grow th factors that improve cell survival and identity.

End user workflow and actual number of units per cryovial

Each cryovial contained 7 x 10⁶ pooled cells frozen in 1 ml. This number is based on optimal cell freezing densities and could range between 1 x 10⁶ to 10 x 10⁶ cells in 1 ml. After spinning down to remove the freezing medium, the end user resuspends this in the assay media to assess cell viability, adds any additional cell ty pes (e.g., adding neurons to JANS), spins down again, and resuspends it in the appropriate volume of extracellular matrix gel to maintain appropriate cell ratios (e.g., in 1 ml of extracellular matrix gel). The number of assays per cryovial depends on the volume of each assay plate. The size of each unit ty pically varies from 5 pl to 50 pl. Therefore, the number of assays will vary depending on the choice of assay size by a user (e.g., from 200 to 20 units). Tables 1 and 2 summarize the number of cells and assays in cryotubes that contain 50 assays of 10 pL each. FIG. 4 summarizes cell viability data.

Note that if microglia are added to the tissue, then it is recommended to grow the tissue for 1-2 weeks and then add microglia progenitors matching the cell to cell ratio shown in Table 2. Microglia progenitors are able to invade the tissue and incorporate into miBrain. miVasC is designed to supply the need of multiple experimental applications that require a microvascular component, including brain and retina 3D tissue and organoids, as well as tissue engineering in regions outside the central nervous system but that are also rich in microvasculature.

Table 1. Number of cells per vial for iBBB and miBrain.

Example 2. Generation of cells.

2.1 Cells

1. Induced pluripotent stem cells. 2.2 Media

2.2.1 iPSC cultures: StemFlex™ Basal Medium

Basal medium with StemFlex™ supplement (StemCell Technologies, Cat. no. A3349401); 1% penicillin-streptomycin.

2.2.2 NPC (neural progenitor cell) Medium

1: 1 DMEM/F12 with Glutamax (Life Technologies, Cat. no.10565-042): Neurobasal media Neurobasal Medium (Thermo Fisher, Cat. no. 21103049), lx N-2 Supplement (Thermo Fisher, Cat. no. 17502048), lx B-27 Serum-Free supplement (Gibco. Cat. no. 17504044), 0.5x GlutaMAX Supplement (Thermo Fisher, Cat. no. 35050079), lx Eagle's Minimum Essential Medium Non-essential Amino Acid Solution (MEM-NEAA) (100*) (Sigma- Aldrich, Cat. no. M7145), 1% penicillin-streptomycin.

2.2.3 Astrocyte Medium

Basal media with supplements and penicillin-streptomycin (ScienCell, Cat. no. 1801).

2.2.4 DeSRl Medium

DMEM/F12 with GlutaMAX, l x MEM-NEAA, l x penicillin-streptomycin.

2.2.5 DeSR2 Medium

DeSRl media, lx N-2, l x B-27.

2.2.6 hECSR Medium

Human endothelial serum-free Medium (Gibco. Cat. no. 11111044), l x MEM-NEAA, l x B- 27. 1 % penicillin-streptomycin.

2.2.7 N2 Medium

DMEM/F12 with GlutaMAX, lx N-2, lx MEM-NEAA, 1% penicillin-streptomycin.

2.2.8 N2B27 Medium

1: 1 DMEM/F12: Neurobasal media, lx B-27, lx N-2, lx MEM-NEAA, lx GlutaMAX, 1% penicillin-streptomycin. 2.2.9 PDGF Medium

N2B27 medium, PDGF, IGF, HGF, NT3, Insulin. Biotin, cAMP, T3

2.2.10 miVasC Medium

500 mL Human Endothelial Serum-free Medium

5 mL Pen/Strep

5 mL NEAA

10 mL B27 Supplement

VEGF - 50ng/mL

FGF - 20ng/mL

EGF - lOng/mL

5 pg/ml doxycycline if required for ETV2 (Ets variant 2) induction

2.2.10 Freezing media for iBBB and miBrain

Any freezing media suitable for cell cryopreservation can be used. Post-thaw cell viability was optimized using the following medium: 60% KSR (KSR, Gibco, Cat. no. 10828028), 30% hECSR, 10% Dimethyl sulfoxide (DMSO, Sigma-Aldrich, Cat. no. D2650), lOpM Y27632 ROCK Inhibitor (PeproTech. Cat. no. 1293823, 50 ng/mL VEGF -A (Mesentier- Louro, L.A. et al. Methods Mol Biol, 2023. 2683: p. 135-151 and Goldman, C. etal. J Vis Exp, 2023(200).

