CN103060196A - Novel efficient micro-fluidic multi-cell co-culturing chip and preparation method thereof - Google Patents
Novel efficient micro-fluidic multi-cell co-culturing chip and preparation method thereof Download PDFInfo
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- CN103060196A CN103060196A CN201210589651XA CN201210589651A CN103060196A CN 103060196 A CN103060196 A CN 103060196A CN 201210589651X A CN201210589651X A CN 201210589651XA CN 201210589651 A CN201210589651 A CN 201210589651A CN 103060196 A CN103060196 A CN 103060196A
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Abstract
The invention relates to a novel efficient micro-fluidic multi-cell co-culturing chip and a preparation method thereof. A microstructure and a micro-channel are arranged on the surface of the micro-fluidic chip; the surface of the micro-channel is decorated by bovine serum albumin; under the driving of gravity generated by liquid level difference between a solution inlet and a solution outlet, three cells are implanted in different regions of the micro-channel by using laminar flow phenomenon between liquid flows; finally, the three cells are co-cultured to lay a basis for studies on cell biology; and the novel efficient micro-fluidic multi-cell co-culturing chip provided by the invention is mainly applied to the related field of cell biology, genetics, drug screening and the like. Through adoption of the micro-fluidic chip, the three cells are co-cultured; the micro-fluidic chip has the advantages of intuitive property, convenience, miniaturization and low consumption of reagents and samples; and a brand new cell culturing and analyzing technology is provided for multi-cell co-culturing.
Description
Technical field
The present invention relates to a kind of new and effective micro-fluidic many cells and cultivate altogether chip and preparation method thereof, there are microstructure and microchannel in this micro-fluidic chip surface, microchannel surface is carried out finishing with bovine serum albumin, under the segregation drive of the poor generation of liquid between solution inlet port and the outlet, utilize the laminar flow phenomenon between the liquid stream to realize that three kinds of cells are in the implantation of microchannel different zones, realize at last the common cultivation of three kinds of cells, for the research cytobiology lays the foundation, be mainly used in the association areas such as cytobiology, genetics and drug screening.
Background technology
The later stage eighties 20th century, in order to set up the culture system that more is similar to internal milieu, external environment and internal milieu are matched, thereby make the iuntercellular information of communicating with each other, mutually support growing multiplication, people have developed Cell Co culturing Techenique on the basis of cell culture technology.Cell Co culturing Techenique be with the cell co-culture more than 2 kinds or 2 kinds in same environment, because it has advantages of and reflect better internal milieu, so this method is widely used in the modern cell research.
At present, Cell Co culturing Techenique is applied at most osteocyte and neurocyte.The co-culture of cells system is mainly set up by two kinds of methods: 1. Co-culture system, and the cell that is about to more than 2 kinds or 2 kinds is inoculated in the same hole at the same time or separately, directly contact between different types of cell; 2. indirect co-culture system, the cell that is about to more than 2 kinds or 2 kinds is inoculated in respectively on the different carriers, then these two kinds of carriers is placed among the same culture environment, makes different types of cell share same culture system and not directly contact.
The co-culture system Main Function: inducing cell is to another kind of cytodifferentiation; Inducing cell self differentiation; Keep cell function and vigor; Regulating cell propagation; Promote early embryonic development and improve meta-bolites output.Since the thirties in last century, cell cultures becomes indispensable important step in researchist's experimentation gradually, its carrier instrument: culture dish/culturing bottle is also progressively approved by everybody, becomes a kind of experiment consumptive material of routine.Although up to the present, a lot of scientists think that this condition of in vitro culture of culture dish/culturing bottle has different significantly from the tumor growth environment, but owing to there not being better culture carrier to change this present situation, the existence of this situation of acquiescence of asking so biologists also can only move back time.But recently, a kind of appearance of Petaka cell culture system obtains the favor of the U.S., European vast bio-science man, and some critical defects of culture dish/culturing bottle are mentioned again again, becomes one of topic that the researchist discusses warmly.
