CN102021116A - Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same - Google Patents
Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same Download PDFInfo
- Publication number
- CN102021116A CN102021116A CN2009101875616A CN200910187561A CN102021116A CN 102021116 A CN102021116 A CN 102021116A CN 2009101875616 A CN2009101875616 A CN 2009101875616A CN 200910187561 A CN200910187561 A CN 200910187561A CN 102021116 A CN102021116 A CN 102021116A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- fluidic chip
- cell culture
- micro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The present invention provides a microfluidic chip and a method for studying non-contact type cell co-cultivation by using the same. The microfluidic chip comprises a sample inlet, a cell cultivation room, a cell migration region and a waste pool. The upper end of the cell cultivation room is connected to the sample inlet and the lower end of the cell cultivation room is connected to the waste pool. Three parallel cell cultivation rooms are joined by cell migration region. The microfluidic chip provides good microenvironment for non-contact type cell co-cultivation, enables to form a concentration gradient in the cell migration region and facilitates observation of cell migration process in real time and research of transdifferentiation. The invention is advantageous in easy operation, simple manufacturing and small dosage of samples.
Description
Technical field
The present invention relates to the micro-fluidic chip technology is applied to the biomedical research field, a kind of micro-fluidic chip specifically is provided and has studied the method for contactless co-culture of cells.
Background technology
Cell interaction is the research focus of current medical science and the research of biological educational circles.Cell is not an isolated living unit, simple mechanical connection between cell and the cell, thereby but exist various forms of mutual interchanges constitute one complete individual and coordinate to finish various vital movements.Explore cell-cell interaction and corresponding signal path, disease treatment etc. is had vital role.
At present, co-culture of cells is the method for research cell interaction the most commonly used.Studying the co-culture of cells method traditionally generally is divided three classes: mixing is cultivated altogether, microcarrier is cultivated altogether and Transwell cultivates altogether.Hybrid cultivation altogether can't be with two or more cellular regions separately; Microcarrier is cultivated altogether, and the cell interaction The limited time is generally less than 4 hours; And, can't monitor cellular response in real time for the co-culture method that adopts this cover ware of Transwell formula, and can only obtain net result, might lack a large amount of biological informations.More than three kinds of methods all exist reagent consumption big, the cell consumption is many, can't realize many shortcomings such as fluid control.
The micro-fluidic chip laboratory claims chip lab (Lab-on-a-Chip) or micro-fluidic chip (Microfluidic) again, refer to specimen preparation, reaction related in chemistry and the field such as biology, separate, basic operation units such as detection, cell cultures, sorting, cracking are integrated or be integrated into substantially on the chip of more than square centimeters (even littler), form network by the microchannel, run through total system with controlled fluid, in order to a kind of technology of the various functions that replace conventional chemical or biology laboratory.The micro-fluidic chip technology is just progressively permeated to biomedical sector, and has shown wide application prospect.Its distinctive micro-dimension feature and cell size are complementary, and the integrated feature of chip flexible design and scale is fit to carry out cell function research very much in addition.
Summary of the invention
The purpose of this invention is to provide a kind of micro-fluidic chip and studied the method for contactless co-culture of cells.Utilize this micro-fluidic chip to can be contactless co-culture of cells good microenvironment is provided, and form stable concentration gradient in cell migration zone, can real-time monitored cell migration process, and change differentiation research.
The invention provides a kind of micro-fluidic chip, this micro-fluidic chip is made up of injection port (1), cell culture chamber (2), cell migration zone (3) and waste liquid pool (4); The upper end of cell culture chamber links to each other with injection port, and the lower end connects waste liquid pool; Three parallel cell culture chambers link to each other by the cell migration zone, and the cell culture chamber height is higher than the cell migration zone.
Micro-fluidic chip provided by the invention, described micro-fluidic chip is formed by two-layer reversible sealing-in up and down, and upper layer of material is the PDMS polymkeric substance, and subsurface material is a glass.
The present invention also provides the method for studying contactless co-culture of cells based on described micro-fluidic chip, and procedure is as follows:
(1) concentration gradient characterizes: the fluorescence dye of fluorescein isothiocyanate (FITC) mark is embedded among the BME, by injection port it is added cell culture chamber C, all the other cell culture chambers only add equivalent α-MEM nutrient solution;
(2) cell application of sample: by the right side injection port first kind of cell added cell culture chamber C, be positioned over CO
2In the incubator; After treating cell attachment 6-24h, second kind of cell added cell culture chamber B by middle injection port; The A of culturing room does not add cell as the blank left side, and cell culture fluid is changed once every day;
(3) the active investigation: cultivate the growth conditions and the activity of cell two days later with Hoechst33342 and propidium iodide sign.
