CN107988070A - A kind of few cells electricity turns micro-fluidic chip, electricity turns sorter and application - Google Patents
A kind of few cells electricity turns micro-fluidic chip, electricity turns sorter and application Download PDFInfo
- Publication number
- CN107988070A CN107988070A CN201711379237.5A CN201711379237A CN107988070A CN 107988070 A CN107988070 A CN 107988070A CN 201711379237 A CN201711379237 A CN 201711379237A CN 107988070 A CN107988070 A CN 107988070A
- Authority
- CN
- China
- Prior art keywords
- electricity
- turns
- micro
- cell
- few cells
- 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
Classifications
-
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- 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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
-
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
Abstract
The present invention relates to a kind of few cells electricity turns micro-fluidic chip, electricity turns sorter and application.Few cells electricity turns sorter and turns unit, display screen, outer case, power supply unit, micro-control unit and master reference including electricity, and electricity, which turns unit, includes the chip.Display screen, which is used to send to micro-control unit, to be instructed, and is received micro-control unit and master reference feedack and is shown；Micro-control unit is used for the instruction for receiving display screen transmission, and turns unit to electricity and power supply unit is controlled；Electricity turns unit and is used to complete cell transfecting process；Master reference turns unit feedack for receiving electricity, and is sent to display screen and micro-control unit.Few cells electricity, which turns micro-fluidic chip, to be included injection port, outlet, negative pressure duct, positive pressure duct and main channel, 96 orifice plates being set after outlet.The present invention can ensure that the transfection state in transfection process in main channel is identical, ensure that the efficiency of transfection.Cell quality is ensure that by 96 orifice plates, easy to the cell culture in later stage.
Description
Technical field
The present invention relates to IPS induced multi-potent stem cells field, and stem cell induction differentiation, is specifically that a kind of few cells electricity turns
Micro-fluidic chip, electricity turn sorter and application.
Background technology
By the induction of external source transcription regulatory factor, make pluripotent cell of the adult cell reprogramming for embryonic stem cell sample,
This cell is known as induced multi-potent stem cell (iPS cells), this method is referred to as iPS technologies.The birth of iPS technologies will be dry thin
Born of the same parents' research has been advanced to a new height, is greatly enriched the research contents of stem cell.In past 3 years, iPS technologies hair
Exhibition is rapid, successively achieves success in mouse, people, macaque, rat and pig.IPS researchs particularly on mouse and people
Increasingly deep, the method for induction is also continuously available optimization, and the various cells of animal body tissue can be induced as iPS cells.
Due to iPS cells have the function of the characteristic similar with ES cells and, although ES cells can't be replaced completely, its success
Ground has got around the puzzlement of immune rejection problems and ethics morals problem, is the preferable seed cell of regenerative medicine, can also be used as and face
Bed drug screening cell model and human disease treatment cell model.Further, since iPS cells can pass steadily in the long term in vitro
It is commissioned to train foster, is preferable seed cell in transgenic technology, as gene targeting recipient cell, in the production of transgenic animals
Have broad application prospects.
Certainly, iPS technologies are not also very perfect, the problem run into and puzzlement at present, as rotaring transfecting mode, carcinoma gene,
Induced efficiency, iPS totipotencies etc., and a large amount of class iPS cells for being produced in Induction Process or incomplete reprogrammed cell are dry
Disturb so that really the iPS cells with totipotency are difficult to be obtained by screening quickly.It is believed that deepening continuously with research,
IPS technologies will be more ripe, and iPS cells will play the effect of bigger in life science.In IPS inductions at this stage
In multipotential stem cell preparation process, in used rotaring transfecting mode, the transfection carrier predominantly virus of use, RNA, plasmid etc.,
The rotaring transfecting mode of use is mainly electroporation transfection method, traditional electroporation transfection mode there are transfection efficiency it is low the drawbacks of,
Cause most cells not to be transfected since voltage is uneven in transfection process to cause to waste, and the cell and not after transfecting
Transfectional cell exists at the same time, and the purifying of cell causes difficulty；In order to ensure that the radix of Transfected cells needs the thin of a myriad of
Born of the same parents (more than 1,000,000), required plasmid high number, causes a large amount of plasmids and is wasted, unavoidably cause into
This rising, it is therefore desirable to which a kind of brand-new IPS cell transfectings training method carries out cell transfecting.
Main problem existing in the prior art has：1st, rotaring transfecting mode is penetrated using existing electricity and there is transfection electric voltage frequency not
The problem of unified, condition during cell transfecting is had differences, causes transfectional cell quality difference；2nd, the electricity of existing rotaring transfecting mode
Turn voltage control trouble, electric transfer efficient is low；3rd, existing electricity turns middle cell to ensure finally to obtain enough transfection adult cells,
Need to use at least more than 1,000,000 adult cells, and the substantial amounts of plasmid of consumption, cost unavoidably to raise；4th, adult cell
Need to carry out purifies and separates after transfection, clone cultivates, and transfectional cell and non-transfected cells exist at the same time in existing method, right
Have difficulties in cell purification, a large amount of manpower and materials are consumed for later stage cell culture.
