CN105316277A - Three-dimensional culture method of adherent cells - Google Patents
Three-dimensional culture method of adherent cells Download PDFInfo
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- CN105316277A CN105316277A CN201510690303.5A CN201510690303A CN105316277A CN 105316277 A CN105316277 A CN 105316277A CN 201510690303 A CN201510690303 A CN 201510690303A CN 105316277 A CN105316277 A CN 105316277A
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Abstract
The invention provides a three-dimensional culture method of adherent cells. The three-dimensional culture method includes: acquiring magnetic beads with cell adherence; placing the magnetic beads as a co-culture medium and to-be-cultured cells in a magnetic field for culture. According to the method, the magnetic beads are used as the cell co-culture medium; compared with glucose or micro-bead culture media of synthetic polymer, in the process of cell culture, distribution of the magnetic beads can be changed by controlling and changing intensity of a magnetic field according to conditions such as space and density required at each culture period, and culture environment can be changed according to requirements of cell growth stage. The magnetic field is externally added to enable the magnetic beads to move, so that a mechanical stirring rod and an airlift stirring device can be replaced to enable a culture solution and cells to flow, so that damage to the cells caused by mechanical impact and shearing stress is reduced.
Description
Technical field
The invention belongs to three-dimensional cell cultivation technical field, be specifically related to the three-dimensional culture method of a kind of adherent sexual cell.
Background technology
Adopt two-dimentional cultural method for the adherent sexual cell such as stem cell or tumour cell, the cell cultivated out is all the Two-dimensional morphology cell adhered on the wall, is unfavorable for the deficiency of the aspects such as short differentiation, cell micro-environment control and application; Therefore the form adopting the three-dimensional structure carrier of differing materials and cell to form cell-three-dimensional carrier compound, co-cultivation in vitro, enables cell move in the three-dimensional space structure of carrier, grows more.
And existing normally two kinds of the three-dimensional structure carrier building Three-dimensional cell culture, one is three-dimensional cell support, another kind is microsphere supported.Wherein, the Three-dimensional cell culture mode of three-dimensional cell support be in culture vessel, set firmly prepared by the materials such as collagen, fibroin, scleroproein, alginates, hyaluronic acid, chitosan be beneficial to the affine stake body of cell; Microsphere supported is the microballon that can be applicable to adherent cell growth, is generally made up of the polymkeric substance of natural dextran or various synthesis.Above-mentioned dimensional culture mode is in the process implemented, and three-dimensional cell support, once after setting, support itself can not move again, is cultivated relatively static; So when needing to carry out the cultivation of cell dynamic environment, adopted is microsphere supported carrying out more.
But adopt microsphere supported when carrying out dimensional culture, microsphere supported calculate according to the density of cell cultures and quantitative requirement in advance after be added in culture vessel, and keep it to be suspended in nutrient solution by modes such as stirrings; But in culturing process except cell quantity and density increase, the environment (microballoon autologous density etc.) of the structure of microballoon self is also constant; And for cell cultures, cell Different growth phases (early stage the cell quantity of inoculation few, need the large promotion of microballoon relative density adherent, after middle and later periods attached cell quantity increases, corresponding reduction Microsphere Density promotes its growing multiplication) on need different environment, the microsphere supported requirement that also cannot meet dynamic environment preferably; Further, microsphere supported alr mode keeps suspension itself to too increase the shear-stress of cell cultures, easily causes large physical abuse to cell, therefore also cannot reach and realize good cell three-dimensional dynamic cultivation environment, reduce the quality of cell cultures.
Summary of the invention
Object of the invention process is to overcome the defect that existing microballon carrier continues to twist three-dimensional cell cultivation, provides a kind of three-dimensional culture method being suitable for the excessive adherent sexual cell of cell culture environment, and realizes the device of the method.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
A three-dimensional culture method for adherent sexual cell, comprising:
Obtain the magnetic bead of cell attachment;
Using described magnetic bead as Dual culture medium with treat that culturing cell is cultivated in magnetic field.
Adopt aforesaid method of the present invention, using magnetic bead as co-culture of cells medium, compare the microballon developing medium of dextran or synthetic polymer, can the condition such as space, density required by each cultivation period in the process of cell cultures, the distribution of magnetic bead can be made to change by the field intensity in control break magnetic field, thus culture environment can be changed according to the demands of Growth of Cells.And, by externally-applied magnetic field, magnetic bead is moved, mechanical stirring rod, air lift type etc. can be substituted and stir and make nutrient solution and cell flowing, reduce mechanical shock and shear-stress to the injury of cell.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is the apparatus structure schematic diagram of the dimensional culture of the adherent sexual cell of the embodiment of the present invention.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Example of the present invention proposes the three-dimensional culture method of a kind of adherent sexual cell, and step and the implementation process of method comprise:
S10, obtains the magnetic bead having cellular affinity, can be used for cell attachment cultivation;
S20, using the magnetic bead of step S10 as Dual culture medium with treat that culturing cell is cultivated in magnetic field.