The method of contactless co-culture of cells provided by the invention, described BME is a basement membrane-like material, is liquid during low temperature, is frozen into colloidal state during room temperature; The concentration gradient in described cell migration zone characterizes with the dextran molecule (FITC-dextran) (molecular weight is 10000D) of marked by fluorescein isothiocyanate.
The method of contactless co-culture of cells provided by the invention, when adding cell, cell can not arrive the migration zone by crossfire; Treat just to enter the migration zone behind the cell attachment.
The method of contactless co-culture of cells provided by the invention can be carried out the migration of two kinds of co-culture of cells simultaneously and be changeed the differentiation detection on chip; This micro-fluidic chip has three cell culture chambers, can add different types of cell respectively, carries out the migration of three kinds of co-culture of cells and changes the differentiation detection.
In a word, the present invention can be on more than one square centimeters chip, for contactless co-culture of cells research provides good microenvironment, generate stable concentration gradient in the cell migration zone, under a kind of effect of cytokines of emiocytosis, can induce another kind of cell that directed moving and the commentaries on classics differentiation taken place.Characteristics such as that the present invention has is simple to operate, it is simple to make and amount of samples is few.Have important biomedical researching value and economic worth.
Description of drawings
Fig. 1 micro-fluidic chip synoptic diagram of the present invention, wherein (a) chip upper strata; (b) chip lower floor, (c) chip one-piece construction figure; (1) injection port, (2) cell culture chamber, (3) cell migration zone, (4) waste liquid pool;
Fig. 2 showed cell is cultivated the fluorescent signal in the migration zone;
The stable fluorescence concentration gradient that Fig. 3 cell migration zone generates;
Fig. 4 inoblast HFL-I, gastric epithelial cells GES-1, liver cancer cell HepG-2 be growth conditions and active the detection on micro-fluidic chip;
Fig. 5 inoblast HFL-I is at blank, gastric epithelial cells GES-1, and liver cancer cell HepG-2 induces down and moves;
The migration area that Fig. 6 inoblast HFL-I takes place under the inducing of gastric epithelial cells GES-1;
The migration area that Fig. 7 inoblast HFL-I takes place under the inducing of liver cancer cell HepG-2;
Fig. 8 inoblast HFL-I induces the migration area that takes place down at cancer cell of oral cavity ACC-2;
The migration area that Fig. 9 inoblast HFL-I takes place under the inducing of cancer cell of oral cavity ACC-M;
The expression of α-SMA under Figure 10 inoblast HFL-I single culture environment;
The expression of Figure 11 inoblast HFL-I α-SMA under the inducing of gastric epithelial cells GES-1;
The expression of Figure 12 inoblast HFL-I α-SMA under the inducing of liver cancer cell HepG-2.
Embodiment
The following examples will give further instruction to the present invention, but not thereby limiting the invention.
Used micro-fluidic chip designs voluntarily for this laboratory and prepares.Chip is formed by two-layer reversible sealing-in up and down, and upper layer of material is the PDMS polymkeric substance, and subsurface material is a glass.As shown in Figure 1, three parallel cell culture chambers are of a size of long 6mm, wide 1mm, and high 50 μ m, cell migration area size is long 1mm, wide 500 μ m, high 50 μ m.By the right side injection port liver cancer cell HepG-2 is added cell culture chamber C, be positioned over 37 ℃ CO gently
2In the incubator; After treating cell attachment, inoblast HFL-I is added cell culture chamber B by middle injection port; The A of culturing room does not add cell as the blank left side, adds and B the cell culture fluid of C equivalent.
Adopt the activity of Hoechst33342 and three kinds of cells of PI joint-detection (HepG-2, GES-1, HFL-I), the result shows that the surviving rate of three kinds of cells on this chip is all greater than 90% (Fig. 4).
Compare with blank side HFL-I cell migration, when 6h and 48h, the HFL-I cell migration more trends towards liver cancer cell HepG-2 (Fig. 5).The HFL-I cell migration area and the control sides of experimental side do not have significant difference (Fig. 6) and induce down at normal cell GES-1.Inducing down at liver cancer cell HepG-2, there are significant difference in the HFL-I cell migration area and the control sides of experimental side.
