WO2013107211A1

WO2013107211A1 - Light-induced cell desorption method and cell culture device therefor

Info

Publication number: WO2013107211A1
Application number: PCT/CN2012/084905
Authority: WO
Inventors: 程逵; 翁文剑; 洪逸
Original assignee: 浙江大学
Priority date: 2012-01-17
Filing date: 2012-11-20
Publication date: 2013-07-25
Also published as: CN102586169A

Abstract

Provided are a light-induced cell desorption method and a cell culture device therefor. The light-induced cell desorption method comprises: before in vitro cell culture is performed, preparing a photosensitive semiconductor structure layer acting as a cell culture surface on a cell contact surface of a cell culture vessel; then, performing in vitro cell culture on the cell culture surface of the cell culture vessel; after the in vitro cell culture is completed, desorbing cells growing on the cell culture surface from the cell culture vessel through irradiation of ultraviolet light or visible light. The method can maximally reduce damage to the cells during cell desorption. The cell culture device used in the method comprises the cell culture vessel, wherein a photosensitive semiconductor structure layer acting as a cell culture surface is prepared on the cell contact surface of the cell culture vessel.

Description

Photoinduced cell desorption method and cell culture apparatus therefor

The present invention belongs to the field of tissue engineering, and in particular relates to a method of photo-induced cell desorption and a cell culture apparatus used therefor. Background technique

In vitro cell culture is an indispensable technology for the research of biomedical technology and biological materials. How to reduce the adverse effects of external uncertainties on cell behavior in in vitro cell culture, enable it to adhere, proliferate, differentiate, and separate from the culture medium when needed, thereby reflecting the response to drugs or materials at the cellular level. Directly affect the development of new drugs and new medical materials.

Many of the cells involved in in vitro cell culture are adherent cells. Cell culture is usually carried out in a cell culture apparatus of polystyrene, and adherent cells are generally firmly attached to the surface of the cell culture apparatus through an extracellular matrix (ECM) and integrin. It has been found that the macroscopic hydrophilic hydrophobic properties on the surface of the substrate material also have a very important influence on the cell attachment and proliferation and differentiation. The cells tend to adhere to the surface of the microporous water and are not easily attached to the hydrophilic surface. When it comes to the characterization of some cell-related enzyme activities, it is necessary to first detach the cells from the surface. One of the existing desorption methods is to mechanically peel the cells by a blade; the other is to digest the connected cells and the extracellular matrix of the culture substrate and the integrin using a digestive enzyme represented by trypsin to make the cells Get rid of the surface of the substrate. In the former method, the mechanical separation of cells will definitely cause some damage to the cells. In the latter method, the amount and duration of trypsin need to be controlled more precisely. Otherwise, the membrane proteins may be digested at the same time, thereby damaging the cells. Moreover, since ECM itself has certain biological functions, it is also questionable whether cells completely separated from ECM can completely and reliably reflect the effects to be characterized.

In recent years, OKA O and the like have used temperature-sensitive polymers to successfully separate cells from cell culture vessels by temperature changes using their hydrophobic hydrophilic properties. [N. Matsuda, T. Shimizu, M. Yamato , T. Okano, Tissue Engineering Based on Cell Sheet Technology, Advanced Materials, 2007, 19, 3089-3099]. However, with temperature as the outside The field still takes a long time, and the required low temperature process can damage the function of the cells to a certain extent. Summary of the invention

The present invention provides a method for photocell desorption in cell culture in vitro and a cell culture apparatus therefor, which can minimize damage to cells upon cell desorption.

