CN109628372A - Paramagnetic particle method culture attached cell - Google Patents
Paramagnetic particle method culture attached cell Download PDFInfo
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- CN109628372A CN109628372A CN201910022715.XA CN201910022715A CN109628372A CN 109628372 A CN109628372 A CN 109628372A CN 201910022715 A CN201910022715 A CN 201910022715A CN 109628372 A CN109628372 A CN 109628372A
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- magnetic bead
- cell
- magnetic
- culture
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 230000005298 paramagnetic effect Effects 0.000 title description 3
- 239000002245 particle Substances 0.000 title description 2
- 230000005291 magnetic effect Effects 0.000 claims abstract description 58
- 239000011324 bead Substances 0.000 claims abstract description 46
- 238000000576 coating method Methods 0.000 claims abstract description 10
- 239000011248 coating agent Substances 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims abstract description 5
- 239000003480 eluent Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 238000001556 precipitation Methods 0.000 claims description 5
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000001079 digestive effect Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 102000008186 Collagen Human genes 0.000 claims description 2
- 108010035532 Collagen Proteins 0.000 claims description 2
- 102000016359 Fibronectins Human genes 0.000 claims description 2
- 108010067306 Fibronectins Proteins 0.000 claims description 2
- 230000001464 adherent effect Effects 0.000 claims description 2
- 244000309466 calf Species 0.000 claims description 2
- 229920001436 collagen Polymers 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 230000001902 propagating effect Effects 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 1
- 230000001419 dependent effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 abstract 9
- 210000001339 epidermal cell Anatomy 0.000 abstract 1
- 210000002950 fibroblast Anatomy 0.000 abstract 1
- 239000012530 fluid Substances 0.000 abstract 1
- 239000002609 medium Substances 0.000 abstract 1
- 235000015097 nutrients Nutrition 0.000 abstract 1
- 210000000130 stem cell Anatomy 0.000 abstract 1
- 239000007787 solid Substances 0.000 description 3
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical group [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 241001474374 Blennius Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- -1 amino, carboxyl Chemical group 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229940096010 iron polysaccharide Drugs 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Classifications
-
- 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
- C12N5/0068—General culture methods using substrates
- C12N5/0075—General culture methods using substrates using microcarriers
-
- 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
- C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
-
- 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
- C12N2531/00—Microcarriers
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Abstract
This patent mainly describes a kind of novel attached cell cultural method.This novel attached cell cultural method dependent on a kind of surface pass through coating buffer processing miniature magnetic bead (50nm), by magnetic bead and mixing with cells in fluid nutrient medium after, cell will be attached at magnetic bead surfaces quickly, will substantially increase the culture area of cell in this way.Magnetic bead has the characteristic of magnetic suck simultaneously, can be convenient by externally-applied magnetic field and quickly separates magnetic bead and culture medium, convenient for the passage and collection of cell.Attached cell cultural method in this patent can be widely applied to the scientific research and industrialized production of stem cell, fibroblast, epidermal cell, DC cell and other attached cells.
Description
Technical field
This patent belongs to field of cell culture.
Background technique
VanWeze in 1967 is that the high yield culture of anchorage-dependent cell proposes the new technology of " microcarrier " culture systems
Concept.Microcarrier refer to diameter at 60~250 μm, suitable for anchorage-dependent cells its surface adherent growth microballon.
Since microcarrier has the advantages that: the advantage of attached cell culture and the culture that suspends is had both, cell local environment is uniform,
Environmental condition (temperature, pH value) easily monitor, specific surface area with higher, culture operation can scale, automation, reduce pollution
Generation.So microcarrier is used widely in large-scale industrial production.But the material of microcarrier is prepared by it
Source can be divided into two major classes: artificial-synthetic copolymer and natural polymer and its derivative.By the form point of microcarrier: solid
Microcarrier and liquid microcarrier are solid microcarriers when application is more at present, and main species have: gelatin, collagen, cellulose, first
Chitin and derivative and seaweed salt；Liquid microcarrier has fluorocarbons liquid film microcarrier.These different microcarriers respectively have excellent
Gesture, while also respective defect, the solid microcarrier later period separate more difficult, mutability, it is unstable the disadvantages of, liquid microcarrier at
This height, complex manufacturing technology, part microcarrier cannot reuse.Miniature magnetic bead is a kind of diameter surpassing for 50nm-2000nm
Grade paramagnetic beads, ingredient is iron oxide and polysaccharide, and surface can mark amino, carboxyl, sulfydryl isoreactivity group.Miniature magnetic bead
These characteristics determine it can in the form of non-covalent bond combination cell, and this binding force can by change pH value and
The ingredient of eluent changes this binding force, realizes the combination and separation of cell, simultaneously because the magnetism of magnetic bead itself, in magnetic
Field is being easily separated, and this unique property determines that miniature magnetic bead can be used as the novel microcarrier of one kind applied to adherent
Cell culture.
