JPWO2019195798A5 - - Google Patents
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- JPWO2019195798A5 JPWO2019195798A5 JP2020554488A JP2020554488A JPWO2019195798A5 JP WO2019195798 A5 JPWO2019195798 A5 JP WO2019195798A5 JP 2020554488 A JP2020554488 A JP 2020554488A JP 2020554488 A JP2020554488 A JP 2020554488A JP WO2019195798 A5 JPWO2019195798 A5 JP WO2019195798A5
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- 238000000034 method Methods 0.000 claims description 67
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 41
- 210000000601 blood cell Anatomy 0.000 claims description 35
- 210000002569 neuron Anatomy 0.000 claims description 35
- 230000008672 reprogramming Effects 0.000 claims description 35
- 210000001130 astrocyte Anatomy 0.000 claims description 33
- 210000004027 cell Anatomy 0.000 claims description 23
- 230000004770 neurodegeneration Effects 0.000 claims description 22
- 208000015122 neurodegenerative disease Diseases 0.000 claims description 22
- 210000000274 microglia Anatomy 0.000 claims description 20
- 239000002609 medium Substances 0.000 claims description 14
- 208000018737 Parkinson disease Diseases 0.000 claims description 12
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 11
- 210000002889 endothelial cell Anatomy 0.000 claims description 10
- 210000005167 vascular cell Anatomy 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 8
- 210000004556 brain Anatomy 0.000 claims description 6
- 210000005064 dopaminergic neuron Anatomy 0.000 claims description 6
- 210000004692 intercellular junction Anatomy 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 6
- 210000002161 motor neuron Anatomy 0.000 claims description 6
- 238000009331 sowing Methods 0.000 claims description 6
- 101150059079 EBNA1 gene Proteins 0.000 claims description 5
- 208000024827 Alzheimer disease Diseases 0.000 claims description 4
- 208000023105 Huntington disease Diseases 0.000 claims description 4
- 238000003501 co-culture Methods 0.000 claims description 4
- 239000000975 dye Substances 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 4
- 210000004925 microvascular endothelial cell Anatomy 0.000 claims description 4
- 238000010899 nucleation Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 230000035699 permeability Effects 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 238000003654 cell permeability assay Methods 0.000 claims description 2
- 238000004891 communication Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 claims description 2
- 230000002025 microglial effect Effects 0.000 claims description 2
- 230000001537 neural effect Effects 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims description 2