2.3. Methods

2.3.1 iPSC cultures

Before thawing iPSCs. plates were coated with Geltrex™ Matrix or Matrigel Matrix diluted at 1: 100 in DMEM medium for at least 20 min at 37 °C. After that, the coating solution was replaced with StemFlex warmed culture medium prior to seeding of cells. iPSCs were cultured as colonies and passaged when colonies were large but still not merged using 0.5 mM EDTA in PBS. For each differentiation protocol, iPSCs were cultured until they reached 60- 70% confluency. At this point, iPSCs were dissociated by incubation with accutase for 5-10 minutes at 37 °C. Dissociated cells were transferred to a falcon tube with a minimum of 1:3 accutase to media ratio and spun down at 300 x g for 3 min at RT. The cell pellet was resuspended in w armed StemFlex™ supplemented with 10 pM Y27632. Cells were replated at the indicated cell number and media for each differentiation. 2.3.2 Preparation of iPSC lines with inducible systems for generation of neurons and endothelial cells.

For the differentiation of neurons or endothelial cells. iPSCs were transfected with an inducible system for expression of NGN2 (neurogenin 2) or ETV2, respectively. The system allows induction of the expression of the gene of interest during the differentiation protocol, but not during maintenance of the iPSCs. A PiggyBac inducible system from AddGene was used. Lentivirus. AAV, and CRISPR systems may be used as well. Gene expression was induced using doxycycline. Tetracycline and other analogs can be used as well. If the inducible system contains a selection cassette, iPSCs may be maintained with the selection antibiotic (e.g., puromycin, neomycin, hygromycin, zeocin, blasticidin, gentamicin, TRP1, LEU2, URA3, HIS3, Basta) to increase purity'. The selection antibiotic can be maintained in the differentiation protocol to increase purity if needed.

2.3.3 Differentiation of human iPSCs into astrocytes

Timing'. 45-50 days (if starting from iPSCs) or 30-35 days (if starting from NPCs).

NPC differentiation was adapted from Chambers, S.M., et al., Nat Biotechnol, 2009. 27(3): p. 275-80). Astrocytes were differentiated as described in TCW, J et al. (Tew, J., et al., Stem Cell Reports, 2017. 9(2): p. 600-614).

Protocol'. Dissociated iPSCs were plated at 100.000 cells/cm² onto Geltrex™-coated plates, in pre-warmed StemFlex™ supplemented with 10 pM Y27632. Cells were fed every other day until they reached >95% confluence (approximately 3-4 days, depending on the cell line). Once cells reached confluence, the medium was replaced with NPC medium supplemented with lOpM SB43152 and lOOnM LDN193189 (day 0). From days 1 to 9, cells were fed daily with NPC media plus lOpM SB43152 and lOOnM LDN193189. On day 10, cells were split with accutase and replated onto fresh Geltrex™-coated plates, in NPC media supplemented with 20ng/mL bFGF and lOpM Y27632. From days 11 to 13, cells were fed with NPC media plus 20ng/mL bFGF. On day 14, cells were split with accutase and re-seeded onto fresh Geltrex™- coated plates in NPC media plus 20ng/mL bFGF and lOpM Y27632. On day 15, NPCs were frozen for stocking. This is day 0 of astrocyte differentiation: cells were fed with Astrocyte Medium (AM) and passaged using accutase once they reached 90% confluence. From this point, NPCs will be fully differentiated into astrocytes in 30 days.

2.3.4 Differentiation of human iPSC into brain microvascular endothelial cells Timing'. 8 days.

Brain endothelial cell differentiation was optimized by a combination of the protocols from Blanchard et al. (Blanchard, J.W., et al.. Nat Med. 2020. 26(6): p. 952-963), Qian et al. (Qian, T., et al., Sci Adv, 2017. 3(11): p. el701679), and Wang et al. (Wang, K., et al., Sci Adv, 2020. 6(30): p. eaba7606). A major modification from Blanchard et al. and Qian et al. was using inducible expression of ETV2 to induce endothelial cell fate. Wang et al. used ETV2 induction, but in conjunction with a different protocol (small molecules used, duration). This protocol uniquely combines a set of small molecules, ETV2 induction, and duration.

