CN1637136A - Laser prepn process of directionally arranged nanometer channel regulating cell - Google Patents
Laser prepn process of directionally arranged nanometer channel regulating cell Download PDFInfo
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- CN1637136A CN1637136A CN 200410084562 CN200410084562A CN1637136A CN 1637136 A CN1637136 A CN 1637136A CN 200410084562 CN200410084562 CN 200410084562 CN 200410084562 A CN200410084562 A CN 200410084562A CN 1637136 A CN1637136 A CN 1637136A
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Abstract
The common medical polymer material to be used as cell culture substrate is treated through laser scanning to form micron/nanometer or submicon/nanometer channels before sterilization. Anchorage dependent cell in condition after trypsinization is inoculated homogeneously to the surface of the substrate for culture. Owing to the regulation and control of the surface channels, cell will grow and orient in the channel direction. The present invention has simple technological process and easy operation, and is suitable for the basic research in cell biology, making of biological sensor and functional device, research of medical implant, tissue engineering, etc.
Description
Technical field
The present invention relates to the method for a kind of regulating cell oriented growth and arrangement, be specifically related to a kind of LIPS (laser induced periodic surface structures) technology of laser of utilizing at cell culture substrate surface preparation nanometer channel, and then carry out cell cultures and induce and control the method that cell directional is grown and arranged, belong to the biomaterial category, can be used for the research of cytobiology, planting body and field of tissue engineering technology and the development of biosensor.
Background technology
The development in the fields such as research of the reparation of organ and tissue and transplanting in the organizational project, cell sensor, drug development, biochip, artificial neural network and life mechanism requires the growth and the arrangement of directed control cell.Cell almost can carry out all functions of life entity, as metabolism, heredity, information transmission etc. as the least unit of life entity.The relevant research of various life technologies is applied in the process of animal and human's body moving towards from theory, based on the research of cell and the cell cultures important step that is absolutely necessary.The zooblast overwhelming majority belongs to the cell of adherent accole growth, and cell has only the stromal surface of attaching ability normal growth and propagation.On traditional cell culture substrate, people often pay attention to the biochemical characteristic of stromal surface, and ignored the physical property (as surface topography, hardness etc.) of stromal surface, cell cultures is almost carried out on two dimensional surface entirely, and being grown in its surperficial cell all is at random to be orientated and to arrange disorderly.Along with the development of the nineties organizational project in last century, require the three-dimensional cell support to come the regulating cell behavior, people recognize the limitation of traditional training method; Simultaneously, the development of biosensor, planting body and tissue repair also proposes higher requirement to the surface topography characteristic of device or utensil, in the hope of realizing the artificially adjustable of microcell cell behavior.Yet traditional technique means can't satisfy this requirement.Utilizing stromal surface to modify the inducing cell oriented growth and arrange is the method that newly emerges.One of the originator of cell cultures Harrison just finds that as far back as 1912 cell is along this phenomenon of spider's thread oriented growth [Anat.Rec., 1912,6:182-193].Weiss was referred to as " contact guidance (Contact guidance) " [J.Exp.Zoo., 100:353-386] to cell along the behavior of groove pattern oriented growth and arrangement afterwards.The means of generally taking are to produce the groove pattern on the culture medium surface, utilize the growth and the arrangement of groove pattern guiding cell.As utilize electron beam or ionic fluid that silicon chip or quartzy etching are made groove, the yardstick of groove is at 1-50 μ m[J.Cell Sci., 99:73-77]; Perhaps utilize fluted silicon chip or quartz to be motherboard, molding on polymkeric substance [Biomaterials 19:1861-1868].The simple utilization has groove silicon chip or quartzy as culture medium, and cost of manufacture height, efficient are low, and silicon chip or quartzyly also be unsuitable for common cell cultures; Utilize molding generation groove on the polymkeric substance, temperature can make other parts of polymer surfaces also deform in moulding process, and also can cause graphic defects or distortion in the knockout course, and this phenomenon is particularly evident to undersized groove influence.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of method of utilizing laser LIPS technology to come regulating cell oriented growth and arrangement at the nanometer channel of cell culture substrate surface preparation is proposed, simplify technology, reduce cost, can realize that cell in culture medium surface localization and oriented and ordered growth and arrangement, satisfies the needs of RESEARCH ON CELL-BIOLOGY, medical planting body, organizational project and pharmacology pathological study.
