Method of cryopreservation of stem cell-derived retinal pigment epithelial cells on polymeric substrate

a technology of stem cells and epithelial cells, which is applied in the field of cryopreservation of stem cells grown on a polymeric substrate, can solve the problems of loss of vision in the center of the visual field, affecting the function of the macula, and compromitting or completely eliminating the individual's ability to perceive visual images, etc., to achieve the effect of improving the storage and long-term storage of cell-containing compositions

Inactive Publication Date: 2014-02-13
UNIV OF SOUTHERN CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]To address the need for improved long term storage of cell-containing compositions for use in cell therapy, there is provided, in some embodiments, a method of cryopreserving stem cells on a substrate comprising exposing a substrate seeded with stem cells to a temperature ramp-down phase having a desired temperature reduction rate, transferring the cell-seeded substrate to a desired intermediate temperature range for a first period of time, and maintaining the stem cells at a desired storage temperature range for a second period of time, resulting in cryopreserved stem cells on a substrate that are suitable for long term storage and later use in cell therapy.

Problems solved by technology

Numerous pathologies can compromise or entirely eliminate an individual's ability to perceive visual images, including trauma to the eye, infection, degeneration, vascular irregularities, and inflammatory problems.
The function of the macula may be adversely affected by age related macular degeneration (wet or dry), diabetic macular edema, idiopathic choroidal neovascularization, high myopia macular degeneration, or advanced retinitis pigmentosa, among other pathologies.
Age related macular degeneration typically causes a loss of vision in the center of the visual field (the macula) because of damage to the retina.
It is a major cause of visual impairment in older adults (>50 years).
In the dry form, cellular debris (drusen) accumulates between the retina and the choroid, which puts pressure on the retina, possibly leading to retinal detachment and loss of vision.
In the more severe wet form, newly formed blood vessels from the choroid infiltrate the space behind the macula, which causes death of photoreceptors and their supporting cells.
In conjunction with the loss of functional cells in the eye, the newly formed blood vessels are fragile and often leak blood and interstitial fluid, which can further damage the macula.

Method used

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  • Method of cryopreservation of stem cell-derived retinal pigment epithelial cells on polymeric substrate
  • Method of cryopreservation of stem cell-derived retinal pigment epithelial cells on polymeric substrate
  • Method of cryopreservation of stem cell-derived retinal pigment epithelial cells on polymeric substrate

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example 1

[0047]Two hours prior to disassociation from a culture vessel, the culture medium of stock H9-RPE cells was changed to fresh X-VIVO 10 medium. The differentiated H9-RPE cells were then washed with calcium-free and magnesium-free DPBS (Mediatech, Inc., VA), pH7.4, once and dissociated with 0.05% trypsin with 0.53 mM EDTA (Mediatech, Inc., VA) for 5 minutes at 37° C. The cells were then thoroughly dissociated by pumping the trypsin-EDTA solution up and down gently over the cells with a pipette (culture plate or dishes) or by transferring the cells by pipette to a culture flask. The trypsin was then neutralized with equal volume of X-VIVO 10 medium (Lozan) containing 10% FBS (Hyclone or other xeno-free neutralization reagents). The cell suspensions were filtered through a sterile 40 μm strainer to remove non-disassociated cells and the disassociated cells were pelleted with a Hirmle Z 300 centrifuge (RPI Corp., IL) and centrifuged at 1000 rpm for 5 minutes.

[0048]The cell pellets were t...

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Abstract

Disclosed herein are methods and compositions for the cryopreservation of stem cells, such as stem-cell derived retinal pigment epithelial cells, that have been seeded onto and cultured on a substrate, such as a polymeric substrate. Such cryopreserved stem cells are useful for cell therapies, such as treatment of ocular damage or disease.

Description

RELATED CASES[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 61 / 481,015, filed on Apr. 29, 2011, the contents of which is expressly incorporated in its entirety by reference herein.BACKGROUND[0002]1. Field of the Invention[0003]The present application relates generally to methods and compositions for the cryopreservation of stem cells grown on a substrate. In particular, methods and compositions for the cryopreservation of retinal pigment epithelial (RPE) cells grown on a polymeric substrate.[0004]2. Description of the Related Art[0005]The scope of human disease that involves loss of or damage to cells is vast and includes, but is not limited to, ocular disease, neurodegenerative disease, endocrine diseases, cancers, and cardiovascular disease. Cellular therapy involves the use of cells, and in some cases stem cells, to treat diseased or damaged tissues. It is rapidly coming to the forefront of technologies that are poised to treat many diseases, i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N1/02
CPCA01N1/0284A47G25/904A41D19/0068A61B42/40A61B42/50
Inventor ZHU, DANHONGHINTON, DAVIDAHUJA, ASHISHHUMAYUN, MARK
Owner UNIV OF SOUTHERN CALIFORNIA
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