Protocol'.

1. Preparation of iPSC lines with inducible expression of ETV2

The iPSCs must be transfected or transduced with an inducible system for expression of ETV2. The system needs to allow induction of the expression of ETV2 during the differentiation protocol, but not during maintenance of the iPSCs. A PiggyBac inducible system from AddGene was used (note that lentivirus, AAV, CRISPR systems could have been used as well). Gene expression was induced using doxycycline. Tetracycline and other analogs could have been used as well. If the inducible system contained a selection cassette, the iPSCs were maintained with the selection antibiotic (e.g., puromycin. neomycin, hygromycin, zeocin, blasticidin, gentamicin, TRP1, LEU2, URA3, HIS3, Basta) to increase purity.

2. Differentiation protocol

Dissociated cells were plated at 20,800 cells/cm² onto Geltrex™-coated plates, in prewarmed StemFlex™ supplemented with 10 pM Y27632 (day 0). On day 1. the medium was replaced with DeSRl supplemented with 10 ng/mL BMP4. 6 pM CHIR99021. and 5 pg/mL doxycycline. On day 3, the medium was replaced with DeSR2 medium with 5 pg/mL doxycycline. On days 5 and 7, the medium was replaced with hECSR medium supplemented with 50 ng/mL VEGF-A, 2 pM Forskolin, and 5 pg/mL doxycycline. On day 8, cells were dissociated using Accutase and re-seeded onto fresh Geltrex™-coated plates, in hECSR supplemented with 50 ng/mL VEGF-A and 5 pg/mL doxycycline. Cells were banked and expanded in hECSR media supplemented with 50 ng/mL VEGF-A and 5 pg/mL doxycycline until ready for iBBB or miBrain cryopreservation. 2.3.5 Differentiation of human iPSCs into pericytes

Timing'. 6 days.

Pericyte differentiation was adapted from Patsch, C. et al. (Patsch, C. et al., Nat Cell Biol, 2015. 17(8): p. 994-1003).

Protocol'. Dissociated iPSCs were plated at 37,000 to 40,000 cells/cm² onto Geltrex™-coated plates, in pre-warmed StemFlex™ supplemented with 10 pM Y27632 (day 0). On day 1, the medium was replaced with N2B27 media supplemented with 25 ng/mL BMP4 and 8 pM CHIR99021. On days 3 and 4, the medium was replaced with N2B27 media supplemented with 2 ng/mL Activin A and 10 ng/mL PDGF-BB. On day 5. pericytes were dissociated with accutase and re-seeded onto fresh 0. 1% gelatin-coated plates at 35,000 cells/cm², in N2B27 supplemented with 10 ng/mL PDGF-BB. This medium was used for every other day medium change for another 5-7 days. Cells were then banked and expanded in N2B27 until they were ready for iBBB or miBrain cryopreservation.

2.3.6 Differentiation of human iPSCs into neurons

Timing: 6 days

Neuron differentiation was adapted from Zhang et al. (Zhang, Y. et al., Neuron, 2013. 78(5): p. 785-98).

Protocol'. Dissociated iPSCs were plated at -80,000 cells/cm² onto Geltrex™-coated plates, in pre-warmed StemFlex™ supplemented with 10 pM Y27632 (day 0). On day 1, the medium was replaced with DeSRl media supplemented with lOpM SB431542, 100 nM LDN, 5 pg/mL doxycycline. On days 3 and 5, the medium was replaced with N2 media supplemented with 5 pg/mL doxycycline. On day 6, neurons were ready to be dissociated in accutase and used for miBrain cryopreservation.

2.3.5 Differentiation of human iPSCs into oligodendrocyte progenitor cells

Timing: 75 days

Neuron differentiation was adapted from Douvaras, P. et al., Stem Cell Reports, 2014. 3(2): p. 250-9.