Conventional Tissue Culture Dish is difficult to finish the common cultivation of various kinds of cell, therefore, develop a kind of convenient, quick, efficiently, many cells culture technique cheaply, be the active demand in the field such as cytobiology.In recent years, the microfluidic chip analysis technology has become important research direction in the analytical chemistry, is wherein most active one, is all to have obtained to pay attention to widely in scientific research or Application Areas.Micro-fluidic chip is as a kind of novel analysis test platform, has high-throughput, integrated, multiple parallel analysis, portable, easy to operate, low cost and other advantages, is applied widely in various fields.Yet, adopt micro-fluidic chip, in its surface preparation microstructure and microchannel, rely on the driving of the laminar flow effect between multilayer liquid sample microfluid in the microchannel, finish simultaneously the implanted prosthetics of various kinds of cell, the Application Areas of cultivating altogether in various kinds of cell at present not yet has substantial breakthrough.
Summary of the invention
The purpose of this invention is to provide a kind of new and effective micro-fluidic many cells and cultivated altogether chip and preparation method thereof, there are microstructure and microchannel in this micro-fluidic chip surface, microchannel surface is carried out finishing with bovine serum albumin, under the segregation drive of the poor generation of liquid between solution inlet port and the outlet, utilize the laminar flow phenomenon between the liquid stream to realize that three kinds of cells are in the implantation of microchannel different zones, realize at last the common cultivation of three kinds of cells, for the research cytobiology lays the foundation, be mainly used in the association areas such as cytobiology, genetics and drug screening.
For achieving the above object, the present invention adopts following operation steps:
(1) microstructure and the microchannel figure of each layer chip in Computer-aided design software design and the drafting micro-fluidic chip.
(2) by micro-processing technology machining needs on each layer micro-fluidic chip substrate surface and adhesive membrane microstructure and microchannel, comprise sample pool, waste liquid pool, micropore and microchannel.
(3) utilize double-deck adhesive membrane, with each leafing core type micro-fluidic chip alignment, bonding, pressurizing and sealing, form the micro-fluidic chip that many cells are cultivated altogether.
(4) three kinds of solution that contain different cells are added from sample well, the laminar velocity of control microfluid is implanted to three kinds of cells in the main microchannel in the micro-fluidic chip.
(5) finish three kinds of cell implantation in the microchannel after, carry out the cultivation of cell.
Among the present invention, the chip substrates that new and effective micro-fluidic many cells are cultivated chip altogether can be PMMA, PC, PVC, COC, copper, aluminium, stainless steel, silicon chip, glass wafer, also commercially available all kinds of common CD CD.
Among the present invention, new and effective micro-fluidic many cells are cultivated the microstructure of chip and adhesive membrane and microchannel altogether can be by numerical control mill quarter, laser ablation, LIGA technology, moulding method, pressure sintering, chemical corrosion preparation, also available soft lithographic technique preparation.
Among the present invention, new and effective micro-fluidic many cells are cultivated altogether chip and are comprised of layers of chips, fit with adhesive membrane between each layer chip, and adhesive membrane can be that double-deck power causes adhesive membrane, also common double faced adhesive tape film.
Among the present invention, new and effective micro-fluidic many cells are cultivated the gravity of the poor generation of driving dependence liquid of the sample solution on the chip altogether.
Among the present invention, the cell implantation that new and effective micro-fluidic many cells are cultivated chip altogether is that the laminar flow phenomenon between many liquid streams is finished in the dependence microchannel.
Among the present invention, new and effective micro-fluidic many cells are cultivated altogether chip and are adopted original position to implant, and microchannel surface is carried out the BSA protein surface and modified fixing.
The new and effective micro-fluidic many cells that the present invention proposes are cultivated chip and preparation method thereof altogether, parallel implantation and common cultivation simple to operate, as to have realized various kinds of cell, reduced the consumption of reagent and sample, simplified the cell implantation process, have portable, economical, fast, efficiently, characteristics accurately, in the association areas such as cytobiology, genetics and drug screening, have a good application prospect.
Description of drawings
Fig. 1. new and effective micro-fluidic many cells are cultivated the structural representation of chip altogether.
A. cytomixis solution filling orifice on the left of, b. intermediate cell mixing solutions filling orifice, c. right side cytomixis solution filling orifice.
Specific embodiments
Embodiment 1
Microstructure and the microchannel figure of the layers of chips of Computer-aided design software design and drafting centrifugal type microfludic chip.Utilize microstructure and the microchannel of numerical control CNC system processing preparation two-layer disc-shaped polymethylmethacrylate (PMMA) chip, clean each layer chip with tap water, distilled water respectively, and with spots such as the residual fingerprint of ethanol chip surface, oil stains.On the double faced adhesive tape film, with required microstructure and the microchannel of carving machine processing preparation.With layers of chips carefully align, bonding, pressurizing and sealing, make the micro-fluidic chip that contains three entrances and a cell cultures main channel that is fit to three kinds of co-culture of cells.From left side cytomixis solution filling orifice, intermediate cell mixing solutions filling orifice, add respectively three kinds of cell culture fluids that contain different cells with right side cytomixis solution filling orifice, the poor height of liquid of control left, center, right side sample filling orifice and waste liquid pool, thereby key-course Flow Velocity, make the cell solution microchannel of slowly flowing through, therefore cell is adsorbed on BSA protein modified fixing microchannel surface, thereby implants when finishing two kinds of cells.
Embodiment 2
Microstructure and the microchannel figure of the layers of chips of Computer-aided design software design and drafting centrifugal type microfludic chip.Utilize microstructure and the microchannel of numerical control CNC system processing preparation two-layer disc-shaped polymethylmethacrylate (PMMA) chip, clean each layer chip with tap water, distilled water respectively, and with spots such as the residual fingerprint of ethanol chip surface, oil stains.On the double faced adhesive tape film, with required microstructure and the microchannel of carving machine processing preparation.With layers of chips carefully align, bonding, pressurizing and sealing, make the micro-fluidic chip that many cells are cultivated altogether.From left side cytomixis solution filling orifice, intermediate cell mixing solutions filling orifice, add respectively three kinds of cell culture fluids that contain different cells with right side cytomixis solution filling orifice, the poor height of liquid of control left, center, right side sample filling orifice and waste liquid pool, thereby key-course Flow Velocity, make the cell solution microchannel of slowly flowing through, therefore cell is adsorbed on BSA protein modified fixing microchannel surface, thereby implants when finishing two kinds of cells.
Claims (10)
1. new and effective micro-fluidic many cells are cultivated chip and preparation method thereof altogether, there are microstructure and microchannel in this micro-fluidic chip surface, microchannel surface is carried out finishing with bovine serum albumin, under the segregation drive of the poor generation of liquid between solution inlet port and the outlet, utilize laminar flow phenomenon between the liquid stream to realize that three kinds of cells in the implantation of microchannel different zones, realize the common cultivation of three kinds of cells at last.
2. cultivate altogether chip and preparation method thereof by new and effective micro-fluidic many cells claimed in claim 1, it is characterized in that, its making step is as follows:
(1) microstructure and the microchannel figure of each layer chip in Computer-aided design software design and the drafting micro-fluidic chip.
(2) by micro-processing technology machining needs on each layer micro-fluidic chip substrate surface and adhesive membrane microstructure and microchannel, comprise sample pool, waste liquid pool, micropore and microchannel.
(3) utilize double-deck adhesive membrane, with each leafing core type micro-fluidic chip alignment, bonding, pressurizing and sealing, form the micro-fluidic chip that many cells are cultivated altogether.
(4) three kinds of solution that contain different cells are added from sample well, the laminar velocity of control microfluid is implanted to three kinds of cells in the main microchannel in the micro-fluidic chip.
(5) finish three kinds of cell implantation in the microchannel after, carry out the cultivation of cell.
3. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, the Core Feature device that this new and effective micro-fluidic many cells are cultivated chip altogether is micro-fluidic chip, this chip is with the gravity of the poor generation of the liquid motivating force as the sample microfluidic flow, can produce in batches, repeatedly utilization, flexible design and assembling.
4. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, this new and effective micro-fluidic many cells cultivate altogether microstructure on the chip and microchannel be micro-processing method by numerical control mill quarter, laser ablation, LIGA technology, moulding method, pressure sintering, chemical corrosion, soft lithographic technique in the preparation of chip substrates surface, size is in micron level.
5. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, it is to be formed by stacking by layers of chips that this new and effective micro-fluidic many cells are cultivated chip altogether, consists of microstructure and the microchannel network of 3 D stereo.
6. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, this new and effective micro-fluidic many cells are cultivated chip altogether can make three solution injection ports at chip piece, can implant simultaneously and cultivate three kinds of different cells, improve the parallelly cultivate ability of unit time.
7. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, this new and effective micro-fluidic many cells are cultivated chip is finished cell in the microchannel by the laminar flow phenomenon between many liquid streams in the microchannel implantation altogether.
8. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, this new and effective micro-fluidic many cells are cultivated altogether chip and are adopted original position to implant, and carry out carrying out after the BSA protein surface is modified the implantation of cell in microchannel surface.
9. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, this new and effective micro-fluidic many cells are cultivated altogether the suitable original position a kind of, two or three cell of chip and are cultivated altogether.
10. cultivate altogether chip and preparation method thereof by claim 1 or 2 described new and effective micro-fluidic many cells, it is characterized in that, this new and effective micro-fluidic many cells are cultivated altogether chip and are convenient to observe, equipment is simple, Direct Sampling, sample and reagent dosage are little, the parallelly cultivate ability is high, sample need not to shift, sample crossed contamination probability is little, can reflect more realistically interacting between the human tissue cell, be conducive to interact between experimenter's observation of cell and the cell, the particularly impact between the different cells, the curative effect and the toxicity that also are conducive to the rapid screening new drug are in cytobiology, the association area such as genetics and drug screening is with a wide range of applications.
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| 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 |
| CN104293666A (en) * | 2014-09-11 | 2015-01-21 | 大连理工大学 | Micro-fluidic chip device for detecting interaction between two different unicells |
| 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 |
| CN104893953A (en) * | 2015-01-30 | 2015-09-09 | 中国科学院电子学研究所 | Adherent cell scratch making and migration observing methods based on micro-fluidic chip |
| CN105713835A (en) * | 2014-12-05 | 2016-06-29 | 中国科学院大连化学物理研究所 | Multi-functional-region cell three-dimensional co-culture method based on micro-fluidic chip |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN104293666A (en) * | 2014-09-11 | 2015-01-21 | 大连理工大学 | Micro-fluidic chip device for detecting interaction between two different unicells |
| CN104293666B (en) * | 2014-09-11 | 2016-06-22 | 大连理工大学 | The micro flow control chip device of the interphase interaction that two kinds of differences are unicellular |
| CN105713835A (en) * | 2014-12-05 | 2016-06-29 | 中国科学院大连化学物理研究所 | Multi-functional-region cell three-dimensional co-culture method based on micro-fluidic chip |
| CN105713835B (en) * | 2014-12-05 | 2018-11-09 | 中国科学院大连化学物理研究所 | A kind of multi-functional region cell three-dimensional co-culture method based on 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 |
| CN104893953A (en) * | 2015-01-30 | 2015-09-09 | 中国科学院电子学研究所 | Adherent cell scratch making and migration observing methods based on micro-fluidic chip |
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Application publication date: 20130424 |