A method for photocell desorption in cell culture in vitro, comprising the steps of:

(1) preparing a photosensitive semiconductor structure layer as a cell culture surface on a cell contact surface of a cell culture vessel before performing in vitro cell culture, the photosensitive semiconductor being a photosensitive semiconductor having a light to hydrophobic-hydrophilic conversion property, The photosensitive semiconductor structure layer is a photosensitive semiconductor film or a photosensitive semiconductor micro/nano dot matrix, wherein the photosensitive semiconductor film has a crystal grain size of 2 to 100 nm and a thickness of 50 nm to 2000 nm, and the semiconductor nanometer in the photosensitive semiconductor micro/nano lattice The density of the dots ranges from 1.0x l 0 ^{1() to} lxl 0 ¹² /cm ² , and the dot size ranges from 10 to 500 cents;

C 2 ) After performing cell culture on the cell culture surface of the cell culture vessel and performing in vitro cell culture, cells grown on the cell culture surface are desorbed from the cell culture vessel by ultraviolet light or visible light irradiation treatment.

In a preferred embodiment, the photosensitive semiconductor is titanium oxide, zinc oxide, tin oxide, iron oxide or zirconium oxide. That is, the photosensitive semiconductor film is a titanium oxide film, a zinc oxide film, a tin oxide film, an iron oxide film or a zirconia film; the photosensitive semiconductor micro-nano lattice is a titanium oxide micro-nano lattice, a zinc oxide micro-nano lattice , tin oxide micro-nano lattice, iron oxide micro-nano lattice or zirconia micro-nano lattice.

Wherein, the process of ultraviolet light or visible light irradiation treatment is:

Ultraviolet light with a wavelength of 300~400 nm is incident from the bottom of the cell culture vessel, and irradiated for 1 to 40 minutes;

Alternatively, visible light having a wavelength of 400 to 700 nm is incident from the cell culture surface or the bottom of the cell culture vessel for 10 to 60 minutes.

A cell culture apparatus for use in photocell desorption in cell culture in vitro, comprising: a cell culture vessel, wherein a photosensitive semiconductor structure layer is prepared as a cell culture surface on a cell contact surface of the cell culture vessel The photosensitive semiconductor is a photosensitive semiconductor having a light-to-hydrophobic-hydrophilic conversion property, and the photosensitive semiconductor structural layer is a photosensitive semiconductor film or a photosensitive semiconductor micro/nano lattice, wherein the photosensitive semiconductor film has a grain size of 2 to 100 nm, thickness 50 nm to 2000 nm, density of semiconductor nanodots in the photosensitive semiconductor micro/nano lattice The range is 1. 0x 10 ^1Q 〜 lxl 0 ¹² /cm ² , and the spot size is 10 nm 〜 5 (X) nm; in a preferred technical solution, the photosensitive semiconductor is titanium oxide, zinc oxide, tin oxide, iron oxide. Or zirconia:: the photosensitive semiconductor film is an oxidized film, a zinc oxide film, a tin oxide film, an iron oxide film or a zirconia film; the photosensitive semiconductor micro-nano lattice is a titanium oxide micro-nano lattice, zinc oxide Micro sodium lattice, oxygen tin micro sodium lattice, iron oxide micro sodium lattice or zirconia micro sodium lattice.

In the present invention:

The cells are adherent cells cultured in a simulated physiological environment in vitro.

The cell culture vessel is a commercially available cell culture vessel, which is commercially available, commonly made of silicate glass, quartz, polystyrene, polylactic acid, polyglycolic acid or polyacrylate.

The photosensitive semiconductor thinning can be prepared by a conventional sol-gel method, chemical vapor deposition or physical vapor deposition.

The photosensitive semiconductor micro-sodium lattice can be prepared by a prior art phase separation self-assembly method, liquid template deposition, vapor phase template deposition, thin film photolithography, hydrothermal method or printing.

In the present invention, a photosensitive semiconductor material which can undergo a transition of hydrophobicity to hydrophilic surface under light irradiation is selected by utilizing characteristics in which cells are liable to adhere to a relatively hydrophobic surface and are not easily attached to a hydrophilic surface, and in cell culture. The cells of the vessel: contact the surface of the photo-sensitized semiconductor film or the micro-sodium lattice as the cell culture surface: Thus, the surface wettability of the cultured cells changes after the light is irradiated, and the cells spontaneously detach from the surface of the culture device. Achieve cell desorption.