Summary of the invention
The configuration of magnetic bead coating buffer: configuration contains 500ng/mlCollagen type and 200ng/ml people's fibronectin splicing variants
PBS solution 20ml, pH value are adjusted to 7.6, and sterilizing filter filtration sterilization is spare.
The coating of magnetic bead: 10mlPBS solution is added in 10g sterile miniature magnetic bead (50nm) and impregnates 30min, is fallen after centrifugation
Fall supernatant, 20ml magnetic bead coating buffer is added in 10g magnetic bead, mix, 37 DEG C of coatings are for 24 hours.
The inoculation of cell: appropriate culture medium is added in the cell for needing to be inoculated with and is mixed, coated Miniature magnetic is then added
Pearl, the ratio of cell and magnetic bead is observed in sampling under the microscope, generally in 2:1 or so, is transferred to culture in culture vessel and is stirred
Culture is mixed, condition of culture is arranged according to cell category.
The passage of cell: when magnetic bead surfaces cell fusion degree reaches 80%, cell liquid is collected, in the effect of externally-applied magnetic field
Or under magnetic-adsorption frame, it is enriched with magnetic bead, remove culture medium, 0.25% pancreatin isometric with magnetic bead be added, digests 1 minute,
2 times of magnetic bead volume terminate liquids (calf serum) are added, digestive juice is added in the Filter column with magnetic field, add 3 times of volumes
Eluent (PBS of pH value 7.6) is eluted, and eluent is carried out 1200rpm and is centrifuged 8 minutes, cell precipitation is obtained, after digestion
Magnetic bead can carry out renaturation again, reuse.Appropriate culture is added cell is resuspended, then adds appropriate miniature magnetic bead
(50nm), the ratio that cell and magnetic bead are observed in sampling under the microscope are transferred in culture vessel and train generally in 2:1 or so
Stir culture is supported, condition of culture is arranged according to cell category.
The collection of cell purifies: collecting cell liquid, in the effect of externally-applied magnetic field or under magnetic-adsorption frame, is enriched with magnetic
Pearl removes culture medium, and 0.25% pancreatin isometric with magnetic bead is added, and digests 1 minute, it is (small that 2 times of magnetic bead volume terminate liquids are added
Cow's serum), digestive juice is added in the Filter column with magnetic field, 3 times of volume eluents is added and is eluted, by eluent into
Row 1200rpm is centrifuged 8 minutes, obtains cell precipitation, notices whether observation centrifugation bottom of the tube has remaining magnetic bead, if containing residual
Magnetic bead is stayed, 5 times of mL normal salines is added, cell is resuspended, be added in the Filter column in magnetic field again, 3 times of volume eluents are added
It is eluted, eluent is subjected to 1200rpm and is centrifuged 8 minutes, obtains cell precipitation again.After confirmation bottom does not remain magnetic bead
The cell of collection can be retained or separately do it and used.
Claims (5)
- It is the method for coating of configuration, miniature magnetic bead including magnetic bead coating buffer, miniature 1. a kind of method of magnetic bead culture attached cell The inoculation method of magnetic bead culture attached cell, the propagating method of miniature magnetic bead culture attached cell and miniature magnetic bead culture are adherent thin The mobile phone purification process of born of the same parents.
- 2. the method for magnetic bead culture attached cell as described in claim 1, it is characterised in that the configuration formula of magnetic bead coating buffer In include collagen and fibronectin splicing variants.