The content of the invention
For defect existing in the prior art, it is an object of the invention to provide a kind of few cells electricity to turn micro-fluidic core
Piece, electricity turn sorter and application.Present invention employs microfluidic chip technology to carry out cell transfecting, and purifies and separates, pass through control
The voltage of micro-fluidic chip processed, can make cell carry out electricity under unified environment and turn, in control transfection process that can be good
Environment, ensure transfection progress it is identical, drastically increase the cell quantity of transfection.Chip can ensure used by the same time
Cell is uniformly distributed, and reduces the quantity of adult cell and the quantitative requirement of transfected plasmids in traditional approach.In micro-fluidic chip
In cell to be transfected can be fixed in micro-fluidic chip further groove, this set can ensure that cell in the orifice plate is all to be turned
The cell of dye, without being isolated and purified again after transfection, simplifies test procedure, and it is convenient to be provided for later stage cell culture,
And it ensure that the quality of inducing cell.
To achieve the above objectives, the present invention adopts the technical scheme that：
A kind of few cells electricity turns micro-fluidic chip, including：For add cell to be transfected and electricity turn buffer solution etc. into
Sample mouth, the final outlet for flowing out transfectional cell, can apply negative pressure (Negative pressure) and be used to adsorb fixed cell
Negative pressure duct, positive pressure (Positive pressure) can be applied and be used to input the positive pressure duct of transfection carrier plasmid, and be used for
Cell proper flow simultaneously completes the main channel that electricity turns over journey；
The few cells electricity, which turns micro-fluidic chip, to be included upper and lower two layers, and few cells electricity turns micro-fluidic chip upper and lower two
It is main channel between layer：The upper strata that the few cells electricity turns micro-fluidic chip is tablet, for connecting electrode, can apply electricity
Pressure；The lower floor that few cells electricity turns micro-fluidic chip is the orifice plate there are 96 grooves, and each groove center is equipped with for connecting
The microchannel in logical negative pressure duct and positive pressure duct, while each bottom portion of groove is laid with electrode contact and is used to apply voltage.
On the basis of such scheme, the height of the main channel is 50um.
On the basis of such scheme, each groove of the orifice plate is the ball that internal diameter is 100um, central angle is 60 °
Connected in star；The internal diameter of the microchannel is 1.5um.
On the basis of such scheme, 96 orifice plates are set in the outlet, for Transfected cells to be turned after dividing shape
Move on to and cell culture is carried out in 96 orifice plates.
On the basis of such scheme, the few cells electricity turns the application method of micro-fluidic chip, including following step
Suddenly：
Step 1, add cell and electricity to be transfected by injection port and turn buffer solution, cell and electricity to be transfected is turned buffer solution and fills
Full main channel；
Step 2, by chip it is upper and lower two layers application voltage, and by negative pressure duct apply negative pressure, by cell to be transfected
Promote and be fixed in chip lower bay；
Step 3, by positive pressure passageway, apply positive pressure, transfection carrier plasmid be transported in chip lower bay, with waiting to turn
Dye cell mixes；
Step 4, apply voltage by chip levels electrode, and formation impulse electric field, leads to the cell membrane of cell to be transfected
Permeability changes, and electricity is turned the transfection carrier plasmid in buffer solution and enters cell to be transfected by cell membrane, completes transfection；
After the completion of step 5, transfection, by controlling negative pressure duct, apply negative pressure, cell is completed into transfection and releases groove, is led to
Cross outlet and be transferred to 96 orifice plates and cultivated.
A kind of few cells electricity turns sorter, and the electricity turns sorter and turns unit, display screen, outer case, power supply including electricity
Unit, micro-control unit and master reference；
For the display location in the upper front end of outer case, electricity turns unit, power supply unit, micro-control unit and master reference
Positioned at the inside of outer case；Display screen is connected with master reference and micro-control unit respectively, and power supply unit turns unit with electricity respectively
Be connected with micro-control unit, electricity turns unit and is also connected with master reference and micro-control unit, master reference also with micro-control unit
It is connected；
Electricity turns unit and turns cell enclosure, fixed seat, electricity including electricity to turn sensor, controllable electrodes I and controllable electrodes II, fixed
Seat turns inside cell enclosure positioned at electricity, and is closely connected with the electric inner wall for turning cell enclosure, and electricity turns sensor, I and of controllable electrodes
Controllable electrodes II turn the inside of cell enclosure positioned at electricity；Supporting placement few cells electricity between controllable electrodes I and controllable electrodes II
Turn micro-fluidic chip.