Description
また、本明細書に記載されているのは、多量の細胞を、１つまたは複数のパラメータを測定する１つまたは複数の試験化合物と接触させる工程と、測定された１つまたは複数のパラメータに基づいて１つまたは複数の試験化合物を選択する工程と、を含む化合物スクリーニングの方法であり、細胞は、神経変性疾患に由来する人工多能性幹細胞（ｉＰＳＣ）から分化する。他の実施形態では、分化した細胞はニューロンである。他の実施形態では、分化した細胞は血管細胞である。他の実施形態では、分化した細胞はアストロサイトである。他の実施形態では、分化した細胞はミクログリアである。他の実施形態では、１つまたは複数のパラメータは、多量の血管細胞を越えた試験化合物の透過性を含む。他の実施形態では、ｉＰＳＣは、多量の血液細胞をリプログラミング因子をコードする１つまたは複数のｏｒｉＰ／ＥＢＮＡ１ベクターと接触させる工程と、多量のリプログラミング因子を該血液細胞に送達する工程と、リプログラミング培地において該血液細胞を培養する工程と、を含む方法によって作られ、該多量の血液細胞は、神経変性疾患に罹患したヒト対象から得られ、さらに、リプログラミング因子の送達及びリプログラミング培地における培養により、血液細胞由来のｉＰＳＣが生成される。
[本発明1001]
細胞を培養する方法であって、
（ｉ）アストロサイト、脳微小血管内皮細胞（ＢＭＥＣ）、またはその両方、
（ｉｉ）ニューロン、
（ｉｉｉ）ミクログリア、
（ｉｖ）上面及び底面を含む膜を含む、マイクロ流体デバイス
を提供する工程と、
前記底面に前記ＢＭＥＣを播種して播種された内皮細胞を作製する、または前記上面に前記アストロサイトを播種して播種されたアストロサイトを作製する、または前記底面に前記ＢＭＥＣを播種して播種された内皮細胞を作製しかつ前記上面に前記アストロサイトを播種して播種されたアストロサイトを作製する工程と、
前記上面にニューロンを播種して播種されたニューロンを作製する工程と、
前記上面にミクログリアを播種して播種されたミクログリアを作製する工程と、
播種された内皮細胞、播種されたアストロサイト、播種されたニューロン、及び播種されたミクログリアのうちの1つまたは複数を、一定の流量で一定期間培養する工程と
を含む、前記方法。
[本発明1002]
前記アストロサイト、ＢＭＥＣ、ニューロン、及びミクログリアが、幹細胞または初代細胞からそれぞれ分化される、本発明1001の方法。
[本発明1003]
ニューロンの播種が、ＢＭＥＣの播種後1日以上である、本発明1003の方法。
[本発明1004]
ニューロンの播種が、ＢＭＥＣの播種後6日である、本発明1003の方法。
[本発明1005]
前記ＢＭＥＣの播種と前記アストロサイトの播種が同時に行われる、本発明1001の方法。
[本発明1006]
前記播種した内皮細胞が、静置培養において培養した同じ細胞と比較して、一定の流量で一定期間培養した後、より成熟した表現型を示す、本発明1001の方法。
[本発明1007]
一定の流量での培養培地の流れが、前記播種された内皮細胞とＢＭＥＣとの間のタイトな細胞間接合の形成を促進する、本発明1001の方法。
[本発明1008]
前記タイトな細胞間接合部を検出する工程をさらに含む、本発明1001の方法。
[本発明1009]
前記タイトな細胞間接合部がＴＥＥＲ測定によって検出される、本発明1008の方法。
[本発明1010]
パッチクランプ測定、細胞外電気生理学測定、カルシウム感受性色素もしくはタンパク質を使用したイメージング、または電圧感受性色素もしくはタンパク質を使用したイメージングのうちの少なくとも1つによって、ニューロンまたはアストロサイト活性を測定する工程をさらに含む、本発明1001の方法。
[本発明1011]
前記タイトな細胞間接合部が、細胞透過性アッセイによって検出される、本発明1010の方法。
[本発明1012]
前記膜の前記上面が上部マイクロ流体チャネルの一部を含み、前記膜の前記底面が下部マイクロ流体チャネルの一部を含む、本発明1001の方法。
[本発明1013]
前記上部マイクロ流体チャネル及び前記下部マイクロ流体チャネルが、少なくとも1つの入口ポート及び少なくとも1つの出口ポートをそれぞれ含み、前記培養培地が前記入口ポートに入り、前記出口ポートから出る、本発明1012の方法。
[本発明1014]
前記ニューロンが、神経変性疾患と診断されたヒト患者由来の人工多能性幹細胞に由来する、本発明1001の方法。
[本発明1015]
前記神経変性疾患が、筋萎縮性側索硬化症（ＡＬＳ）、パーキンソン病（ＰＤ）、ハンチントン病（ＨＤ）、またはアルツハイマー病（ＡＤ）である、本発明1014の方法。
[本発明1016]
前記ニューロンが、脊髄運動ニューロンまたはドーパミン作動性ニューロンである、本発明1001の方法。
[本発明1017]
前記脊髄運動ニューロンまたはドーパミン作動性ニューロンが、少なくとも3週間、一定の流量での前記培養培地の流れを含む条件下で培養される、本発明1016の方法。
[本発明1018]
脳微小血管内皮細胞（ＢＭＥＣ）、アストロサイト、及びニューロンを含む共培養物を含む、マイクロ流体デバイス。
[本発明1019]
前記ニューロンが、脊髄運動ニューロン及びドーパミン作動性ニューロンである、本発明1018のマイクロ流体デバイス。
[本発明1020]
前記共培養物が、ミクログリア細胞をさらに含む、本発明1018のマイクロ流体デバイス。
[本発明1021]
前記ミクログリアが、人工多能性幹細胞（ｉＰＳＣ）由来のミクログリアである、本発明1020のマイクロ流体デバイス。
[本発明1022]
前記ニューロンが、ｉＰＳＣ由来のニューロンである、本発明1018のマイクロ流体デバイス。
[本発明1023]
前記ＢＭＥＣが、ｉＰＳＣ由来のＢＭＥＣである、本発明1018のマイクロ流体デバイス。
[本発明1024]
前記アストロサイトが、ｉＰＳＣ由来のアストロサイトである、本発明1018のマイクロ流体デバイス。
[本発明1025]
前記ＢＭＥＣ、アストロサイト、及びニューロンが、マイクロ流体チップのマイクロチャネル内または膜上にある、本発明1018のマイクロ流体デバイス。
[本発明1026]
