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Method for constructing stem cell micro-tissues

A construction method and stem cell technology, applied in the field of stem cell microtissue construction, can solve the problems of membrane thickness and layer combination limitations, and difficult to break through the thickness of cell membranes, so as to shorten the time for microtissue construction and culture in vitro, and accurately measure the size. , the effect of improving cell survival

Pending Publication Date: 2020-07-31
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the limitation of contact inhibition and other reasons in the process of cell culture in vitro, it is difficult to break through the thickness of the spontaneously formed cell membrane.
The method of magnetic control can stack multiple layers of cells in vitro, however, how to improve the overall rapid vascularization and cell survival after microtissue transplantation in vivo is still a challenge
[0004] The combined use of endothelial cells is one of the conventional methods used to improve the effect of vascularization. Yan and other scholars inoculated endothelial cells on pre-constructed mesenchymal stem cell membranes in vitro, and co-cultured to form endothelial cells with a microvascular network structure. -Composite membrane of mesenchymal stem cells, but it takes 2-3 weeks to construct and culture the membrane in vitro, and affected by the two-dimensional culture of cells in vitro, the thickness and layer combination of the membrane are limited

Method used

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  • Method for constructing stem cell micro-tissues
  • Method for constructing stem cell micro-tissues
  • Method for constructing stem cell micro-tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Optimization of time for stem cells and endothelial cells to label magnetic nanoparticles

[0049] Step 1: Co-culture of stem cells and endothelial cells with magnetic nanoparticles solution

[0050]Dental pulp stem cells (DPSC) were extracted from clinically extracted impacted teeth. Endothelial cells were extracted from human umbilical vein (human umbilical vein endothelial cells, human umbilical vein endothelial cells, HUVEC). Dental pulp stem cells and endothelial cells were co-cultured with magnetic nanoparticle solution at a concentration of 100 μg / ml for 0, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 8 hours, and observed by Prussian blue staining. Happening. like figure 1 As shown, the amount of magnetic nanoparticles phagocytized by dental pulp stem cells and endothelial cells increases with the increase of co-culture time, and rapid magnetic labeling of stem cells and endothelial cells can be achieved only by co-culture for 30 minutes to...

Embodiment 2

[0053] Example 2: Microtissue Construction of Magnetically Controlled Laminated Stem Cells and Endothelial Cells

[0054] Step 1: If Figure 4 As shown, add dental pulp stem cells labeled with magnetic nanoparticles to the culture dish at an interval of 5 to 10 minutes. The endothelial cells labeled with particles form the second layer structure, and so on, gradually alternately superimposed to form a multi-layered microtissue, continue to culture for 1 to 2 days, and transplant the microtissue into the body after it is initially stabilized.

Embodiment 3

[0055] Example 3: Construction of microtissues of stem cell layers and endothelial cell layers combined by magnetic control lamination with different thicknesses and layers

[0056] like Figure 5 As shown, the dental pulp stem cell layer and the endothelial cell layer are superimposed in sequence to construct 2 layers (2layers, a layer of dental pulp stem cells and a layer of endothelial cells), 3 layers (3layers, the middle layer is the endothelial cell layer, and the upper and lower layers). layer is dental pulp stem cell layer), 5 layers (5layers, 3 layers of dental pulp stem cell layer and 2 layers of endothelial cell layer, two kinds of cells are superimposed alternately) and 7 layers of cell layer (7layers, 4 layers of dental pulp stem cell layer and 3 layers of endothelial cell layer Cell layer, a microtissue formed by alternating superimposition of two types of cells. Microtissues have different numbers of cell layers and therefore different thicknesses. Figure 5 A...

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Abstract

The invention discloses a method for constructing stem cell micro-tissues. The method comprises the following steps: S1, co-culturing stem cells with magnetic nanoparticles and co-culturing endothelial cells with magnetic nanoparticles to mark the stem cells and the endothelial cells with magnetic nanoparticles; S2, taking a culture dish to which the magnetic field is applied, adding the stem cells marked with magnetic nanoparticles and the endothelial cells marked with magnetic nanoparticles into the culture dish by several times, and stacking the cells to the bottom of the culture dish underaction of the magnetic field to form a cell layer, wherein a same kind of cells is added every time, a cell layer is formed every time the cells are added, and at least one stem cell layer and one endothelial cell layer are laminated at the bottom of the culture dish; and S3, culturing the stem cells and the endothelial cells in a culture dish to form a microstructure with a multilayer compositestructure. According to the invention, stem cells and endothelial cells are laminated by adopting a magnetic control method to construct micro-tissues, so that the gradation and position of the cellscan be accurately controlled, the construction time is shortened, the thickness is effectively increased, and the overall rapid vascularization and cell survival after the micro-tissues are transplanted into a body are promoted.

Description

technical field [0001] The invention relates to the technical fields of tissue engineering and regenerative medicine, in particular to a method for constructing microtissues, and more specifically, to a method for constructing stem cell microtissues. Background technique [0002] Stem cell transplantation has great potential in the treatment of a variety of diseases and injuries. Pulp tissue is located in the pulp cavity inside the tooth and is the only soft tissue in the tooth tissue. Dental pulp stem cells (DPSC) are a kind of stem cells with multi-directional differentiation potential. Under in vitro techniques, they can differentiate into human tissue cells such as fat, bone, cartilage, muscle, vascular endothelium, liver, and nerves. , and then differentiate into corresponding human tissues, which can be derived from deciduous teeth or adult wisdom teeth, and have higher activity than stem cells from other sources. [0003] Cell sheet or microtissue technology can eff...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61L27/38
CPCC12N5/0664A61L27/3808A61L27/3834A61L27/3886A61L27/3895C12N2502/28C12N2513/00
Inventor 蒋欣泉张文杰陆跃智孙宁佳
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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