Simple limbal stem cell separating and in-vitro culture kit and method

An in vitro culture and stem cell technology, applied in the direction of non-embryonic pluripotent stem cells, animal cells, vertebrate cells, etc., can solve the problems of poor proliferation ability of stem cells, loss of proliferation ability, poor reproducibility, etc., and achieve less damage to primary stem cells, Strong value-added ability and high success rate

Pending Publication Date: 2015-08-26
SHANXI MEDICAL UNIV
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  • Application Information

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Problems solved by technology

However, the current methods of isolation and in vitro culture of primary limbal stem cells are relatively complicated, with low success rate and poor reproducibility, and the obtained stem cells have poor proliferation ability or even lose their proliferation ability.

Method used

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  • Simple limbal stem cell separating and in-vitro culture kit and method

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Embodiment 1

[0019] Embodiment 1: A simple primary corneal limbal stem cell isolation and in vitro culture kit, said kit includes: tissue cleaning solution 100ml, cold digestion solution 50ml, hot digestion solution 50ml and culture solution 1000ml, its preparation method is described as follows :

[0020] 1) Tissue cleaning solution: Dissolve penicillin and streptomycin in 0.1% fluconazole injection, and make 100ml of final concentration: 100IU / ml penicillin, 100IU / ml streptomycin and 0.1% fluconazole Liquid, sterile filtered and refrigerated.

[0021] 2) Cold digestion solution: Use PBS buffer to prepare 50ml of the neutral protease Dispase II to a concentration of 1.2U / ml, refrigerate after sterile filtration.

[0022] 3) Heat digestion solution: Prepare 50ml of trypsin (Trypsin) with physiological saline to a concentration of 0.125%, refrigerate after sterile filtration.

[0023] 4) Culture medium: Add appropriate amount of fetal bovine serum (FBS) 100ml, epithelial growth factor (EG...

Embodiment 2

[0026] Embodiment 2: a kind of simple primary generation rabbit corneal limbus stem cell isolation and in vitro culture method, described method comprises the following steps:

[0027] 1) Limbal tissue extraction from rabbits: the animals were anesthetized with 3% pentobarbital sodium 40 mg / kg intraperitoneally, and fixed in lateral position. Wash the conjunctival sac with tissue cleaning solution, disinfect the eye area with povidone iodine, anesthetize the eyeball with 2% lidocaine, cut the bulbar conjunctiva and subconjunctival fascia circularly along the corneal limbus, and cut 1 mm outside the corneal limbus and corneal limbus circularly. and 2 mm of corneal epithelial tissue within the limbus (about 150 μm in depth); put the excised lamellar corneal tissue into a tube filled with 10ml of tissue cleaning solution, and store it in an ice box temporarily;

[0028] 2) Cold digestion: put the lamellar corneal tissue in 10ml of cold digestion solution, digest at 4°C and 90rpm ...

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Abstract

The invention relates to a simple limbal stem cell separating and in-vitro culturing kit and method. The simple limbal stem cell separating and in-vitro culturing kit comprises tissue washing liquid, cold digestive juice, hot digestive juice and culturing liquid. The simple limbal stem cell separating and in-vitro culturing method includes the following steps: limbus tissue extracting, digesting and in-vitro culturing. By means of the simple limbal stem cell separating and in-vitro culturing kit and method, limbal stem cell separating and in-vitro culturing are carried out, operation is simple, the stem cell yield is high, damage to obtained stem cells is small, the appreciation capacity is high, and the in-vitro culturing success rate is high, and the reproducibility is good.

Description

technical field [0001] The invention relates to the field of in vitro culture of stem cells, in particular to a simple kit and method for the isolation and in vitro culture of limbal stem cells. Background technique [0002] Corneal disease is one of the major blinding diseases at present. Corneal epithelial tissue, especially limbal tissue transplantation can obtain good postoperative results in the treatment of stubborn corneal surface diseases. However, due to the limited number of corneal donors, it cannot meet the treatment needs of patients. In addition, the allogeneic corneal epithelium is highly immunogenic, and the high incidence of immune rejection after surgery is also a problem. Using autologous limbal stem cells as seed cells, culturing and constructing tissue-engineered corneas in vitro, and then transplanting them back into the patient's eyes is an important direction for the current research and treatment of corneal diseases. Limbal stem cells are a special...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
Inventor 马蕾程潇于花
Owner SHANXI MEDICAL UNIV
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