前記マイクロ流体チップが、第1及び第2の表面を有する多孔質膜によって分離された2つのマイクロチャネルを含み、前記ニューロンが前記第1の表面で培養され、前記ＢＭＥＣが前記第2の表面で培養される、本発明1025のマイクロ流体デバイス。
[本発明1027]
前記脳内皮細胞及び前記ニューロンが、流動培養培地と接触している、本発明1018のマイクロ流体デバイス。
[本発明1028]
多量の血液細胞を、リプログラミング因子をコードする1つまたは複数のベクターと接触させる工程と、
多量のリプログラミング因子を前記血液細胞に送達する工程と、
リプログラミング培地において前記血液細胞を培養する工程と
を含む方法であって、
前記多量の血液細胞が、神経変性疾患に罹患したヒト対象から得られ、さらに前記リプログラミング因子の送達及びリプログラミング培地における培養により、血液細胞由来の人工多能性幹細胞（ｉＰＳＣ）が生成される、
前記方法。
[本発明1029]
前記神経変性疾患が、パーキンソン病（ＰＤ）である、本発明1028の方法。
[本発明1030]
前記神経変性疾患が、筋萎縮性側索硬化症（ＡＬＳ）である、本発明1028の方法。
[本発明1031]
前記ｉＰＳＣが、アストロサイト、ミクログリア、及び血管細胞のうちの1つまたは複数と流動的に連通してさらに培養される、本発明1028の方法。
[本発明1032]
前記1つまたは複数のベクターが、ｏｒｉＰ／ＥＢＮＡ1ベクターである、本発明1028の方法。
[本発明1033]
前記ｉＰＳＣをニューロンに分化させる工程を含む、本発明1028の方法。
[本発明1034]
前記ｉＰＳＣを血管細胞に分化させる工程を含む、本発明1028の方法。
[本発明1035]
前記ｉＰＳＣをアストロサイトに分化させる工程を含む、本発明1028の方法。
[本発明1036]
前記ｉＰＳＣをミクログリアに分化させる工程を含む、本発明1028の方法。
[本発明1037]
多量の血液細胞をリプログラミング因子をコードする1つまたは複数のベクターと接触させる工程と、
多量のリプログラミング因子を前記血液細胞に送達する工程と、
リプログラミング培地において前記血液細胞を培養する工程と
を含む方法により作られた、多量の神経変性疾患に由来する人工多能性幹細胞（ｉＰＳＣ）であって、
前記多量の血液細胞が、神経変性疾患に罹患したヒト対象から得られ、さらに前記リプログラミング因子の送達及びリプログラミング培地における培養により、血液細胞由来のｉＰＳＣが生成される、
前記ｉＰＳＣ。
[本発明1038]
前記神経変性疾患が、パーキンソン病（ＰＤ）である、本発明1037の方法。
[本発明1039]
前記神経変性疾患が、筋萎縮性側索硬化症（ＡＬＳ）である、本発明1037の方法。
[本発明1040]
多量の細胞を1つまたは複数の試験化合物と接触させる工程と、
1つまたは複数のパラメータを測定する工程と、
前記測定された1つまたは複数のパラメータに基づいて1つまたは複数の試験化合物を選択する工程と
を含む、化合物スクリーニングの方法であって、
細胞が神経変性疾患由来の人工多能性幹細胞（ｉＰＳＣ）から分化する、
前記方法。
[本発明1041]
前記分化細胞がニューロンである、本発明1040の方法。
[本発明1042]
前記分化細胞が血管細胞である、本発明1040の方法。
[本発明1043]
前記分化細胞がアストロサイトである、本発明1040の方法。
[本発明1044]
前記分化細胞がミクログリアである、本発明1040の方法。
[本発明1045]
前記1つまたは複数のパラメータが、多量の血管細胞を通る前記試験化合物の透過性を含む、本発明1040の方法。
[本発明1046]
前記ｉＰＳＣが、
多量の血液細胞を、リプログラミング因子をコードする1つまたは複数のｏｒｉＰ／ＥＢＮＡ1ベクターと接触させる工程と、
多量のリプログラミング因子を前記血液細胞に送達する工程と、
リプログラミング培地において前記血液細胞を培養する工程と
を含む方法によって作られ、
前記多量の血液細胞が、神経変性疾患に罹患したヒト対象から得られ、さらに前記リプログラミング因子の送達及びリプログラミング培地における培養により、血液細胞由来のｉＰＳＣが生成される、
本発明1040の方法。
Also described herein are the steps of contacting a large number of cells with one or more test compounds measuring one or more parameters, and one or more measured parameters. A method of compound screening comprising selecting one or more test compounds based on the cells differentiate from induced pluripotent stem cells (iPSCs) derived from neurodegenerative diseases. In another embodiment, the differentiated cell is a neuron. In another embodiment, the differentiated cell is a vascular cell. In other embodiments, the differentiated cells are astrocytes. In other embodiments, the differentiated cells are microglia. In other embodiments, one or more parameters include permeability of the test compound across large numbers of vascular cells. In another embodiment, the iPSC comprises contacting a large number of blood cells with one or more oriP / EBNA1 