CN109554333A - A method of unicellular sorting is carried out using the unicellular separator of Namocell - Google Patents
A method of unicellular sorting is carried out using the unicellular separator of Namocell Download PDFInfo
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Abstract
CHO-K1 cell to be sorted: being diluted to the viable cell density of 1E5cells/mL by a kind of method that the present invention discloses unicellular sorting, comprising the following steps: 1) sample prepares with secondary culture base, and calcein reagent dyeing is added；After the completion of dyeing, cell is diluted to 5000cells/mL using secondary culture base；2) sample loads: cell is injected into chip cavity, then loads chip to be detected by the starting unicellular separator of Namocell, and 3) position correction: adjustment laser facula to microfluidic channel center；4) unicellular sorting parameter setting: setting fluorescent value are as follows: FITCH=500, FITCL=30, PEH=3, PEL=0；5) unicellular sorting: starting to sort unicellular, accurately captures competent cell by high-velocity electrons valve, and ultimately form the drop of 1 μ L volume, sorts into orifice plate, and every hole sorts 1 cell.Method of the invention can make the recovery rate for improving the unicellular efficiency of separation and monoclonal, other sortings of CHO-K1 cell and condition of culture compared to the prior art have significant progress.
Description
Technical field
The invention belongs to bio-pharmaceuticals and field of biotechnology, more particularly to use the unicellular separator of Namocell into
The method of the unicellular sorting of row, can be applied to the building of Cells for production strain.
Background technique
Monoclonal antibody has the characteristics that targeting is strong, specificity is high low with toxic side effect, has been used successfully to control at present
Treat a variety of disease areas such as immune, cancer.For producing the stable cell line of monoclonal antibody, generally by mammal table
It is screened up to system, wherein Chinese hamster ovary cell (Chinese Hamster Ovary, CHO) is most common
One of host cell.In order to guarantee the safety of biological medicament, there is very the monoclonicity of Cells for production strain in supervision department
Strict requirements, therefore, cell monoclonal are a steps very crucial in entire Cells for production strain building process.
Cell monoclonal frequently with means include the following: semisolid culturemedium selecting method, limiting dilution assay, streaming are thin
The unicellular separator separating method of born of the same parents' instrument separating method, Namocell.
Semisolid culturemedium selecting method: cell inoculation (be joined into the fluorescence of identification target product in semisolid culturemedium
Labelled antibody) in, individual cells will form a group after a couple of days.In conjunction with ClonePix (Molecular Devices) etc.
Instrument can achieve higher monoclonal rate by the experiment of at least two-wheeled, but increase time and economic cost.
Traditional limiting dilution assay: cell is gradually diluted to very low density and is inoculated in 96 orifice plates, in conjunction with Cell
The Image-forming instruments such as Metric (Solentim) can obtain higher monoclonal rate by a wheel experiment.But due to this method
Non- slender hilum is more when bed board, and cell restores limited, so limiting dilution assay is also a kind of lower monocloning method of efficiency.
Selected by flow cytometry apoptosis method: this method is carried out by means of intracellular fluorescent marker, or to cell secretory product
Fluorescent staining can effectively sub-elect the high clone of expression quantity, greatly improve the efficiency of separation and monoclonal rate.Shortcoming
It is that the pressure of sheath fluid has certain injury to cell, used consumptive material is expensive, and the cleaning process complexity of instrument is cumbersome
It is and time-consuming, it is also necessary to which that special messenger is responsible for maintenance, expends economy, time and human cost.