On the basis of such scheme, the display screen is used to send control instruction to micro-control unit, and receives micro-control
Unit and master reference feedack processed and operation display interface and experimental state information；
The micro-control unit is used for the control instruction for receiving display screen transmission, and turns unit and power supply unit progress to electricity
Control；
The electricity turns unit and is used to complete cell transfecting process, and the electricity turns the transfection process that sensor is used to track cell
And send master reference to；
The master reference turns unit feedack for receiving electricity, and sends information to display screen and microcontroller list
Member.
On the basis of such scheme, controllable electrodes I are located at the top that few cells electricity turns micro-fluidic chip, controllable electrodes
II turns the lower section of micro-fluidic chip positioned at few cells electricity, and electricity turns the lower section that sensor is located at controllable electrodes II；
Top one end that electricity turns cell enclosure is equipped with note sample mouth I and note sample mouth II, and the note sample mouth I turns with few cells electricity
The injection port of micro-fluidic chip is corresponding, and note sample mouth II is corresponding with the positive pressure duct that few cells electricity turns micro-fluidic chip.
A kind of few cells electricity turns the application process of sorter, comprises the following steps：
Step 1, turn the note sample mouth I at the top of cell enclosure by electricity and inject the electricity containing cell to be transfected turn buffering to injection port
Liquid, turns buffer solution by noting sample mouth II to positive pressure duct electricity of the injection comprising transfection carrier plasmid；By display screen arrange parameter,
Micro-control unit turns unit to electricity according to the parameter of setting and power supply unit is controlled；
Step 2, according to the parameter set in step 1, in control note sample mouth I cell electricity to be transfected turn buffer solution enter it is micro
Cell electricity turns micro-fluidic chip main channel, cell to be transfected is uniformly full of in whole main channel；
Step 3, according to the parameter set in step 1, applying micro voltage by controllable electrodes I and controllable electrodes II will be free
Cell to be transfected push-in groove in, while negative pressure duct applies the negative pressure of appropriate intensity, concentrates absorption to exist in cell to be transfected
Each groove center, each groove one cell to be transfected of interior absorption；
Step 4, according to the parameter set in step 1, the electricity of plasmid containing transfection carrier turns buffer solution and leads in control note sample mouth II
Positive pressure duct is crossed to inject in chip lower bay by microchannel；
Step 5, control few cells electricity turns the voltage and frequency of upper and lower two layers of micro-fluidic chip, forms impulse electric field, makes
The cell membrane penetration of cell to be transfected sexually revises, and the electric transfection carrier plasmid turned in buffer solution is entered by cell membrane and treats
Transfectional cell, completes transfection；
In transfection process, the master reference receives electricity and turns unit feedack, and send information to display screen and
Micro-control unit, and real-time operation interface and status information are shown in display screen；
Step 6, after transfection, apply micro voltage by controllable electrodes I and controllable electrodes II and be pushed into Transfected cells
In main channel, and electricity is persistently filled by injection port and turns buffer solution, Transfected cells is turned buffer solution with electricity and pass through outlet stream
Go out, the sorted formation of Transfected cells is unicellular, it is transferred in 96 orifice plates and carries out cell culture.
On the basis of such scheme, the parameter includes cell sample size to be transfected, electricity turns buffer solution sample size, electricity turns
Voltage, frequency, sample introduction flow and flow rate etc..
It is an advantage of the current invention that can ensure that the condition in whole transfection process in main channel is completely the same, ensure
Transfection state in transfection is identical, while also ensure that the efficiency of transfection.The consumptive material and cell used in the process
It is micro Deng all, saves consumables cost.Under micro-fluidic state the process of transfection can be controlled to ensure that the efficiency of transfection,
The content of transfectional cell can greatly be improved.And during cell sorting, acted on by the retention of orifice plate, it is ensured that
It is high that cell content is transfected in assorting room, the purification degrees of transfectional cell can be made higher, easy to the cell culture in later stage.Chip
Cell after transfection can be directly transferred to 96 orifice plates of culture cell, simplified by inside there are 96 grooves after undue shape
Operating process, ensure that cell quality.
Brief description of the drawings
The present invention has drawings described below：
Fig. 1 few cells electricity turns the structure chart of micro-fluidic chip；
Fig. 2 few cells electricity turns micro-fluidic chip and uses schematic diagram one；
Fig. 3 few cells electricity turns micro-fluidic chip and uses schematic diagram two；
Fig. 4 few cells electricity turns micro-fluidic chip and uses schematic diagram three；
Fig. 5 few cells electricity turns the external structure schematic diagram of sorter；
Fig. 6 few cells electricity turns the internal structure schematic diagram of sorter；
Fig. 7 electricity turns the internal structure schematic diagram of unit；
Fig. 8 few cells electricity turns the attachment structure schematic diagram of sorter.
