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Cell co-culture system for promoting cartilage differentiation and preparation method thereof

A co-cultivation system and co-cultivation technology, applied in the field of cell co-cultivation system and its preparation, can solve problems such as no optimization yet

Inactive Publication Date: 2016-07-13
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research on the optimization of the co-culture system between meniscus cells and synovial mesenchymal stem cells to meet its application in tissue engineering meniscus

Method used

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  • Cell co-culture system for promoting cartilage differentiation and preparation method thereof
  • Cell co-culture system for promoting cartilage differentiation and preparation method thereof
  • Cell co-culture system for promoting cartilage differentiation and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Obtain synovial mesenchymal stem cells from SPF grade 5-week-old male Sprague Dawley (SD) rats, wash the cells twice with PBS solution, and place them in a 25cm 2 Plate on the petri dish, add 3ml DMEM medium (containing 10% FBS, 1% penicillin-streptomycin);

[0051] In order to further isolate synovium-derived stem cells, the cells were passaged 3 times to remove non-adherent A-type macrophage-like cells, and the rest were synovium-derived mesenchymal stem cells (SMSCs), which grew to 80% confluence , subcultured, and cell flow cytometry confirmed that the cell purity was greater than 90%.

[0052] In this study, synovial mesenchymal stem cells were used in the experiments of P2-P5 generation cells.

[0053] Obtain meniscus cells from SPF grade 5-week-old male Sprague Dawley (SD) rats, wash the cells twice with PBS solution, and place them in a 25cm 2 Plate on the culture dish, add 3ml DMEM medium (containing 10% FBS, 1% penicillin-streptomycin); when the growth reach...

Embodiment 2

[0056] Cell proliferation after SMSCs and MCs are co-cultured in flow cytometry embodiment 1

[0057] Before starting co-culture, SMSCs were labeled with CFDASE fluorescent probe, and then resuspended three times in PBS to wash away the floating color. Unlabeled MCs and fluorescently labeled SMSCs were mixed evenly with low-sugar DMEM medium in different ratios, according to 30000 cells / cm 2 Density access Petri dish. Pure MCs and fluorescently labeled pure SMSCs at the same density served as controls for MCs and SMSCs in the co-culture group. On the 3rd day of co-cultivation, the cell ratio of the respective cell cycles of MCs and SMSCs in the co-culture system was detected by flow cytometry ( figure 1 ), where G2+M+S / G1 is used to calculate the cell proliferation rate ( figure 2 ). Such as figure 2 showed that the G2+M+S / G1 ratio of MCs in the co-culture group was increased (22.43±3.58%) compared with that of the pure MCs group; the G2+M+S / G1 ratio of SMSCs in the co-...

Embodiment 3

[0059] Cell proliferation after SMSCs and MCs co-culture in assay detection embodiment 1

[0060] SMSCs and MCs were co-cultured with Alamarblue assay at different time points. According to the kit operation steps, the cells were stained and incubated at 37°C for 3 hours, and then the fluorescence intensity was compared under a fluorescence spectrophotometer. Such as image 3 The results showed that the number of cells in the co-culture group with a ratio of 1:1 was higher than that of the other groups on the 10th day and 14th day. All co-culture groups had higher cell numbers than the MCs-only group on day 5 and 10, while the cell numbers in the 1:1 or 3:1 co-culture groups had no significant difference from the SMSCs-only group. And with the extension of culture time, the number of cells in all co-culture groups and SMSCs-only group was higher than that in MCs-only group.

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Abstract

The invention relates to a cell co-culture system for promoting cartilage differentiation.The cell co-culture system is composed of synovial-derived mesenchymal stem cells and meniscus cells, the proportion of the synovial-derived mesenchymal stem cells to the meniscus cells is 1: (0.33-3), the synovial-derived mesenchymal stem cells are 2-5 -generation cells which are subjected to primary culture, and the meniscus cells are 2-5 -generation cells which are subjected to primary culture; the system can jointly form a meniscus transplantation system with demineralization cortical bone and a BMP-4-PBS solution; the invention further provides application of the cell co-culture system to meniscus transplantation of allogeneic or xenogenic animals; the cell co-culture system effectively promotes cartilage differentiation, and the success rate of meniscus transplantation surgery is raised.

Description

technical field [0001] The invention relates to a cell co-cultivation system and a preparation method thereof, in particular to a cell culture system in which two types of seed cells in a specific ratio are co-cultivated to promote mutual chondrogenic differentiation of seed cells and a preparation method thereof. Background technique [0002] The meniscus is an important structure of the knee joint, and its functions are as follows: 1. Strengthen the coordination of the knee joint; 2. Transfer load; 3. Maintain joint stability; 4. Absorb shock; 5. Lubricate the joint; 6. Reduce contact stress; 7. Prevent knee hyperextension and hyperflexion. The mechanical function of the knee joint under the load of the meniscus is the reason why the meniscus injury is the most common sports injury. If the knee joint loses the protection of the meniscus, it will lead to premature and progressive degeneration of articular cartilage, which will eventually lead to the occurrence of knee oste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077A61L27/38A61L27/50
CPCC12N5/0668A61L27/3852A61L27/3886A61L27/50A61L2430/06C12N5/0652C12N2502/13C12N2502/1388
Inventor 敖英芳谢兴胡晓青朱敬先
Owner PEKING UNIV THIRD HOSPITAL
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