The unicellular separator separating method of Namocell: using microflow control technique, can be in a mild low pressure force environment
Under, while laser excitation is utilized, and the testing principle of fluorescence reception quickly finishes the sorting of cell, the injury to cell is reduced,
Ensure the activity and function of cell.The instrument has efficient sorting capability, and one piece of 96 orifice plate can be separated in 1 minute.It adopts
With disposable sterilized chip, it is ensured that sterile sorting avoids the cross contamination between sample.And the instrument price is suitable
In, it is easy to operate, it is safeguarded without special messenger.To sum up, unicellular using the unicellular separator sorting of Namocell, there is efficient, nothing
Bacterium, it is easy to operate, economical and practical the advantages that.But the unicellular efficiency of separation and monoclonal of the unicellular separator of Namocell
Recovery rate influenced by cell sorting and condition of culture very big, the sorting of different cells and condition of culture differentiation are significant,
Therefore, it is suitably sorted for the exploitation of certain specific cells and condition of culture is to improve the unicellular efficiency of separation and monoclonal
Recovery rate, be those skilled in the art's problem to be solved.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of slender using the unicellular separator progress of Namocell
The method of born of the same parents' sorting, to further increase the recovery rate of the unicellular efficiency of separation and monoclonal.
In order to solve the above technical problems, the present invention provides a kind of method of unicellular sorting, comprising the following steps:
1) sample prepares: CHO-K1 cell to be sorted is diluted to the living cells of 1E5cells/mL with secondary culture base
Calcein reagent is added to the final concentration of 10nM of calcein, by cell dyeing 5 minutes in density；After the completion of dyeing, biography is used
Cell is diluted to 5000cells/mL for culture medium, it is contemplated that separation velocity 1.6cells/s is used for unicellular sorting；
2) sample loads: the cell that step 1) obtains is injected into chip cavity by the starting unicellular separator of Namocell
In, chip to be detected is then loaded, hole sheet tray is moved to immediately below chip the drop for receiving to come out from nozzle；
3) position correction: adjustment laser facula to microfluidic channel center；
4) unicellular sorting parameter setting: setting fluorescent value are as follows: FITCH=500, FITCL=30, PEH=3, PEL=0；
5) unicellular sorting: starting to sort unicellular, and unicellular stream flows through laser detection region in microfluidic channel, right
Cell is identified and is got rid of cell fragment, dead cell and the poor cell of state, is accurately caught by high-velocity electrons valve
Competent cell is obtained, and ultimately forms the drop of 1 μ L volume, is sorted into orifice plate, every hole sorts 1 cell.
Specifically, the CHO-K1 cell to be sorted first is filtered with cell strainer before step 1), cell is then used
Calculating instrument counts.
Specifically, the calcein reagent is the DMSO solution of calcein in step 1), concentration is 100 μM.
Specifically, cell is injected into chip cavity by disposable chip upper end well in step 2).
Specifically, in step 2), when the unicellular separator of starting Namocell, hole sheet tray is mobile and goes back to original position,
Air pump is opened, and waste liquid line ports have drop outflow.
Specifically, the orifice plate is 96 orifice plates in step 5), preparatory every hole joined 100 μ L culture mediums.
Specifically, the method also includes cell culture after sorting: after the completion of sorting, by cell in carbon dioxide culture
Case stationary culture is taken pictures behind 2h, 1 day, 2 days, 5 days and 9 days using Cell Metric monoclonal cell strain analyzer respectively,
After clonal growth recovery, by clonal expansion, to carry out subsequent screening.
Preferably, the condition of culture of carbon dioxide incubator is 36.5 DEG C, 6%CO2。
The present invention provides points that the unicellular separator of Namocell carries out unicellular sorting and Cells for production strain building
Choosing and condition of culture, solve the problems, such as to further increase the recovery rate of the unicellular efficiency of separation and monoclonal.It is existing to have
Limit dilution cloning process, it is assumed that according to 0.4 cells/well bed board, the percentage in every piece of 96 orifice plate monoclonal holes is generally 20% left side
The right side, monoclonal recovery rate are usually less than 50%.Using method of the invention, the unicellular separator of Namocell carries out unicellular point
Choosing, the unicellular efficiency of separation are 80.5%, and the average recovery rate of monoclonal is up to 66.6%, has all reached very high level.Phase
Compared with semisolid culturemedium selecting method and limiting dilution assay, the efficiency of monoclonal is greatly improved.Simultaneously compared to fluidic cell
Instrument sorting, it is easy to operate without carrying out the cleaning and maintenance of instrument using disposable sterilized chip, further improve efficiency.