Reference numeral：
1- outer cases, 2- display screens, 3- master references, 4- electricity turn unit, 5- power supply units, 6- micro-control units, and 7- is micro-
Amount cell electricity turns micro-fluidic chip, and 8- electricity turns cell enclosure, 9- fixed seats, 10- controllable electrodes I, 11- controllable electrodes II, 12- electricity
Turn sensor.
Embodiment
Below in conjunction with attached drawing, 1~8 couple of present invention is described in further detail.
A kind of few cells electricity of the present invention turns micro-fluidic chip, including：Turn for adding cell to be transfected and electricity
The injection port A of buffer solution, the final outlet B for flowing out transfectional cell, can apply negative pressure (Negative pressure) and be used for
The negative pressure duct C of the fixed cell of absorption, can apply positive pressure (Positive pressure) and be used to input transfection carrier plasmid just
Duct D is pressed, and for cell proper flow and completes the electric main channel E for turning over journey；
The few cells electricity turns micro-fluidic chip 7 including being main channel E between upper and lower two layers, upper and lower two layers：It is described
The upper strata that few cells electricity turns micro-fluidic chip 7 is tablet, for connecting electrode, can apply voltage；Few cells electricity turns miniflow
The lower floor of chip 7 is controlled there are 96 grooves, each groove center is equipped with for connecting the micro- of negative pressure duct C and positive pressure duct D
Duct, while each bottom portion of groove is laid with electrode contact and is used to apply voltage.
On the basis of such scheme, the height of the main channel E is 50um.
On the basis of such scheme, each groove of the orifice plate is the ball that internal diameter is 100um, central angle is 60 °
Connected in star；The internal diameter of the microchannel is 1.5um.
On the basis of such scheme, the outlet B set 96 orifice plates, for by Transfected cells after undue shape
It is transferred in 96 orifice plates and carries out cell culture.
On the basis of such scheme, the few cells electricity turns the application method of micro-fluidic chip, including following step
Suddenly：
Step 1, cell and electricity to be transfected are added by injection port and turns buffer solution, cell and electricity to be transfected is turned buffer solution and fill
Full main channel；
Step 2, by upper and lower two layers of application voltage of chip, and by negative pressure duct application negative pressure, by cell to be transfected
Promote and be fixed in chip lower bay；
Step 3, by positive pressure passageway, apply positive pressure, transfection carrier plasmid is transported in chip lower bay, with waiting to turn
Dye cell mixes；
Step 4, voltage is applied by chip levels electrode, forms impulse electric field, lead to the cell membrane of cell to be transfected
Permeability changes, and electricity is turned the transfection carrier plasmid in buffer solution and enters cell to be transfected by cell membrane, completes transfection；
Step 5, after the completion of transfection, by controlling negative pressure duct, apply negative pressure, cell is completed into transfection and releases groove, is led to
Cross outlet and be transferred to 96 orifice plates and cultivated.
A kind of few cells electricity turns sorter, and the electricity turns sorter and turns unit 4, display screen 2, outer case 1, electricity including electricity
Source unit 5, micro-control unit 6 and master reference 3；
The display screen 2 is located at the upper front end of outer case 1, and electricity turns unit 4, power supply unit 5, micro-control unit 6 and master
Sensor 3 is located at the inside of outer case 1；Display screen 2 is connected with master reference 3 and micro-control unit 6 respectively, and power supply unit 5 divides
Do not turn unit 4 with electricity and micro-control unit 6 is connected, electricity turns unit 4 and is also connected with master reference 3 and micro-control unit 6, main sensing
Device 3 is also connected with micro-control unit 6；
Electricity turns unit 4 and turns cell enclosure 8, fixed seat 9, electricity including electricity to turn sensor 12, controllable electrodes I 10 and controllable electrodes
II 11, fixed seat 9 is located at electricity and turns inside cell enclosure 8, and is closely connected with the electric inner wall for turning cell enclosure 8, and electricity turns sensor
12nd, controllable electrodes I 10 and controllable electrodes II 11 turn the inside of cell enclosure 8 positioned at electricity；Controllable electrodes I 10 and controllable electrodes II 11
Between it is supporting placement few cells electricity turn 7 and 96 orifice plate of micro-fluidic chip.
On the basis of such scheme, the display screen 2 is used to send control instruction to micro-control unit 6, and receives micro-
Control unit 6 and 3 feedack of master reference and operation display interface and experimental state information；
The micro-control unit 6 is used for the control instruction for receiving the transmission of display screen 2, and turns unit 4 and power supply unit 5 to electricity
It is controlled；
The electricity turns unit 4 and is used to complete cell transfecting process, and the electricity turns the transfection that sensor 12 is used to track cell
Process simultaneously sends master reference 3 to；
The master reference 3 turns 4 feedack of unit for receiving electricity, and sends information to display screen 2 and micro-control
Unit 6 processed.
