CN108034632A - A kind of method that cell monoclonal is carried out using flow cytometer - Google Patents
A kind of method that cell monoclonal is carried out using flow cytometer Download PDFInfo
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- CN108034632A CN108034632A CN201711281781.6A CN201711281781A CN108034632A CN 108034632 A CN108034632 A CN 108034632A CN 201711281781 A CN201711281781 A CN 201711281781A CN 108034632 A CN108034632 A CN 108034632A
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Abstract
The present invention discloses a kind of method that cell monoclonal is carried out using flow cytometer, including：Step 1: cell sorting：Cell sorting is carried out using the flow cytometer after cleaning, cytoadherence body removal is carried out using the signal of FSC and SSC, 1 target cell is sorted per hole in orifice plate.Step 3: cell culture：By the target cell after sorting in CO2gas incubator quiescent culture, condition of culture is 36.5 DEG C and 5~6%CO2, cultivate 5~15 days, treat that clone recovers.Method using the present invention, monoclonal rate reach 99.5%.
Description
Technical field
The invention belongs to bio-pharmaceuticals and biological technical field, and in particular to a kind of to carry out cell point using flow cytometer
The method of the structure of choosing, monoclonal and Cells for production strain.
Background technology
Bio-pharmaceuticals refers to the principle for comprehensively utilizing the science such as microbiology, chemistry, biochemistry, biotechnology, pharmacy
One kind with method manufacture is main using monoclonal antibody, recombinant protein, enzyme, vaccine for the product for preventing, treating and diagnosing
Represent.The production of the biological medicaments such as monoclonal antibody is generally by means of mammalian expression systems (such as CHO, i.e. Chinese hamster ovary
Cell), i.e., the expression vector of monoclonal antibody is transfected into mammalian host cell line, screening stability and high efficiency expression target
The cell line of product.
Production generally includes following steps with the structure of stable cell line, (1) transfection, i.e., by the expression vector of destination protein
It is transfected into host cell by the method for liposome or electroporation, on expression vector in addition to objective gene sequence, is also contained
Antibiotic or the relevant screening-gene of metabolism；(2) screen, i.e., pressurization sieve is carried out to the cell after transfection using antibiotic etc.
Choosing, only the cell of express express target protein can just survive in theory；(3) monoclonal, the cell obtained after the completion of screening
Group need to carry out monoclonal, and for each monoclonal cell strain, all cells derive from same mother cell.(4) gram
Grand screening, after obtaining numerous monoclonal cell strains, need to assess its expression quantity, product quality, cell line stability, reactor can put
Big property etc., finally select one be used for produce stable cell line.Cell monoclonal is in stable cell line building process
Committed step.The process of monoclonal, on the one hand needs efficiently to filter out the high clone of expression quantity, on the other hand needs to ensure
Monoclonal rate reaches sufficiently high level.At present, supervision department is thin with stablizing to production in order to ensure the security of biologics
The monoclonicity of born of the same parents' strain has strict requirements.
The common method of cell monoclonal includes limiting dilution assay, semisolid culturemedium bed board, flow cytometer point
Choosing etc..Limiting dilution assay is method the most traditional, and common operating method is to dilute cell gradient, with very low density
(generally 0.3~0.8 cells/well) is inoculated into 96 orifice plates (or 384 orifice plates).At present, it is limited by means of Image-forming instrument, a wheel
The i.e. reachable higher monoclonal rate of dilution.In limiting dilution experiment, cell meets Poisson distribution in 96 orifice plates.Work as bed board
When density is very low, there are substantial amounts of emptying aperture and non-monoclonal hole, while in view of the recovery situation of cell, final every piece of 96 holes
The alternative well-grown monoclonal boring ratio of plate is relatively limited.Therefore, limiting dilution assay is a kind of less efficient monoclonal
Change mode, compares and takes time and effort.Semisolid culturemedium bed board, is seeded cells into semisolid culturemedium, after growing a couple of days,
Individual cells form a group.If the anti-of the fluorescent marker of identification target product is previously added in semisolid culturemedium
Body, by means of instruments such as ClonePix, can effectively pick out the high clone of expression quantity.However, semisolid culturemedium bed board
Typically at least need two-wheeled experiment just to reach sufficiently high monoclonal rate, therefore add time and financial cost.
The unicellular sorting of flow cytometer is a kind of means of efficient monoclonal, it is by by means of intracellular
Fluorescent marker, or fluorescent staining is carried out to cell secretory product, to sub-elect the high clone of expression quantity.Use flow cytometer
By unicellular sorting to 96 orifice plates, relative to limiting dilution assay, void content substantially reduces, while monoclonal rate gets a promotion.Mesh
Before, the unicellular sorting technology of flow cytometer has been obtained for being widely applied in stable cell line building process.Due to prison
Pipe portion door has strict requirements for cell line monoclonicity, therefore in practical applications, it is necessary to optimize and verify that streaming is thin
The monoclonal rate of born of the same parents' instrument sorting.
