CN102586169A - Photon stimulated cell desorption method and cell culture implement used by same - Google Patents
Photon stimulated cell desorption method and cell culture implement used by same Download PDFInfo
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- CN102586169A CN102586169A CN2012100136680A CN201210013668A CN102586169A CN 102586169 A CN102586169 A CN 102586169A CN 2012100136680 A CN2012100136680 A CN 2012100136680A CN 201210013668 A CN201210013668 A CN 201210013668A CN 102586169 A CN102586169 A CN 102586169A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
Abstract
The invention discloses a photon stimulated cell desorption method and a cell culture implement used by the same. The method comprises the following steps: preparing a photosensitive semiconductor structure layer on the cell contact surface of a cell culture vessel as the cell culture surface; then performing in-vitro cell culture on the cell culture surface of the cell culture vessel; and after completing the in-vitro cell culture, performing ultraviolet light or visible light irradiation treatment to desorb cells growing on the cell culture surface from the cell culture vessel. According to the method disclosed by the invention, injuries to the cells can be reduced to the greatest extent during cell desorption. The cell culture implement used in the method comprises the cell culture vessel, and the photosensitive semiconductor structure layer is prepared on the cell contact surface of the cell culture vessel as the cell culture surface. The implement can be realized by performing very small improvement on the basis of the prior art and has the advantages of low cost and easiness in popularization and application.
Description
Technical field
The invention belongs to field of tissue engineering technology, be specifically related to a kind of method and employed cell cultures utensil thereof of photic cell desorption.
Background technology
It is the requisite technology of research of present biological medicine technology, biomaterial aspect that cell in vitro is cultivated (in vitro cell culture).In cell in vitro is cultivated, how to reduce the behavior of extraneous uncertain factor pair cell disadvantageous effect, be effective and adhere to, breed, break up; And in needs, separate with substratum; Thereby embody the response to medicine or material on the cell levels, these directly influence the research and development of novel drugs and new medical material.
Cultivating in the related cell at cell in vitro, much is attached cell.Usually be employed in the cell cultures utensil of PS and carry out cell cultures, attached cell generally through extracellular matrix (ECM) with to integrate element etc. surperficial attached to the cell cultures utensil comparatively securely.Discover that can the macroscopical hydrophilic hydrophobic character of substrate material surface also pair cell adhere to and behaviors such as propagation and differentiation have very important influence.Cell tends to attached on little hydrophobic surface, and is not easy to be attached on the hydrophilic surface.When relating to, need cell is separated from the surface to some cell related enzyme activity signs.Existing desorption method is a kind of to be to peel off cell through perching knife with mechanical force; Another kind is that to adopt with the pancreatin be that the digestive ferment of representative will connect digestion such as cell and the extracellular matrix of cultivating base material and integration element, makes the cell detachment substrate surface.Before peel off cell with mechanical force in a kind of mode and be certain to pair cell and cause certain damage, the consumption of pancreatin and action time then need control comparatively accurately in then a kind of mode, otherwise maybe be simultaneously with epicyte protein digestion, thus damaging cells.And, because ECM etc. itself promptly has certain biological function, the cell that has broken away from ECM fully can reflect complete, reliably the effect that will characterize, yet have query.
In recent years, employing temperature sensitive polymers such as OKANO utilize its hydrophobic water-wet behavior temperature variant characteristic successfully to have realized that through temperature variation cell separates [N.Matsuda, T.Shimizu from the cell cultures vessel; M.Yamato, T.Okano, Tissue Engineering Based on Cell Sheet Technology; Advanced Materials; 2007,19,3089-3099].But, still need the long time with temperature as the outfield, but and required chilling process in the function of damaging cells to a certain degree.
Summary of the invention
The invention provides a kind of method and employed cell cultures utensil thereof that cell in vitro is cultivated photic cell desorption that be used for, can at utmost reduce the damage of pair cell when the cell desorption.