On the basis of such scheme, controllable electrodes I 10 are located at the top that few cells electricity turns micro-fluidic chip 7, controllable
Electrode II 11 is located at the lower section that few cells electricity turns micro-fluidic chip 7, and electricity turns the lower section that sensor 12 is located at controllable electrodes II 11；
Top one end that electricity turns cell enclosure 8 is equipped with note sample mouth I and note sample mouth II, the note sample mouth I and few cells electricity
Turn that the injection port A of micro-fluidic chip 7 is corresponding, note sample mouth II is opposite with the positive pressure duct D that few cells electricity turns micro-fluidic chip 7
Should.
A kind of few cells electricity turns the application process of sorter, comprises the following steps：
Step 1, the note sample mouth I for turning the top of cell enclosure 8 by electricity turns slow to electricity of the injection port A injections containing cell to be transfected
Fliud flushing, turns buffer solution by noting sample mouth II to positive pressure duct electricity of the injection comprising transfection carrier plasmid；Set and joined by display screen 2
Number, micro-control unit 6 turns unit 4 to electricity according to the parameter of setting and power supply unit 5 is controlled；
Step 2, according to the parameter set in step 1, in control note sample mouth I cell electricity to be transfected turn buffer solution enter it is micro
Cell electricity turns micro-fluidic chip main channel E, cell to be transfected is uniformly full of in whole main channel E；
Step 3, according to the parameter set in step 1, applying micro voltage by controllable electrodes I and controllable electrodes II will be free
Cell to be transfected push-in groove in, while negative pressure duct C applies the negative pressure of appropriate intensity, cell to be transfected absorption that will be free
Entreat in each recess, one cell to be transfected of absorption in each groove；
Step 4, according to the parameter set in step 1, transfection carrier matter is included to positive pressure duct D injections by noting sample mouth II
The electricity of grain turns buffer solution and applies the positive pressure of appropriate intensity, the electricity comprising transfection carrier plasmid is turned buffer solution and is noted by microchannel
In the groove for entering chip lower floor；
Step 5, control few cells electricity turns the voltage and frequency of 7 upper and lower two layers of micro-fluidic chip, forms impulse electric field,
The cell membrane penetration of cell to be transfected is sexually revised, the transfection carrier plasmid that electricity turns in buffer solution is entered by cell membrane
Cell to be transfected, completes transfection；
In transfection process, the master reference 3 receives electricity and turns 4 feedack of unit, and sends information to display screen 2
With micro-control unit 6, and real-time operation interface and status information are shown in display screen 2；
Step 6, after transfection, apply micro voltage by controllable electrodes I and controllable electrodes II and be pushed into Transfected cells
In the E of main channel, electricity is persistently filled by injection port A and turns buffer solution, Transfected cells is turned buffer solution with electricity and flowed by outlet B
Go out, sorted be transferred in 96 orifice plates of Transfected cells is subjected to cell culture.
On the basis of such scheme, the parameter includes cell sample size to be transfected, electricity turns buffer solution sample size, electricity turns
Voltage, frequency, sample introduction flow and flow rate etc..
Few cells electricity of the present invention turns micro-fluidic chip product, which is formed by upper and lower two layers, and upper strata is flat
Plate, connects electrode, and lower floor is orifice plate, and containing 96 spherical grooves, there are microchannel, microchannel connection positive and negative pressure in groove center
Duct, the chip include injection port, main channel and outlet at the same time.
During electricity turns, the voltage of electricity turn and frequency etc. can be adjusted according to different cells.It is described micro
Cell electricity turns micro-fluidic chip connection micro-control unit, and the voltage and frequency needed for adjustment are controlled by the micro-control unit
Deng.Turning micro-fluidic chip using few cells electricity described herein can all cell category of electricity turn.
The content not being described in detail in this specification belongs to the prior art known to professional and technical personnel in the field.
Claims (10)
1. a kind of few cells electricity turns micro-fluidic chip, it is characterised in that：Including turning buffering for adding cell to be transfected and electricity
The injection port of liquid, can apply the negative pressure duct that negative pressure is used to adsorb fixed cell, can apply positive pressure and be used to input transfection carrier matter
The positive pressure duct of grain, and the main channel that electricity turns over journey is flowed and completed for cell, the final outlet for flowing out Transfected cells；
The few cells electricity, which turns micro-fluidic chip, to be included upper and lower two layers, few cells electricity turn upper and lower two layers of micro-fluidic chip it
Between be main channel：The upper strata that the few cells electricity turns micro-fluidic chip is tablet, can connect electrode and apply voltage；Few cells
Electricity turn micro-fluidic chip lower floor be there are 96 grooves orifice plate, each groove center be equipped with for connect negative pressure duct and
The microchannel in positive pressure duct, each bottom portion of groove, which is laid with microelectrode contact pin, can apply voltage.