Protocol'. To initiate differentiation, iPSC media was replaced with KSR containing 10 pM SB431542 and 200 ng/ml Noggin (or 100 nM LDN) with 100 nM all-trans RA (day 0). On day 4, the medium was replaced with KSR/N2 media (3: 1) supplemented with lOpM SB431542 and 200 ng/ml Noggin. On day 6, the medium was replaced with KSR/N2 (1: 1) with lOpM SB431542 and 200 ng/ml Noggin. On day 8, the medium was replaced with KSR/N2 (1:3) with IpM SAG, 10gM SB431542, and 200 ng/ml Noggin. On day 12, adherent cells were lifted and seeded in low-attachment plates to favor sphere aggregation. Spheres were cultured in the presence of RA and SAG. On day 30, spheres were plated into poly-L-omithine/laminin-coated dishes and cells were allowed to migrate out of the sphere. At this stage, PDGF medium was used to promote OPC formation. On day 75, cells were banked and expanded as OPC-like cells using N2/B27 media + 10 ng/ml PDGFAA, 10 ng/ml bFGF, 10 ng/ml NT3 until ready for miBrain cry opreservation.

2.3.6 Differentiation of human iPSCs into microglia

Timing-, generation of hematopoietic progenitors (12 days); generation of microglia (28-34 days).

Microglia differentiation is obtained using commercial kits based on the protocol from Abud, E.M. et al., Neuron, 2017. 94(2): p. 278-293 e9.

Protocol'. To generate hematopoietic progenitors from iPSC, the kit STEMdifl™ Hematopoietic Kit (Catalog #05310) was used following manufacturer's recommendations. To generate microglia from the hematopoietic progenitors, the STEMdifl™ Microglia Culture System comprising STEMdifl™ Microglia Differentiation Kit and STEMdiff™ Microglia Maturation Kit was used following manufacturer’s recommendations. Microglia were added to miBrain at different stages of differentiation.

Example 3. iBBB, miVasC, JAN, and miBrain cryopreservation.

Timing-. 2-4h.

This cryopreservation protocol can be performed using mammalian primary, iPSC- or embryonic stem cell-derived neurons, glia, and vascular cells. It was optimized for iPSC- derived human neurons, astrocytes, oligodendrocyte progenitor cells (OPCs). endothelial cells and pericytes.

3.1 iBBB Medium 1. Astrocyte media (basal media with supplements and penicillin-streptomycin (ScienCell. Cat. no. 1801) and

2. 10 ng/mL VEGF-A (R&D Systems, Cat. no. 293-VE-050/CF). Supplement astrocyte media with VEGF-A immediately before feeding cells.

3.2 mi Brain Medium

500mL Human Endothelial Serum-free Medium

5mL Pen/Strep

5mL NEAA

5mL CD Lipids

5mL Astrocyte Growth Supplement (AGS)

1 OmL B27 Supplement

500pL Insulin cAMP-dibutyl - 1 pM

Ascorbic acid - 50pg/mL

NT3 - lOng/mL

IGF - lOng/mL

Biotin - lOOng/mL

T3 - 60 ng/mL

VEGF - lOng/mL - WEEK 1 ONLY

SAG - 1 pM - WEEK 1 ONLY

Protocol'. All cell types were dissociated using accutase (neurons, OPCs, endothelial cells, pericytes, microglia) or TryplE Select (astrocytes). Each dissociated cell type was transferred to a separate tube with a minimum of 1:3 accutase to media ratio. Cells were spun down (at 300 x g for 3 min at RT for all cells except neurons that were spun at 200 x g for 5 min at RT) and resuspended in corresponding media. Using an automated cell counter, each cell type was counted. For each iBBB cryovial, a tube was prepared containing 5 x 10⁶ endothelial cells, 1 x 10⁶ astrocytes and 1 x 10⁶ pericytes (Table 2). For each miBrain cry ovial, a tube was prepared to contain 2.5 x 10⁶ neurons, 2.5 x 10⁶ endothelial cells, 0.5 x 10⁶ astrocytes, 0.5 x 10⁶ OPCs, 0.5 x 10⁶ microglia, and 0.5 x 10⁶ pericytes. Pooled cells were spun down at 200 x g for 5 min at RT. Media was aspirated carefully, leaving the cell pellet undisturbed. For each tube, 1 ml of miBrain or iBBB pre-chilled freezing medium was added to resuspend the cells and transfer the mixture to a cryovial. Cryovials were placed in a Mr. Frosty pre-chilled at 4 °C and transferred to -80 °C for 24h. Cryovials were then stored in liquid nitrogen until the day of using miBrains or iBBB for assays. Note that for each cryovial for other assays described herein, a tube was prepared containing a combination of cell ty pes similar to the preparation of iBBB or miBrain cryovials (Table 2).