vectors encoding a reprogramming factor, and delivering a large amount of the reprogramming factor to the blood cells. Produced by a method comprising culturing the blood cells in a reprogramming medium, the large amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and is further delivered with a reprogramming factor and a reprogramming medium. By culturing in, iPSC derived from blood cells is produced.
[Invention 1001]
A method of culturing cells
(I) Astrocytes, brain microvascular endothelial cells (BMECs), or both,
(Ii) Neuron,
(Iii) Microglia,
(Iv) Microfluidic device, including membranes including top and bottom surfaces
And the process of providing
The BMEC is seeded on the bottom surface to prepare seeded endothelial cells, or the astrocytes are seeded on the top surface to prepare seeded astrocytes, or the BMEC is seeded and seeded on the bottom surface. A step of producing astrocytes and seeding the astrocytes on the upper surface to prepare the seeded astrocytes.
The step of sowing neurons on the upper surface to produce the seeded neurons, and
A step of sowing microglia on the upper surface to prepare seeded microglia, and
A step of culturing one or more of seeded endothelial cells, seeded astrocytes, seeded neurons, and seeded microglia at a constant flow rate for a period of time.
The method described above.
[Invention 1002]
The method of the present invention 1001 in which the astrocytes, BMECs, neurons, and microglia are differentiated from stem cells or primary cells, respectively.
[Invention 1003]
The method of the present invention 1003, wherein the dissemination of neurons is at least 1 day after dissemination of BMEC.
[Invention 1004]
The method of the present invention 1003, wherein the dissemination of neurons is 6 days after dissemination of BMEC.
[Invention 1005]
The method of the present invention 1001 in which the seeding of BMEC and the seeding of the astrocyte are performed at the same time.
[Invention 1006]
The method of the present invention 1001 in which the seeded endothelial cells show a more mature phenotype after being cultured at a constant flow rate for a certain period of time as compared with the same cells cultured in static culture.
[Invention 1007]
The method of the present invention 1001 in which a flow of culture medium at a constant flow rate promotes the formation of tight cell-cell junctions between the seeded endothelial cells and BMEC.
[Invention 1008]
The method of the present invention 1001 further comprising the step of detecting the tight intercellular junction.
[Invention 1009]
The method of the present invention 1008, wherein the tight intercellular junction is detected by TEER measurement.
