Development of spinal cord on a microfluidic chip

Patent metadata

Field	Value
Publication number	US-12091650-B2
Application number	US-201916286185-A
Country	US
Kind code	B2
Filing date	Feb 26, 2019
Priority date	Aug 29, 2016
Publication date	Sep 17, 2024
Grant date	Sep 17, 2024

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

The invention claimed is:

1. A method of culturing cells, comprising:
a) providing:
i) a dual microchannel microfluidic device comprising a first microchannel and a second microchannel separated by a membrane;
ii) human ventral spinal neuron progenitor cells seeded within said first channel; and
iii) brain microvascular endothelial cells seeded within said second microchannel;

b) differentiating at least a portion of said human ventral spinal neuron progenitor cells with a flowing culture media in said first microchannel; and
c) culturing said human ventral spinal neuron progenitor cells to express at least one marker of a spinal motor neuron.

2. The method of claim 1 , wherein said at least one marker is selected from the group consisting of SMI32, nuclear marker islet 1 (ISL1), Beta 3 tubulin (TUBB3), NIOC6.1, neurofilament marker microtubule-associated protein 2 (MAP2), and synaptic marker synaptophysin (SYNP).

3. The method of claim 1 , wherein said first microchannel is fluidically coupled to a top chamber comprising an open region.

4. The method of claim 1 , wherein said ventral spinal neuron progenitor cells seeded within said first microchannel are disposed within a gel or gel-precursor.

5. The method of claim 1 , wherein said ventral spinal neuron progenitor cells are seeded on top of a gel present within a portion of said first microchannel.

6. The method of claim 1 , wherein said ventral spinal neuron progenitor cells are derived from induced pluripotent stem cells.

7. The method of claim 1 , wherein said ventral spinal neuron progenitor cells are derived from embryonic stem cells.

8. The method of claim 1 , further comprising seeding glial cells within said first microchannel.

9. The method of claim 8 , wherein said glial cells comprise one or more cell types selected from the list comprising astrocytes, oligodendrocytes, ependymal cells, Schwann cells, microglia, and satellite cells.

10. The method of claim 6 , wherein said induced pluripotent stem cells, prior to step a), are differentiated using a Wingless/Integrated (WNT) agonist and SMAD inhibitors to neural ectoderm.

11. The method of claim 10 , wherein said neural ectoderm cells, prior to step a), are exposed to retinoic acid and sonic hedgehog agonist to pattern said ventral spinal neural progenitor cells.

12. The method of claim 1 , wherein said ventral spinal neural progenitor cells were frozen, banked, and thawed before step a).

13. A method of culturing cells, comprising:
a) providing induced pluripotent human stem cells, brain microvascular endothelial cells, and a dual microchannel microfluidic device comprising a first microchannel and second microchannel separated by a membrane;
b) differentiating said induced pluripotent stem cells to neural ectoderm;
c) exposing said neural ectoderm to retinoic acid and sonic hedgehog agonist to pattern ventral spinal neural progenitor cells;
d) seeding said ventral spinal neural progenitor cells in said first microchannel on said membrane and seeding said brain microvascular endothelial cells in said second microchannel on said membrane of said microfluidic device so as to create seeded cells; and
e) culturing said ventral spinal neural progenitor cells with a flowing culture media such that at least a portion of said patterned ventral spinal neural progenitor cells and said brain microvascular endothelial cells are in direct physical contact with each other through said membrane.

14. The method of claim 13 , wherein said cultured cells express a marker selected from the group consisting of SMI32, nuclear marker islet 1 (ISL1), Beta 3 tubulin (TUBB3), NKX6.1, neurofilament marker microtubule-associated protein 2 (MAP2), and synaptic marker synaptophysin (SYNP).

15. The method of claim 13 , wherein said ventral spinal neural progenitor cells were frozen, banked and thawed before step d).

16. The method of claim 13 , wherein said first and second microchannels each comprise a surface that is parallel to said membrane, and each comprise side walls, wherein said first and second microfluidic microchannels comprise polydimethylsiloxane.

17. The method of claim 13 , further comprising seeding glial cells within said first microchannel.

18. The method of claim 17 , wherein said glial cells comprise one or more cell types selected from the list comprising astrocytes, oligodendrocytes, ependymal cells, Schwann cells, microglia, and satellite cells.

19. A method of culturing cells, comprising:
a) providing human ventral spinal neuron progenitor cells, brain microvascular endothelial cells, glial cells and a dual microchannel microfluidic device comprising a first microchannel and a second microchannel separated by a membrane;
b) seeding said human ventral spinal neuron progenitor cells and said glial cells within said first microchannel and seeding said brain microvascular endothelial cells within said second microchannel so as to create seeded cells; and
c) differentiating said seeded human ventral spinal neuron progenitor cells with a culture media flowing over said human ventral spinal neuron progenitor cells such that at least a portion of said human ventral spinal neuron progenitor cells and brain microvascular endothelial cells are in direct physical contact with each other.

20. The method of claim 19 , wherein said glial cells comprise one or more cell types selected from the list comprising astrocytes, oligodendrocytes, ependymal cells, Schwann cells, microglia, and satellite cells.

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

• C12N2506/02

  from embryonic cells · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N5/0068

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

• C12N2539/00

  Supports and/or coatings for cell culture characterised by properties · CPC title

• C12N2502/28

  Vascular endothelial cells · CPC title