The fluorescence dye of fluorescein isothiocyanate (FITC) mark is embedded among the BME, by injection port it is added cell culture chamber C, all the other cell culture chambers add equivalent α-MEM nutrient solution, after for some time, fluorescent signal (Fig. 2) appears in the cell migration zone, and data analysis showed cell migration zone forms stable fluorescence concentration gradient (Fig. 3).
By the right side injection port normal cell GES-1 is added cell culture chamber C, be positioned over 37 ℃ CO gently
2In the incubator; After treating cell attachment, inoblast HFL-I is added cell culture chamber B by middle injection port; The A of culturing room does not add cell as the blank left side, adds and B the cell culture fluid of C equivalent.Compare with blank side HFL-I cell migration, when 6h and 48h, basic identical under HFL-I cell migration number and area and the blank (Fig. 5).Inducing down at normal cell GES-1, the HFL-I cell migration area and the control sides of experimental side do not have significant difference (Fig. 7).
By the right side injection port cancer cell of oral cavity ACC-2/ACC-M is added cell culture chamber C, be positioned over 37 ℃ CO gently
2In the incubator; After treating cell attachment, inoblast HFL-I is added cell culture chamber B by middle injection port; The A of culturing room does not add cell as the blank left side, adds and B the cell culture fluid of C equivalent.Compare with blank side HFL-I cell migration, cancer cell of oral cavity ACC-2/ACC-M induce the HFL-I cell migration area of following experimental side and control sides do not have significant difference (Fig. 8, Fig. 9).
After cultivating for some time altogether, take upper strata PDMS chip off, the cell of glass negative is carried out immunofluorescence assay, detect the expression of smooth muscle actin α-SMA in the HFL-I cell.The expression amount of α-SMA seldom (Figure 10) under the inoblast HFL-I single culture environment, expression amount also less (Figure 11) under the inducing of gastric epithelial cells GES-1, but the expression amount showed increased (Figure 12) of inoblast HFL-I α-SMA under the inducing of liver cancer cell HepG-2.
Claims (8)
1. micro-fluidic chip, it is characterized in that: this micro-fluidic chip is made up of injection port (1), cell culture chamber (2), cell migration zone (3) and waste liquid pool (4);
The upper end of cell culture chamber links to each other with injection port, and the lower end connects waste liquid pool, and three parallel cell culture chambers link to each other by the cell migration zone.
2. according to the described micro-fluidic chip of claim 1, it is characterized in that: described micro-fluidic chip is formed by two-layer reversible sealing-in up and down, and upper layer of material is the PDMS polymkeric substance, and subsurface material is a glass.
3. according to the described micro-fluidic chip of claim 1, it is characterized in that: described cell culture chamber height is higher than the cell migration zone.
4. study the method for contactless co-culture of cells based on the described micro-fluidic chip of claim 1, it is characterized in that: procedure is as follows:
(1) concentration gradient characterizes: the fluorescence dye of FITC mark is embedded among the BME, by injection port it is added cell culture chamber C, all the other cell culture chambers only add equivalent α-MEM nutrient solution;
(2) cell application of sample: by the right side injection port first kind of cell added cell culture chamber C, be positioned over CO
2In the incubator; After treating cell attachment 6-24h, second kind of cell added cell culture chamber B by middle injection port; The A of culturing room does not add cell as the blank left side, and cell culture fluid is changed once every day;
(3) the active investigation: cultivate the growth conditions and the activity of cell two days later with Hoechst33342 and propidium iodide sign.
5. according to the method for the described contactless co-culture of cells of claim 4, it is characterized in that: described BME is a basement membrane-like material, is liquid during low temperature, is frozen into colloidal state during room temperature.
6. according to the method for the described contactless co-culture of cells of claim 4, it is characterized in that: the concentration gradient FIEC-dextran characterization of molecules in described cell migration zone.
7. according to the method for the described contactless co-culture of cells of claim 4, it is characterized in that: when adding cell, cell can not arrive the migration zone by crossfire; Treat just to enter the migration zone behind the cell attachment.
