Microfluidic Model Of The Blood Brain Barrier

Patent metadata

Field	Value
Publication number	US-2018298331-A1
Application number	US-201815955335-A
Country	US
Kind code	A1
Filing date	Apr 17, 2018
Priority date	Oct 19, 2015
Publication date	Oct 18, 2018
Grant date	—

Abstract

The invention relates to culturing brain endothelial cells, and optionally astrocytes and neurons in a fluidic device under conditions whereby the cells mimic the structure and function of the blood brain barrier. Culture of such cells in a microfluidic device, whether alone or in combination with other cells, drives maturation and/or differentiation further than existing systems.

First claim

1 . A method of culturing cells, comprising: a) providing a fluidic device comprising a membrane, said membrane comprising a top surface and a bottom surface; b) seeding endothelial cells on said bottom surface so as to created seeded endothelial cells; c) seeding neurons on said top surface to create seeded neurons and, d) exposing said seeded endothelial cells and said seeded neurons to a flow of culture media for a period of time, wherein said neurons exhibit a more mature electrophysiology after exposure to said flow as compared to the same neurons cultured in a static culture.

2 . The method of claim 1 , wherein said endothelial cells are selected from the group consisting of stem cell-derived cells, cells differentiated from stem cells and primary cells.

3 . The method of claim 2 , wherein said cells differentiated from stem cells are brain microvascular endothelial cells.

4 . The method of claim 2 , wherein said cells differentiated from stem cells are iBMECs.

5 . The method of claim 1 , further comprising e) seeding astrocytes to create seeded astrocyte cells.

6 . (canceled)

7 . The method of claim 5 , further comprising culturing said astrocytes under conditions such that an astrocyte or portion thereof transmigrates said membrane and contacts one or more brain microvascular endothelial cells on said bottom surface.

8 . The method of claim 5 , wherein said neurons are derived or extracted from EZ spheres, iNPCs or iMNPs.

9 . The method of claim 2 , wherein said stem cells are human induced pluripotent stem cells.

10 . (canceled)

11 . The method of claim 1 , wherein prior to step b) at least one of said top or bottom surface are coated with one or more extracellular matrix proteins.

12 . The method of claim 11 , wherein said top surface is coated with laminin.

13 . The method of claim 11 , wherein said bottom surface is coated with a mixture of collagen and fibronectin, and lacks laminin.

14 . The method of claim 5 , wherein said cells cultured on said top surface further comprise pericytes.

15 . (canceled)

16 . The method of claim 1 , wherein said flow promotes the maturation of brain microvascular endothelial cells.

17 . The method of claim 1 , wherein said flow promotes the formation of tight cell-to-cell junctions among said brain microvascular endothelial cells.

18 . The method of claim 17 , further comprising detecting said tight cell-to-cell junctions.

19 . The method of claim 18 , wherein said tight cell-to-cell junctions are detected by TEER measurements.

20 . The method of claim 5 , further comprising step e) measuring of neuron or astrocyte activity by at least one of patch clamp measurements, extracellular electrophysiology measurements, imaging using calcium-sensitive dyes or proteins, or imaging using voltage-sensitive dyes or proteins.

21 . The method of claim 18 , wherein said tight cell-to-cell junctions are detected by cell permeability assays.

22 . The method of claim 1 , wherein said brain microvascular endothelial cells express the marker Glut 1.

23 . The method of claim 1 , wherein said culturing of step c) is performed for at least four days.

24 . The method of claim 23 , wherein said culturing of step c) is performed for at least seven days.

25 . The method of claim 1 , wherein said fluidic device further comprises at least one inlet port and at least one outlet port, and said culture media enters said inlet port and exits said outlet port.

26 . The method of claim 5 , wherein said membrane comprises a nanopatterned surface which promotes extended and directed neurite growth.

27 - 80 . (canceled)

Assignees

• Emulate Inc
• Cedars Sinai Medical Center

Inventors

• Kerns S Jordan
• Wen Norman
• Lucchesi Carolina
• Hinojosa Christopher David
• Fraser Jacob
• Hamilton Geraldine
• Vatine Gad
• Sances Samuel
• Svendsen Clive
• Levner Daniel
• Sareen Dhruv

Classifications

• C12N2502/088

  neural stem cells · CPC title

• C12N5/0623

  Stem cells · CPC title

• C12N5/0068

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title

• B01L2300/123

  Flexible; Elastomeric · CPC title

• B01L2300/0858

  Side walls · CPC title