Culture medium, application thereof and method for inducing differentiation of tendon stem cells to adipocytes

Abstract

The invention relates to the technical field of stem cell culture, in particular to a culture medium, application thereof and a method for inducing differentiation of tendon stem cells into adipocytes. TGF-beta1, EGF and G-CSF are added to a basal medium so as to obtain an induction culture liquid for differentiation of the tendon stem cells into adipocytes, the number of the tendon stem cells which are differentiated into adipocytes is increased, and the time of differentiation is shortened. It is shown through experiments that the tendon stem cells which are induced by the culture liquid canexhibit the characteristics of adipocytes within 21 days, the cell morphology is changed, and the number of Oil red O stained cells is increased; and after 28 days of induction, the cell morphology has gradual change and a polygonal shape, calcified nodules formed through mineral deposition are contained in intercellular substances, and the staining characteristic of oil red O is positive.

Description

technical field

[0001] The invention relates to the technical field of stem cell culture, in particular to a culture medium and its application and a method for inducing tendon stem cells to differentiate into fat cells. Background technique

[0002] Tendinopathy is common in people who exercise heavily, and is characterized clinically by localized pain, swelling, and dysfunction. The common disease sites are the rotator cuff, supraspinatus muscle, patellar tendon, and Achilles tendon. At present, the pathogenesis of tendinopathy has not been fully studied, and the treatment methods for tendinopathy are limited and the curative effect is not clear, which is related to the lack of in-depth understanding of the pathological mechanism of the disease.

[0003] In 2007, American scholars isolated a kind of cells with self-renewal ability from human and rabbit tendons, and named them tendon stem cells or progenitor cells (tendon-derived stem cells, TDSCs). In 2010, Hong Kong scho...

Examples

Embodiment 1

[0034] Human Achilles tendon tissue was collected under sterile conditions, rinsed with PBS three times, part of the connective tissue on the surface was cut off, and the Achilles tendon tissue was separated. The Achilles tendon was cut into pieces of 1mm×1mm×1mm, digested with 3mg / mL type Ⅰ collagenase at 37°C for 2h, and filtered through a 70μm filter to form a single cell suspension. Centrifuge the single cell suspension at 300×g for 5 min, discard the supernatant, and resuspend the cell pellet in complete medium (L-DMEM medium containing 10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin) , at 500 pieces / cm 2 The cell density was seeded into a 10cm-diameter petri dish, cultured for 10 days, digested with 0.25% trypsin, mixed, and labeled as primary cells. Subculture after the cells cover 90% of the bottom of the bottle, discard the culture medium, wash twice with sterile PBS, digest with 0.25% trypsin for 3 minutes, add complete medium to stop digestion; centrifuge the ...

Embodiment 2

[0038] Adipogenic induction: Take the 3rd passage cells and use 5×10 4 The cells / well density were inoculated in 6-well plates, and the experiments were divided into the following groups:

[0039] The blank control group continued to be cultured with L-DMEM containing 10% FBS.

[0040] The positive control group was cultured in the culture medium of L-DMEM containing 10% FBS, 500nmol / L dexamethasone, 500 μmol / L isobutylmethylxanthine, 50 μmol / L indomethacin, and 10 μg / mL insulin;

technical field

[0001] The invention relates to the technical field of stem cell culture, in particular to a culture medium and its application and a method for inducing tendon stem cells to differentiate into fat cells. Background technique

[0002] Tendinopathy is common in people who exercise heavily, and is characterized clinically by localized pain, swelling, and dysfunction. The common disease sites are the rotator cuff, supraspinatus muscle, patellar tendon, and Achilles tendon. At present, the pathogenesis of tendinopathy has not been fully studied, and the treatment methods for tendinopathy are limited and the curative effect is not clear, which is related to the lack of in-depth understanding of the pathological mechanism of the disease.

[0003] In 2007, American scholars isolated a kind of cells with self-renewal ability from human and rabbit tendons, and named them tendon stem cells or progenitor cells (tendon-derived stem cells, TDSCs). In 2010, Hong Kong scho...

Examples

Embodiment 1

[0034] Human Achilles tendon tissue was collected under sterile conditions, rinsed with PBS three times, part of the connective tissue on the surface was cut off, and the Achilles tendon tissue was separated. The Achilles tendon was cut into pieces of 1mm×1mm×1mm, digested with 3mg / mL type Ⅰ collagenase at 37°C for 2h, and filtered through a 70μm filter to form a single cell suspension. Centrifuge the single cell suspension at 300×g for 5 min, discard the supernatant, and resuspend the cell pellet in complete medium (L-DMEM medium containing 10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin) , at 500 pieces / cm 2 The cell density was seeded into a 10cm-diameter petri dish, cultured for 10 days, digested with 0.25% trypsin, mixed, and labeled as primary cells. Subculture after the cells cover 90% of the bottom of the bottle, discard the culture medium, wash twice with sterile PBS, digest with 0.25% trypsin for 3 minutes, add complete medium to stop digestion; centrifuge the ...

Embodiment 2

[0038] Adipogenic induction: Take the 3rd passage cells and use 5×10 4 The cells / well density were inoculated in 6-well plates, and the experiments were divided into the following groups:

[0039] The blank control group continued to be cultured with L-DMEM containing 10% FBS.

[0040] The positive control group was cultured in the culture medium of L-DMEM containing 10% FBS, 500nmol / L dexamethasone, 500 μmol / L isobutylmethylxanthine, 50 μmol / L indomethacin, and 10 μg / mL insulin;

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