Organotypic slices of the central nervous system

• US 8,198,083 B1
• Filed: 10/31/2008
• Issued: 06/12/2012
• Est. Priority Date: 10/31/2007
• Status: Active Grant

• Current Assignee: Brent A. Dethlefs, Shengwen Li, William Gunter Loudon
• Original Assignee: Brent A. Dethlefs, Shengwen Li, William Gunter Loudon
• Inventors: Li, Shengwen, Loudon, William Gunter, Dethlefs, Brent A.
• Primary Examiner(s): Li, Q. Janice
• Application Number:
  US12/263,347
• Time in Patent Office: 1,320 Days
• Field of Search: None
• US Class Current: 435/347
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0618 Cells of the nervous system

Abstract

An organotypic slice and a method of preparing an organotypic slice from a central nervous system tissue, wherein the organotypic slice comprises a brain slice obtained from a brain wherein mature synapses have not been established and the organotypic slice is seeded with a population of stem cells; wherein the organotypic slice has enhanced viability as compared to an organotypic slice comprising a similar brain slice not seeded with a population of stem cells.

Claims

1. An organotypic slice comprising a brain slice obtained from a brain wherein mature synapses have not been established, wherein the organotypic slice is seeded with a population of stem cells and is further disposed over a polyacrylamide gel (PAA) crosslinked with extracellular matrix (ECM) attached to a culture dish via tissue culture grade glue;

wherein the organotypic slice has enhanced viability as compared to an organotypic slice comprising a similar brain slice not seeded with a population of stem cells and is disposed over PAA crosslinked with ECM attached to a culture dish via tissue culture grade glue.

2. The organotypic slice of claim 1, wherein the brain is obtained from a rodent of an age between three to nine days.

3. The organotypic slice of claim 1, wherein the enhanced viability allows the organotypic slice to live for more than about 6 months.

4. The organotypic slice of claim 1, wherein the enhanced viability allows the organotypic slice to live for more than about 12 months.

5. A method of preparing an organotypic slice from a central nervous system tissue, said method comprising:

(a) obtaining a brain from a rodent, wherein mature synapses have not been established in the brain;

(b) preparing a brain slice from the brain; and

(c) disposing the brain slice over a polyacrylamide gel (PAA) crosslinked with extracellular matrix (ECM) attached via tissue culture grade glue to a culture dish.

6. The method of claim 5, wherein the brain is obtained from a rodent of an age between three to nine days.

7. The method of claim 5, wherein the method further comprises seeding the brain slice with a population of stem cells.

8. The method of claim 5, wherein the step of obtaining a brain from a rodent comprises:

(a) euthanizing the rodent and removing the brain;

(b) placing the brain in a dish of ice-cold Hank'"'"'s Balanced Salt Solution (HBSS) on ice; and

(c) embedding the brain in a dish of 38°

C. low melting point agarose (LMP) and allowing the LMP agarose to harden.

9. The method of claim 5, wherein the step of preparing the brain slice comprises:

(a) placing the LMP agarose-embedded brain in a vibratome; and

(b) slicing sections of the brain between about 250 to 300 μ

M in thickness.

10. The method of claim 5, wherein method further comprises microdissecting the brain slice.