And without consuming expensive consumptive material, time, economy and human cost are reduced.
Specific embodiment
Clear, complete description will be carried out to technical solution of the present invention below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art exist
Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.
The unicellular separator of the Namocell that the present invention is developed carries out unicellular method for separating, can be applied to CHO-K1 etc.
The production building of stable cell line.
Embodiment one
A kind of specific implementation method is as follows:
1, the preparation of fluorescent dyeing reagent: calcein (Calcein-AM) is a kind of fluorescent dyeing reagent, excitation wavelength
It is 495nm and 515nm respectively with launch wavelength, living cells can be dyed.It will using sterile dimethyl sulfoxide (DMSO)
Calcein-AM (ThermoFisher, C3100MP) is configured to the solution that concentration is 100 μM (10,000 ×), in case using.
2, sample prepares: taking out 3~5mL CHO-K1 cell to be sorted, is filtered with 40 μm of cell strainers, then used
Vi-cell is counted.The viable cell density of 1E5cells/mL is diluted to using secondary culture base, according to 1:10,000 ratio adds
The Calcein-AM for entering 100 μM made the final concentration of 10nM of Calcein-AM, by cell dyeing 5 minutes.After the completion of dyeing, make
Cell is diluted to 5,000cells/mL (it is expected that separation velocity is 1.6cells/s) with secondary culture base, is used for unicellular point
Choosing.
3, instrument starts: opening the switch of the unicellular separator of Namocell, opens software, software interface lower left state
Item becomes green and illustrates online success.The starting of click system, hole sheet tray can move and go back to original position, and air pump can be opened, and give up
Liquid line ports have drop outflow.
4, sample loads: by cell by disposable chip upper end well, being injected into chip cavity.Then it loads
Chip to be detected, hole sheet tray are moved to immediately below chip the drop for receiving to come out from nozzle.
5, position correction: adjustment laser facula to microfluidic channel center.
6, unicellular sorting parameter setting: clicking Sorting (sorting) menu, starts Analysis (analysis), selection
FITC is Trigger (trigger condition), start recording cell signal.Analysis is again tapped on, stops analysis, in operating software
The upper suitable fluorescent value range of setting, such as FITCH=500, FITCL=30, PEH=3, PEL=0.
7, unicellular sorting: 96 orifice plates are put into instrument, 100 μ L culture mediums are added in every hole to 96 orifice plates in advance.It is soft in instrument
All holes in 96 orifice plates are chosen on part, selected hole will appear white and beat hook mark.Dispensing is clicked, instrument starts point
Menu cell.Unicellular stream flows through laser detection region in microfluidic channel, and machine can identify cell, gets rid of thin
Born of the same parents' fragment, dead cell and the poor cell of state, so that the cell eventually fallen in orifice plate all has good activity.At a high speed
Electronic valve can accurately capture each competent cell, ultimately form the drop of 1 μ L volume, sort every hole into 96 orifice plates
Sort 1 cell.
8, after sorting terminates, cell cell culture after sorting: is placed on carbon dioxide incubator stationary culture (36.5
DEG C, 6%CO2), it is taken pictures after 2h, 1 day, 2 days, 5 days and 9 days using Cell Metric respectively, it, will after clonal growth recovery
Clonal expansion, to carry out subsequent screening.
Embodiment two
Method based on embodiment one carries out the unicellular separator sorting clone recovery rate assessment of amocell:
100 μ L culture mediums are added in the every hole of 96 orifice plates in advance, carry out unicellular point using the unicellular separator of Namocell
Choosing, every hole sort 1 cell.After sorting, cell exists respectively at carbon dioxide incubator stationary culture (36.5 DEG C, 6%CO2)
It is taken pictures behind 2h, 1 day, 2 days, 5 days and 9 days using Cell Metric.After 9 days, the recovery situation of cell is assessed.Assess cell
Recovery situation is as shown in table 1.
