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NEW YORK – A self-assembling model brain neurovascular unit showed that it emulated in vivo behavior of the human blood-brain barrier under a variety of conditions, including hypoxia and histamine exposure.
Goodwell Nzou, a doctoral student at Wake Forest University, Winston-Salem, N.C., discussed findings published earlier this year in Scientific Reports showing that the three-dimensional brain organoid has promise for rapid in vitro testing of central nervous system drugs.
The model contains all the primary cell types in the human brain cortex, said Mr. Nzou, speaking at the International Conference for Parkinson’s Disease and Movement Disorders. These include human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes, and neurons. Human endothelial cells enclose the parenchymal cells in the model.
The human neurovascular unit (NVU) organoid model was developed using induced pluripotent stem cells for the microglial, oligodendrocyte, and neuron cell components. Human primary cells were used for the remaining components.
First, Mr. Nzou and his collaborators constructed a four-cell model. By placing the cells in a hanging drop culture environment and culturing for 96 hours, the investigators were able to induce assembly of the organoids. Since the cells had been pretreated with a durable labeling dye, the investigators could confirm anatomically appropriate self-assembly using confocal microscopy. Blood-brain barrier (BBB) tight junctions were confirmed by testing for the tight junction protein ZO-1 via immunofluorescent labeling, said Mr. Nzou.
From this experience, they were able to conduct a staged assembly using all six cell types, yielding a neurovascular unit that was durable, maintaining “very high cell viability for up to 21 days in vitro,” Mr. Nzou said, with both core and outer cells showing good viability.
Mr. Nzou and his colleagues at the Wake Forest Institute for Regenerative Medicine tested the model’s function against several emulated physical states: In one, they flooded the field with histamine, finding that the junctions lost integrity, accurately mimicking the “leaky” tissue state that occurs in vivo with histamine release.
The histamine-treated organoids allowed IgG permeability that was largely absent in the control organoids. “In the control system we did not see much of the IgG going in. We did see a lot more going in after we treated the organoids with histamine,” said Mr. Nzou.
However, IgG is a large molecule, and much CNS drug discovery right now is focused on small molecules, so Mr. Nzou and his colleagues also wanted to see whether the NVU’s BBB integrity would hold up against a small molecule.
Using exposure to a molecule called MPTP, Mr. Nzou and his collaborators compared how much MPTP entered two different types of organoids: One was the six-cell organoid, and the other was made up of neurons only.
The neuron-only organoid would not be expected to prevent influx of MPTP since it lacked the BBB-like composition of the full organoid, explained Mr. Nzou. Once past the BBB, MPTP is converted to an active substance that interferes with adenosine triphosphate (ATP) production . The investigators did see a significant drop in APT production with MPTP exposure in the neuron-only, but not the full, organoid, said Mr. Nzou.
In another trial, they exposed the model to an atmosphere with lowered oxygen tension and saw resultant changes consistent with ischemia. The model “showed normal physiologic responses under hypoxic conditions,” they said. These included increased proinflammatory cytokine production and decreased integrity of the BBB.
The in vitro hypoxia was profound – oxygen exposure was dropped to 1% from normal atmospheric composition of 21%. Still, the organoids maintained good viability despite the hypoxia-induced changes in physiology, making them appropriate candidates for testing such hypoxic conditions as ischemic stroke and conditions that elevate intracranial pressure, Mr. Nzou said.
In addition to drug discovery uses, the model could allow for rapid and safe toxicology research and for accelerated investigation of neurologic diseases, including Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis. The research group, said Mr. Nzou, has largely achieved its model of “forming a better blood-brain barrier–equivalent model through the concerted interactions of all cell types with the endothelial layer,” he said.
NEW YORK – A self-assembling model brain neurovascular unit showed that it emulated in vivo behavior of the human blood-brain barrier under a variety of conditions, including hypoxia and histamine exposure.
Goodwell Nzou, a doctoral student at Wake Forest University, Winston-Salem, N.C., discussed findings published earlier this year in Scientific Reports showing that the three-dimensional brain organoid has promise for rapid in vitro testing of central nervous system drugs.
The model contains all the primary cell types in the human brain cortex, said Mr. Nzou, speaking at the International Conference for Parkinson’s Disease and Movement Disorders. These include human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes, and neurons. Human endothelial cells enclose the parenchymal cells in the model.
The human neurovascular unit (NVU) organoid model was developed using induced pluripotent stem cells for the microglial, oligodendrocyte, and neuron cell components. Human primary cells were used for the remaining components.
First, Mr. Nzou and his collaborators constructed a four-cell model. By placing the cells in a hanging drop culture environment and culturing for 96 hours, the investigators were able to induce assembly of the organoids. Since the cells had been pretreated with a durable labeling dye, the investigators could confirm anatomically appropriate self-assembly using confocal microscopy. Blood-brain barrier (BBB) tight junctions were confirmed by testing for the tight junction protein ZO-1 via immunofluorescent labeling, said Mr. Nzou.
From this experience, they were able to conduct a staged assembly using all six cell types, yielding a neurovascular unit that was durable, maintaining “very high cell viability for up to 21 days in vitro,” Mr. Nzou said, with both core and outer cells showing good viability.
Mr. Nzou and his colleagues at the Wake Forest Institute for Regenerative Medicine tested the model’s function against several emulated physical states: In one, they flooded the field with histamine, finding that the junctions lost integrity, accurately mimicking the “leaky” tissue state that occurs in vivo with histamine release.