Table 2. Cell ratio and composition of each off-the-shelf tissue vial.

3.3 Assay assembly by the end user

Timing: 1 to 2h.

Protocol: The appropriate volume of Geltrex^1M or any other extracellular matrix is thawed overnight at 2-8 °C (1 ml per cryovial) (Table 2). As an alternative to extracellular matrix gels, hydrogels can be synthesized in-house for immediate use. Cryovials are removed from liquid nitrogen and quickly placed in a warm bath at 37 °C for thawing. Upon thaw of -90% of the volume, the cell suspension is transferred to 15 ml tubes with pre-warmed miBrain, iBBB, or miVasC media and spun down at 200 x g for 5 min at RT. Media is carefully aspirated to preserve the undisturbed cell pellet with minimum remaining liquid left. Cell pellet is placed on ice and resuspended in 1 ml of Geltrex™ (or other gel of choice), avoiding air bubbles. The cell suspension must remain on ice before seeding onto glass bottom plates. Failure to do so will result in premature Geltrex™ polymerization and inability to properly seed the tissue. Cells are seeded at the appropriate volume of assay. It is recommended that 10 pL of gel-encapsulated tissue is used for a 96-well plate or 50 pL for a 48-well MatTek plate or 24-well transwell. For example, 50 pL Geltrex™ and encapsulated cell mixture cover the entire inner well of a 48-well MatTek plate. After cultures are seeded, they are transferred into a 37 °C 95%/5% Air/CCh incubator for 30-45 minutes to allow the Geltrex™ to polymerize. After polymerization of the gel, iBBB, miVasC, or miBrain medium is added on each well, ensuring complete submersion of the culture in media. Media change should be performed every 2-3 days. Cultures should be ready for downstream assays in 1-2 weeks. Cultures can be used for experiments, and subsequently fixed, stained, and imaged.

Example 4. Applications.

The above-described off-the-shelf tissue can be used in several ways. Users interested in utilizing human brain tissue or BBB constructs to perform assays such as drug screening can utilize full miBrain or BBB constructs. Considering that users may have a specific cell ty pe they wish to manipulate (e.g., gene editing approaches, fluorescent reporters, etc.), a tissue was designed that lacks neurons, but contains glia and vascular cells (JANs). allowing the user to add their specific neuronal cell type. A miVasC was also generated to provide users with an option to add microvasculature to their co-culture, 3D tissue, and organoids, taking advantage of their ability to invade extracellular matrix to form tubular networks resembling primary' vascular structures.

Claims

CLAIMS What is claimed is:

1. A method of cry opreserving cells for modeling a human brain or a human blood-brain barrier (BBB), the method comprising: a) combining cells comprising mammalian primary, induced pluripotent stem cells (iPSC)- or embryonic stem cell-derived neurons, glia, and vascular cells at a predetermined cell ratio to obtain a cell mixture; b) resuspending the cell mixture in a freezing medium; c) transferring the resuspended cell mixture into a single container; d) pre-chilling the container at a first temperature followed by storing the container at a second temperature for a predetermined period of time, wherein the second temperature is lower than the first temperature and causes the cell mixture to freeze; and e) storing the container at a third temperature.

2. The method of claim 1, wherein the cell mixture comprises iPSC-derived cells, embryonic stem cell-derived cells or cells derived from primary cells.

3. The method of claim 1, further comprising: prior to combining the cells, differentiating iPSC- or embryonic stem cells into neurons, glia, and vascular cells.

4. The method of claim 3, further comprising differentiating the neurons, glia, and vascular cells into astrocytes, oligodendrocyte progenitor cells, brain endothelial cells, microglia, and pericytes.

5. The method of claim 1, wherein the cell mixture comprises brain endothelial cells, astrocytes, and pericytes.

6. The method of claim 1, wherein the cell mixture comprises human neurons, astrocytes, oligodendrocyte progenitor cells, endothelial cells, and pericytes.

