CN205188309U - Three -dimensional culture apparatus of adherence sexual cell - Google Patents
Three -dimensional culture apparatus of adherence sexual cell Download PDFInfo
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- CN205188309U CN205188309U CN201520821904.0U CN201520821904U CN205188309U CN 205188309 U CN205188309 U CN 205188309U CN 201520821904 U CN201520821904 U CN 201520821904U CN 205188309 U CN205188309 U CN 205188309U
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Abstract
The utility model provides a three -dimensional culture apparatus of adherence sexual cell, including magnetic field generator and set up the cell culture chamber in this magnetic field generator produced magnetic field. Adopt the utility model discloses a magnetic bead that supplies the cell adherence can be put into in the implementation to above -mentioned device in cultivateing the chamber, the field intensity that produces magnetic field through control change magnetic field generator can make the distribution of magnetic bead change to can require according to cell growth's stage to change cultivates the density environment. And, make the magnetic bead do exercises through applied magnetic field, can replace stirrings such as mechanical stirring stick, gas lift formula and make culture solution and cell flow, reduced the injury of mechanical shock and shear stress pair cell.
Description
technical field
The utility model belongs to three-dimensional cell cultivation technical field, is specifically related to the three-dimensional cultivation device 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.
utility model content
The object that the utility model is implemented is to overcome the defect that existing microballon carrier continues to twist three-dimensional cell cultivation, provides a kind of three-dimensional cultivation device being suitable for the excessive adherent sexual cell of cell culture environment.
In order to realize above-mentioned utility model object, the technical scheme of the utility model embodiment is as follows:
A three-dimensional cultivation device for adherent sexual cell, the cell culture chamber in comprising magnetic field generator and being arranged at magnetic field that this magnetic field generator produces.
Adopt said apparatus of the present utility model, the magnetic bead for cell attachment can be inserted in culture chamber in enforcement, the field intensity being produced magnetic field by control break magnetic field generator can make the distribution of magnetic bead change, thus can change culture density environment 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 utility model is described in further detail, in accompanying drawing:
Fig. 1 is the apparatus structure schematic diagram of the dimensional culture of the adherent sexual cell of the utility model embodiment.
embodiment
In order to make the purpose of this utility model, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the utility model is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the utility model, and be not used in restriction the utility model.
The utility model example 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, the change in size or direction all can change the stressed of magnetic bead and cause the motion of magnetic bead) 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 utility model, the utility model 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 utility model; not in order to limit the utility model; all do within spirit of the present utility model and principle any amendment, equivalent to replace and improvement etc., all should be included within protection domain of the present utility model.
Claims (4)
1. a three-dimensional cultivation device for adherent sexual cell, is characterized in that, the cell culture chamber in comprising magnetic field generator and being arranged at magnetic field that this magnetic field generator produces.
2. the three-dimensional cultivation device of adherent sexual cell as claimed in claim 1, is characterized in that, described magnetic field generator is the parallel plate capacitor that vertical direction is arranged; Described cell culture chamber is between two pieces of flat boards of this parallel plate capacitor.
3. the three-dimensional cultivation device of adherent sexual cell as claimed in claim 2, is characterized in that, the truncated cone-shaped shape that described cell culture chamber increases gradually in vertically upper section diameter.
4. the three-dimensional cultivation device of the adherent sexual cell as described in any one of claims 1 to 3, is characterized in that, the magnetic bead for cell attachment is housed in described cell culture chamber.
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| CN201520821904.0U CN205188309U (en) | 2015-10-22 | 2015-10-22 | Three -dimensional culture apparatus of adherence sexual cell |
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| CN201520821904.0U CN205188309U (en) | 2015-10-22 | 2015-10-22 | Three -dimensional culture apparatus of adherence sexual cell |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055210A (en) * | 2018-07-05 | 2018-12-21 | 曾小敏 | The device of inexpensive cell culture |
| CN109097275A (en) * | 2018-07-05 | 2018-12-28 | 曾小敏 | High-purity stem cell culture device |
| CN110914402A (en) * | 2018-02-15 | 2020-03-24 | 富有干细胞株式会社 | Cell culture device |
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2015
- 2015-10-22 CN CN201520821904.0U patent/CN205188309U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110914402A (en) * | 2018-02-15 | 2020-03-24 | 富有干细胞株式会社 | Cell culture device |
| CN110914402B (en) * | 2018-02-15 | 2023-04-25 | 富有干细胞株式会社 | Cell culture apparatus |
| CN109055210A (en) * | 2018-07-05 | 2018-12-21 | 曾小敏 | The device of inexpensive cell culture |
| CN109097275A (en) * | 2018-07-05 | 2018-12-28 | 曾小敏 | High-purity stem cell culture device |
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Effective date of registration: 20200106 Address after: 518045 101, 201, 301, 401, building 8, Biomedical Innovation Industrial Park, No. 14, Jinhui Road, Pingshan New District, Shenzhen City, Guangdong Province Patentee after: ISTEM REGENERATIVE MEDICINE SCI-TECH CO., LTD. Address before: 518057, room 2, building 10, Shenzhen biological incubation center, No. 302, Nanshan District hi tech, Shenzhen, Guangdong Patentee before: SHENZHEN HUAYU HEMATOPOIETIC STEM CELL RESEARCH CO., LTD. |