Therefore, in the present invention, the photosensitive semiconductor film or the photosensitive semiconductor micro/nano lattice may also be replaced by other surface nanostructures of the photosensitive semiconductor, such as a sodium bar array or a nanorod film, a nanoflower array or a sodium popcorn film. The photosensitive semiconductor is preferably titanium oxide zinc oxide, tin oxide, iron oxide or zirconium oxide.

Compared with the prior art, the present invention has the following beneficial technical effects _:

The method of the invention has the advantages of high cell detachment efficiency, small damage to cells, simple operation, wide application range and the like, and has strong practicability. The cell culture apparatus of the present invention only needs to be slightly modified from the prior art cell culture vessels, has low cost, and is easy to implement and is convenient for popularization and application. DRAWINGS

Fig. 1 is an inverted micrograph of the cultured cells of Example 1 before ultraviolet light irradiation using an inverted biological microscope. Fig. 2 is an inverted micrograph of the cultured cells of Example 1 after ultraviolet light observation using an inverted biological microscope.

Fig. 3 is a graph showing the comparison of MTS activity values of the detached cells in Example 1 and the control group obtained by the MTS assay. detailed description

The invention will be described in detail below with reference to the embodiments and the drawings, but the invention is not limited thereto. Example 1

The titanium oxide micro-nano lattice was prepared by the phase separation self-assembly method on the cell contact surface of the quartz glass cell culture vessel as follows: Ethanol: Deionized water: Acetylacetone = 0.3: 1 1 (in molar ratio) And adding tetra-n-butyl titanate at 0.02 mol/L, adding poly(ethylene) alkenylpyrrolidone (K-30) to 40 g of L to form a precursor solution, which is added dropwise to the cell contact surface of the quartz glass cell culture vessel. Spin coating for 40 s at 6000 rpm, followed by heat treatment at 50 CTC for two hours gave a titanium oxide micro-nano lattice as the cell culture surface. The nanodots in the titanium oxide micro-nano lattice have a density of 1.0 x 10 1 ^Q / cm ² and a dot size of 200 nm 500

The bone cells were cultured in vitro on the cell culture surface of the above quartz glass cell culture vessel. After three days of culture, ultraviolet light having a wavelength of 350 nm was incident from the bottom of the cell culture vessel, and irradiation for 30 minutes allowed 91% of the cells to be detached from the surface. .

Fig. 1 and Fig. 2 are inverted micrographs of the cultured cells of Example 1 observed before and after ultraviolet irradiation, respectively, observed by an inverted biological microscope. Comparing Fig. 1 and Fig. 2, it can be seen that a large number of cells changed from fusiform to spheroidal after ultraviolet light irradiation, indicating that a large number of cells are about to detach.

The activity of the detached cells in the control group cultured on the standard cell culture plate of Example 1 and the same conditions was examined by MTS assay, and the MTS activity values of the detached cells in Example 1 and the control group were compared. As shown in Fig. 3, it can be seen from Fig. 3 that the activity value (OD value, optical density value, also referred to as absorbance) of the cells detached in Example 1 is significantly higher than that of the control cultured on the standard cell culture plate under the same conditions. The activity values of the cells detached from the group indicate that the photodetachment has good safety, and the detached cells still have a good activity, and the activity is even higher than that of the cells detached from the control group.

The above MTS assay is a commonly used method for detecting cell viability in the art, and the MTS reagent used is also a conventional reagent, which is commercially available from manufacturers such as Promega. The chemical names of MTS are as follows: 3-(4,5-dime&ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny l)-2H-tetrazolium, inner salt. Example 2

A zinc oxide film is prepared as a cell culture surface by chemical vapor deposition on a cell contact surface of a silicate glass cell culture vessel. The zinc oxide film has a grain size of 2 to 10 nm and a thickness of SOnrtio.