2. few cells electricity as claimed in claim 1 turns micro-fluidic chip, it is characterised in that：The height of the main channel is
50um。
3. few cells electricity as claimed in claim 1 turns micro-fluidic chip, it is characterised in that：Each groove of the orifice plate is equal
The spherical groove that for internal diameter be 100um, central angle is 60 °；The internal diameter of the microchannel is 1.5um.
4. few cells electricity as claimed in claim 1 turns micro-fluidic chip, it is characterised in that：In the outlet, 96 holes are set
Plate, cell culture is carried out for being transferred directly to after transfectional cell is sorted in 96 orifice plates.
5. few cells electricity as claimed in claim 1 turns the application method of micro-fluidic chip, it is characterised in that including following step
Suddenly：
Step 1, cell and electricity to be transfected are added by injection port and turns buffer solution, cell and electricity to be transfected is turned buffer solution full of master
Passage；
Step 2, by upper and lower two layers of application voltage of chip, and by negative pressure duct application negative pressure, cell to be transfected is promoted
And it is fixed in chip lower bay；
Step 3, by positive pressure passageway, apply positive pressure, transfection carrier plasmid is transported in chip lower bay, it is and to be transfected thin
Born of the same parents mix；
Step 4, voltage is applied by chip levels electrode, forms impulse electric field, make the permeability of cell membrane of cell to be transfected
Change, electricity is turned the transfection carrier plasmid in buffer solution and cell to be transfected is entered by cell membrane, complete transfection；
Step 5, after the completion of transfection, by controlling negative pressure duct, apply negative pressure, cell is completed into transfection and releases groove, by going out
Sample mouth is transferred to 96 orifice plates and is cultivated.
6. a kind of few cells electricity turns sorter, turn micro-fluidic chip using the few cells electricity described in claim 1, its feature
It is：Turn unit, display screen, outer case, power supply unit, micro-control unit and master reference including electricity；
In the upper front end of outer case, electricity turns unit, power supply unit, micro-control unit and master reference and is located at the display location
The inside of outer case；Display screen is connected with master reference and micro-control unit respectively, and power supply unit turns unit and micro- with electricity respectively
Control unit is connected, and electricity turns unit and is also connected with master reference and micro-control unit, and master reference is also connected with micro-control unit；
Electricity turns unit and turns cell enclosure, fixed seat, electricity including electricity to turn sensor, controllable electrodes I and controllable electrodes II, fixed seating
Turn in electricity inside cell enclosure, and turn the inner wall of cell enclosure with electricity and be closely connected, electricity turns sensor, controllable electrodes I and controllable
Electrode II turns the inside of cell enclosure positioned at electricity；Supporting placement few cells electricity turns micro- between controllable electrodes I and controllable electrodes II
Fluidic chip and 96 orifice plates.
7. few cells electricity as claimed in claim 6 turns sorter, it is characterised in that：The display screen is used for microcontroller list
Member sends control instruction, receives micro-control unit and master reference feedack and operation display interface and experimental state are believed
Breath；
The micro-control unit is used for the control instruction for receiving display screen transmission, and turns unit to electricity and power supply unit is controlled
System；
The electricity turns unit and is used to complete cell transfecting process, and the electricity turns the transfection process and biography that sensor is used to track cell
Give master reference；
The master reference turns unit feedack for receiving electricity, and sends information to display screen and micro-control unit.
8. few cells electricity as claimed in claim 6 turns sorter, it is characterised in that：Controllable electrodes I are located at few cells electricity
Turn the top of micro-fluidic chip, controllable electrodes II are located at the lower section that few cells electricity turns micro-fluidic chip, and electricity turns sensor and is located at
The lower section of controllable electrodes II；
Top one end that electricity turns cell enclosure is equipped with note sample mouth I and note sample mouth II, and the note sample mouth I turns miniflow with few cells electricity
It is corresponding to control the injection port of chip, note sample mouth II is corresponding with the positive pressure duct that few cells electricity turns micro-fluidic chip.