A kind of method that is used for the photic cell desorption of cell in vitro cultivation may further comprise the steps:
(1) before carrying out the cell in vitro cultivation; Preparation photosensitive semiconductor structural sheet is as cell culturing surfaces on the cells contacting surface of cell cultures vessel; Said photosensitive semiconductor is the photosensitive semiconductor with light to hydrophobic-hydrophilic conversion characteristic; Said photosensitive semiconductor structural sheet is photosensitive semiconductor film or photosensitive semiconductor micro-nano dot matrix, and wherein, the grain-size of said photosensitive semiconductor film is 2～100nm; Thickness is 50nm～2000nm, and the density range of the semiconductor nano point in the said photosensitive semiconductor micro-nano dot matrix is 1.0 * 10
10～1 * 10
12/ cm
2, the spot size scope is at 10nm～500nm;
(2) on the cell culturing surfaces of said cell cultures vessel, carry out and the outer cell cultures of perfect aspect after, make the cell that grows in said cell culturing surfaces from said cell cultures vessel desorption through UV-light or radiation of visible light processing.
In the optimized technical scheme, said photosensitive semiconductor is titanium oxide, zinc oxide, White tin oxide, red stone or zirconium white.That is, said photosensitive semiconductor film is thin film of titanium oxide, zinc-oxide film, SnO 2 thin film, sull or zirconia film; Said photosensitive semiconductor micro-nano dot matrix is titanium oxide micro-nano dot matrix, zinc oxide micro-nano dot matrix, White tin oxide micro-nano dot matrix, red stone micro-nano dot matrix or zirconium white micro-nano dot matrix.
Wherein, the process of described UV-light or radiation of visible light processing is:
The UV-light that with wavelength is 300～400 nanometers shone 1～40 minute from the bottom incident of cell cultures vessel;
Perhaps, the visible light that with wavelength is 400～700 nanometers shone 10～60 minutes from cell cultures vessel cell culturing surfaces or bottom incident.
A kind of employed cell cultures utensil when being used for cell in vitro and cultivating photic cell desorption; Comprise: the cell cultures vessel, wherein, preparation has the photosensitive semiconductor structural sheet as cell culturing surfaces on the cells contacting surface of said cell cultures vessel; Said photosensitive semiconductor is the photosensitive semiconductor with light to hydrophobic-hydrophilic conversion characteristic; Said photosensitive semiconductor structural sheet is photosensitive semiconductor film or photosensitive semiconductor micro-nano dot matrix, and wherein, the grain-size of said photosensitive semiconductor film is 2～100nm; Thickness is 50nm～2000nm, and the density range of the semiconductor nano point in the said photosensitive semiconductor micro-nano dot matrix is 1.0 * 10
10～1 * 10
12/ cm
2, the spot size scope is at 10nm～500nm;
In the optimized technical scheme, said photosensitive semiconductor is titanium oxide, zinc oxide, White tin oxide, red stone or zirconium white.That is: said photosensitive semiconductor film is thin film of titanium oxide, zinc-oxide film, SnO 2 thin film, sull or zirconia film; Said photosensitive semiconductor micro-nano dot matrix is titanium oxide micro-nano dot matrix, zinc oxide micro-nano dot matrix, White tin oxide micro-nano dot matrix, red stone micro-nano dot matrix or zirconium white micro-nano dot matrix.
Among the present invention:
The attached cell of said cell under in-vitro simulated physiological environment, cultivating.
Said cell cultures vessel can be buied from market for cell cultures vessel commonly used, are processed by silicate glass, quartz, PS, POLYACTIC ACID, Sodium bromoacetate homopolymer, SRU or polyacrylic ester usually.
Said photosensitive semiconductor film can adopt conventional methods such as sol-gel method, chemical vapour deposition or physical vapor deposition preparation.
Said photosensitive semiconductor micro-nano dot matrix can prepare through methods such as phase-splitting self-assembly method of the prior art, liquid phase template deposition, gas phase template deposition, thin film photolithography method, hydrothermal method or printings.