- 3. the method for magnetic bead culture attached cell as described in claim 1, it is characterised in that the method for coating of magnetic bead is 37 DEG C Coating is for 24 hours.
- 4. the method for magnetic bead culture attached cell as described in claim 1, it is characterised in that be inoculated in the method for cell inoculation The ratio of cell and magnetic bead is 2:1.
- 5. the method for magnetic bead culture attached cell as described in claim 1, it is characterised in that cell collects the method purified and is Cell liquid is collected, in the effect of externally-applied magnetic field or under magnetic-adsorption frame, magnetic bead is enriched with, removes culture medium, addition and magnetic bead 0.25% isometric pancreatin digests 1 minute, 2 times of magnetic bead volume terminate liquids (calf serum) is added, digestive juice is added to band It in the Filter column in magnetic field, adds 3 times of volume eluents and is eluted, eluent is subjected to 1200rpm and is centrifuged 8 minutes, is obtained Cell precipitation, notices whether observation centrifugation bottom of the tube has remaining magnetic bead, if 5 times of mL normals are added containing residual magnetic bead Cell is resuspended in salt water, is added in the Filter column in magnetic field again, 3 times of volume eluents are added and are eluted, eluent is carried out 1200rpm is centrifuged 8 minutes, obtains cell precipitation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910022715.XA CN109628372A (en) | 2019-01-10 | 2019-01-10 | Paramagnetic particle method culture attached cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910022715.XA CN109628372A (en) | 2019-01-10 | 2019-01-10 | Paramagnetic particle method culture attached cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109628372A true CN109628372A (en) | 2019-04-16 |
Family
ID=66060452
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910022715.XA Pending CN109628372A (en) | 2019-01-10 | 2019-01-10 | Paramagnetic particle method culture attached cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109628372A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1850970A (en) * | 2006-05-12 | 2006-10-25 | 浙江大学 | Method for sorting primary human marrow mesenchy malstem cell by monoclonal antibody ZUB4 immunomagnetic bead |
| CN102206611A (en) * | 2011-04-27 | 2011-10-05 | 西北农林科技大学 | Isolation and culture method of amniotic-fluid-derived neural stem cells |
| CN103237886A (en) * | 2010-08-24 | 2013-08-07 | 明尼苏达大学董事会 | Non-static suspension culture of cell aggregates |
| CN105316277A (en) * | 2015-10-22 | 2016-02-10 | 深圳华毓造血干细胞研究有限公司 | Three-dimensional culture method of adherent cells |
| CN106754672A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cultural method of attached cell |
| CN108192867A (en) * | 2017-12-27 | 2018-06-22 | 重庆斯德姆生物技术有限公司 | A kind of preparation method of clinic cord blood monocyte-macrophage |
-
2019
- 2019-01-10 CN CN201910022715.XA patent/CN109628372A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1850970A (en) * | 2006-05-12 | 2006-10-25 | 浙江大学 | Method for sorting primary human marrow mesenchy malstem cell by monoclonal antibody ZUB4 immunomagnetic bead |
| CN103237886A (en) * | 2010-08-24 | 2013-08-07 | 明尼苏达大学董事会 | Non-static suspension culture of cell aggregates |
| CN102206611A (en) * | 2011-04-27 | 2011-10-05 | 西北农林科技大学 | Isolation and culture method of amniotic-fluid-derived neural stem cells |
| CN105316277A (en) * | 2015-10-22 | 2016-02-10 | 深圳华毓造血干细胞研究有限公司 | Three-dimensional culture method of adherent cells |
| CN106754672A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cultural method of attached cell |
| CN108192867A (en) * | 2017-12-27 | 2018-06-22 | 重庆斯德姆生物技术有限公司 | A kind of preparation method of clinic cord blood monocyte-macrophage |
Non-Patent Citations (2)
| Title |
|---|
| 作者：程燕主编: "《生命科学实验仪器设备与使用》", 31 August 2014, 出版发行：北京：科学技术文献出版社 * |
| 陈万涛主编: "《口腔临床免疫学实验技术》", 30 September 2009, 出版发行：上海：上海交通大学出版社 * |
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| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190416 |