8. according to the method for the described contactless co-culture of cells of claim 6, it is characterized in that: the molecular weight of described FIEC-dextran molecule is 10000D.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101875616A CN102021116A (en) | 2009-09-23 | 2009-09-23 | Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101875616A CN102021116A (en) | 2009-09-23 | 2009-09-23 | Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102021116A true CN102021116A (en) | 2011-04-20 |
Family
ID=43862901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101875616A Pending CN102021116A (en) | 2009-09-23 | 2009-09-23 | Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102021116A (en) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102728422A (en) * | 2012-06-11 | 2012-10-17 | 清华大学 | Microfluidic chip apparatus and application thereof |
| CN102746986A (en) * | 2012-07-13 | 2012-10-24 | 中国科学院大连化学物理研究所 | Tumor cell migration dynamics monitoring method based on microfluidic chip |
| CN102816682A (en) * | 2011-06-08 | 2012-12-12 | 大连医科大学 | Micro-fluidic chip and method for studying exosmosis of tumor cell cluster |
| CN102978109A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip |
| CN103087912A (en) * | 2011-10-27 | 2013-05-08 | 中国科学院大连化学物理研究所 | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method |
| CN103361263A (en) * | 2013-07-11 | 2013-10-23 | 北京大学 | Cell chemotaxis analysis chip and device as well as use method and preparation method of cell chemotaxis analysis chip |
| CN103667057A (en) * | 2013-12-30 | 2014-03-26 | 中国科学院苏州纳米技术与纳米仿生研究所 | Micro-fluidic chip-based method for monitoring cell migration biological behavior after cells are wounded |
| CN104630059A (en) * | 2015-01-16 | 2015-05-20 | 中国科学院深圳先进技术研究院 | Microfluidic chip and method for establishing in-vitro co-culture model of three kinds of cells |
| CN106520698A (en) * | 2016-01-05 | 2017-03-22 | 苏州汶颢芯片科技有限公司 | Cell non-contact culture method based on micro-fluidic chip |
| CN107904168A (en) * | 2017-11-27 | 2018-04-13 | 中国科学院理化技术研究所 | A kind of micro-fluidic chip and method for studying cell chemotaxis |
| CN107955783A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of construction method based on the external diabetes Glomerulus model of micro-fluidic chip |
| CN107955784A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of three-dimensional cell ball migration monitoring method based on microfluidic chip technology |
| CN108117989A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of nano particle injury of lungs evaluation method based on organ chip technology |
| CN108117990A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of construction method of the bionical blood barrier model based on microflow control technique |
| CN109957597A (en) * | 2017-12-14 | 2019-07-02 | 深圳先进技术研究院 | Detect the method and its application and micro-fluidic chip of biofilm formation |
| CN110029059A (en) * | 2019-04-22 | 2019-07-19 | 清华大学深圳研究生院 | A kind of cell co-culture system based on nano fibrous membrane |
-
2009
- 2009-09-23 CN CN2009101875616A patent/CN102021116A/en active Pending
Cited By (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816682A (en) * | 2011-06-08 | 2012-12-12 | 大连医科大学 | Micro-fluidic chip and method for studying exosmosis of tumor cell cluster |
| CN102816682B (en) * | 2011-06-08 | 2014-06-18 | 大连医科大学 | Micro-fluidic chip and method for studying exosmosis of tumor cell cluster |
| CN103087912A (en) * | 2011-10-27 | 2013-05-08 | 中国科学院大连化学物理研究所 | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method |
| CN102728422A (en) * | 2012-06-11 | 2012-10-17 | 清华大学 | Microfluidic chip apparatus and application thereof |
| CN102728422B (en) * | 2012-06-11 | 2015-02-18 | 清华大学 | Microfluidic chip apparatus and application thereof |
| CN102746986A (en) * | 2012-07-13 | 2012-10-24 | 中国科学院大连化学物理研究所 | Tumor cell migration dynamics monitoring method based on microfluidic chip |
| CN102746986B (en) * | 2012-07-13 | 2014-07-16 | 中国科学院大连化学物理研究所 | Tumor cell migration dynamics monitoring method based on microfluidic chip |
| CN102978109A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip |
| CN103361263A (en) * | 2013-07-11 | 2013-10-23 | 北京大学 | Cell chemotaxis analysis chip and device as well as use method and preparation method of cell chemotaxis analysis chip |
| CN103667057A (en) * | 2013-12-30 | 2014-03-26 | 中国科学院苏州纳米技术与纳米仿生研究所 | Micro-fluidic chip-based method for monitoring cell migration biological behavior after cells are wounded |
| CN103667057B (en) * | 2013-12-30 | 2015-10-28 | 中国科学院苏州纳米技术与纳米仿生研究所 | The monitoring method of cell migration biological behaviour after being wound based on the cell of micro-fluidic chip |
