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Production of extracellular matrix, conditioned media and uses thereof

a technology of conditioned media and extracellular matrix, which is applied in the direction of manufacturing tools, drug compositions, skeletal/connective tissue cells, etc., can solve the problems of affecting post-attachment events, cell death or inability of cells to differentiate, and existing therapy has significant limitations

Inactive Publication Date: 2012-08-30
RUTGERS THE STATE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]In another aspect, the invention provides a method of increasing survival and / or differentiation of target cells on a base matrix through providing a soluble formulation of the extracellular matrix or conditioned media to the apical surface of the cells to stimulate self-assembly and deposition of extracellular matrix and / or stimulation of mechanisms for cell survival and differentiation.
[0021]In yet another aspect, the invention provides a kit for improving survival and differentiation of target cells on a matrix. Generally, the kit includes at least an active fraction of the conditioned media or solubilized extracellular matrix according to any embodiments described herein. The kit may also include a base matrix. In another set of embodiments, the kit comprises a modified base matrix. Further, in any embodiments of this aspect of the invention, the target cells may be provided.

Problems solved by technology

Disease-related changes may mask extracellular matrix ligand availability to transplanted cells, impairing post-attachment events and leading, in turn, to cell death or inability of the cells to differentiate.
In addition, disease-related changes in the extracellular matrix can promote cell death, leading to the clinical situation in which cell transplantation is contemplated.
Existing therapy has significant limitations.
Antioxidants, for example, do not seem to be effective in the prevention of early AMD (i.e., drusen, retinal pigmentary changes).

Method used

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  • Production of extracellular matrix, conditioned media and uses thereof
  • Production of extracellular matrix, conditioned media and uses thereof
  • Production of extracellular matrix, conditioned media and uses thereof

Examples

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Effect test

example 1

Long-Term Survival of Fetal RPE on Aged Submacular Human Bruch's Membrane is Impaired

[0129]Fetal RPE (3164 cells / mm2) were seeded on aged human submacular Bruch's membrane debrided to expose the superficial surface of the inner collagenous layer. To create surfaces exposing the RPE basement membrane, RPE were gently wiped off the RPE / choroid / sclera explant using a wet surgical sponge. To create surfaces exposing the surface of the inner collagenous layer beneath the RPE basement membrane (i.e., superficial ICL), following RPE removal as indicated previously, a moistened surgical sponge was use to abrade the RPE basement membrane. In general, the area of RPE basement membrane debridement was created by approximately 5 wipes of the moistened sponge in each of 4 directions (rotating the explant 90 degrees after each series of 5 wipes). (V. K. Gullapalli, et al., Exp Eye Res 2005; 80(2):235-248). Cells were seeded onto the sclera / choroid explant and cultured for 21 days and evaluated fo...

example 2

Fetal RPE Resurfacing on Aged Bruch's Membrane Resurfaced with Bovine Corneal Endothelial Matrix (BCE-ECM)

[0130]BCE (3164 cells / mm2) were cultured on the inner collagenous layer of aged human submacular Bruch's membrane (65 yr. old donor) for 14 days to allow ECM deposition. Cells were culture in the same way as cells cultured for ECM deposition on culture dishes (see paragraph 0056). Following BCE removal with NH4OH to expose the newly deposited ECM and extensive washing with PBS, explants were seeded with fetal RPE (3164 cells / mm2) and cultured for 21 days. The results of these experiments are illustrated in FIG. 2.

[0131]FIG. 2A is a scanning electron micrograph (SEM), showing that fetal RPE fully resurfaced the treated explant with large, flat polymorphic cells. Cells showed varying amounts of short apical processes on their surfaces (insert). Mag. bar 50 μm; insert mag bar 10 μm.

[0132]FIGS. 2B and 2C are light micrographs (LMs). As shown in FIG. 2B, cells fully resurfaced the tr...

example 3

Resurfacing Bruch's Membrane with a Biologically Deposited Extracellular Matrix (ECM) Improves Cell Survival

[0136]Bovine corneal endothelial cells (BCE, passage-2) were seeded onto human submacular superficial ICL of Caucasian donors over 55 years old at a density of 3164 cells / mm2 and cultured for 14 days to allow ECM deposition or treated for 14 days with serum-free media only. Following BCE removal with NH4OH and extensive rinsing, fetal RPE (passage-2-5) were seeded at the same density onto the treated Bruch's membrane surface and cultured for 21 days. The fellow eye was treated similarly except no BCE were seeded.

[0137]RPE seeding density was 3164 cells / mm2 for 21-day incubations to determine long-term survival and morphology. FIG. 3 shows the cumulative data from 9 explant pairs. Counts are mean fetal RPE nuclei / mm Bruch's membrane (±SEM).

[0138]A statistically significant 230% (p=0.006) increase in cell density is seen at day-21 on treated explants compared to explants treated...

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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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority of U.S. Provisional Application No. 61 / 471,951 filed on Apr. 5, 2011 and U.S. Provisional Application No. 61 / 561,224 filed on Nov. 17, 2011. This application is a continuation-in-part of U.S. patent application Ser. No. 12 / 738,839, which is a 35 U.S.C. §371 National Phase application of International Application Serial No. PCT / US2008 / 080408, filed Oct. 19, 2008, which claims benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60 / 999,601 filed on Oct. 19, 2007. The disclosures of the aforementioned applications are incorporated herein by reference.GOVERNMENTAL SUPPORT[0002]The Research leading to the present invention was supported in part, by National Institutes of Health Grant No. NIH RO3 EY013690. Accordingly, the U.S. Government has certain rights in this invention.FIELD OF INVENTION[0003]This invention relates to the production of an extracellular matrix, conditioned media, ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): B32B1/06A61P27/02C12N5/0735C12N5/0775C12N5/071A61K35/12
CPCA61K35/00C12N5/0621Y10T428/1348C12N2533/90C12N2501/115A61P27/02A61K35/30
Inventor SUGINO, ILENEGULLAPALLI, VAMSIZARBIN, MARCO
Owner RUTGERS THE STATE UNIV
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