The content of the invention
The technical problems to be solved by the invention are, there is provided a kind of to carry out cell monoclonal using flow cytometer
Method, can solve the problems, such as that monoclonal rate is not high in the prior art.In order to solve the above technical problems, the present invention provides a kind of profit
The method that cell monoclonal is carried out with flow cytometer, including step：
Step 1: cell sorting：Cell sorting is carried out using the flow cytometer after cleaning, utilizes the signal of FSC and SSC
Cytoadherence body removal is carried out, 1 cell is sorted per hole in orifice plate.
Step 2: cell culture：It is in CO2gas incubator quiescent culture, condition of culture by the target cell after sorting
36.5 DEG C and 5~6%CO2, cultivate 5~15 days, treat that clone recovers.
The present invention provides a kind of method that cell sorting is carried out using flow cytometer, to solve existing single cell clone skill
Monoclonal rate is not high in art, it is difficult to meets the defects of supervision department is for cell line monoclonicity requirement.
The method for carrying out cell sorting in the present invention using flow cytometer, including step：
1. sort cell to prepare：Target cell is centrifuged, is hanged using secondary culture base weight, it is 1E5 to make cell density
~1E6cells/mL, by loading after cell strainer filtering.
2. sort parameter setting：It is 85 or 100 μm to set jet size, and Window extension are arranged to 1~3ms,
Loading flow velocity is arranged to 1~11.
3. unicellular sorting：Cytoadherence body removal is carried out using the signal of FSC and SSC, is sorted in orifice plate per hole 1 thin
Born of the same parents.
Specifically, step 1 target cell is the CHO-K1 cells of secondary culture.
Preferably, step 1 cell density is 1E5~1E6cells/mL.
Specifically, adjusting the deflection angle of side liquid stream and the position in orifice plate A1 holes in the step 2, it is located at side liquid stream
The center position in A1 holes.
Specifically, need to adjust FSC (forward scattering light) and SSC (lateral scatterings when sorting parameter setting in the step 2
Light) magnitude of voltage, the area factor of FSC and blue laser.
Specifically, step 3 aperture plate adds 50~200 μ L culture mediums per hole；Preferably, 100 μ L trainings are added per hole
Support base.
Specifically, the orifice plate is 96 orifice plates.
The method for separating provided using invention carries out unicellular sorting, and 80.3% hole is monoclonal, and 19.3% hole is
Emptying aperture, 0.4% hole contains 2 or more than 2 cells, monoclonal rate reach 99.5%.
The beneficial effects of the present invention are, unicellular sorting is carried out using flow cytometer, 80.3% hole is monoclonal,
19.3% hole is emptying aperture, and 0.4% hole contains 2 or more than 2 cells, monoclonal rate reach 99.5%.Relative to existing
The monoclonal rate of technology greatly improves.
Brief description of the drawings
Fig. 1 is FSC-A (forward scattering light-area) and SSC-A (side scattered light-area) scatter diagram, passes through Scatter
Gate excludes dead cell and cell fragment.
Fig. 2 is SSC-H (side scattered light-height) and SSC-W (side scattered light-width) scatter diagram, passes through SSC
Gate excludes cytoadherence body.
Fig. 3 is FSC-H (forward scattering light-height) and FSC-W (forward scattering light-width) scatter diagram, passes through FSC
Gate further excludes cytoadherence body.
Embodiment
Clear, complete description is carried out to technical scheme below in conjunction with attached drawing, it is clear that described implementation
Example is the part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this area
Art personnel all other embodiments obtained on the premise of creative work is not made, belong to the model that the present invention protects
Enclose.
Embodiment 1
The method that cell monoclonal is carried out using flow cytometer that the present invention is developed, it is raw to can be applied to CHO-K1 etc.
The structure of production stable cell line, using FACSAriaII (BD Biosciences) flow cytometer as representative, it is embodied
Method is as follows：
1st, instrument performance detection is adjusted with liquid stream：Flow cytometer main power source and laser power supply are opened, opens computer, most
FACSDiva softwares are opened afterwards.Start liquid stream, adjusting amplitude and frequency values, stablize liquid stream, Drop 1 and Gap values are suitable.Make
With BD FACSDivaTMCS＆T Beads carry out performance detection to instrument.Use BD FACSTMAccudrop Beads are adjusted
The value of Drop delay.
2nd, cell is sorted to prepare：The CHO-K1 cells of secondary culture are centrifuged, is hanged using secondary culture base weight, makes cell close
Spend for 1E5~1E6cells/mL.Loading sorts after cell is used strainer filtering.
3rd, parameter setting is sorted：Jet size is 85 or 100 μm.Cell sample detection, adjust FSC (forward scattering light) and
The area factor of the magnitude of voltage of SSC (side scattered light), FSC and blue laser.Window extension are arranged to 1~3ms, on
Sample flow velocity is arranged to 1~11.
4th, 96 orifice plate positions are adjusted：The deflection angle of side liquid stream, and the position in 96 orifice plate A1 holes are adjusted, makes side liquid stream position
Center position in A1 holes.
5th, unicellular sorting：Cytoadherence body removal is carried out using the signal of FSC and SSC.96 orifice plates are added per hole in advance
50~200 μ L culture mediums, 1 cell is sorted per hole.