Adopt above-mentioned magnetic bead as co-culture of cells medium in this case, compare the microballon developing medium of dextran or synthetic polymer, can the condition such as space, density required by each cultivation period in the process of cell cultures, by the field intensity in control break magnetic field, (field intensity is vector, include size and Orientation) distribution of magnetic bead can be made to change, thus culture environment can be changed according to the demands of Growth of Cells.And, by externally-applied magnetic field, magnetic bead is moved, mechanical stirring rod, air lift type etc. can be substituted and stir and make nutrient solution and cell flowing, reduce mechanical shock and shear-stress to the injury of cell.
In the implementation process that above-mentioned magnetic bead is cultivated, magnetic bead itself is wanted to be beneficial to the adherent of adherent sexual cell, and its surperficial affinity of the magnetic bead of common iron, cobalt material is poor, be unfavorable for the adherent of cell and easily corroded by nutrient solution and cell metabolite, so the magnetic bead of cell attachment in step S10, can adopt the compound magnetic bead that the coating layers such as dextran, synthetic polymer, affine resin are coated, the magnetic core that inside is wrapped by is for providing magnetic; The coating layer on its surface be used on the one hand cell affine attaching, can coated iron, cobalt magnetic core do not corroded by nutrient solution on the other hand.
And in the employing process of magnetic bead, magnetic bead itself is as Dual culture medium, the zooblast of acellular wall is only attached to solid substrate and could breeds, therefore cell is the key sprawled further and grow in the attaching of micro-carrier surface; Increase unit volume internal surface area (S/F) growth to cell highly beneficial, but the magnetic field force that when particle diameter is too small, magnetic bead is relatively suffered is unfavorable for controlling; So preferably adopt the magnetic bead of particle diameter 200 ~ 500 μm in cultivation.
Due to the zooblast of adherent property, all acellular wall, responsive to shearing force, very easily dead, thus best mode need not add mechanical stirring rod to increase contact probability.Externally-applied magnetic field in this case is all generally with the alternating magnetic field that magnet builds or alternating-current produces, by the distance of moving magnet or the size of change exchange current, produce motion after the magnetic field force that magnetic bead is subject to changes, thus replace alr mode to increase cells contacting probability.
And further in the process implemented, the microcarrier of existing usual employing is same density material, so be suspended in the uniform height position in nutrient solution; Compare in this case and adopt the magnetic bead of at least 3 kinds of different densities to carry out (density is different can be realized by adopting different magnetic core material or different magnetic cores and coating thickness ratio) as far as possible, because the magnetic bead of different densities weight is on the one hand suspended in different height in nutrient solution, the magnetic bead of different densities and volume is because self gravitation, buoyancy, magnetic force is different and be in different height; Such magnetic bead distribution all can not be gathered in sustained height, can present density difference, be conducive to the cultivation of cell like this; Because cell is on the different stages, these environment of needs are different, and after inoculation, bead density assembles many regions, can directly be more conducive to early stage growth.After cell enters mid-term, cell slowly moves or is dispersed to the lower region of bead density gathering and grows, and by changing additional magnetic field, can adjust the position that magnetic bead suspends, change the intensity of magnetic bead, the requirement of cell at different steps growing environment can be met respectively.
Further, after above-mentioned magnetic bead etc. is all determined, cell is cultivated by step S20 in magnetic field, and the condition in magnetic field controls to carry out selecting and changing according to the position of required magnetic bead and aggregation extent.
And most preferred mode in the present case, magnetic field preferably adopts uniform magnetic field, and the direction of uniform magnetic field is set to the straight up direction contrary with magnetic bead gravity direction, and when magnetic bead is cultivated in nutrient solution so like this, suffered power is gravity, the buoyancy contrary with gravity direction and the magnetic force contrary with gravity direction straight down all the time.Owing to itself being the magnetic bead with the magnetic core such as iron, cobalt, density certainly much larger than nutrient solution in density, so buoyancy is certainly much smaller than gravity, then can be made by magnetic force additional on this direction that magnetic bead energy in the vertical direction is stressed to be reached balance and be suspended in nutrient solution as a supplement.Further, further when adjusting magnitude of field intensity, the motion of magnetic bead is all move up and down at vertical direction, there will not be multidirectional mixed and disorderly collision, avoids impacting damage to cell.
The magnetic field of certain employing other types also can meet makes magnetic bead in a certain position balance in inside, but this type may compare the not too convenient vibrations track controlling magnetic bead in force, than being easier to, mutual collision may occur.And adopting the relatively good enforcement of above-mentioned uniform magnetic field, employing and two pieces of parallel plate capacitors can produce above-mentioned uniform magnetic field after connecting direct current between parallel plate; Be beneficial to very much the above-mentioned purpose and effect that realize this case.
After above-mentioned cell cultivation process completes, first drain nutrient solution, at least use damping fluid rinsing 1 time, then add corresponding enzyme and carry out adherent digestion, dissociate afterwards and get final product collecting cell and products thereof.
The method of Three-dimensional cell culture is carried out based on above-mentioned employing magnetic bead of the present invention, the present invention also proposes a kind of three-dimensional cultivation device realizing the adherent sexual cell of aforesaid method further, apparatus structure with further reference to shown in accompanying drawing 1, can comprise round table-like culture chamber 10 and magnetic field generator 20; Wherein, magnetic field generator 20, for providing magnetic field to culture chamber 10, finally makes culture chamber 10 be arranged in the magnetic field of magnetic field generator 20 generation.