Among the present invention, utilize cell to be easy to be attached to comparatively hydrophobic surface and be not easy to be attached to characteristics such as hydrophilic surface, the light-sensitive semiconductor material of hydrophobic to hydrophilic transformation can take place to be chosen in the rayed lower surface; And form photosensitive semiconductor film or micro-nano dot matrix as cell culturing surfaces on the cells contacting of cell cultures vessel surface; Like this, institute's cultured cells is after rayed, and surface wettability changes; Cell is spontaneous to break away from from cultivating the utensil surface, realizes the cell desorption.
Therefore, among the present invention, said photosensitive semiconductor film or photosensitive semiconductor micro-nano dot matrix also can be substituted by other surface nano-structures of photosensitive semiconductor, like nanometer stick array or nano-rod film, nanometer flower array or nanometer flower film etc.Described photosensitive semiconductor is preferably titanium oxide, zinc oxide, White tin oxide, red stone or zirconium white.
Compared with prior art, the present invention has following beneficial technical effects:
The inventive method has characteristics such as cell detachment efficient is high, little, easy and simple to handle, the suitable cell scope of pair cell damage is wide, has very strong practicality.Cell cultures utensil of the present invention only needs cell cultures vessel of the prior art are carried out very little improvement, and cost is low, is easy to realize, and is easy to utilize.
Description of drawings
Fig. 1 observes the inversion microgram of culturing cell before ultraviolet lighting among the embodiment 1 for adopting inverted biologic microscope.
Fig. 2 observes the inversion microgram of culturing cell behind ultraviolet lighting among the embodiment 1 for adopting inverted biologic microscope.
Fig. 3 is the MTS activity value comparison diagram of the cell of the disengaging in the embodiment 1 that adopts the MTS detection method and obtain and the control group.
Embodiment
Specify the present invention below in conjunction with embodiment and accompanying drawing, but the present invention is not limited to this.
Embodiment 1
On the cells contacting surface of silica glass cell cultures vessel, prepare titanium oxide micro-nano dot matrix with the phase-splitting self-assembly method; Process is following: press ethanol: deionized water: methyl ethyl diketone=0.3: 1: 1 (with molar ratio computing) obtain solution; And press 0.02mol/L and add tetra-n-butyl titanate, press 40g/L adding PVP K120 (K-30); Form precursor solution; Be added drop-wise on the cells contacting surface of silica glass cell cultures vessel,, obtained titanium oxide micro-nano dot matrix in two hours as cell culturing surfaces 500 ℃ of following thermal treatments then with the speed spin coating 40s of 6000 revolution per seconds.The density of nano dot is 1.0 * 10 in this titanium oxide micro-nano dot matrix
10/ cm
2, spot size is 200nm～500nm.
On the cell culturing surfaces of above-mentioned silica glass cell cultures vessel, carry out the osteocyte vitro culture, cultivate after three days, the UV-light that with wavelength is 350 nanometers shone 30 minutes from the bottom incident of cell cultures vessel, and 91% cell is broken away from from the surface.
Fig. 1 and Fig. 2 are respectively and adopt among the viewed embodiment 1 of inverted biologic microscope culturing cell at the forward and backward inversion microgram of ultraviolet lighting.Comparison diagram 1 and Fig. 2 can find out that a large amount of cells are spherical by fusiformis type of becoming behind UV-irradiation, explain that a large amount of cells are about to break away from.
Adopt the MTS detection method that the cell activity of the disengaging in the control group of on the standard cell lines culture plate, cultivating under embodiment 1 and the similarity condition is detected; The MTS activity value contrast of the cell of the disengaging in embodiment 1 and the control group is as shown in Figure 3; As can be seen from Figure 3, the cell activity value (OD value, the optical density value that break away among the embodiment 1; Claim light absorption value again) the cell activity value that broken away from the control group on the standard cell lines culture plate, cultivated under the similarity condition; The good security that p0hotodetachment has is described, the cell of disengaging still has good active, the cell activity that it is active even be higher than in the control group to be broken away from.