The fibroblasts were cultured in vitro on the cell culture surface of the above silicate glass cell culture vessel, and after three days of culture, ultraviolet light having a wavelength of 300 nm was incident from the bottom of the cell culture vessel, and irradiation was performed for 1 minute, so that 70% of the cells were obtained. The surface is separated. Example 3

A titanium oxide film was prepared as a cell culture surface by physical vapor deposition on the cell contact surface of the polylactic acid cell culture vessel, and the titanium oxide film had a crystal grain size of 10 to 30 nm and a thickness of 1000 Å.

The fibroblasts were cultured in vitro on the cell culture surface of the above polylactic acid cell culture vessel. After three days of culture, ultraviolet light having a wavelength of 365 nm was incident from the bottom of the cell culture vessel, and irradiation for 25 minutes allowed 74% of the cells to be detached from the surface. . Example 4

An iron oxide micro-nano lattice was prepared as a cell culture surface on the cell contact surface of the silicate glass cell culture vessel by liquid template deposition method, and the density of the semiconductor nanodots in the iron oxide micro-nano lattice was in the range of 1.0×10. ¹² / cm ² , point size range from 10 to 100.

The vascular endothelial cells were cultured in vitro on the cell culture surface of the above silicate glass cell culture vessel, and after three days of culture, visible light having a wavelength of 700 nm was incident from the bottom of the cell culture vessel, and irradiation was performed for 25 minutes to make 90% of the cells from the surface. Get rid of. Example 5

A tin oxide film was prepared as a cell culture surface by a vapor phase template deposition on a cell contact surface of a polyglycolic acid cell culture vessel, and the tin oxide film had a grain size of 20 to 80 nm and a thickness of 2000 Å. Epithelial cells were cultured in vitro on the cell culture surface of the above-mentioned polyglycolic acid cell culture vessel. After three days of culture, ultraviolet light having a wavelength of 300 nm was incident from the bottom of the cell culture vessel, and irradiation for 5 minutes allowed 80% of the cells to be detached from the surface. . Example 6

An iron oxide film was prepared as a cell culture surface on a cell contact surface of a quartz glass cell culture vessel by a sol-gel method, and the iron oxide film had a grain size of 50 to 100 nm and a thickness of ■ leg.

The fibroblasts were cultured in vitro on the cell culture surface of the above quartz glass cell culture vessel, and after three days of culture, visible light having a wavelength of 420 nm was incident from the bottom of the cell culture vessel, and irradiation was carried out for 10 minutes to detach 72% of the cells from the surface. Example 7

A titanium oxide micro-nano lattice is prepared on the cell contact surface of the silicate glass cell culture vessel as a cell culture surface by a printing method, and the density of the semiconductor nanodots in the titanium oxide micro-nano lattice is in the range of l. lx l0 ^1Q / Cm ² , the dot size ranges from 400 nm to 500 nm.

Cardiomyocytes were cultured in vitro on the cell culture surface of the above silicate glass cell culture vessel. After three days of culture, ultraviolet light having a wavelength of 365 nm was incident from the bottom of the cell culture vessel, and after irradiation for 30 minutes, 66% of the cells were detached from the surface. Example 8

A zirconia film was prepared as a cell culture surface on a cell contact surface of a polyacrylate cell culture vessel by a sol-gel method, and the zirconia film had a crystal grain size of 2 to 20 nm and a thickness of 1,500 Å.

The fibroblasts were cultured in vitro on the cell culture surface of the above polyacrylate cell culture vessel. After three days of culture, ultraviolet light having a wavelength of 365 nm was incident from the bottom of the cell culture vessel, and after irradiation for 30 minutes, 85% of the cells were detached from the surface. Example 9

A titanium oxide micro-nano lattice was prepared as a cell culture surface on a cell contact surface of a quartz glass cell culture vessel having a titanium oxide film by thin film photolithography, and half of the titanium oxide micro-nano lattice The density of the conductor nanodots ranges from 1.0x10 ^{1 ()} / cm ² , and the dot size ranges from 300 to 500 nm.

