CN109554332A - A method of unicellular sorting is carried out using unicellular printer - Google Patents
A method of unicellular sorting is carried out using unicellular printer Download PDFInfo
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- CN109554332A CN109554332A CN201811382635.7A CN201811382635A CN109554332A CN 109554332 A CN109554332 A CN 109554332A CN 201811382635 A CN201811382635 A CN 201811382635A CN 109554332 A CN109554332 A CN 109554332A
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- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
Abstract
The present invention discloses a kind of method for carrying out unicellular sorting using unicellular printer, include: 1) sample preparation: the CHO-K1 cell of secondary culture is centrifuged 5min, it is outstanding using sterile Dulbecco's phosphate buffer or secondary culture base weight, make cell density 1E6cells/mL；2) drop adjusting parameter is arranged: starting the drop mass control of unicellular printer, setting trigger delay is 3ms, and depth is 10 μm, and speed is 120 μm/ms；3) sort parameter setting: 12 μm -18 μm of cell dia of setting, cell circularity 55%-100% and cell detection radius are 60 μm；4) it sorts: the cell sample in the print cartridge of unicellular printer is sorted into orifice plate, 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, unicellular more particularly to using unicellular printer to carry out
The method of sorting can be applied to the building of Cells for production strain.
Background technique
The biological medicaments such as monoclonal antibody are to research and develop increasingly burning hoter field in recent years, and the following relevant laws and regulations are wanted
Ask is also to be increasingly stringenter.Production is constructed with stable cell line, Chinese hamster ovary cell (Chinese Hamster
Ovary, CHO) it is one of most common host cell.In order to guarantee the safety of biological medicament, supervision department is to Cells for production
The requirement of the monoclonicity of strain is very high, and therefore, cell monoclonal is step very crucial in entire cell strain building process
Suddenly.
Currently, the common two methods of cell monoclonalization are limiting dilution assay and selected by flow cytometry apoptosis.Limiting dilution
Method is a kind of more conventional method, by the way that cell to be gradually diluted to very low density and is plated in 96 orifice plates, in combination with
The Image-forming instruments such as Cell Metric (Solentim) can obtain higher monoclonal rate.Due to low-density bed board, one piece 96
Effective number of perforations in orifice plate is less, needs to increase bed board number, compares and expends time and manpower, so the effect of limiting dilution assay
Rate is relatively low.It is a kind of method of relative efficiency that selected by flow cytometry apoptosis is unicellular, by intracellular or secrete to cell
Product carries out fluorescent staining, thus effectively by unicellular sorting to 96 orifice plates.This method reduces compared to limiting dilution assay
The number of non-slender hilum, improves monoclonal rate.Simultaneously flow cytometer cleaning process it has been experienced that avoid pollution and
Cross contamination can guarantee the monoclonicity of cell strain.But the cleaning process of flow cytometer is cumbersome, and it is complicated for operation, when same
When thering are multiple samples to carry out unicellular sorting, after the unicellular sorting and the complete cleaning step that need to complete a sample
The sorting that just can be carried out next sample causes the operating efficiency of sorting to reduce.And consumables associated therewith parts price used is expensive,
Instrument needs special messenger to be responsible for maintenance.
Unicellular printer (Single-Cell Printer, Cytena) technology is a kind of novel unicellular sorting skill
Art.Unicellular printer is to sort individual cells using micro-fluidic technologies and real-time image analysis and deposit to standard microplate
In.The innovation print cartridge similar with inkjet printing technology is for unidirectionally distributing.Five images are captured when cell distribution, are provided slender
The evidence of born of the same parents' separation.It can quickly distinguish unicellular, many cells by high-resolution imaging, cell-free and fragment, easily
It distinguishes unicellular.This method carries out unicellular sorting and has higher efficiency than limiting dilution assay, and has than flowing point
Select higher vigor.Unicellular printer uses disposable sterilized print cartridge, independent packaging, to avoid the intersection between sample simultaneously
Pollution.Unicellular printer does not need cleaning instrument with respect to flow cytometer in use process, operation is more quickly and simple,
Without the expensive consumptive material of consumption, it is not necessarily to special messenger's maintenance tool, reduces time, economy and human cost.But it unicellular beats
The unicellular efficiency of separation of print machine and the recovery rate of monoclonal are influenced very big, different cells by cell sorting and condition of culture
Sorting and condition of culture differentiation it is significant, therefore, seek certain specific cells it is suitable sorting and condition of culture to improve
The recovery rate of the unicellular efficiency of separation and monoclonal is 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 and a kind of carry out unicellular sorting using unicellular printer
Method, 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: the CHO-K1 cell of secondary culture is centrifuged 5min, it is slow using sterile Dulbecco's phosphate
Fliud flushing (D-PBS, Hyclone SH30028.02) or secondary culture base weight are outstanding, make cell density 1E6cells/mL；Then
It reuses 40 μm of filter to filter the cell suspension after resuspension, for use；
2) the drop adjusting parameter setting of unicellular printer: start the drop mass control of unicellular printer, setting
Trigger delay is 3ms, and depth is 10 μm, and speed is 120 μm/ms；
3) the sorting parameter setting of unicellular printer: setting 12 μm -18 μm of cell dia, cell circularity 55%-100%
And cell detection radius is 60 μm；
4) it sorts: the cell sample in the print cartridge of unicellular printer is sorted into orifice plate, every hole sorts 1 cell.