[Invention 1010]
Further comprising measuring neuronal or astrosite activity by at least one of patch clamp measurements, extracellular electrophysiological measurements, imaging with calcium-sensitive dyes or proteins, or imaging with voltage-sensitive dyes or proteins. , The method of the present invention 1001.
[Invention 1011]
The method of the present invention 1010, wherein the tight intercellular junction is detected by a cell permeability assay.
[Invention 1012]
The method of the present invention 1001 wherein the upper surface of the membrane comprises a portion of the upper microfluidic channel and the bottom surface of the membrane comprises a portion of the lower microfluidic channel.
[Invention 1013]
The method of the invention 1012, wherein the upper microfluidic channel and the lower microfluidic channel each include at least one inlet port and at least one outlet port, with the culture medium entering and exiting the inlet port.
[Invention 1014]
The method of the present invention 1001 in which the neurons are derived from induced pluripotent stem cells derived from a human patient diagnosed with a neurodegenerative disease.
[Invention 1015]
The method of the present invention 1014, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Huntington's disease (HD), or Alzheimer's disease (AD).
[Invention 1016]
The method of the present invention 1001, wherein the neuron is a spinal motor neuron or a dopaminergic neuron.
[Invention 1017]
The method of the invention 1016, wherein the spinal motor or dopaminergic neurons are cultured for at least 3 weeks under conditions comprising a flow of the culture medium at a constant flow rate.
[Invention 1018]
A microfluidic device comprising a co-culture containing brain microvascular endothelial cells (BMECs), astrocytes, and neurons.
[Invention 1019]
The microfluidic device of the present invention 1018, wherein the neurons are spinal motor neurons and dopaminergic neurons.
[Invention 1020]
The microfluidic device of the present invention 1018, wherein the co-culture further comprises microglial cells.
[Invention 1021]
The microfluidic device of the present invention 1020, wherein the microglia are microglia derived from induced pluripotent stem cells (iPSC).
[Invention 1022]
The microfluidic device of the present invention 1018, wherein the neuron is an iPSC-derived neuron.
[Invention 1023]
The microfluidic device of the present invention 1018, wherein the BMEC is a BMEC derived from iPSC.
[Invention 1024]
The microfluidic device of the present invention 1018, wherein the astrocytes are iPSC-derived astrocytes.
[Invention 1025]
The microfluidic device of the present invention 1018, wherein the BMEC, astrocytes, and neurons are in the microchannel or on the membrane of a microfluidic chip.
[Invention 1026]
The microfluidic chip contains two microchannels separated by a porous membrane with first and second surfaces, the neurons are cultured on the first surface, and the BMEC is on the second surface. The microfluidic device of the present invention 1025 to be cultured.
[Invention 1027]
The microfluidic device of the present invention 1018, wherein the brain endothelial cells and neurons are in contact with a fluid culture medium.
[Invention 1028]
The process of contacting large numbers of blood cells with one or more vectors encoding reprogramming factors, and
The step of delivering a large amount of reprogramming factor to the blood cells,
The step of culturing the blood cells in a reprogramming medium
Is a method that includes
The large amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and further delivery of the reprogramming factor and culture in a reprogramming medium produce induced pluripotent stem cells (iPSC) derived from blood cells. ,
The method.
[Invention 1029]
The method of the present invention 1028, wherein the neurodegenerative disease is Parkinson's disease (PD).
[Invention 1030]
The method of the present invention 1028, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
[Invention 1031]
The method of the invention 1028, wherein the iPSC is further cultured in fluid communication with one or more of astrocytes, microglia, and vascular cells.
[Invention 1032]
The method of the present invention 1028, wherein the one or more vectors are the oriP / EBNA1 vector.
[Invention 1033]
The method of the present invention 1028 comprising the step of differentiating the iPSC into neurons.
[Invention 1034]
The method of the present invention 1028 comprising the step of differentiating the iPSC into vascular cells.
[Invention 1035]
The method of the present invention 1028 comprising the step of differentiating the iPSC into astrocytes.
[Invention 1036]
The method of the present invention 1028 comprising the step of differentiating the iPSC into microglia.