MT reconnaissance S detection method is a cytoactive detection method commonly used in this area, and the MTS reagent that is wherein adopted also for conventional reagent, can be buied from manufacturers such as Promega companies.The chemical name of MTS is following:
3-(4，5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny?l)-2H-tetrazolium，inner?salt。
Embodiment 2
On the cells contacting surface of silicate glass cell cultures vessel, prepare zinc-oxide film as cell culturing surfaces with chemical Vapor deposition process, this zinc-oxide film grain-size is 2～10nm, and thickness is 50nm.
Cell culturing surfaces at above-mentioned silicate glass cell cultures vessel is carried out to the fibrocyte vitro culture, cultivates after three days, and the UV-light that with wavelength is 300 nanometers shone 1 minute from the bottom incident of cell cultures vessel, and 70% cell is broken away from from the surface.
Embodiment 3
On the cells contacting surface of POLYACTIC ACID cell cultures vessel, prepare thin film of titanium oxide as cell culturing surfaces with physical vaporous deposition, this thin film of titanium oxide grain-size is 10～30nm, and thickness is 1000nm.
Cell culturing surfaces at above-mentioned POLYACTIC ACID cell cultures vessel is carried out to the fibrocyte vitro culture, cultivates after three days, and the UV-light that with wavelength is 365 nanometers shone 25 minutes from the bottom incident of cell cultures vessel, and 74% cell is broken away from from the surface.
Embodiment 4
On the cells contacting surface of silicate glass cell cultures vessel, prepare red stone micro-nano dot matrix as cell culturing surfaces with the liquid phase templated deposition method, the density range of the semiconductor nano point in this red stone micro-nano dot matrix is 1.0 * 10
12/ cm
2, the spot size scope is at 10nm～100nm.
Cell culturing surfaces at above-mentioned silicate glass cell cultures vessel carries out the vascular endothelial cell vitro culture; Cultivate after three days; The visible light that with wavelength is 700 nanometers shone 25 minutes from the bottom incident of cell cultures vessel, and 90% cell is broken away from from the surface.
Embodiment 5
Be deposited on the gas phase template that the preparation SnO 2 thin film is as cell culturing surfaces on the cells contacting surface of PGTA cell cultures vessel, this SnO 2 thin film grain-size is 20～80nm, and thickness is 2000nm.
Cell culturing surfaces at above-mentioned PGTA cell cultures vessel carries out the epithelial cell vitro culture, cultivates after three days, and the UV-light that with wavelength is 300 nanometers shone 5 minutes from the bottom incident of cell cultures vessel, and 80% cell is broken away from from the surface.
Embodiment 6
On the cells contacting surface of silica glass cell cultures vessel, prepare sull as cell culturing surfaces with sol-gel method, this sull grain-size is 50～100nm, and thickness is 800nm.
Cell culturing surfaces at above-mentioned silica glass cell cultures vessel is carried out to the fibrocyte vitro culture, cultivates after three days, and the visible light that with wavelength is 420 nanometers shone 10 minutes from the bottom incident of cell cultures vessel, and 72% cell is broken away from from the surface.
Embodiment 7
On the cells contacting surface of silicate glass cell cultures vessel, prepare titanium oxide micro-nano dot matrix as cell culturing surfaces with impact system, the density range of the semiconductor nano point in this titanium oxide micro-nano dot matrix is 1.1 * 10
10/ cm
2, the spot size scope is at 400nm～500nm.
Cell culturing surfaces at above-mentioned silicate glass cell cultures vessel carries out cardiomyocytes cultured.Cultivate after three days, the UV-light that with wavelength is 365 nanometers shone 30 minutes from the bottom incident of cell cultures vessel, and 66% cell is broken away from from the surface.
Embodiment 8
On the cells contacting surface of polyacrylic ester cell cultures vessel, prepare zirconia film as cell culturing surfaces with sol-gel method, this zirconia film grain-size is 2～20nm, and thickness is 1500nm.
Cell culturing surfaces at above-mentioned polyacrylic ester cell cultures vessel is carried out to the fibrocyte vitro culture.Cultivate after three days, the UV-light that with wavelength is 365 nanometers shone 30 minutes from the bottom incident of cell cultures vessel, and 85% cell is broken away from from the surface.