The histamine-treated organoids allowed IgG permeability that was largely absent in the control organoids. “In the control system we did not see much of the IgG going in. We did see a lot more going in after we treated the organoids with histamine,” said Mr. Nzou.
However, IgG is a large molecule, and much CNS drug discovery right now is focused on small molecules, so Mr. Nzou and his colleagues also wanted to see whether the NVU’s BBB integrity would hold up against a small molecule.
Using exposure to a molecule called MPTP, Mr. Nzou and his collaborators compared how much MPTP entered two different types of organoids: One was the six-cell organoid, and the other was made up of neurons only.
The neuron-only organoid would not be expected to prevent influx of MPTP since it lacked the BBB-like composition of the full organoid, explained Mr. Nzou. Once past the BBB, MPTP is converted to an active substance that interferes with adenosine triphosphate (ATP) production . The investigators did see a significant drop in APT production with MPTP exposure in the neuron-only, but not the full, organoid, said Mr. Nzou.
In another trial, they exposed the model to an atmosphere with lowered oxygen tension and saw resultant changes consistent with ischemia. The model “showed normal physiologic responses under hypoxic conditions,” they said. These included increased proinflammatory cytokine production and decreased integrity of the BBB.
The in vitro hypoxia was profound – oxygen exposure was dropped to 1% from normal atmospheric composition of 21%. Still, the organoids maintained good viability despite the hypoxia-induced changes in physiology, making them appropriate candidates for testing such hypoxic conditions as ischemic stroke and conditions that elevate intracranial pressure, Mr. Nzou said.
In addition to drug discovery uses, the model could allow for rapid and safe toxicology research and for accelerated investigation of neurologic diseases, including Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis. The research group, said Mr. Nzou, has largely achieved its model of “forming a better blood-brain barrier–equivalent model through the concerted interactions of all cell types with the endothelial layer,” he said.
NEW YORK – A self-assembling model brain neurovascular unit showed that it emulated in vivo behavior of the human blood-brain barrier under a variety of conditions, including hypoxia and histamine exposure.
Goodwell Nzou, a doctoral student at Wake Forest University, Winston-Salem, N.C., discussed findings published earlier this year in Scientific Reports showing that the three-dimensional brain organoid has promise for rapid in vitro testing of central nervous system drugs.
The model contains all the primary cell types in the human brain cortex, said Mr. Nzou, speaking at the International Conference for Parkinson’s Disease and Movement Disorders. These include human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes, and neurons. Human endothelial cells enclose the parenchymal cells in the model.
The human neurovascular unit (NVU) organoid model was developed using induced pluripotent stem cells for the microglial, oligodendrocyte, and neuron cell components. Human primary cells were used for the remaining components.
First, Mr. Nzou and his collaborators constructed a four-cell model. By placing the cells in a hanging drop culture environment and culturing for 96 hours, the investigators were able to induce assembly of the organoids. Since the cells had been pretreated with a durable labeling dye, the investigators could confirm anatomically appropriate self-assembly using confocal microscopy. Blood-brain barrier (BBB) tight junctions were confirmed by testing for the tight junction protein ZO-1 via immunofluorescent labeling, said Mr. Nzou.
From this experience, they were able to conduct a staged assembly using all six cell types, yielding a neurovascular unit that was durable, maintaining “very high cell viability for up to 21 days in vitro,” Mr. Nzou said, with both core and outer cells showing good viability.
Mr. Nzou and his colleagues at the Wake Forest Institute for Regenerative Medicine tested the model’s function against several emulated physical states: In one, they flooded the field with histamine, finding that the junctions lost integrity, accurately mimicking the “leaky” tissue state that occurs in vivo with histamine release.
The histamine-treated organoids allowed IgG permeability that was largely absent in the control organoids. “In the control system we did not see much of the IgG going in. We did see a lot more going in after we treated the organoids with histamine,” said Mr. Nzou.
However, IgG is a large molecule, and much CNS drug discovery right now is focused on small molecules, so Mr. Nzou and his colleagues also wanted to see whether the NVU’s BBB integrity would hold up against a small molecule.
Using exposure to a molecule called MPTP, Mr. Nzou and his collaborators compared how much MPTP entered two different types of organoids: One was the six-cell organoid, and the other was made up of neurons only.
The neuron-only organoid would not be expected to prevent influx of MPTP since it lacked the BBB-like composition of the full organoid, explained Mr. Nzou. Once past the BBB, MPTP is converted to an active substance that interferes with adenosine triphosphate (ATP) production . The investigators did see a significant drop in APT production with MPTP exposure in the neuron-only, but not the full, organoid, said Mr. Nzou.
In another trial, they exposed the model to an atmosphere with lowered oxygen tension and saw resultant changes consistent with ischemia. The model “showed normal physiologic responses under hypoxic conditions,” they said. These included increased proinflammatory cytokine production and decreased integrity of the BBB.
The in vitro hypoxia was profound – oxygen exposure was dropped to 1% from normal atmospheric composition of 21%. Still, the organoids maintained good viability despite the hypoxia-induced changes in physiology, making them appropriate candidates for testing such hypoxic conditions as ischemic stroke and conditions that elevate intracranial pressure, Mr. Nzou said.
In addition to drug discovery uses, the model could allow for rapid and safe toxicology research and for accelerated investigation of neurologic diseases, including Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis. The research group, said Mr. Nzou, has largely achieved its model of “forming a better blood-brain barrier–equivalent model through the concerted interactions of all cell types with the endothelial layer,” he said.
REPORTING FROM ICPDMD 2018