[Invention 1037]
The process of contacting large numbers of blood cells with one or more vectors encoding reprogramming factors, and
The step of delivering a large amount of reprogramming factor to the blood cells,
The step of culturing the blood cells in a reprogramming medium
Induced pluripotent stem cells (iPSC) derived from a large amount of neurodegenerative diseases produced by a method containing
The large amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and further delivery of the reprogramming factor and culture in a reprogramming medium produce blood cell-derived iPSCs.
The iPSC.
[Invention 1038]
The method of the present invention 1037, wherein the neurodegenerative disease is Parkinson's disease (PD).
[Invention 1039]
The method of the present invention 1037, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
[Invention 1040]
The process of contacting large numbers of cells with one or more test compounds, and
The process of measuring one or more parameters, and
With the step of selecting one or more test compounds based on the measured one or more parameters
A method of compound screening, including
Cells differentiate from induced pluripotent stem cells (iPSCs) derived from neurodegenerative diseases,
The method.
[Invention 1041]
The method of the present invention 1040, wherein the differentiated cells are neurons.
[Invention 1042]
The method of the present invention 1040, wherein the differentiated cells are vascular cells.
[Invention 1043]
The method of the present invention 1040, wherein the differentiated cells are astrocytes.
[Invention 1044]
The method of the present invention 1040, wherein the differentiated cells are microglia.
[Invention 1045]
The method of the invention 1040, wherein the one or more parameters include the permeability of the test compound through a large number of vascular cells.
[Invention 1046]
The iPSC
The step of contacting a large number of blood cells with one or more oriP / EBNA1 vectors encoding reprogramming factors,
The step of delivering a large amount of reprogramming factor to the blood cells,
The step of culturing the blood cells in a reprogramming medium
Made by methods including
The large amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and further delivery of the reprogramming factor and culture in a reprogramming medium produce blood cell-derived iPSCs.
The method of the present invention 1040.
Claims (15)
（ｉ）アストロサイト、脳微小血管内皮細胞（ＢＭＥＣ）、またはその両方、
（ｉｉ）ニューロン、
（ｉｉｉ）ミクログリア、
（ｉｖ）上面及び底面を含む膜を含む、マイクロ流体デバイス
を提供する工程と、
前記底面に前記ＢＭＥＣを播種して播種された内皮細胞を作製する、または前記上面に前記アストロサイトを播種して播種されたアストロサイトを作製する、または前記底面に前記ＢＭＥＣを播種して播種された内皮細胞を作製しかつ前記上面に前記アストロサイトを播種して播種されたアストロサイトを作製する工程と、
前記上面にニューロンを播種して播種されたニューロンを作製する工程と、
前記上面にミクログリアを播種して播種されたミクログリアを作製する工程と、
播種された内皮細胞、播種されたアストロサイト、播種されたニューロン、及び播種されたミクログリアのうちの1つまたは複数を、一定の流量で一定期間培養する工程と
を含む、前記方法。 A method of culturing cells
(I) Astrocytes, brain microvascular endothelial cells (BMECs), or both,
(Ii) Neuron,
(Iii) Microglia,
(Iv) A step of providing a microfluidic device comprising a membrane including a top surface and a bottom surface.
The BMEC is seeded on the bottom surface to prepare seeded endothelial cells, or the astrocytes are seeded on the top surface to prepare seeded astrocytes, or the BMEC is seeded and seeded on the bottom surface. A step of producing astrocytes and seeding the astrocytes on the upper surface to prepare the seeded astrocytes.
The step of sowing neurons on the upper surface to produce the seeded neurons, and
A step of sowing microglia on the upper surface to prepare seeded microglia, and
The method comprising culturing one or more of seeded endothelial cells, seeded astrocytes, seeded neurons, and seeded microglia at a constant flow rate for a period of time.
