Production of extracellular matrix, conditioned media and uses thereof

• US 9,598,672 B2
• Filed: 12/24/2014
• Issued: 03/21/2017
• Est. Priority Date: 10/19/2007
• Status: Active Grant

• Current Assignee: Rutgers University
• Original Assignee: Rutgers University
• Inventors: Sun, Qian, Birge, Raymond B., Sugino, Ilene, Zarbin, Marco
• Primary Examiner(s): Schultz, Doug
• Application Number:
  US14/582,851
• Publication Number:
  US 20150118200A1
• Time in Patent Office: 818 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 35/00 Medicinal preparations containing materials or reaction products thereof with undetermined constitution
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: Y10T 428/1348 Cellular material derived from plant or animal source [e.g., wood, cotton, wool, leather, etc.]

Abstract

Provided is a matrix for promoting survival and differentiation of cells transplanted thereon, comprising a base matrix and a cell-made matrix thereon. Methods and means for making and using same are also provided. Also provided are conditioned media, related compositions, related methods, and related packaging products.

Claims

1. A kit comprising a first active fraction of conditioned media and a second active fraction of conditioned media, wherein the first active fraction and the second active fraction are prepared by a method comprising the steps of:

(a) culturing cells capable of generating extracellular matrix on a base matrix in a media for culturing said cells;

(b) harvesting an unfractionated conditioned media from said cells, wherein the source of said cells is selected from the group consisting of corneal endothelial cells and retinal pigment epithelial (RPE) cells;

(c) separating said first active fraction and said second active fraction from said unfractionated conditioned media by a first centrifugal filtration and a second centrifugal filtration, wherein said first centrifugal filtration uses a 50 kDa molecular weight cutoff filter for said first active fraction and said second centrifugal filtration uses a <

3 kDa molecular weight cutoff filter for said second active fraction, and wherein said first centrifugal filtration is applied directly to said unfractionated conditioned media and said second centrifugal filtration is applied to either one of (i) directly to said unfractionated conditioned media or (ii) to said first active fraction; and

(d) collecting said first active fraction and said second active fraction,

wherein the kit does not comprise unfractionated conditioned media.

2. The kit of claim 1, wherein the first active fraction comprises Gas6.

3. The kit of claim 1, wherein the first active fraction further comprises a pharmaceutically acceptable carrier and wherein the second active fraction further comprises a pharmaceutically acceptable carrier.

4. The kit of claim 1, wherein said kit further comprises instructions for growing said source of cells on said base matrix.

5. The kit of claim 1, wherein the source of said cells is corneal endothelial cells.

6. The kit of claim 5, wherein the corneal endothelial cells are bovine corneal endothelial cells (BCE).

7. The kit of claim 1, wherein said first active fraction undergoes an additional centrifugal filtration, wherein said additional centrifugal filtration uses a 10 kDa molecular weight cutoff filter.

8. The kit of claim 1, further comprising (e) combining said first active fraction is combined with said second active fraction.

9. The kit of claim 7, further comprising (e) combining said first active fraction is combined with said second active fraction.