For realizing above-mentioned target, the present invention selects for use general medical polymer material as cell culture substrate, adopt the method for laser scanning irradiation, the culture medium surface is handled, make it form micrometer/nanometer or submicron surface groove structures, pair cell culture medium disinfection then, select the cell of the good adherent accole growth of growth conditions, after the trysinization, with certain density, evenly be inoculated in the culture medium surface, put into incubator and cultivate, regulate culture condition according to cell type.Because the regulating and controlling effect of culture medium Surface Groove pattern cell growth makes direction oriented growth and the arrangement of cell along little groove.
Concrete steps of the present invention comprise:
1, culture medium Surface Groove processing
The present invention adopts polymeric substrate to make cell culture substrate, forms periodic nanometer or sub-micron trench structures with laser means at polymer surfaces.The laser that laser apparatus is produced forms polarization laser through polaroid, handles the culture medium surface by the mode of scanning and irradiation.The pulse width of laser is 5-20ns, and pulse-repetition is 5-10Hz, and the incident angle of incoming beam can change to 30 ° from 0 °, and the incoming beam power density is 5-30mJ/cm
2, the polarization direction is an in-plane polarization, scanning speed is 0.01-30mm/s.Through 20-50/cm
2Energy, the polarization laser radiation that wavelength is 266nm, form periodic nanometer or sub-micron trenches at polymer surfaces.The direction of groove can be regulated arbitrarily, changes the angle of laser polarization direction and sample, can realize direction variation from 0 ° to 90 ° of groove, prepares the nanometer channel of different directions at sample surfaces.Enlarge sweep limit, can realize the big areaization of sample surfaces process range.It is the prerequisite that realizes that vessel surface is effectively handled that polymer materials has absorption to employed optical maser wavelength.Optical maser wavelength can be selected ultraviolet region or visible region, selects according to the light absorption wavelength of polymer materials.
2, culture medium disinfects
To select for use uv light irradiation 1-2 hour through the polymkeric substance culture medium of above-mentioned processing, or adopt ethylene oxide gas sterilization 30 minutes, or after utilizing 75% medical alcohol to soak disinfection in 24 hours, as cell cultures.
3, cell cultures
Select the cell of the good adherent accole growth of growth conditions, after the trysinization, with 1.0 * 10
4Individual/cm
2, evenly be inoculated in stromal surface, after incubator is cultivated 6 hours, under inverted phase contrast microscope, can observe direction oriented growth and the arrangement of cell along groove.
The reason that the induced with laser polymer surfaces forms nanometer or sub-micron trenches is that scattering wave and incident light interfere, thereby cause the periodic distribution of energy and temperature at polymer surfaces when incident illumination is mapped to the polymer samples surface.Polymer molecular chain under the effect of stress field from the high zone of temperature to the low regional movement of temperature, form periodically nanometer channel through the laser pulse post polymerization thing surface of some amount.The scale size of nanometer channel is relevant with the character of laser irradiation energy, incident angle and material itself.
Polymer materials culture medium of the present invention can be made the sample of various size shape, comprises polymeric film, support or Tissue Culture Dish etc.Institute draws materials and comprises polycarbonate (PC), polystyrene (PS), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polyimide macromolecular materials such as (PI).
The present invention adopts laser directly to prepare the nanometer channel structure at surface of polymer substrates, does not need other intermediate steps or process of surface treatment, and method is simplified, cost is low, but but not only big area but also microcell localization making nanometer channel structure; The nanometer channel direction can arbitrarily be regulated and control, and the size of nanometer channel is the (cycle: 200-300nm in certain scope; The degree of depth: 30-70nm) can regulate and control.
The cell culture substrate that the present invention selects for use can be cell and the most general medical polymer material of tissue culture, selection face width, applied range.Technology of the present invention is simple, and is workable, and the surface of preparation has the channel form culture medium and have that cost is low, light transmission good, microscopic examination is convenient, be suitable for various biology characterizes the wide characteristics of suitability that make things convenient for.The cell of media surface localization and oriented growth, arrangement can be used as the RESEARCH ON CELL-BIOLOGY model, is suitable for the research of various kinds of cell Basic of Biology; Can be used for the development of cell and organization chip; Can be used for making biosensor based on viable cell; Can be used as liner, the reparation of induce neural tissue and be applied to Tissue Engineering Study etc. as cytoskeleton.