Embodiment 9
On the cells contacting surface of the silica glass cell cultures vessel with thin film of titanium oxide, prepare titanium oxide micro-nano dot matrix as cell culturing surfaces with the thin film photolithography method, the density range of the semiconductor nano point in this titanium oxide micro-nano dot matrix is 1.0 * 10
10/ cm
2, the spot size scope is at 300～500nm.
Cell culturing surfaces at above-mentioned silica glass cell cultures vessel is carried out to the fibrocyte vitro culture, cultivates after three days, and the UV-light that with wavelength is 320 nanometers shone 10 minutes from the bottom incident of cell cultures vessel, and 76% cell is broken away from from the surface.
Embodiment 10
On the cells contacting surface of PS cell cultures vessel, prepare thin film of titanium oxide as cell culturing surfaces with hydrothermal method, this thin film of titanium oxide grain-size is 20～70nm, and thickness is 1200nm.
Cell culturing surfaces at above-mentioned PS cell cultures vessel is carried out to the fibrocyte vitro culture, cultivates after three days, and the UV-light that with wavelength is 300 nanometers shone 15 minutes from the bottom incident of cell cultures vessel, and 70% cell is broken away from from the surface.
Embodiment 11
On the cells contacting surface of silicate glass cell cultures vessel, prepare zinc-oxide film as cell culturing surfaces with sol-gel method, this zinc-oxide film grain-size is 10～70nm, and thickness is 1800nm.
Cell culturing surfaces at above-mentioned silicate glass cell cultures vessel is carried out to the fibrocyte vitro culture, cultivates after three days, and the UV-light that with wavelength is 300 nanometers shone 1 minute from the bottom incident of cell cultures vessel, and 70% cell is broken away from from the surface.
Claims (5)
1. one kind is used for the method that cell in vitro is cultivated photic cell desorption, it is characterized in that, may further comprise the steps:
(1) before carrying out the cell in vitro cultivation; Preparation photosensitive semiconductor structural sheet is as cell culturing surfaces on the cells contacting surface of cell cultures vessel; Said photosensitive semiconductor is the photosensitive semiconductor with light to hydrophobic-hydrophilic conversion characteristic; Said photosensitive semiconductor structural sheet is photosensitive semiconductor film or photosensitive semiconductor micro-nano dot matrix, and wherein, the grain-size of said photosensitive semiconductor film is 2～100nm; Thickness is 50nm～2000nm, and the density range of the semiconductor nano point in the said photosensitive semiconductor micro-nano dot matrix is 1.0 * 10
10～1 * 10
12/ cm
2, the spot size scope is at 10nm～500nm;
(2) on the cell culturing surfaces of said cell cultures vessel, carry out and the outer cell cultures of perfect aspect after, make the cell that grows in said cell culturing surfaces from said cell cultures vessel desorption through UV-light or radiation of visible light processing.
2. the method that is used for the photic cell desorption of cell in vitro cultivation as claimed in claim 1 is characterized in that said photosensitive semiconductor is titanium oxide, zinc oxide, White tin oxide, red stone or zirconium white.
According to claim 1 or claim 2 be used for the method that cell in vitro is cultivated photic cell desorption, it is characterized in that the process that described UV-light or radiation of visible light are handled is:
The UV-light that with wavelength is 300～400 nanometers shone 1～40 minute from the bottom incident of cell cultures vessel;
Perhaps, be of cell culturing surfaces or the bottom incident of the visible light of 400～700 nanometers with wavelength from the cell cultures vessel, shone 10～60 minutes.
4. employed cell cultures utensil when being used for cell in vitro and cultivating photic cell desorption; Comprise: the cell cultures vessel is characterized in that preparation has the photosensitive semiconductor structural sheet as cell culturing surfaces on the cells contacting surface of said cell cultures vessel; Said photosensitive semiconductor is the photosensitive semiconductor with light to hydrophobic-hydrophilic conversion characteristic; Said photosensitive semiconductor structural sheet is photosensitive semiconductor film or photosensitive semiconductor micro-nano dot matrix, and wherein, the grain-size of said photosensitive semiconductor film is 2～100nm; Thickness is 50nm～2000nm, and the density range of the semiconductor nano point in the said photosensitive semiconductor micro-nano dot matrix is 1.0 * 10
10～1 * 10
12/ cm
2, the spot size scope is at 10nm～500nm.