Magnetic field generator 20 can adopt the permanent magnetism object such as more unobstructed power frequency magnetic field generator or some magnet, and adjusts its intensity to magnetic bead can be made to suspend in the culture chamber 10 being added with nutrient solution.
Meanwhile, the culture chamber 10 in device adopts the proterties of truncated cone-shaped to design, and the proterties of truncated cone-shaped is direction diameter of section from from bottom to top increases gradually.This shape compares common columniform cell culture chamber, early stage in cell inoculation culture, can the intensity in magnetic field that sends of controlling magnetic field producer 20 in relatively little degree, make magnetic bead neutral buoyancy in the underlying space of culture chamber 10, intensity between such magnetic bead is higher, is suitable for cell adhesion and the amplification of early stage comparatively small amt; When cell start amplification and adherent firmly after, adjustment strengthens the intensity of electromagnetic field; Under the influence of a magnetic field, magnetic bead can carry this stem cell or tumour cell is floated to higher position.According to cell quantity and the demand of cultivation, adjust the height that magnetic bead suspends in a reservoir, to guarantee that cell can not adhere to bottom of culture vessel, but the correct position floated on a liquid is cultivated.The cycle of the growth configuration of cell can be shortened so further, and the cell injury caused in cell cultivation process can be reduced, compare common microcarrier training method better effects if some.
Therefore, based on the preferred implementation result of the above-mentioned uniform magnetic field of this case, above-mentioned magnetic field generator 20 preferably adopts parallel plate capacitor to realize, this mode is in accompanying drawing 1, because two of parallel plate capacitor flat boards can produce uniform electric field and the uniform magnetic field with E-field normal after turning on galvanic positive and negative electrode respectively between two pieces of flat boards.So, based on object and the effect of this case, two of parallel plate capacitor pieces of flat boards are vertically be arranged in parallel, the magnetic field of vertical direction can be produced.Then by the size and Orientation (upwards or downwards) that can change magnetic field of galvanic cathode and anode directions and size, the aforesaid method device for carrying out said of this case can be built.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. a three-dimensional culture method for adherent sexual cell, is characterized in that, comprising:
Obtain the magnetic bead of cell attachment;
Using described magnetic bead as Dual culture medium with treat that culturing cell is cultivated in magnetic field.
2. the three-dimensional culture method of adherent sexual cell as claimed in claim 1, is characterized in that, described magnetic field is uniform magnetic field, and the direction in magnetic field is contrary with magnetic bead gravity direction.
3. the three-dimensional culture method of adherent sexual cell as claimed in claim 1 or 2, is characterized in that, described magnetic bead is made up of the magnetic bead of at least three kinds of different densities.
4. the three-dimensional culture method of adherent sexual cell as claimed in claim 1 or 2, is characterized in that, the particle diameter of described magnetic bead is 200 ~ 500 μm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671029A (en) * | 2016-03-08 | 2016-06-15 | 万香波 | Establishment method of three-dimensional cell model |
| CN109055210A (en) * | 2018-07-05 | 2018-12-21 | 曾小敏 | The device of inexpensive cell culture |
| CN109628372A (en) * | 2019-01-10 | 2019-04-16 | 武汉济源高科技有限公司 | Paramagnetic particle method culture attached cell |
| CN110331097A (en) * | 2019-08-02 | 2019-10-15 | 齐鲁工业大学 | The integrated multi-modal movement Three Dimensional Cavities intestines organ chip of enteron aisle and method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020090741A1 (en) * | 2001-01-08 | 2002-07-11 | Jurgensen Stewart Russell | Method of separating cells from a sample |
| CN104694474A (en) * | 2015-03-31 | 2015-06-10 | 南京新诺丹生物技术有限公司 | Cell culture method |
-
2015
- 2015-10-22 CN CN201510690303.5A patent/CN105316277A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020090741A1 (en) * | 2001-01-08 | 2002-07-11 | Jurgensen Stewart Russell | Method of separating cells from a sample |
| CN104694474A (en) * | 2015-03-31 | 2015-06-10 | 南京新诺丹生物技术有限公司 | Cell culture method |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671029A (en) * | 2016-03-08 | 2016-06-15 | 万香波 | Establishment method of three-dimensional cell model |
| CN109055210A (en) * | 2018-07-05 | 2018-12-21 | 曾小敏 | The device of inexpensive cell culture |
| CN109628372A (en) * | 2019-01-10 | 2019-04-16 | 武汉济源高科技有限公司 | Paramagnetic particle method culture attached cell |
| CN110331097A (en) * | 2019-08-02 | 2019-10-15 | 齐鲁工业大学 | The integrated multi-modal movement Three Dimensional Cavities intestines organ chip of enteron aisle and method |
| CN110331097B (en) * | 2019-08-02 | 2023-05-16 | 齐鲁工业大学 | Integrated intestinal multi-mode movement three-dimensional cavity intestinal organ chip and method |
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