6th, cell culture after sorting：After sorting, cell CO2gas incubator quiescent culture (36.5 DEG C, 5~6%
CO2), take pictures after sorting.After clone recovers, by clonal expansion, follow-up screening is carried out.
The unicellular efficiency of separation of 2 flow cytometer of embodiment
The culture medium that 100 μ L contain ethanol is added in the every hole of 96 orifice plates in advance, unicellular point is carried out using flow cytometer
Choosing, 1 cell is sorted per hole.Culture medium adds ethanol, it is therefore an objective to cell is no longer divided.After the completion of sorting, 96 orifice plates stand 2
~3h makes cell fully settle, and then takes pictures, to judge whether the every hole of 96 orifice plates is monoclonal.Testing result is as follows：
| Sort number | 96 orifice plate numbers | Emptying aperture number | Unicellular hole count | 2 or more than 2 cell hole counts |
| 1 | 4 | 73 | 301 | 2 |
| 2 | 2 | 46 | 142 | 0 |
| 3 | 2 | 67 | 121 | 0 |
| 4 | 4 | 84 | 289 | 3 |
| 5 | 2 | 29 | 158 | 1 |
| 6 | 2 | 12 | 175 | 1 |
| 7 | 2 | 15 | 173 | 0 |
| Collect | 18 | 326 (19.3%) | 1359 (80.3%) | 7 (0.4%) |
Result of the test shows, adopts the method for separating of the present invention, and available 80.3% hole is monoclonal, 19.3% hole
For emptying aperture, 0.4% hole contains 2 or more than 2 cells, monoclonal rate reach 99.5%, relative to the Dan Ke of the prior art
Grand rate greatly improves.
In conclusion the various embodiments described above and attached drawing are only presently preferred embodiments of the present invention, not limiting this
The protection domain of invention, within the spirit and principles of the invention, any modification, equivalent substitution, improvement and etc. done, all should
Comprising within the scope of the present invention.
Claims (8)
- A kind of 1. method that cell monoclonal is carried out using flow cytometer, it is characterised in that including step：1.1 sorting cells prepare：Target cell is centrifuged, is hanged using secondary culture base weight, it is 1 E5~1 to make cell density E6cells/mL, by loading after cell strainer filtering；1.2 sorting parameter settings：It is 85 or 100 μm to set jet size, and Window extension are arranged to 1~3ms, loading Flow velocity is arranged to 1~11；1.3 unicellular sortings：Cytoadherence body removal is carried out using the signal of FSC and SSC, 1 cell is sorted per hole in orifice plate.
- 2. the method as described in claim 1, it is characterised in that step 2.1 target cell is the CHO-K1 of secondary culture Cell.
- 3. method as claimed in claim 2, it is characterised in that the cell density of the target cell is 1 E5~1 E6cells/mL。
- 4. the method as described in claim 1, it is characterised in that in the step 2.2 adjust side liquid stream deflection angle and The position in orifice plate A1 holes, makes side liquid stream be located at the center position in A1 holes.
- 5. the method as described in claim 1, it is characterised in that need to adjust FSC when sorting parameter setting in the step 2.2 The area factor of the magnitude of voltage of (forward scattering light) and SSC (side scattered light), FSC and blue laser.
- 6. the method as described in claim 1, it is characterised in that step 2.3 aperture plate adds 50~200 μ L cultures per hole Base.
- 7. method as claimed in claim 6, it is characterised in that the orifice plate is 96 orifice plates.
- 8. the method as described in claim 1, it is characterised in that stand the target cell sub-elected in CO2gas incubator Culture.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711281781.6A CN108034632A (en) | 2017-12-07 | 2017-12-07 | A kind of method that cell monoclonal is carried out using flow cytometer |
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| CN201711281781.6A CN108034632A (en) | 2017-12-07 | 2017-12-07 | A kind of method that cell monoclonal is carried out using flow cytometer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109554333A (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 the unicellular separator of Namocell |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106834271A (en) * | 2017-03-23 | 2017-06-13 | 中国科学院合肥物质科学研究院 | A kind of high-throughput screening method of deletion mutant |
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2017
- 2017-12-07 CN CN201711281781.6A patent/CN108034632A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834271A (en) * | 2017-03-23 | 2017-06-13 | 中国科学院合肥物质科学研究院 | A kind of high-throughput screening method of deletion mutant |
Non-Patent Citations (1)
| Title |
|---|
| 闵智慧等: "第三代流式细胞分选仪及96孔板分选单个细胞的方法及参数优化", 《中国临床医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109554333A (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 the unicellular separator of Namocell |
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Address after: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant after: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant after: Wuxi Yaoming Biotechnology Co., Ltd. Address before: 200131 701, 7 floor, 2 Hua Jing Road, Pudong New Area, Shanghai. Applicant before: SHANGHAI YAOMING BIOTECHNOLOGY CO., LTD. Applicant before: WUXI APPTEC BIOPHARMACEUTICALS CO., LTD. |
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Application publication date: 20180515 |