Method for acquiring in-vitro cultured cell/thin cell layer by utilizing visible light irradiation

A technology of in vitro culture and in vitro cell culture, applied in the field of cell/cell thin layer, can solve the problems of cytotoxic gene and mutation, and achieve the effect of simple operation, easy implementation and good signal transmission

Active Publication Date: 2016-11-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, ultraviolet light has certain toxicity to cells and may cause gene mutation, so this method also has certain defects.

Method used

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  • Method for acquiring in-vitro cultured cell/thin cell layer by utilizing visible light irradiation
  • Method for acquiring in-vitro cultured cell/thin cell layer by utilizing visible light irradiation
  • Method for acquiring in-vitro cultured cell/thin cell layer by utilizing visible light irradiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] On the surface of the above-mentioned monocrystalline silicon substrate (p / n junction depth 0.3 μm, phosphorus diffusion), osteoblasts were cultured in vitro, and the seeding density was 3×10 4 piece / cm 2 , placed in a cell incubator with a constant temperature of 37 degrees Celsius and 5% carbon dioxide for 1 day, the wavelength was 400-800 nanometers, and the intensity was 30mW / cm 2 95% of the cells can be detached from the surface after 20 minutes of irradiation with visible light from the top of the cell culture vessel. figure 1 and figure 2 Confocal laser micrographs of cells cultured in Example 1 before and after visible light were observed by confocal laser microscopy. Compared figure 1 and figure 2 , it can be seen that a large number of cells detach after being irradiated by visible light, indicating that visible light achieves the detachment of single cells.

Embodiment 2

[0029] On the surface of the above polysilicon substrate (p / n junction depth 0.5 μm, phosphorus diffusion), osteoblasts were cultured in vitro, and the seeding density was 1×10 5 piece / cm 2 , placed in a cell incubator with a constant temperature of 37 degrees Celsius and 5% carbon dioxide and cultured for 5 days, the cells formed a membrane, with a wavelength of 400-800 nanometers and an intensity of 50mW / cm 2 Visible light is incident from the top of the cell culture vessel and irradiated for 10 minutes to detach the cell sheet from the surface. image 3 It is the photos before and after the cultured cell sheet in Example 2 observed by Nikon camera under visible light. It can be seen that the entire cell sheet is completely desorbed after being irradiated with visible light.

Embodiment 3

[0031] On the surface of the above amorphous silicon substrate (p / n junction depth 0.9 μm, phosphorus diffusion), osteoblasts were cultured in vitro at a seeding density of 5×10 5 piece / cm 2 After being cultured in a cell incubator with a constant temperature of 37 degrees Celsius and 5% carbon dioxide for 3 days, the cells form a membrane, with a wavelength of 400-800 nanometers and an intensity of 70mW / cm 2 Visible light is incident from the top of the cell culture vessel and irradiated for 8 minutes to detach the cell sheet from the surface. Figure 4 It is the migratory fluorescent image of the cultured cell sheet in Example 3 observed by a fluorescence microscope after being desorbed under visible light and then cultured for 2 days. It can be seen that the cell sheet desorbed by visible light can crawl out new cells well after re-cultivation, and maintain good cell shape and vitality, indicating that the cell sheet desorbed by visible light maintains good re-migration p...

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Abstract

The invention discloses a method for acquiring an in-vitro cultured cell / thin cell layer by utilizing visible light irradiation. The method comprises the following steps: implementing cell in-vitro cultivation on the surface of a photosensing material; performing photoelectric transformation on the material surface by applying a light field after in-vitro cultivation is completed so as to realize cell detachement and avoid injury on cell functions caused by a traditional enzymolysis method. The photosensing material is a photosensing semiconductor sheet or film thereof with p / n junctions. Important extracellular matrix proteins, such as ion channels and connexins, are preserved in the cell layer acquired by utilizing visible light, signal transfer can be performed among cells, and harmonization of functions can be kept, so that the cell utilization and transfer cell activity can be greatly improved. The experimental method is simple in flow, mild in experiment condition and low in cost, has excellent operability, and is convenient for popularization and implementation.

Description

technical field [0001] The invention belongs to the field of tissue engineering, and in particular relates to a method for obtaining in vitro cultured cells / cell thin layers by irradiating visible light. Background technique [0002] In recent years, due to the limitations of existing tissue engineering methods, cell sheet tissue engineering technology has shown great potential as a new constructable tissue-like and organoid structure in the process of tissue repair and reconstruction. The biggest advantage of the cell sheet is that it can preserve the integrity of the cell surface proteins, extracellular matrix environment and intercellular connections necessary for the tissue. At present, cell sheet technology is used to treat esophageal cancer, heart, cornea, liver, periodontal and urinary system diseases, etc., and shows bright prospects. Therefore, how to obtain a complete cell sheet in a mild and non-invasive way is a key issue for cell sheet tissue engineering. At p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/077C12N5/0775
CPCC12N5/0602C12N5/0625C12N5/0654C12N5/0656C12N5/0658C12N2539/00
Inventor 程逵王小召翁文剑
Owner ZHEJIANG UNIV
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