Stent-free periodontal ligament stem cell sphere as well as preparation method and application thereof

A periodontal ligament stem cell and scaffold-free technology, which is applied in the field of scaffold-free periodontal ligament stem cell spheres and its preparation, can solve problems such as differences in number and shape, limited cultured cell types, and difficulties in mass production, and achieve uniform size, Preparation method is reproducible, promoting interaction effect

Pending Publication Date: 2021-12-07
SHANGHAI STOMATOLOGICAL HOSPITAL FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods often have problems such as difficult mass production, differences in the size, number an

Method used

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  • Stent-free periodontal ligament stem cell sphere as well as preparation method and application thereof
  • Stent-free periodontal ligament stem cell sphere as well as preparation method and application thereof
  • Stent-free periodontal ligament stem cell sphere as well as preparation method and application thereof

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preparation example Construction

[0048] The invention provides a method for preparing scaffold-free periodontal ligament stem cell spheres, comprising the following steps:

[0049] (1) Preparation of cell spheroid culture plate: add agarose solution to the wells of a two-dimensional cell culture six-well plate, place the mold in the agarose solution in the six-well plate, and pull out the mold after the agarose solution is solidified, so that Well holes are formed in the agarose gel to obtain a cell spheroid culture plate;

[0050] (2) Rinse the wells: Rinse each well of the cell spheroid culture plate with PBS;

[0051] (3) Periodontal ligament stem cell inoculation: add the periodontal ligament stem cell suspension into the wells of the cell sphere culture plate, add culture medium into the well holes, and then put the cell sphere culture plate into the cell incubator;

[0052] (4) Cultivation of scaffold-free periodontal ligament stem cell spheres: after culturing for 1 day, replace the culture medium, an...

Embodiment 1

[0067] This embodiment provides a method for preparing scaffold-free periodontal ligament stem cell spheres, comprising the following steps:

[0068] Such as figure 1 Shown:

[0069] (1) Preparation of cell spheroid culture plate: select a cell spheroid plate mold with a specific number of holes and density as required. The main material of the cell spheroid culture plate is agarose gel, which uses its hydrophobicity to prevent the cells from adhering to the wall and make the cells grow in suspension. Prepare agarose solution, heat the solution in a microwave oven, stop heating after the liquid bubbles 3 times, and when the solution is in a liquid state, slowly put the mold along the edge of one side of the hole to the edge of the other side to avoid bubbles and wait for the agarose solution After solidification and forming, pull out the mold vertically to ensure that each hole has a uniform depth and volume; each hole contains 176 or 76 small holes (with two kinds of molds)...

Embodiment 2

[0076] This embodiment provides a method for detecting the bioactivity of scaffold-free periodontal ligament stem cell spheres.

[0077] (1) Apoptosis detection of scaffold-free periodontal ligament stem cell spheroids

[0078] Scaffold-free periodontal ligament stem cell spheres were prepared according to the above method, and cultured continuously for 8 days, and the cells were incubated overnight with serum-free medium on the day before the samples were collected. Select 3 wells of scaffold-free periodontal ligament stem cell spheres every day, wash them twice with PBS buffer, then blow out the cell spheres in the wells with PBS buffer, collect them in 15ml centrifuge tubes, and digest the stent-free teeth with EDTA-free trypsin. Perimembranous stem cell spheres (digested with 0.25% trypsin for 80 seconds, supplemented by gentle pipetting), normal culture medium to stop digestion, centrifuged at 800 rpm for 3 minutes, and discarded the supernatant. Wash twice with PBS, dis...

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Abstract

The invention relates to the technical field of cell culture, in particular to a stent-free periodontal ligament stem cell sphere as well as a preparation method and application thereof. The stent-free periodontal ligament stem cell spheres are prepared by utilizing a cell sphere culture plate three-dimensional culture technology and are applied to tissue engineering related to osteogenic differentiation. The stent-free periodontal ligament stem cell sphere disclosed by the invention has good stability and biological activity and relatively strong osteogenic differentiation efficiency, and can be used as a cell bone substitute material in the fields of periodontal tissue engineering and bone tissue engineering or used in combination with a scaffold material to improve the osteogenic efficiency. The stent-free periodontal ligament stem cell spheres cultured and collected by the method are uniform and consistent in size, and the cell activity can be maintained for a long time; and meanwhile, the preparation method disclosed by the invention is repeatable, simple, convenient and feasible, does not need to depend on large-scale equipment or devices, and is beneficial to quickly and effectively obtaining the cell sphere structure.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a scaffold-free periodontal ligament stem cell sphere and a preparation method and application thereof. Background technique [0002] At present, the traditional cell culture is mainly based on the two-dimensional culture mode represented by culture flask or culture dish. This culture mode does not have the spatial form of living tissue, and cells often lose certain tissue properties during the culture process. In the real human environment, adjacent cells and extracellular matrix communicate with each other to form a three-dimensional spatial organization and a complex biochemical signal network. This structure is of great significance to the normal physiological activities of cells. In most cases, the results of in vitro studies of two-dimensional cell culture methods are not consistent with in vivo experiments. On the one hand, it is related to the gradual loss of the or...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04C12N5/0775A61L27/38
CPCC12N11/10C12N11/04C12N5/0668A61L27/3834A61L27/3847A61L27/3895A61L2430/02
Inventor 陈静刘月华
Owner SHANGHAI STOMATOLOGICAL HOSPITAL FUDAN UNIV
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