Clone's recovery rate of the 1 unicellular sorting of unicellular printer of table
| 96 orifice plate numbers | Unicellular hole count | The unicellular efficiency of separation | The unicellular hole count of growth recovery | Monoclonal recovery rate |
| 6 | 464 | 80.5% | 309 | 66.6% |
In conclusion the various embodiments described above are only presently preferred embodiments of the present invention, it is not of the invention to limit
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be all included in
In protection scope of the present invention.
Claims (8)
1. a kind of method of unicellular sorting, which comprises the following steps:
1) sample prepares: CHO-K1 cell to be sorted is diluted to the viable cell density of 1E5cells/mL with secondary culture base,
Calcein reagent is added to the final concentration of 10nM of calcein, by cell dyeing 5 minutes；After the completion of dyeing, trained using passage
It supports base and cell is diluted to 5000cells/mL, be used for unicellular sorting；
2) sample loads: the cell that step 1) obtains is injected into chip cavity, so by the starting unicellular separator of Namocell
After load chip to be detected, hole sheet tray is moved to immediately below chip the drop for receiving to come out from nozzle；
3) position correction: adjustment laser facula to microfluidic channel center；
4) unicellular sorting parameter setting: setting fluorescent value are as follows: FITCH=500, FITCL=30, PEH=3, PEL=0；
5) unicellular sorting: starting to sort unicellular, and unicellular stream flows through laser detection region in microfluidic channel, to cell
It is identified, competent cell is accurately captured by high-velocity electrons valve, and ultimately form the drop of 1 μ L volume, sorting to orifice plate
In, every hole sorts 1 cell.
2. the method as described in claim 1, which is characterized in that before step 1), the CHO-K1 cell to be sorted first is used
Cell strainer filtering, is then counted using cell counter.
3. the method as described in claim 1, which is characterized in that in step 1), the calcein reagent is calcein
DMSO solution, concentration are 100 μM.
4. the method as described in claim 1, which is characterized in that in step 2), cell is loaded by disposable chip upper end
Hole is injected into chip cavity.
5. the method as described in claim 1, which is characterized in that in step 2), when the starting unicellular separator of Namocell, hole
Sheet tray is mobile and goes back to original position, and air pump is opened, and waste liquid line ports have drop outflow.
6. the method as described in claim 1, which is characterized in that in step 5), the orifice plate is 96 orifice plates, and every hole is added in advance
100 μ L culture mediums.
7. the method as described in claim 1, which is characterized in that the method also includes cell culture after sorting: wait sort
Cheng Hou uses Cell by cell in carbon dioxide incubator stationary culture behind 2h, 1 day, 2 days, 5 days and 9 days respectively
Metric monoclonal cell strain analyzer is taken pictures, after clonal growth recovery, by clonal expansion, to carry out subsequent screening.
8. the method for claim 7, which is characterized in that the condition of culture of carbon dioxide incubator be 36.5 DEG C, 6%
CO2。
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Cited By (5)
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| CN109554332A (en) * | 2018-11-20 | 2019-04-02 | 上海药明生物技术有限公司 | A method of unicellular sorting is carried out using unicellular printer |
| CN111175112A (en) * | 2020-01-13 | 2020-05-19 | 浙江卫未生物医药科技有限公司 | Improved microcarrier living cell fluorescent staining method |
| CN111239028A (en) * | 2020-02-10 | 2020-06-05 | 浙江大学 | High-yield high-activity single cell sorting method |
| CN112080430A (en) * | 2020-09-22 | 2020-12-15 | 长春长光辰英生物科学仪器有限公司 | Method for processing cell sample in single cell sorting process |
| WO2024244454A1 (en) * | 2023-05-31 | 2024-12-05 | 南京凌芯生物科技有限公司 | Cell sorting apparatus |
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