Description of drawings
The nanometer channel shape characteristic that Fig. 1 characterizes for atomic force microscope.
Among Fig. 1, the PS stromal surface pattern of A for not having to handle; After B is laser treatment, has the PS stromal surface pattern of ordered nano groove.
Fig. 2 is the orientation characteristic photo of C6 cell under the opticmicroscope on the PS surface with nanometer channel.
The cell cultures time is 24 hours among Fig. 2, and the nanometer channel direction is a direction shown in the arrow.
Cell process and nanometer channel concerned photo when Fig. 3 was orientated on the C6 cell nano groove for atomic force microscope characterizes.
Among Fig. 3, A is a 2 d plane picture, and B is a 3 dimensional drawing.
Fig. 4 is induced to differentiate into osteoblastic oriented growth photo for nanometer channel regulating bone marrow stroma stem cell (MSCs).
Among Fig. 4, A is the optical photograph of cell cultures after 4 days, and B is the photo of cell cultures after 20 hours, and C is that scleroblast is cultivated the SEM photo after 14 days.
Fig. 5 utilizes the variation of culture medium surface microcell nanometer channel direction to regulate and control the orientation of grown cell, prepares the photo of the cell figure of different orientations.
Among Fig. 5, A is for having trench region groove direction and not having trench region vertical, B has trench region groove direction not become 30 ° angle with there being trench region, C is separated by the wide trench region that do not have of 100 μ m in the middle of two mutually perpendicular microcells of groove direction, and D is that two mutually perpendicular microcells of groove direction are close together.
Embodiment
Below by accompanying drawing and specific embodiment technical scheme of the present invention is further described.
The most frequently used in the cell culture medium material is polystyrene (PS) material, and itself is cheap, and pair cell or organize nontoxicity is easy to make the sample of various size size and shape.In addition, PS has good light transmission, can utilize various opticmicroscopes, growth cell is thereon observed the influence of research nanometer channel pair cell behavior under static state or dynamic condition.Therefore be the base material of example with PS among the present invention as cell cultures.But embodiment does not constitute the qualification that substratum of the present invention is drawn materials.
Embodiment 1:
Growth of nanometer channel regulating rat neurospongioma (C6) cell directional and arrangement.
1, PS polymer surfaces nanometer channel preparation: select the device of Nd:YAG laser 4 frequencys multiplication for use, making laser apparatus output Wavelength of Laser is 266nm; Simultaneously, the speculum group with the 266nm wavelength makes laser vertical ground arrive the mobile platform surface.The PS culture dish ( 35mm) of cleaning is fixed on the support on the X-Y move operation platform.Open laser apparatus, preheating is after 40 minutes, and laser produces and stable output.The pulse width of laser is 6ns, and frequency is 10Hz.Detect and the adjusting laser energy with energy meter, making its energy that arrives sample surfaces is 30mJ/cm
2Simultaneously, the incident angle of regulating laser is 20 °, and incident light is the S polarized light.Open the sequence of control of mobile platform in the computer, platform is accurately moved in X-Y plane, wherein platform is 0.01mm/s in the translational speed of directions X, is 15mm/s in the translational speed of Y direction.Like this, laser carries out scanning and irradiation to PS culture dish surface, promptly can make nanometer channel at sample surfaces.Change the translational speed of Y direction and can obtain the nanometer channel zone of different area size sweep time.To not having nanometer channel and having the shape characteristic on the PS surface of nanometer channel to characterize, see Fig. 1 with atomic force microscope.The preparation nanometer channel shown in Figure 1B, about 210nm of the cycle of nanometer channel, the about 30-40nm of the degree of depth; And the PS surface of process laser treatment is not the micron not of uniform size, irregular and the structure of nanoscale blend, sees Figure 1A.
2, culture medium is immersed in 75% the medical alcohol 24 hours and carries out disinfection, and takes out then to place the aseptic technique platform to dry.
3, cell cultures: the C6 cell is cultivated in containing the high sugared DMEM culture medium culturing liquid of 10% (Vol/Vol) foetal calf serum, utilizes it to keep clone going down to posterity of culturing bottle.Get the growth conditions good cell with 0.25% trypsin Sigma company) after the digestion, with 1 * 10
4Individual cell/cm
2Be inoculated in the PS sample surfaces that laser treatment is crossed.Placing temperature then is 37 ℃ and CO
2Concentration is to cultivate in 5% the incubator.