ニューロンの播種が、ＢＭＥＣの播種後1日以上、任意で6日である、かつ／または
前記ＢＭＥＣの播種と前記アストロサイトの播種が同時に行われる、かつ／または
前記播種した内皮細胞が、静置培養において培養した同じ細胞と比較して、一定の流量で一定期間培養した後、より成熟した表現型を示す、かつ／または
一定の流量での培養培地の流れが、前記播種された内皮細胞とＢＭＥＣとの間のタイトな細胞間接合の形成を促進する、
請求項1に記載の方法。 The astrocytes, BMECs, neurons, and microglia are differentiated from stem cells or primary cells, respectively, and / or
Neuron dissemination is at least 1 day and optionally 6 days after BMEC dissemination and / or
The BMEC sowing and the astrocyte sowing are performed simultaneously and / or
The seeded endothelial cells exhibit a more mature phenotype and / or after being cultured at a constant flow rate for a period of time as compared to the same cells cultured in static culture.
Flow of culture medium at a constant flow rate promotes the formation of tight intercellular junctions between the seeded endothelial cells and BMEC.
The method according to claim 1.
パッチクランプ測定、細胞外電気生理学測定、カルシウム感受性色素もしくはタンパク質を使用したイメージング、もしくは電圧感受性色素もしくはタンパク質を使用したイメージングのうちの少なくとも1つによって、ニューロンもしくはアストロサイト活性を測定する工程をさらに含む、かつ／または
前記膜の前記上面が上部マイクロ流体チャネルの一部を含み、前記膜の前記底面が下部マイクロ流体チャネルの一部を含む、任意で前記上部マイクロ流体チャネル及び前記下部マイクロ流体チャネルが、少なくとも1つの入口ポート及び少なくとも1つの出口ポートをそれぞれ含み、前記培養培地が前記入口ポートに入り、前記出口ポートから出る、かつ／または
前記ニューロンが、脊髄運動ニューロンもしくはドーパミン作動性ニューロンである、任意で前記脊髄運動ニューロンもしくはドーパミン作動性ニューロンが、少なくとも3週間、一定の流量での前記培養培地の流れを含む条件下で培養される、
請求項1または2に記載の方法。 The tight intercellular junction is optionally detected by TEER measurement or cell permeability assay and / or, further comprising the step of detecting the tight intercellular junction.
It further comprises measuring neuronal or astrocyte activity by at least one of patch clamp measurements, extracellular electrophysiological measurements, imaging with calcium-sensitive dyes or proteins, or imaging with voltage-sensitive dyes or proteins. , And / or
The upper surface of the membrane comprises a portion of the upper microfluidic channel and the bottom surface of the membrane comprises a portion of the lower microfluidic channel, optionally the upper microfluidic channel and the lower microfluidic channel at least one. Each includes an inlet port and at least one outlet port, wherein the culture medium enters and exits the inlet port and / or
The neurons are spinal motor neurons or dopaminergic neurons, optionally the spinal motor neurons or dopaminergic neurons are cultured for at least 3 weeks under conditions comprising a flow of the culture medium at a constant flow rate. ,
The method according to claim 1 or 2 .
前記共培養物が、ミクログリア細胞をさらに含む、かつ／または
前記ミクログリア、前記ニューロン、前記ＢＭＥＣ、及び／もしくは前記アストロサイトが、人工多能性幹細胞（ｉＰＳＣ）由来のものである、
請求項6に記載のマイクロ流体デバイス。 The neurons are spinal motor neurons and dopaminergic neurons and / or
The co-culture further comprises microglial cells and / or
The microglia, the neurons, the BMEC, and / or the astrocytes are derived from induced pluripotent stem cells (iPSCs).
The microfluidic device of claim 6 .
前記脳内皮細胞及び前記ニューロンが、流動培養培地と接触している、
請求項6または7に記載のマイクロ流体デバイス。 The BMEC, astrocytes, and neurons are in or on the microchannel of the microfluidic chip, optionally the microfluidic chip is separated by a porous membrane with first and second surfaces. Containing channels, the neuron is cultured on the first surface, the BMEC is cultured on the second surface, and / or
The brain endothelial cells and the neurons are in contact with the fluid culture medium,
The microfluidic device of claim 6 or 7 .