5. cell cultures utensil as claimed in claim 4 is characterized in that, said photosensitive semiconductor is titanium oxide, zinc oxide, White tin oxide, red stone or zirconium white.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100136680A CN102586169A (en) | 2012-01-17 | 2012-01-17 | Photon stimulated cell desorption method and cell culture implement used by same |
| PCT/CN2012/084905 WO2013107211A1 (en) | 2012-01-17 | 2012-11-20 | Light-induced cell desorption method and cell culture device therefor |
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| CN2012100136680A CN102586169A (en) | 2012-01-17 | 2012-01-17 | Photon stimulated cell desorption method and cell culture implement used by same |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013107211A1 (en) * | 2012-01-17 | 2013-07-25 | 浙江大学 | Light-induced cell desorption method and cell culture device therefor |
| CN104087928A (en) * | 2013-12-25 | 2014-10-08 | 周婧 | Photoresponsive nanostructure film with high visible light transmittance and application thereof |
| CN104175680A (en) * | 2014-07-21 | 2014-12-03 | 浙江大学 | Titanium dioxide based composite thin film with classified nano point structure and preparation method thereof |
| CN106085948A (en) * | 2016-06-16 | 2016-11-09 | 浙江大学 | A kind of method of the cell/cell thin utilizing radiation of visible light to obtain In vitro culture |
| CN106963987A (en) * | 2017-03-29 | 2017-07-21 | 浙江大学 | A kind of conductive extracellular matrix laminated film obtained via cell sheets and preparation method thereof |
| CN107142206A (en) * | 2017-03-29 | 2017-09-08 | 浙江大学 | A kind of silicon/graphene-based composite surface of cell/cell thin harvesting for visible photic in vitro culture and preparation method thereof |
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| WO2016115333A1 (en) * | 2015-01-15 | 2016-07-21 | President And Fellows Of Harvard College | Optical selection of cells |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013107211A1 (en) * | 2012-01-17 | 2013-07-25 | 浙江大学 | Light-induced cell desorption method and cell culture device therefor |
| CN104087928A (en) * | 2013-12-25 | 2014-10-08 | 周婧 | Photoresponsive nanostructure film with high visible light transmittance and application thereof |
| CN104175680A (en) * | 2014-07-21 | 2014-12-03 | 浙江大学 | Titanium dioxide based composite thin film with classified nano point structure and preparation method thereof |
| CN104175680B (en) * | 2014-07-21 | 2016-08-17 | 浙江大学 | Titanium dioxide based coextruded film with classifying nano dot structure and preparation method thereof |
| CN106085948A (en) * | 2016-06-16 | 2016-11-09 | 浙江大学 | A kind of method of the cell/cell thin utilizing radiation of visible light to obtain In vitro culture |
| CN106085948B (en) * | 2016-06-16 | 2019-10-29 | 浙江大学 | A method of obtaining cell/cell thin of in vitro culture using radiation of visible light |
| CN106963987A (en) * | 2017-03-29 | 2017-07-21 | 浙江大学 | A kind of conductive extracellular matrix laminated film obtained via cell sheets and preparation method thereof |
| CN107142206A (en) * | 2017-03-29 | 2017-09-08 | 浙江大学 | A kind of silicon/graphene-based composite surface of cell/cell thin harvesting for visible photic in vitro culture and preparation method thereof |
| CN106963987B (en) * | 2017-03-29 | 2020-05-29 | 浙江大学 | Conductive extracellular matrix composite film obtained through cell sheet layer and preparation method thereof |
| CN107142206B (en) * | 2017-03-29 | 2021-04-06 | 浙江大学 | Silicon/graphene-based composite surface for visible light induced in-vitro culture cell/cell thin layer harvesting and preparation method thereof |
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| WO2013107211A1 (en) | 2013-07-25 |
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Application publication date: 20120718 |