9. the few cells electricity as described in claim 6~8 any claim turns the application process of sorter, its feature exists
In：Comprise the following steps：
Step 1, turn the note sample mouth I at the top of cell enclosure by electricity and turn buffer solution to electricity of the injection port injection containing cell to be transfected,
Turn buffer solution to positive pressure duct electricity of the injection comprising transfection carrier plasmid by noting sample mouth II；It is micro- by display screen arrange parameter
Control unit turns unit to electricity according to the parameter of setting and power supply unit is controlled；
Step 2, according to the parameter set in step 1, cell electricity to be transfected turns buffer solution and enters few cells in control note sample mouth I
Electricity turns micro-fluidic chip main channel, cell to be transfected is uniformly full of in whole main channel；
Step 3, according to the parameter set in step 1, apply micro voltage by controllable electrodes I and controllable electrodes II and treat free
In transfectional cell push-in groove, while negative pressure duct applies the negative pressure of appropriate intensity, and cell to be transfected is concentrated absorption each
Groove center, each groove one cell to be transfected of interior absorption；
Step 4, according to the parameter set in step 1, the electricity of plasmid containing transfection carrier turns buffer solution and passes through just in control note sample mouth II
Duct is pressed to be injected by microchannel in chip lower bay；
Step 5, control few cells electricity turns the voltage and frequency of upper and lower two layers of micro-fluidic chip, forms impulse electric field, makes to wait to turn
The cell membrane penetration of dye cell sexually revises, and the electric transfection carrier plasmid turned in buffer solution is entered by cell membrane to be transfected
Cell, completes transfection；
In transfection process, the master reference receives electricity and turns unit feedack, and sends information to display screen and micro-control
Unit processed, and real-time operation interface and status information are shown in display screen；
Step 6, after transfection, micro voltage is applied by controllable electrodes I and controllable electrodes II Transfected cells push-in is main logical
In road, and electricity is persistently filled by injection port and turns buffer solution, Transfected cells is turned buffer solution with electricity and flowed out by outlet, will
The sorted formation of Transfected cells is unicellular, is transferred in 96 orifice plates and carries out cell culture.
10. few cells electricity as claimed in claim 9 turns the application process of sorter, it is characterised in that：The parameter includes
Cell sample size to be transfected, electricity turns buffer solution sample size, electricity turns voltage, frequency, sample introduction flow and flow rate.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710456836 | 2017-06-13 | ||
| CN2017104568366 | 2017-06-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107988070A true CN107988070A (en) | 2018-05-04 |
Family
ID=62039178
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711379237.5A Pending CN107988070A (en) | 2017-06-13 | 2017-12-13 | A kind of few cells electricity turns micro-fluidic chip, electricity turns sorter and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107988070A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728328A (en) * | 2018-05-30 | 2018-11-02 | 中北大学 | The micro-current controlled cell for integrating unicellular capture sorts chip |
| CN110468048A (en) * | 2019-09-16 | 2019-11-19 | 许中伟 | Micromation device and method based on microfluidic system control production engineering immunocyte |
| WO2020047504A3 (en) * | 2018-08-31 | 2020-04-09 | The Charles Stark Draper Laboratory, Inc. | Method and apparatus for high throughput high efficiency transfection of cells |
| WO2020096690A3 (en) * | 2018-09-04 | 2020-08-27 | The Charles Stark Draper Laboratory, Inc. | Method and device for high field strength electrotransfection of microvescicles and cells |
| CN114196532A (en) * | 2021-11-01 | 2022-03-18 | 上海盟德生物科技有限公司 | Multi-channel cell electrotransformation instrument |
| CN116656744A (en) * | 2023-06-08 | 2023-08-29 | 上海科技大学 | Use of microfluidic devices in cell electroporation or exogenous material introduction into cells |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080081372A1 (en) * | 2006-09-30 | 2008-04-03 | Yong Huang | High-throughput cell transfection device and methods of using thereof |
| CN101486964A (en) * | 2009-03-06 | 2009-07-22 | 北京大学 | Cell electroporation method based on micro-fluidic chip and special chip therefor |
| CN101541945A (en) * | 2006-11-29 | 2009-09-23 | 皇家飞利浦电子股份有限公司 | Cell array or matrix assembly and electroporation |
| CN102656260A (en) * | 2009-10-19 | 2012-09-05 | 瑞生生物技术有限公司 | Method, device and apparatus for inducing self-adjusting cell electroporation |
| CN103320319A (en) * | 2013-07-05 | 2013-09-25 | 博奥生物有限公司 | Alternative high-throughput electroporation device |