Specifically, first starting unicellular printer before the step 2), is completed to software initialization, restart drop
Quality control.
Specifically, the result of the drop mass control stores in the form of images.
Specifically, the cell detection radius is either automatically or manually to be configured.
Specifically, the cell sample in the print cartridge is 10-80 μ L.
Specifically, the orifice plate is 96 orifice plates, preparatory every hole joined 100 μ L culture mediums.
Specifically, the method also includes assessing monoclonal rate step: after orifice plate is previously added culture medium, using Cell
Metric (Solentim) monoclonal cell strain analyzer scans culture medium, is then sorted；After the completion of sorting, orifice plate is quiet
Setting settles cell sufficiently, reuses Cell Metric monoclonal cell strain analyzer and is imaged, to judge that the every hole of orifice plate is
No is monoclonal；The culture medium is the culture medium containing ethyl alcohol.
Preferably, the culture medium is 75% ethyl alcohol of secondary culture base+volume fraction 10% of volume fraction 90%.
Specifically, the method also includes assessments to clone recovery rate step: after the completion of sorting, by cell in carbon dioxide
Incubator stationary culture is clapped behind 2h, 1 day, 2 days, 5 days and 9 days using Cell Metric monoclonal cell strain analyzer respectively
According to assessing the recovery situation of cell.
Preferably, the condition of culture of carbon dioxide incubator is 36.5 DEG C, 6%CO2。
The present invention provides sortings and culture that unicellular printer carries out unicellular sorting and Cells for production strain building
Condition solves the problems, such as to further increase the recovery rate of the unicellular efficiency of separation and monoclonal.Existing limiting dilution gram
Grand method, 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% or so, monoclonal
Recovery rate is usually less than 50%.Using method of the invention, unicellular printer carries out unicellular sorting, 83.8% Kong Weidan
Clone, 8.3% hole is emptying aperture, and 2 or 2 or more cells are contained in 7.9% hole.Cell, monoclonal are sorted using the present invention
Average recovery rate be up to 82.7%.The unicellular efficiency of separation and the recovery rate of monoclonal is set to have reached very high level, phase
Other sortings of CHO-K1 cell than in the prior art and condition of culture have significant progress, compared to limiting dilution assay, Dan Ke
The efficiency of Longhua greatly improves, and further improves operation effect without carrying out the cleaning of instrument compared to selected by flow cytometry apoptosis
Rate.And without consuming expensive consumptive material, it is not necessarily to special messenger's maintenance tool, reduces time, economy and human cost.
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 flow cytometer monoclonal method for separating developed of the present invention can be applied to the productions such as CHO-K1 with stablizing cell
The building of strain.
Embodiment one
A kind of specific implementation method is as follows:
1, instrument starts: opening the main power switch of unicellular printer, starts instrument.Software is clicked, executes ROI automatically
(Region of Interest defines the volume sprayed when generating drop from chip nozzle) detection, waits software initialization
It completes.
2, sample prepares: the CHO-K1 cell 300g of secondary culture being centrifuged 5min, uses sterile Dulbecco's phosphoric acid
Salt buffer (D-PBS, Hyclone SH30028.02) or secondary culture base weight are outstanding, make cell density 1E6cells/mL.
Then it reuses 40 μm of filter to filter the cell suspension after resuspension, for use.
3, instrument prepares: starting drop mass control first, presses " control of starting quality " button, starts drop QC.Drop
The result of QC can store in the form of images.Then drop adjustment is carried out, trigger delay is set as 3ms, and depth is 10 μm, speed
Degree is 120 μm/ms, to confirm that drop is single and stable.Carry out vacuum shutter inspection again, vacuum can be by using " beating
Opening/closing vacuum " button switchs.
4, sort parameter setting: 12 μm -18 μm of cell dia, cell circularity 55%-100% and cell detection radius are
60μm.Cell detection radius can be either automatically or manually configured, and manual setting is by click " manual setting ROI " button
Come what is realized.It will appear an image window in the background of display nozzle.Window manuallys locate ROI.Clicking will be arranged
The center of ROI.ROI circle will be drawn, the positioning of ROI is carried out.
5, unicellular sorting: the cell sample of 10-80 μ L is added into print cartridge, and print cartridge is installed.Preparatory every hole is added
96 orifice plates for having entered 100 μ L culture mediums are placed on instrument, after setting completed to parameters, start to sort.The every hole of 96 orifice plates
Sort 1 cell.
6, 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 unicellular printer sorting monoclonal rate assessment:
Culture medium (+10%75% second of 90% secondary culture base that 100 μ L contain ethyl alcohol is added in the every hole of 96 orifice plates in advance
Alcohol), unicellular sorting is carried out using unicellular printer, every hole sorts 1 cell.Ethyl alcohol is added in culture medium, it is therefore an objective to make
Cell no longer divides.After the completion of sorting, 96 orifice plates, which stand 2h, settles cell sufficiently, then uses Cell Metric
(Solentim) it is imaged, to judge whether the every hole of 96 orifice plates is monoclonal.It is miscellaneous in 96 orifice plates and culture medium in order to distinguish
Matter, 96 orifice plates are added after culture medium and are scanned in advance using Cell Metric.Sorting for 24 hours, is rescaned using Cell Metric
96 orifice plates, to reaffirm the monoclonicity in the every hole of 96 orifice plates.It is as shown in table 1 to sort monoclonal rate assessment result.
The 1 unicellular efficiency of separation of unicellular printer of table
| 96 orifice plate numbers | Emptying aperture number | Unicellular hole count | 2 or 2 or more cell hole counts |
| 5 | 40 (8.3%) | 402 (83.8%) | 38 (7.9%) |
Embodiment three
Method based on embodiment one carries out unicellular printer sorting clone recovery rate assessment:
100 μ L culture mediums are added in the every hole of 96 orifice plates in advance, carry out unicellular sorting, every hole point using unicellular printer
Select 1 cell.After sorting, cell is in carbon dioxide incubator stationary culture (36.5 DEG C, 6%CO2), respectively in 2h, 1 day, 2
It, 5 days and taken pictures after 9 days using Cell Metric.After 9 days, the recovery situation of cell is assessed.Assess the recovery situation of cell
As shown in table 2.
Clone's recovery rate of the 2 unicellular sorting of unicellular printer of table
| 96 orifice plate numbers | Unicellular hole count | The unicellular hole count of growth recovery | Recovery rate |
| 1 | 81 | 67 | 82.7% |
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 (10)
1. a kind of method of unicellular sorting, which comprises the following steps:
1) sample prepares: the CHO-K1 cell of secondary culture being centrifuged 5min, uses sterile Dulbecco's phosphate buffer
Or secondary culture base weight is outstanding, makes cell density 1E6cells/mL；
2) the drop adjusting parameter setting of unicellular printer: start the drop mass control of unicellular printer, setting triggering
Delay is 3ms, and depth is 10 μm, and speed is 120 μm/ms；
3) the sorting parameter setting of unicellular printer: setting 12 μm -18 μm of cell dia, cell circularity 55%-100% and
Cell detection radius is 60 μm；
4) it sorts: the cell sample in the print cartridge of unicellular printer is sorted into orifice plate, every hole sorts 1 cell.
2. the method as described in claim 1, which is characterized in that in the step 1), reusing 40 μm of filter will be resuspended
Cell suspension filtering afterwards, for use.
3. the method as described in claim 1, which is characterized in that before the step 2), first start unicellular printer, to soft
Part initialization is completed, and drop mass control is restarted.
4. the method as described in claim 1, which is characterized in that in the step 2), the result of drop mass control is with image
Form storage.
5. the method as described in claim 1, which is characterized in that in the step 3), cell detection radius is automatic or hand
It is dynamic to be configured.
6. the method as described in claim 1, which is characterized in that in the step 4), the cell sample in print cartridge is 10-80 μ
L。
7. the method as described in claim 1, which is characterized in that in the step 3), the orifice plate is 96 orifice plates, preparatory every hole
It joined 100 μ L culture mediums.
8. the method according to claim 1 to 7, which is characterized in that the method also includes assessment monoclonal rate steps
It is rapid: after orifice plate is previously added culture medium, using Cell Metric monoclonal cell strain analyzer scan culture medium, then into
Row sorting；After the completion of sorting, orifice plate standing settles cell sufficiently, reuses the analysis of Cell Metric monoclonal cell strain
Instrument is imaged, to judge whether the every hole of orifice plate is monoclonal；The culture medium is the culture medium containing ethyl alcohol.
9. method according to claim 8, which is characterized in that the culture medium be volume fraction 90% secondary culture base+
75% ethyl alcohol of volume fraction 10%.
10. the method according to claim 1 to 7, which is characterized in that the method also includes assessments to clone recovery rate
Step: after the completion of sorting, by cell in carbon dioxide incubator stationary culture, respectively behind 2h, 1 day, 2 days, 5 days and 9 days
It is taken pictures using Cell Metric monoclonal cell strain analyzer, assesses the recovery situation of cell.
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Application publication date: 20190402 |