7. The method of claim 1, wherein the cell mixture comprises brain endothelial cells and pericytes.

8. The method of claim 1, wherein the first temperature is about 4 °C.

9. The method of claim 1, wherein the second temperature is about -80 °C.

10. The method of claim 1, wherein the third temperature is from about -80 °C to about - 196 °C.

1 1. The method of claim 1, wherein the predetermined period time is from about 12 hours to about 36 hours.

12. The method of claim 11, wherein the predetermined period of time is about 24 hours.

13. The method of claim 1, wherein the predetermined ratio is about 5: 1 : 1 by cell density of brain endothelial cells, astrocytes, and pericytes.

14. The method of claim 1, wherein the cell mixture comprises about 1 x 10⁶ to about 10 x 10⁶ brain endothelial cells/mL, about 1 x 10⁶ to about 2 x 10⁶ astrocytes/mL, and about 1 x 10⁶ to about 2 x 10⁶ pericytes/mL.

15. The method of claim 14. wherein the cell mixture comprises about 5 x 10⁶ brain endothelial cells/mL, about 1 x 10⁶ astrocytes/mL, and about 1 x 10⁶ pericytes/mL.

16. The method of claim 1 , wherein the cell mixture comprises about 1 x 10⁶ to about 10 x 10⁶ neurons/mL, about 1 x 10⁶ to about 10 x 10⁶ brain endothelial cells/mL, about 0.1 x 10⁶ to about 2 x 10⁶ astrocytes/mL, about 0.1 x 10⁶ to about 2 x 10⁶ OPCs/mL, and about 0.1 x 10⁶to about 2 x 10⁶ pericytes/mL.

17. The method of claim 1, wherein the cell mixture comprises about 0.1 x 10²to about 2 x 10¹⁰ pericytes/mL and about 1 x 10² to about 2.5 x 10¹⁰ brain endothelial cells/mL.

18. The method of claim 1 , wherein the predetermined ratio is about 5 : 1 : 1 : 1 : 1 by cell density of brain endothelial cells, astrocytes, OPCs, microglia, and pericytes.

19. The method of claim 17, wherein the cell mixture comprises about 2.5 x 10⁶ neurons/mL, about 2.5 x 10⁶ brain endothelial cells/mL, about 0.5 x 10⁶ astrocytes/mL, about 0.5 x 10⁶ OPCs/mL, about 0.5 x 10⁶ microglia/mL, and about 0.5 x 10⁶ pericytes/mL.

20. The method of claim 1, wherein the step of combining comprises combining the cells in a combination medium comprising:

(a) astrocyte media and 10 ng/mL VEGF-A;

(b) 500 mL human endothelial serum-free (hECSR) media, 5 mL penicillinstreptomycin, 5 mL NEAA, 5 mL CD lipids, 5 mL astrocyte grow th supplement, 10 mL serum-free supplement for neuronal cells, 500 pl insulin, 1 pM dibutyl-cAMP. 50 pg/mL ascorbic acid, 10 ng/mL NT3, 10 ng/mL IGF, 100 ng/mL biotin, 60 ng/mL T3, 10 ng/mL VEGF, and 1 pM SAG; or

(c) 500 mL hECSR media, 5 mL penicillin-streptomycin, 5 mL NEAA, 10 mL B27 supplement, 50 ng/mL VEGF, 20 ng/mL FGF, and 10 ng/mL FGF.

21. The method of claim 20, wherein the combination medium of (c) further comprises 5 pg/ml doxycycline.

22. The method of claim 1, wherein the freezing medium comprises 60% FBS-free serum replacement, 30% hECSR, 10% DMSO, 10 pM Y27632 ROCK Inhibitor, and 50 ng/mL VEGF-A.

23. The method of claim 1, comprising resuspending the cell mixture in 1 mL of the freezing medium.

24. The method of claim 1, wherein the cell mixture comprises 3 to 6 different cell ty pes.

25. The method of claim 1, wherein the cell mixture has about 1 x 10⁶ to about 10 x 10⁶ cells/mL.

26. The method of claim 25, wherein the cell mixture has about 7 x 10⁶ cells/mL.

27. An assay comprising the cells produced by the method of claim 1 .

8. The assay of claim 27, wherein the assay is an assay based on induced blood-brain barrier (iBBB), just add neurons (JANs), a multicellular integrated human brain tissue (miBrain), or an induced microvascular cell combo (miVasC).