| CN104357323A (en) * | 2014-12-02 | 2015-02-18 | 陈剑 | Cell electroporation apparatus |
-
2017
- 2017-12-13 CN CN201711379237.5A patent/CN107988070A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080081372A1 (en) * | 2006-09-30 | 2008-04-03 | Yong Huang | High-throughput cell transfection device and methods of using thereof |
| CN101541945A (en) * | 2006-11-29 | 2009-09-23 | 皇家飞利浦电子股份有限公司 | Cell array or matrix assembly and electroporation |
| CN101486964A (en) * | 2009-03-06 | 2009-07-22 | 北京大学 | Cell electroporation method based on micro-fluidic chip and special chip therefor |
| CN102656260A (en) * | 2009-10-19 | 2012-09-05 | 瑞生生物技术有限公司 | Method, device and apparatus for inducing self-adjusting cell electroporation |
| CN103320319A (en) * | 2013-07-05 | 2013-09-25 | 博奥生物有限公司 | Alternative high-throughput electroporation device |
| CN104357323A (en) * | 2014-12-02 | 2015-02-18 | 陈剑 | Cell electroporation apparatus |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728328A (en) * | 2018-05-30 | 2018-11-02 | 中北大学 | The micro-current controlled cell for integrating unicellular capture sorts chip |
| CN108728328B (en) * | 2018-05-30 | 2021-11-16 | 中北大学 | Microfluidic cell sorting chip integrated with single cell capture |
| WO2020047504A3 (en) * | 2018-08-31 | 2020-04-09 | The Charles Stark Draper Laboratory, Inc. | Method and apparatus for high throughput high efficiency transfection of cells |
| US11859162B2 (en) | 2018-08-31 | 2024-01-02 | The Charles Stark Draper Laboratory, Inc. | Method and apparatus for high throughput high efficiency transfection of cells |
| WO2020096690A3 (en) * | 2018-09-04 | 2020-08-27 | The Charles Stark Draper Laboratory, Inc. | Method and device for high field strength electrotransfection of microvescicles and cells |
| US11878300B2 (en) | 2018-09-04 | 2024-01-23 | The Charles Stark Draper Laboratory, Inc. | Method and device for high field strength electrotransfection of microvescicles and cells |
| CN110468048A (en) * | 2019-09-16 | 2019-11-19 | 许中伟 | Micromation device and method based on microfluidic system control production engineering immunocyte |
| CN110468048B (en) * | 2019-09-16 | 2022-09-02 | 许中伟 | Micro-scale equipment and method for controlling production of engineered immune cells based on micro-fluidic system |
| CN114196532A (en) * | 2021-11-01 | 2022-03-18 | 上海盟德生物科技有限公司 | Multi-channel cell electrotransformation instrument |
| CN116656744A (en) * | 2023-06-08 | 2023-08-29 | 上海科技大学 | Use of microfluidic devices in cell electroporation or exogenous material introduction into cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988070A (en) | A kind of few cells electricity turns micro-fluidic chip, electricity turns sorter and application | |
| Ao et al. | One-stop microfluidic assembly of human brain organoids to model prenatal cannabis exposure | |
| Takebe et al. | Synergistic engineering: organoids meet organs-on-a-chip | |
| Duzagac et al. | Microfluidic organoids-on-a-chip: Quantum leap in cancer research | |
| KR102402037B1 (en) | Bioreactor and method of use thereof | |
| Ghaemmaghami et al. | Biomimetic tissues on a chip for drug discovery | |
| Kokovay et al. | The incredible elastic brain: how neural stem cells expand our minds | |
| CN106544271A (en) | A kind of many cells 3D co-culture devices and method of research tumor invasion blood vessel | |
| Faber et al. | Through the looking glass: in vitro models for inhalation toxicology and interindividual variability in the airway | |
| CN102124096A (en) | Organ mimic device with microchannels and methods of use and manufacturing thereof | |
| Borrell | In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation | |
| CN104640971A (en) | Formation and use of neuronal network, and neuron seeding device | |
| CN107354094A (en) | The equally distributed micro-current controlled cell culture chip of cell and cell sample injection method | |
| CN103877631A (en) | Bioartificial liver system | |
| US20120315694A1 (en) | Cell culture unit and cell culture device including the same | |
| Jayasooriya et al. | mRNA-based CAR T-cells manufactured by miniaturized two-step electroporation produce selective cytotoxicity toward target cancer cells | |
| CN207958390U (en) | A kind of few cells electricity turns micro-fluidic chip and electricity turns sorter | |
| CN112852627A (en) | Method for co-culturing pluripotent stem cells from human liver and pancreatic islets based on multi-organ chip | |
| Yu et al. | Emerging strategies of engineering retinal organoids and organoid-on-a-chip in modeling intraocular drug delivery: Current progress and future perspectives | |
| CN103131635B (en) | Cavity-type dynamic-filling bioreaction device | |
| CN116103152B (en) | Organoid chip model for drug testing | |
| CN106244416A (en) | Cell microinjection chip based on dielectrophoresis, injection device and method of work thereof | |
| ES2936343T3 (en) | Device for dividing objects and dividing procedure | |
| CN107937340A (en) | Catalpol is promoting application of the neural stem cell into oligodendrocyte differentiation | |
| CN206799584U (en) | Micro-current controlled cell sorting unit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |