Preparation, induced differentiation and application of self-assembled mesenchymal stem cell aggregate

A stem cell and self-assembly technology, applied in the field of stem cells, can solve the problems of unsatisfactory transplantation and differentiation of three-dimensional constructs, and achieve the effects of not easy apoptosis and necrosis, uniform differentiation degree, and increased phenotype maintenance ability

Inactive Publication Date: 2015-10-07
广东佰鸿干细胞再生医学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the unsatisfactory technical problems of mesenchymal stem cells wrapping scaffold materials for three-dimensional culture, three-dimensional construct transplantation and chondrocyte differentiation, the present invention provides a method for preparing self-assembled mesenchymal stem cell clusters and inducing mesenchymal stem cells Method for differentiation of stem cell aggregates into chondrocytes

Method used

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  • Preparation, induced differentiation and application of self-assembled mesenchymal stem cell aggregate
  • Preparation, induced differentiation and application of self-assembled mesenchymal stem cell aggregate
  • Preparation, induced differentiation and application of self-assembled mesenchymal stem cell aggregate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, inducing bone marrow mesenchymal stem cells to differentiate into chondrocytes

[0036] Step 1. Coating of multi-well cell culture plate

[0037] Weigh 2 g of agarose powder, place it in a conical flask, add 100 ml of sterile deionized water to it, and stir thoroughly to form an agarose solution with a concentration of 2% (W / V). Place the flask in a microwave oven and heat it to boiling. Take it out and let it stand under sterile conditions. When it is cooled to 45°C, add 50 μl of agarose gel into each well of a multi-well culture plate with a pore size of 6.35 mm and a depth of 10.03 mm. Gel, let it stand until the agarose solidifies and use it for later use.

[0038] Step 2. Isolation and cultivation of bone marrow mesenchymal stem cells

[0039] New Zealand white rabbits aged 4 to 5 months were anesthetized with 1% pentobarbital sodium at a rate of 3.5ml / Kg by ear vein injection, and the skin was prepared on the femur of the rabbits. A 1 cm incision was...

Embodiment 2

[0048] Example 2, inducing the differentiation of adipose-derived mesenchymal stem cells into chondrocytes

[0049] Step 1. Coating of multi-well cell culture plate

[0050] Take 5.4 ml of sterile deionized water, 2.0 ml of acrylamide-bisacrylamide mixture with a concentration of 30% (W / V), 2.5 ml of 1.5M Tris solution with pH 8.8, 10% (W / V) ammonium persulfate 0.1ml, TEMED 8μl and mix thoroughly; after making 10ml of acrylamide solution with a concentration of 6% (W / V), quickly add 60μl of the above solution to each well of a multi-well culture plate with a pore size of 6.35mm and a depth of 10.03mm. Let it stand until the polyacrylamide gel solidifies and use it for later use.

[0051] Step 2. Isolation and cultivation of adipose-derived mesenchymal stem cells

[0052] Obtain adipose tissue from the animal skin, remove the adherent membrane and blood stains, wash with phosphate buffered saline (PBS), disperse the adipose tissue with scissors, and place it in a sterile 50ml...

Embodiment 3

[0060] Example 3, Bone Marrow Mesenchymal Stem Cell Agglomerates Repair Rabbit Articular Cartilage Defects After Induction of Chondrogenic Cells

[0061] The resulting chondrocyte-induced bone marrow mesenchymal stem cell aggregates were collected ( image 3 A), use PBS to wash 3 times and set aside.

[0062] New Zealand white rabbits were anesthetized according to the method described in Example 1, placed in a supine position, and the skin was prepared on the right knee joint. A medial parapatellar approach was taken to expose the femoral end of the knee joint, and an electric bone drill was used to create an articular cartilage defect with a diameter of 4.5 mm and a depth of 3 mm at the trochlea. The above-mentioned bone marrow mesenchymal stem cell clumps were filled into the defect site, close to 1 / 3 of the articular surface, sealed with fibrin glue, the patella was reset, the wound was sutured in layers and disinfected.

[0063] Three months later, the experimental anim...

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Abstract

The invention discloses preparation, induced differentiation and application of a self-assembled mesenchymal stem cell aggregate. The mesenchymal stem cell aggregate is prepared through the following steps of (1), coating a porous culture plate by using aquogel under aseptic conditions; (2), carrying out amplification cultivation on mesenchymal stem cells to obtain the third generation; and (3), preparing cell suspension from the third generation of mesenchymal stem cells by using aggregate forming induced liquid, adding the cell suspension into each pore of the coated porous culture plate so that the number of cells in each pore is 1*10<5> to 1*10<6>, changing the aggregate every day to form induced liquid for one time, and putting the porous culture plate in a cell culture box to culture so as to form the mesenchymal stem cell aggregate with the diameter of 500-600mu m. According to the invention, high-density three-dimensional culture of mesenchymal stem cells can be realized without a bracket material; the uniformity of chondroblast induced mesenchymal stem cells is increased; and the mesenchymal stem cell aggregate can be directly used for repairing articular cartilage defects.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a method for preparing a self-assembled mesenchymal stem cell mass, a method for differentiating into chondrocytes, and its application in repairing articular cartilage defects. Background technique [0002] Autologous Chondrocytes Implantation (ACI) is currently the most advanced method for the treatment of large cartilage defects. It is to expand the patient's autologous chondrocytes in vitro, and then compound them with scaffold materials for three-dimensional culture. Finally, the "cells" —Material”composite transplanted to the cartilage defect site. However, chondrocytes are a small number of terminally differentiated cells, which are difficult to subculture and easily lose the cartilage phenotype. The disadvantages of small number of chondrocytes, difficult proliferation and dedifferentiation limit the clinical application of autologous chondrocyte transplan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077A61K35/28A61P19/02
Inventor 魏菁
Owner 广东佰鸿干细胞再生医学有限公司
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