多量のリプログラミング因子を前記血液細胞に送達する工程と、
リプログラミング培地において前記血液細胞を培養する工程と
を含む方法であって、
前記多量の血液細胞が、神経変性疾患に罹患したヒト対象から得られ、さらに前記リプログラミング因子の送達及びリプログラミング培地における培養により、血液細胞由来の人工多能性幹細胞（ｉＰＳＣ）が生成される、
前記方法。 The process of contacting large numbers of blood cells with one or more vectors encoding reprogramming factors, and
The step of delivering a large amount of reprogramming factor to the blood cells,
A method comprising the step of culturing the blood cells in a reprogramming medium.
The large amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and further delivery of the reprogramming factor and culture in a reprogramming medium produce induced pluripotent stem cells (iPSC) derived from blood cells. ,
The method.
前記1つまたは複数のベクターが、ｏｒｉＰ／ＥＢＮＡ1ベクターである、かつ／または
前記方法が、前記ｉＰＳＣをニューロン、血管細胞、アストロサイト、及び／もしくはミクログリアに分化させる工程をさらに含む、
請求項9または10に記載の方法。 The iPSC is in fluid communication with one or more of astrocytes, microglia, and vascular cells and is further cultured and / or .
The one or more vectors are oriP / EBNA1 vectors and / or
The method further comprises the step of differentiating the iPSC into neurons, vascular cells, astrocytes, and / or microglia.
The method of claim 9 or 10 .
多量のリプログラミング因子を前記血液細胞に送達する工程と、
リプログラミング培地において前記血液細胞を培養する工程と
を含む方法により作られた、多量の神経変性疾患に由来する人工多能性幹細胞（ｉＰＳＣ）であって、
前記多量の血液細胞が、神経変性疾患に罹患したヒト対象から得られ、さらに前記リプログラミング因子の送達及びリプログラミング培地における培養により、血液細胞由来のｉＰＳＣが生成される、
前記ｉＰＳＣ。 The process of contacting large numbers of blood cells with one or more vectors encoding reprogramming factors, and
The step of delivering a large amount of reprogramming factor to the blood cells,
An induced pluripotent stem cell (iPSC) derived from a large amount of neurodegenerative disease produced by a method including the step of culturing the blood cells in a reprogramming medium.
The large amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and further delivery of the reprogramming factor and culture in a reprogramming medium produce blood cell-derived iPSCs.
The iPSC.
1つまたは複数のパラメータを測定する工程と、
前記測定された1つまたは複数のパラメータに基づいて1つまたは複数の試験化合物を選択する工程と
を含む、化合物スクリーニングの方法であって、
細胞が神経変性疾患由来の人工多能性幹細胞（ｉＰＳＣ）から分化する、
前記方法。 The process of contacting large numbers of cells with one or more test compounds, and
The process of measuring one or more parameters, and
A method of compound screening comprising the step of selecting one or more test compounds based on the measured one or more parameters.
Cells differentiate from induced pluripotent stem cells (iPSCs) derived from neurodegenerative diseases,
The method.
前記1つまたは複数のパラメータが、多量の血管細胞を通る前記試験化合物の透過性を含む、かつ／または
前記ｉＰＳＣが、
多量の血液細胞を、リプログラミング因子をコードする1つまたは複数のｏｒｉＰ／ＥＢＮＡ1ベクターと接触させる工程と、
多量のリプログラミング因子を前記血液細胞に送達する工程と、
リプログラミング培地において前記血液細胞を培養する工程と
を含む方法によって作られ、
前記多量の血液細胞が、神経変性疾患に罹患したヒト対象から得られ、さらに前記リプログラミング因子の送達及びリプログラミング培地における培養により、血液細胞由来のｉＰＳＣが生成される、
請求項14に記載の方法。 The differentiated cells are neurons , vascular cells, astrocytes, and / or microglia and / or
The one or more parameters include the permeability of the test compound through a large number of vascular cells and / or
The iPSC
The step of contacting a large number of blood cells with one or more oriP / EBNA1 vectors encoding reprogramming factors,
The step of delivering a large amount of reprogramming factor to the blood cells,
The step of culturing the blood cells in a reprogramming medium
Made by methods including
The large amount of blood cells is obtained from a human subject suffering from a neurodegenerative disease, and further delivery of the reprogramming factor and culture in a reprogramming medium produce blood cell-derived iPSCs.
The method of claim 14 .
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