Experimental result: Fig. 2 is a C6 cell cultures after 24 hours, the photo that viable cell obtains with observation by light microscope; Fig. 3 fixes with glutaraldehyde for the C6 cell, after 50%, 75%, 90% and 100% alcohol progressively dewaters, adopts atomic force microscope observation (contact mode), obtains the image of cell process and nanometer channel relation; (A) 2 d plane picture wherein, (B) 3 dimensional drawing.The direction of the orientation of C6 cell and arrangement is consistent with the direction of nanometer channel as can be seen, and also surfacewise nanometer channel direction of the projection of cell.
Embodiment 2:
Nanometer channel regulating bone marrow stroma stem cell (MSC) is induced to differentiate into osteoblastic oriented growth.
1, nanometer channel is made: the energy of laser is 50mJ/cm
2, the incident angle of regulating laser is 30 °, platform is 0.015mm/s in the translational speed of directions X, is 30mm/s in the translational speed of Y direction.Other are with example 1.
2, culture medium is immersed in 75% the medical alcohol 24 hours and carries out disinfection, and takes out then to place the aseptic technique platform to dry.
3, MSCs cell cultures: induce the bone marrow stroma stem cell directed differentiation to become osteoblastic nutrient solution to form: to contain 100nmol/L dexamethasone (Sigma), 50 μ g/ml vitamins C (Sigma, cultivate pure), 10mmol/L β-phospho-glycerol, the DMEM (Sigma) of high sugar, 15% foetal calf serum.
Get one of the new zealand white rabbit at 4 monthly ages, 1% carbrital (1ml/kg) auricular vein injecting anesthetic is operated under strict aseptic condition.Getting the femur stage casing is point of puncture, extracts 4ml marrow, join in the PS culture dish (Corning) of 90mm, with the full marrow method of nutrient solution that contains inductor carry out former be commissioned to train foster.Changed liquid once every three days, the cell of dehematizing when changing liquid.After changing liquid three times, hemocyte has not existed, has been formed into the fibrocyte colony in the culture dish, and cell is the shuttle type, and cell is arranged with certain directivity, and the part is the growth of whirlpool sample, and the cell boundary line is unclear.At this moment cell can go down to posterity.After going down to posterity, continue to cultivate with the nutrient solution that contains inductor.Go down to posterity after three times, cell phenotype tends towards stability, and the growing ability of cell is also the most vigorous.At this moment cell is used 0.25% tryptic digestion, with 5.0 * 10
3Individual cell/cm
2Be inoculated into laser treatment and contain on the PS substrate of nanostructure, cultivate with the DMEM nutrient solution (containing 10% foetal calf serum) of the high sugar that does not contain inductor.Fig. 4 is seen in microscopic examination in the cell cultivation process.Among Fig. 4, (A) be the optical photograph of cell cultures after 4 days, most as can be seen cells are arranged along the direction of nanometer channel; (B) be the photo of cell cultures after 20 hours, by laser co-focusing microscopic examination cytoskeleton, orientations has taken place in cell Actin muscle fiber as can be seen, and beginning is extended and growth along the nanometer channel direction; (C) photo of cell cultures after 14 days found out have the bone tubercle to occur in the cell from photo, and the collegen filament of emiocytosis are also arranged along the nanometer channel direction.
Embodiment 3:
Utilize the variation of nanometer channel direction to prepare the cell figure
1. can control cell in the characteristic of determining direction oriented growth and arrangement according to nanometer channel, the nanometer channel microcell that has prepared different directions on same matrix is with the cell figure (cell patterning) of this regulating cell orientation formation different orientation direction.
2. cell cultures: cell is selected the C6 cell for use, and cultural method is with embodiment 1.When the nanometer channel direction changed, the direction of cell figure also changed thereupon.In Fig. 5 A and B, figure lower part is not have the nanometer channel zone in contrast, and upper part is that the nanometer channel zone is arranged.Among Fig. 5 A, the nanometer channel direction is vertical with untreated areas, and the direction of the cell figure also cell with non-oriented is vertical; When the angle of nanometer channel direction and untreated areas was 30 °, corresponding variation (Fig. 5 B) also took place in the direction of cell figure.Further, handle two mutually perpendicular nanometer channel zones at same PS culture dish, the centre stays the wide zone of about 100 μ m and does not deal with; Behind the culturing cell, find that the nanometer channel direction that cell is still followed separately forms the cell figure, the cell orientation is also vertical mutually; The cell of untreated areas also more or less is subjected to the influence (Fig. 5 C) of both sides orientation cell simultaneously.Together adjacent when mutually perpendicular trench region, cell is grown in the above and is still arranged by the groove direction, forms close mutually and orthogonal cell graphics field (Fig. 5 D).But laser is regulated at will, can select different microcells to prepare the nanometer channel of different directions in the PS stromal surface, and cell is grown by the direction of design in advance in the above.This has the meaning of particularly important for organizational project and Study on Biosensor.
