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Limbal stem cells and preparation method thereof

A corneal limbal stem cell and cornea technology, applied in the field of tissue engineering, can solve problems such as structural disorder, poor cell stability, and inability to maintain stem cell activity well, and achieve the effect of large nuclear-to-cytoplasmic ratio and slow renewal cycle

Active Publication Date: 2019-08-02
广州市亮目生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing cell preservation solution has simple nutrients, no fixed ratio, and a single preservation method, which leads to low cell survival rate of stem cells, poor cell stability, and cannot maintain the activity of stem cells well
For corneal tissue that is not used for a short period of time, it is often stored in corneal preservation solution at 2-2°C for 2 weeks. When the storage time exceeds 2 weeks, the method used is the organ culture preservation method. and cryopreservation method, both methods require special preservation solution and preservation equipment, because with the increase of preservation time, the number of corneal apoptotic cells is increasing, and the structure is gradually disordered, so the cornea is seldom subjected to long-term preservation in practical applications. save
[0007] The above-mentioned cultivation, screening and preservation methods of limbal stem cells have a common feature in that the culture, screening and preservation of limbal stem cells must be performed after the cells are obtained from fresh corneal tissue. All steps may cause the loss of limbal stem cells, and it is difficult to successfully obtain a large number of highly active limbal stem cells

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  • Limbal stem cells and preparation method thereof
  • Limbal stem cells and preparation method thereof
  • Limbal stem cells and preparation method thereof

Examples

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preparation example Construction

[0066] One embodiment of the present invention provides a method for preparing limbal stem cells, comprising the following steps:

[0067] Obtain corneal tissue and pretreat the corneal tissue to be used;

[0068] Adding a cell cryopreservation solution containing a cell inducer to the pretreated corneal tissue;

[0069] Freezing corneal tissue with cell freezing solution;

[0070] The cell-inducing agent is a reagent that can induce local inflammation or stress response of cells in the corneal tissue, but does not directly cause cell death.

[0071] In the above method, the cells are screened and preserved by adding a cryopreservation solution containing a cell-inducing agent to the corneal tissue, and the added cell-inducing agent can induce local inflammation or apoptosis of the cells in the corneal tissue, and the low-temperature Freezing conditions can induce apoptosis or necrosis of adult cells in corneal tissue, while maintaining and preserving the activity of limbal ...

Embodiment 1

[0088] Example 1: Preparation of Rabbit Limbal Stem Cells

[0089] (1) Obtain corneal tissue and pretreat the corneal tissue to be used:

[0090] The eyeballs removed from New Zealand rabbits were washed 3 times with phosphate buffered saline (PBS) added with 1% penicillin-streptomycin solution, and the excess tissue around the outside of the eyeballs was removed with tissue scissors. 5mm of sclera and cut off the rest, remove the contents at the same time, scrape off the iris and other parts, and then wash 3 times with PBS.

[0091] (2) Add cell cryopreservation solution containing cell inducer to the pretreated corneal tissue:

[0092] First prepare the cell culture medium KCM for culturing limbal stem cells in vitro: high-glucose DMEM+10% fetal bovine serum+1% glutamine+1% non-essential amino acids+1% penicillin-streptomycin solution+10ng / ml epidermal growth factor (EGF)+5μg / ml transferrin+5μg / ml insulin, and add 4μg / ml prednisolone acetate and 20ng / ml staurosporine (STS)...

Embodiment 2

[0101] Example 2: Preparation of human limbal stem cells

[0102] (1) Obtain corneal tissue and pretreat the corneal tissue to be used:

[0103] The human eyes were washed with Kolabito eye drops for 5 to 6 times, and then washed with Ⅲ Aner Iodine and normal saline with gentamicin respectively, and the eyeballs were removed with tissue scissors to remove the excess tissue around the outside of the eyeballs. Keep 1-5mm of the sclera along the limbus and cut off the rest, remove the contents, scrape off the iris and other parts, and then clean the cornea with phosphate buffered saline (PBS) with 1% penicillin-streptomycin solution Wash 3 times.

[0104] (2) Add cell cryopreservation solution containing cell inducer to the pretreated corneal tissue:

[0105] First prepare the cell culture medium KCM for culturing limbal stem cells in vitro: high-glucose DMEM+10% fetal bovine serum+1% glutamine+1% non-essential amino acids+1% penicillin-streptomycin solution+10ng / ml epidermal g...

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Abstract

The invention relates to limbal stem cells and a preparation method thereof. The preparation method comprises the following steps: obtaining corneal tissues, and pre-treating the to-be-used corneal tissues; adding a cell cryopreservation solution containing a cell inducer into the pre-treated corneal tissues; freezing the corneal tissues with the cell cryopreservation solution, wherein the cell inducer is a reagent which can induce inflammation or stress response of part of cells in the corneal tissues, but does not cause cell death directly. The method can achieve screening of the limbal stemcells and various kinds of maturely differentiated corneal cells in the corneal tissues through simple low-temperature refrigeration operation before cell culture, can achieve long-term cryopreservation of the high-activity limbal stem cells at the same time, does not need specific instruments or equipment, is simple to operate, high in screening efficiency and high in purity of screened cells, and can improve the ratio and activity of the limbal stem cells.

Description

technical field [0001] The invention relates to the field of tissue engineering, in particular to a method for preparing corneal limbal stem cells. Background technique [0002] The corneal limbus is the transition zone between the cornea and the sclera. Since the transparent cornea is embedded in the opaque sclera and gradually transitions to the sclera, there is no clear dividing line on the surface of the eyeball and histology. The corneal limbus is a natural storage location for adult stem cells responsible for corneal epithelial regeneration and repair. The corneal limbal stem cells are arranged in a palisade at the base of the epithelial layer in the corneal limbal tissue. Cell source, limbal stem cell transplantation is considered to be one of the most promising treatments for ocular surface diseases. [0003] Limbal stem cells play an important role in the reconstruction of the corneal ocular surface. However, because limbal stem cells are easy to differentiate in v...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/079
CPCC12N5/0621C12N5/0623C12N2501/25C12N2501/998
Inventor 武征王颖薇徐李玲何程智李国正
Owner 广州市亮目生物科技有限公司
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