The fibroblasts were cultured in vitro on the cell culture surface of the quartz glass cell culture vessel. After three days of culture, ultraviolet light having a wavelength of 320 nm was incident from the bottom of the cell culture vessel, and irradiation for 10 minutes allowed 76% of the cells to be detached from the surface. . Example 10

A titanium oxide film was prepared by hydrothermal method on a cell contact surface of a polystyrene cell culture vessel as a cell culture surface having a crystal grain size of 20 70 nm and a thickness of 1200 nm. Cell culture in the above polystyrene cell culture vessel The surface was cultured in vitro for fibroblasts. After three days of culture, ultraviolet light having a wavelength of 300 nm was incident from the bottom of the cell culture vessel for 15 minutes, and 70% of the cells were detached from the surface. Example 11

A zinc oxide film was prepared as a cell culture surface on a cell contact surface of a silicate glass cell culture vessel by a sol-gel method, and the zinc oxide film had a grain size of 10 70 nm and a thickness of 1800.

The fibroblasts were cultured in vitro on the cell culture surface of the above silicate glass cell culture vessel, and after three days of culture, ultraviolet light having a wavelength of 300 nm was incident from the bottom of the cell culture vessel, and irradiation was performed for 1 minute, so that 70% of the cells were obtained. The surface is separated.

Claims

Claim

(1) preparing a photosensitive semiconductor structure layer as a cell culture surface on a cell contact surface of a cell culture vessel before performing in vitro cell culture, the photosensitive semiconductor being a photosensitive semiconductor having a light to hydrophobic-hydrophilic conversion property, The photosensitive semiconductor structure layer is a photosensitive semiconductor film or a photosensitive semiconductor micro/nano dot matrix, wherein the photosensitive semiconductor film has a crystal grain size of 2 to 100 nm and a thickness of 50 nm to 2000 nm, and the semiconductor nanometer in the photosensitive semiconductor micro/nano lattice The density of the dots ranges from 1.0xl 0 ^{1() to} lxl 0 ¹² /cm ² , and the dot size ranges from 10 to 500 cents;

(2) After performing cell culture on the cell culture surface of the cell culture vessel and completing in vitro cell culture, cells grown on the cell culture surface are desorbed from the cell culture vessel by ultraviolet light or visible light irradiation treatment.

The method for photocell desorption in cell culture in vitro according to claim 1, wherein the photosensitive semiconductor is titanium oxide, zinc oxide, tin oxide, iron oxide or zirconium oxide.

The method for photocell desorption in cell culture in vitro according to claim 1 or 2, wherein the ultraviolet light or visible light irradiation treatment process is - a wavelength of 300 to 400 nm Ultraviolet light is incident from the bottom of the cell culture vessel and irradiated for 1 to 40 minutes;

Alternatively, visible light having a wavelength of 400 to 700 nm is incident from the cell culture surface or the bottom of the cell culture vessel, and irradiated for 10 to 60 minutes.

4. A cell culture apparatus for use in photocell desorption in cell culture in vitro, comprising: a cell culture vessel, characterized in that a photosensitive semiconductor structural layer is prepared on a cell contact surface of the cell culture vessel. As a cell culture surface, the photosensitive semiconductor is a photosensitive semiconductor having a light-to-hydrophobic-hydrophilic conversion property, and the photosensitive semiconductor structural layer is a photosensitive semiconductor film or a photosensitive semiconductor micro/nano lattice, wherein the photosensitive semiconductor film The grain size is 2~100 nm and the thickness is 50nn! ~ 2000 nm, the density of the semiconductor nano-dots in the photosensitive semiconductor micro-nano lattice ranges from 1.0 x 10 ^{1Q to} lx 10 ¹² /cm ² , and the dot size ranges from 10 nm to 500 nm.

The cell culture apparatus according to claim 4, wherein the photosensitive semiconductor is titanium oxide, zinc oxide, tin oxide, iron oxide or zirconium oxide.