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Cell culture fluid, application thereof and method for inducing bone cell differentiation of skeletal muscle stem cells

A cell culture and culture medium technology, applied in the field of stem cell culture, can solve problems such as unsatisfactory induction effect, and achieve the effects of high speed, increased expression and increased quantity

Inactive Publication Date: 2017-02-22
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on the induction of osteogenic differentiation of MDSCs mainly focuses on the different effects of different cytokines on their biological behavior, and the induction effect is not ideal.

Method used

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  • Cell culture fluid, application thereof and method for inducing bone cell differentiation of skeletal muscle stem cells
  • Cell culture fluid, application thereof and method for inducing bone cell differentiation of skeletal muscle stem cells
  • Cell culture fluid, application thereof and method for inducing bone cell differentiation of skeletal muscle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Isolation and passage of MDSCs

[0067] 1. Materials:

[0068] Take adult normal temporal muscle specimens (taken from craniotomy patients), rinse with PBS buffer containing double antibodies, transfer them to sterile petri dishes, and cut them into 1mm with ophthalmic scissors 3 Fragments of about the size, and then transferred to a 50mL centrifuge tube, blown and washed with PBS buffer 3 times, left to stand for 1 minute, then discarded the supernatant and floating tissues.

[0069] 2. Enzyme hydrolysis:

[0070] Add 2 times the volume of mixed enzymes to the muscle fragments obtained above, including 2.4u / mL Dispase II, 1% type I collagenase, 2.5mmol / L CaCl2, mix well and place it in a constant temperature shaker at 37°C for about 60min. Until the muscle fragments in the test tube are digested into minced muscle, the muscle mass cannot be seen with the naked eye. After digestion, centrifuge at 1000r / min for 5min and discard the supernatant. Add 2 to 3 t...

Embodiment 2

[0078] Example 2 Differentiation of MDSCs

[0079] Preparation of platelet-rich plasma (PRP): collect peripheral blood from volunteers, transfer blood samples into 15mL centrifuge tubes in an ultra-clean bench, 10mL per tube, centrifuge at 2500rpm / min for 10min. After centrifugation, the blood sample was divided into a light yellow supernatant layer, an intermediate white blood cell layer and a bottom layer of red blood cells. Aspirate all supernatant, white cell layer and upper 1-2mm of red cell layer, transfer to new 15ml centrifuge tube, centrifuge at 2500rpm / min for 5min. After centrifugation, absorb the supernatant layer and white blood cell layer, transfer to a new 15mL centrifuge tube, centrifuge at 1200g / min for 5min. Discard the supernatant after centrifugation, and keep the bottom 2.5mL, that is, platelet-rich plasma. Transfer to a coagulation-promoting tube containing calcium chloride, overnight at 4°C, and centrifuge at 4000 rpm / min for 12 minutes after the blood...

Embodiment 2～4

[0082]

[0083] Get the 3rd generation MDSCs prepared in Example 1, press 1×10 4 Cell / mL density was inoculated in 6-well culture plates, and when the cells reached 60%-70% confluence, each test group was replaced with the osteogenic induction medium shown in Table 1. After induction of differentiation and culture for 14-21 days, the differentiation of cells in each test group was detected.

[0084] 1. Alizarin red staining

[0085] The cells in each group were stained with Alizarin Red, and the specific method was as follows:

[0086] Discard the culture medium, wash the cells with PBS 2 to 3 times; add 4% paraformaldehyde to fix for 10-15min; aspirate and discard the paraformaldehyde, wash with PBS 3 times; add 2% alizarin red, and incubate at 37°C for 30min. After the incubation, the residual solution was discarded, and washed 2 to 3 times with 37°C preheated deionized water, 20s each time; after drying, observe and collect images under an inverted microscope. The sta...

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Abstract

The invention relates to the field of stem cell cultivation and especially relates to a cell culture fluid, an application thereof and a method for inducing bone cell differentiation of skeletal muscle stem cells. The cell culture fluid provided by the invention comprises a base culture solution, a vitamin C, dexamethasone, beta-sodium glycerophosphate, IGF-1, BMP-2, PRP and FBS. The cell culture fluid provided by the invention can be used as an inducing culture fluid for bone differentiation of skeletal muscle stem cells. The quantity of bone cell differentiation of the skeletal muscle stem cells is increased, and the differentiation time is shortened. A test proves that when the cell culture fluid provided by the invention is used for inducing the skeletal muscle stem cells, the characteristics of bone cells can appear within 7d, the cellular morphology is changed within 14d, and the quantity of cells dyed by alizarin red is increased. After the cells are induced for 21 days, the expression quantity of OPN and Col-I genes in the cells is increased and is (p<0.05) obviously higher than the expression quantity of OPN and Col-I genes in comparison examples.

Description

technical field [0001] The invention relates to the field of stem cell culture, in particular to a cell culture solution and its application and a method for inducing differentiation of skeletal muscle stem cells into bone cells. Background technique [0002] Bone grafting (bone grafting) is necessary for the treatment of bone defects, nonunion fractures and spinal fusion. Traditional bone grafting materials are autologous bone, allogeneic bone and artificial bone materials. Except for a few osteoblasts in autologous cancellous bone, they can only provide filling and scaffolding functions; the body needs to gradually absorb them and replace them with bone. Slowly, there is a high incidence of nonunion, bone defect or intervertebral pseudarthrosis, which affects the effect of bone grafting. In recent years, the rapid development of tissue engineering technology provides an effective solution to solve the technical difficulties such as slow bone formation and poor new bone fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0654C12N2500/38C12N2500/84C12N2501/105C12N2501/155C12N2501/30C12N2501/999C12N2506/1323
Inventor 葛啸虎陈海佳王一飞戚康艺
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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