| CN104630059A (en) * | 2015-01-16 | 2015-05-20 | 中国科学院深圳先进技术研究院 | Microfluidic chip and method for establishing in-vitro co-culture model of three kinds of cells |
| CN106520698A (en) * | 2016-01-05 | 2017-03-22 | 苏州汶颢芯片科技有限公司 | Cell non-contact culture method based on micro-fluidic chip |
| CN107955784B (en) * | 2016-10-14 | 2021-10-15 | 中国科学院大连化学物理研究所 | Three-dimensional cell sphere migration monitoring method based on micro-fluidic chip technology |
| CN107955783A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of construction method based on the external diabetes Glomerulus model of micro-fluidic chip |
| CN107955784A (en) * | 2016-10-14 | 2018-04-24 | 中国科学院大连化学物理研究所 | A kind of three-dimensional cell ball migration monitoring method based on microfluidic chip technology |
| CN107955783B (en) * | 2016-10-14 | 2021-11-30 | 中国科学院大连化学物理研究所 | Method for constructing in-vitro diabetic glomerulus model based on microfluidic chip |
| CN108117990B (en) * | 2016-11-29 | 2021-06-11 | 中国科学院大连化学物理研究所 | Construction method of bionic qi-blood barrier model based on microfluidic technology |
| CN108117989B (en) * | 2016-11-29 | 2021-06-11 | 中国科学院大连化学物理研究所 | Nanoparticle lung injury evaluation method based on organ chip technology |
| CN108117990A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of construction method of the bionical blood barrier model based on microflow control technique |
| CN108117989A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of nano particle injury of lungs evaluation method based on organ chip technology |
| CN107904168B (en) * | 2017-11-27 | 2021-04-06 | 中国科学院理化技术研究所 | Micro-fluidic chip and method for researching cell chemotaxis |
| CN107904168A (en) * | 2017-11-27 | 2018-04-13 | 中国科学院理化技术研究所 | A kind of micro-fluidic chip and method for studying cell chemotaxis |
| CN109957597A (en) * | 2017-12-14 | 2019-07-02 | 深圳先进技术研究院 | Detect the method and its application and micro-fluidic chip of biofilm formation |
| CN110029059A (en) * | 2019-04-22 | 2019-07-19 | 清华大学深圳研究生院 | A kind of cell co-culture system based on nano fibrous membrane |
| CN110029059B (en) * | 2019-04-22 | 2022-04-01 | 清华大学深圳研究生院 | Cell co-culture system based on nanofiber membrane |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102021116A (en) | Microfluidic chip and method for studying non-contact type cell co-cultivation by using the same | |
| CN102071138B (en) | Microfluidic chip and application thereof | |
| Meyvantsson et al. | Cell culture models in microfluidic systems | |
| Zhou et al. | Recent advances in microfluidic devices for bacteria and fungus research | |
| CN102746986B (en) | Tumor cell migration dynamics monitoring method based on microfluidic chip | |
| CN102580794B (en) | Micro-fluidic chip capable of positioning cells and organisms and application thereof | |
| CN103981096B (en) | A kind of two-layer cell culture system organ chip and preparation method thereof | |
| CN102071139A (en) | Microfluidic chip-based cell three-dimensional co-culture method | |
| CN103087912A (en) | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method | |
| US6943008B1 (en) | Bioreactor for cell culture | |
| CN204097450U (en) | A kind of multidimensional many concentration susceptibility detects micro-fluidic chip | |
| CN102140422B (en) | Device for controlling interaction of various cells as well as preparation method and application thereof | |
| CN103257213B (en) | A kind of fully integrated high-flux cell horizontal micro-fluidic chip drug evaluation system | |
| CN103952300B (en) | A kind of micro-fluidic chip and cell chemotaxis motion study method | |
| Huang et al. | A dynamic hanging-drop system for mesenchymal stem cell culture | |
| CN102978109A (en) | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip | |
| WO2017175236A1 (en) | Microfluidic platform for developing in-vitro co-cultures of mammalian tissues. | |
| CN106544270A (en) | A kind of micro-fluidic chip and its cell culture processes for co-culture of cells | |
| CN103627635A (en) | Multifunctional micro-fluidic chip for cell migration and invasion assay | |
| Strieth et al. | A semi-continuous process based on an ePBR for the production of EPS using Trichocoleus sociatus | |
| CN109868256A (en) | A kind of kidney organ's chip and kidney organ's drug test model | |
| CN107881106A (en) | A kind of array cell dynamic cultivation handles micro-fluidic chip and its preparation method and application with compartmentalization | |
| Wen-Ming et al. | Diversification of microfluidic chip for applications in cell-based bioanalysis | |
| CN116445285A (en) | Organ-like co-culture chip, construction method and co-culture method | |
| CN106520698A (en) | Cell non-contact culture method based on micro-fluidic chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110420 |