Claims (3)
1, the method that aligns of a kind of nanometer channel regulating cell that utilizes laser preparation is characterized in that comprising following concrete steps:
1) culture medium Surface Groove processing: utilize incident polarization laser scanning polymkeric substance culture medium surface, the pulse width of laser is 5-20ns, pulse-repetition is 5-10Hz, and the incident angle of incoming beam is from 0 ° to 30 °, and the incoming beam power density is 5-30mJ/cm
2, the polarization direction is an in-plane polarization, scanning speed is 0.01-30mm/s, through 20-50/cm
2Energy, the polarization laser radiation that wavelength is 266nm, form periodic submicron or nanometer channel structure at polymer surfaces;
2) disinfecting of culture medium: will be through the polymkeric substance culture medium of above-mentioned processing, selected for use uv light irradiation 1-2 hour, or adopt ethylene oxide gas sterilization 30 minutes, or after utilizing 75% medical alcohol to soak disinfection in 24 hours, as cell cultures;
3) cell cultures: select the cell of the good adherent accole growth of growth conditions, after the trysinization, with 1.0 * 10
4Individual/cm
2, evenly be inoculated in culture surface, after incubator is cultivated 6 hours, can be observed direction oriented growth and the arrangement of cell along groove.
2, the method that aligns according to the nanometer channel regulating cell that utilizes laser preparation of claim 1 is characterized in that the employed optical maser wavelength of culture medium Surface Groove processing selects ultraviolet region or visible region according to the light absorption wavelength of polymer materials.
3, the method that aligns according to the nanometer channel regulating cell that utilizes laser preparation of claim 1, it is characterized in that described polymkeric substance culture medium is polymeric film, support or Tissue Culture Dish, draw materials is polycarbonate, poly(lactic acid), polystyrene, polyethylene terephthalate, polybutylene terephthalate or polyimide.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285083B (en) * | 2008-05-30 | 2011-06-29 | 华中科技大学 | Process for preparing patterned cellulosic by micro-fluidic chip |
| CN103436491A (en) * | 2013-09-05 | 2013-12-11 | 东南大学 | Method for carrying out cell density and arrangement controllable culture based on insect wing base |
| CN110230058A (en) * | 2019-05-31 | 2019-09-13 | 上海交通大学 | Promote the method for the medical titanium alloy surface building of growth of marrow mesenchyme stem cell differentiation |
| CN114984311A (en) * | 2022-05-11 | 2022-09-02 | 上海市第六人民医院 | Piezoelectric conductive composite support and preparation method thereof |
-
2004
- 2004-11-25 CN CN 200410084562 patent/CN1298842C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285083B (en) * | 2008-05-30 | 2011-06-29 | 华中科技大学 | Process for preparing patterned cellulosic by micro-fluidic chip |
| CN103436491A (en) * | 2013-09-05 | 2013-12-11 | 东南大学 | Method for carrying out cell density and arrangement controllable culture based on insect wing base |
| CN103436491B (en) * | 2013-09-05 | 2015-10-28 | 东南大学 | A kind of based on the cell density at the bottom of insect wing base and the method arranging controlled cultivation |
| CN110230058A (en) * | 2019-05-31 | 2019-09-13 | 上海交通大学 | Promote the method for the medical titanium alloy surface building of growth of marrow mesenchyme stem cell differentiation |
| CN110230058B (en) * | 2019-05-31 | 2020-08-14 | 上海交通大学 | Method for constructing medical titanium alloy surface for promoting growth and differentiation of bone marrow mesenchymal stem cells |
| CN114984311A (en) * | 2022-05-11 | 2022-09-02 | 上海市第六人民医院 | Piezoelectric conductive composite support and preparation method thereof |
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