Method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells

A technology for the isolation and culture of skin stem cells, which is applied in the field of high-efficiency isolation and culture of adult porcine skin stem cells in vitro, can solve the problems of difficulty in isolation and culture, lack of methods for separation and culture of stem cells, and hinder the further development of research, and achieve good repeatability and easy operation. Effect

Inactive Publication Date: 2013-12-25
QINGDAO AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The outer side of adult pig skin is covered with hard hair, and the inner side is densely covered with fat and connective tissue. The number of stem cells is limited, so it is difficult to isolate and culture them in vitro. In addition, due to the lack of effective isolation and culture methods for adult skin stem cells in tissues in current research at home and abroad, impedes further development of research in this field

Method used

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  • Method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells
  • Method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells
  • Method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells

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Effect test

Embodiment 1

[0014] Embodiment 1, in vitro isolation and culture of adult porcine skin stem cells

[0015] In this method, skin stem cells (SDSCs) are derived from adult porcine skin.

[0016] The first step is to separate and clean the skin of adult pigs: cut off the skin on the back and belly of adult sows, cut off the hair on the outside of the skin, and use curved tweezers to scrape off the dirt in the pores, and cut off the subcutaneous fat and connective tissue as much as possible. Clean, soak in 75% alcohol for 5 minutes for sterilization, wash with phosphate buffer saline (PBS, pH7.0) three times to remove residual alcohol, cut the skin into 2-3mm 2 Small pieces were digested overnight at 4°C in 0.25% Trypsin (Hyclone) to loosen the skin;

[0017] The second step, the primary culture of adult porcine skin stem cells: the skin block was washed 3 times with DMEM / F12 (Hyclone Company), placed in 0.25% Trypsin-0.04% EDTA (Hyclone Company), digested at 37°C for 30 minutes, pipette Blo...

Embodiment 2

[0020] Embodiment 2, the alkaline phosphatase staining of adult porcine skin stem cells

[0021] After washing adult porcine skin stem cells in PBS for 3 times, fix them in 4% paraformaldehyde (Solebo) at room temperature for 15 minutes, and prepare the phosphatase staining working solution as follows:

[0022]

[0023] After fixation, wash with PBS 3 times. After the last wash, remove the washing solution and add an appropriate amount of BCIP / NBT staining working solution to ensure that the sample can be fully covered. Incubate at room temperature in the dark for 5-30 minutes or longer (up to 24 hours) until the color develops to the expected depth. Remove the BCIP / NBT staining solution and wash with distilled water 1-2 times to terminate the color reaction. For tissue sections or cell samples, after the chromogenic reaction is terminated, stain with neutral red staining solution (neutral red staining solution) to facilitate observation if necessary. image 3 Alkaline ph...

Embodiment 3

[0024] Example 3, Immunofluorescence chemical analysis of specific gene expression in adult porcine skin stem cells

[0025] Adult porcine skin stem cells that had been passaged twice were taken out, digested into single cells, washed twice with PBS, and fixed by adding 4% paraformaldehyde (Solaibo) for 15 minutes at room temperature. Or wash with PBS 3 times, 5 minutes each time. Permeabilize with PBST (PBS+0.5% Trition-100) solution, room temperature for 10 minutes. Wash with PBS (phosphate buffered saline, pH 7.0) once for 5 minutes. Add PBST (PBS+0.5% Trition-100) containing 10% goat serum or horse serum (Solabo Company), and block at room temperature for 30-60 minutes. Discard the blocking solution, add the primary antibody (1:200, abcam company) diluted in the blocking solution, and incubate overnight at 4°C or 37°C for 2 hours. Wash in PBS with 1% BSA (bovine serum albumin, Solebol Company) 3 times, 5 minutes each time. Add secondary antibody (1:200, Biyuntian Compa...

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Abstract

The invention relates to a method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells. The method comprises the following steps of cutting off adult porcine skin, removing hair, fat and dirt of the adult porcine skin, immersing the treated adult porcine skin in alcohol having the content of 75%, cleaning the adult porcine skin by PBS, cutting the adult porcine skin into small porcine skin pieces, carrying out digestion in 0.25%Trypsin at a temperature of 4 DEG C until the next day, cleaning the porcine skin pieces by a DMEM high-glucose culture solution, carrying out digestion by 0.25%Trypsin-0.04%EDTA, carrying out blowing beating, carrying out screening by a cell sieve of 400 meshes to obtain a cell suspension, transferring the cell suspension into a porcine skin-derived stem cell culture solution, carrying out culture, cleaning the adult porcine skin cells subjected to primary culture for 3-4 days by PBS, adding 0.25%Trypsin-0.04%EDTA into the culture products, carrying out rinsing and digestion, tapping a side wall of a dish so that the cells fall off, transferring the cell suspension subjected to digestion into a centrifuge tube, carrying out centrifugation, removing a supernatant, carrying out cell re-suspension, and carrying out passage. The method has simple and convenient processes and good repeatability. The porcine skin-derived stem cells obtained by the method can express beta1-integrin and Nestin skin-derived stem cell specific proteins and are alkaline phosphatase-positive cells.

Description

Technical field: [0001] The invention relates to a technical method for efficiently separating and culturing adult pig skin stem cells in vitro, and belongs to the technical field of stem cells and tissue engineering in biomedicine. Background technique: [0002] Stem cells (SC) are a kind of pluripotent cells with self-replication ability, which can differentiate into a variety of functional cells under certain conditions. Stem cells are divided into embryonic stem cells (ESC) and adult stem cells (somatic stem cells) according to their developmental stages. Embryonic stem cells are totipotent in development, but they only exist in the early stages of embryonic development, so their availability and ethical issues limit their further research and application. [0003] Although adult stem cells do not have the advantages of embryonic stem cells in terms of developmental totipotency, they are easy to obtain, have a wide range of sources, and still differentiate into various ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 孙源超王俊杰葛伟李兰沈伟
Owner QINGDAO AGRI UNIV
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