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Composition and method for preserving or culturing ocular cells

A composition and cell technology, applied in cell culture supports/coatings, biochemical equipment and methods, animal cells, etc., can solve the problems of insufficient donors, rejection, surgical invasion, etc., to improve work efficiency, cell The effect of increasing the number

Pending Publication Date: 2021-06-29
DOSHISHA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many problems to be solved in current corneal transplantation, such as surgical invasion, rejection, and shortage of donors.

Method used

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  • Composition and method for preserving or culturing ocular cells
  • Composition and method for preserving or culturing ocular cells
  • Composition and method for preserving or culturing ocular cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0282] (Example 1: Screening of frozen preservation solution)

[0283] In this example, the cryopreservation solution suitable for cryopreservation of corneal endothelial cells was screened.

[0284] (A cell survival rate)

[0285] Thaw the cryopreservation tube containing human corneal endothelial cells in a water bath at 37°C for 1-2 minutes. Human corneal endothelial cells were recovered with basal medium preheated to 37°C. Subsequently, the human corneal endothelial cells were centrifuged at 190G for 5 minutes, the supernatant was removed, and the human corneal endothelial cells were suspended in the culture medium. Subsequently, dead cells were stained with 0.5%-Trypan Blue Stain solution (Nacalai Tesque, 29853-34) for 10 minutes. Use a hemocytometer to measure the number of viable cells and dead cells. For cells that have been frozen and preserved, divide the number of viable cells by the number of preserved cells (5 × 10 5 ), to calculate the cell survival rate, and...

Embodiment 2

[0296] (Example 2: Liquid volume research of preservation solution)

[0297] In this example, the appropriate amount of cryopreservation solution used for cryopreservation was studied.

[0298] (A cell survival rate)

[0299] The medium was removed from the culture dish for culturing human corneal endothelial cells, and 1×PBS(-) (Nissui Fen Co., Ltd., 05913) preheated to 37° C. was added for washing. This operation was repeated 2 times. Add 1×PBS(-) again, at 37°C (5% CO 2 ) for 3 minutes. After removing PBS(-), add TrypLE TM Select Enzyme (10X) (ThermoFisher Scientific, A1217701), at 37°C (5% CO 2 ) for 15 minutes. Subsequently, the cells were recovered by being suspended in a medium and centrifuged at 280G for 3 minutes. After counting the number of cells, the suspension was dispensed into 15ml centrifuge tubes and centrifuged at 280G for 3 minutes. After removing the supernatant, add 0.5ml, 1ml, 1.5ml of frozen storage reagents to reach 5×10 per 1ml respectively. 5...

Embodiment 3

[0310] (Example 3: Evaluation of cryopreservation of high-density cells)

[0311] (A cell survival rate)

[0312] In order to evaluate the feasibility of the cryopreservation protocol, as cells for clinical use, the number of passages is 3 to 5 and the cell density is 2000 cells / mm 2 The above cells were based on the quality that can be used in clinical practice, and the effect of Bambanker hRM on cell preservation was studied. The medium was removed from the culture dish for culturing human corneal endothelial cells, and 1×PBS(-) (Nissui Fen Co., Ltd., 05913) preheated to 37° C. was added for washing. This operation was repeated 2 times. Add 1×PBS(-) again, at 37°C (5% CO 2 ) for 3 minutes. After removing PBS(-), add TrypLE TM SelectEnzyme (10X) (Thermo Fisher Scientific, A1217701), at 37°C (5% CO 2 ) for 15 minutes. Subsequently, the cells were recovered by being suspended in a medium and centrifuged at 280G for 3 minutes. After counting the number of cells, the huma...

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Abstract

The present disclosure provides a composition and method for preserving ocular cells. More specifically, the present disclosure provides: a composition for preserving ocular cells, or culturing the same after preservation, the composition containing albumin and dimethyl sulfoxide; a cell preparation containing said ocular cells, albumin, and dimethyl sulfoxide; and a therapeutic / preventive method using the same. The present disclosure also provides a method for preserving corneal endothelial cells, the method including a step of suspending ocular cells in said composition to provide a suspension, and a step of freezing the suspension. In some embodiments, the ocular cells are corneal cells (for example, corneal endothelial cells).

Description

technical field [0001] The present invention relates to techniques for preservation or post-preservation culture of eye cells. Background technique [0002] Light entering from the cornea, the transparent tissue at the front of the eyeball, reaches the retina and excites nerve cells in the retina, and the electrical signals generated are transmitted to the visual cortex of the brain via the optic nerve, whereby visual information is recognized. For good vision, the cornea needs to be transparent. The transparency of the cornea is maintained by keeping the water content constant through the pump function and barrier function of the corneal endothelial cells. [0003] Human corneal endothelial cells exist at a density of about 3,000 per 1 square centimeter at birth, but their ability to regenerate is limited. If they are severely damaged, they cannot maintain their function and the cornea loses its transparency. In bullous keratopathy due to endothelial corneal dystrophy, co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02A61K9/19A61K35/30A61K47/02A61K47/20A61K47/26A61K47/42A61L27/38A61P27/02A61P43/00C12N1/04C12N5/077C07K14/76
CPCA61K35/30A61P27/02A61P43/00A61L2430/16A61L27/3808C12N5/0621C12N2500/34C12N2500/14A01N1/0226C12N2500/10C12N2523/00C12N2537/00
Inventor 小泉范子奥村直毅
Owner DOSHISHA CO LTD
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