CN106554937B - A kind of external three-dimensional culture method of liver cell - Google Patents
A kind of external three-dimensional culture method of liver cell Download PDFInfo
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- CN106554937B CN106554937B CN201610951267.8A CN201610951267A CN106554937B CN 106554937 B CN106554937 B CN 106554937B CN 201610951267 A CN201610951267 A CN 201610951267A CN 106554937 B CN106554937 B CN 106554937B
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Abstract
The present invention discloses a kind of poly- ball culture substrate of cell comprising cultivation region and the enclosing for being connected to the cultivation region periphery；The cultivation region is equipped with the poly- ball that several lower half portion are segment-shaped cavity.A kind of substrate mould is also disclosed, forms the first template and the second template of the plate of the poly- ball culture substrate form chamber of cell including mutually nested；The groove being circumferentially arranged in first template equipped with boss and the neighbouring boss, the boss surface are equipped with the salient point that several top halfs are spheroid；Second template offers the through-hole with the outer profile cooperation of cell wall on the outside of the groove.A kind of method carrying out the three-dimensional poly- ball culture of animal cells in vitro using the poly- ball culture substrate of the cell is also disclosed.Cost is relatively low, scale is controllable for the external three-dimensional culture method of liver cell of the present invention, process is simple, and culture effect is significantly better than two-dimensional flat plate culture.
Description
Technical field
The present invention relates to a kind of animal cells in vitro culture technique more particularly to a kind of external dimensional culture sides of liver cell
Method.
Background technique
Liver cell is widely used in the fields such as drug screening, bioartificial liver, liver transplant research as liver substitute.It passes
The cell in vitro plate two dimension culture of system, hepatic cell growth culture vessel surface, cell be in adherent island growth, cell protein and
Urea and drug-metabolizing function are not easy to maintain, and have very big difference with internal liver cell, can not substitute internal liver cell and complete
Liver function, for liver cell physiological status in better analogue body, increase between cell and cell and extracellular matrix it is mutual
Effect, to enhance the function of Cultured Hepatocytes in vitro, makes liver cell polarity be restored, and has been developed at present several external thin
Born of the same parents' three-dimensional culture method, including: the sandwich culture of culture dish, liver cell is overlay using extracellular matrix, the poly- ball of liver cell, is added
Enter the stimulants such as growth factor, and uses biomaterial scaffolds repopulating cell.The wherein poly- ball culture of liver cell is to study at present
One of cultural method of greatest concern.
The poly- ball method of liver cell common at present has: suspension spinner culture method, adds polymer substance in culture medium with shape
The cultural method of glomeration microcarrier forms poly- ball culture mold etc. using photolithographic techniques.Wherein hang spinner culture, cell
The balling-up time is long, and balling ratio is low, it is different to obtain the big small-bore of cell ball, while instrument is during the shaking process, can not accurate simulation
The intracorporal physiological status of cell；By adding polymer substance, so that cell is reached polymerization state, but there is sky between cell
Gap makes to exchange existing defects between cell and cell, if while obtain cell ball polymerization diameter it is excessive, cell ball center has
Meronecrosis happens；Then use cost is high for photolithographic techniques, expensive, complex procedures etc..
Summary of the invention
The purpose of the present invention is in order to overcome the deficiencies of the prior art, providing one kind, cost is relatively low, and scale is controllable, and process is simple
The efficiently poly- ball method of hepatocyte.
To reach the above technical purpose, The technical solution adopted by the invention is as follows:
A kind of poly- ball culture substrate of cell is provided first comprising cultivation region and is connected to enclosing for the cultivation region periphery
Gear；The cultivation region is equipped with the poly- ball that several lower half portion are segment-shaped cavity.
Further, the top half of the poly- ball to opening is cylindrical cavity, the cylindrical cavity and segment
Shape cavity connects.
Preferably, the diameter of the cylindrical cavity is identical as the diameter of the segment-shaped cavity.
It is highly preferred that the depth of the poly- ball is greater than the diameter of the segment-shaped cavity.
More preferably, the diameter of the segment-shaped cavity is 400um, and the depth of the poly- ball is 500um.
It is further preferred that the distance between adjacent centre of sphere of the segment-shaped cavity is the segment-shaped cavity diameter
2 times.
Preferably, the poly- ball is evenly distributed in the cultivation region.
It is highly preferred that the end face of the enclosing is higher than the end face of the cultivation region.
It is further preferred that the cultivation region and enclosing are integrally formed.
Next provides a kind of substrate mould, and the plate of the poly- ball culture substrate form chamber of cell is formed including mutually nested
The first template and the second template of shape；The groove being circumferentially arranged in first template equipped with boss and the neighbouring boss, institute
State salient point of the boss surface equipped with several top halfs for spheroid；Second template offers and cell wall on the outside of the groove
Outer profile cooperation through-hole.
Further, the lower half portion of the salient point is cylindrical body, the cylindrical body and the segment body by integral forming.
Preferably, the diameter of the cylindrical body is identical as the diameter of the spheroid.
It is highly preferred that the height of the salient point is greater than the diameter of the spheroid.
More preferably, the diameter of the spheroid is 400um, and the height of the salient point is 500um.
It is further preferred that the distance between adjacent centre of sphere of the spheroid is 2 times of the spheroid diameter.
Preferably, the salient point is evenly distributed on the boss.
Further, the shape of the through-hole of second template is identical as the shape of outer profile of the groove lateral wall.
Selectively, the size of the through-hole of second template is equal to or more than the big of the outer profile of the groove lateral wall
It is small.
Further, first template is respectively provided with the mutually nested positioning knot with cooperation with the second template
Structure.
A kind of method of three-dimensional poly- ball culture of animal cells in vitro is provided again comprising following steps:
Preparation is used to form the substrate mould of the poly- ball culture substrate of cell；
The poly- ball culture substrate of the cell is prepared using the substrate mould；
The poly- ball culture substrate of the cell is placed in cell culture container and carries out the poly- nodularization culture of zooblast；
Wherein, the poly- ball culture substrate of the cell is the poly- ball culture substrate of foregoing cell, or
The substrate mould is foregoing substrate mould.
Preferably, the substrate mould is obtained using 3D printing technique.
It is further preferred that the substrate mould is made using photosensitive resin.
Preferably, the poly- ball culture substrate of cell is made by agarose.
Further, the poly- ball culture substrate of the cell the preparation method comprises the following steps:
It will be heated to by autoclaved agarose after cooling in molten condition；
The agarose of molten condition is filled into while hot in the assembled substrate mould；
It is demoulded cooling in gelatinous agarose, obtains the poly- ball culture substrate of the cell.
Preferably, the concentration of the agarose is 1.5%~2%.
Preferably, the heating temperature of the agarose is 80 DEG C.
Further, the process of the poly- nodularization culture are as follows:
Cell with the density inoculation of each 500 cells of poly- ball by passage；
Continuous stationary culture extremely forms the poly- ball of cell.
Further, the cell culture container includes Tissue Culture Dish, 6 orifice plates, 12 orifice plates, 24 orifice plates and 48 holes
Plate.
Compared with prior art, the present invention has the advantage that
(1) the poly- ball culture substrate of cell of the invention provides the bracket or matrix of similar tumor growth environment for cell, makes
Cell establishes contacting between iuntercellular and cell and extracellular matrix by the connection types such as closely connection and gap connection, forms one
Fixed three-dimensional structure is conducive to the integrality for improving cell physiological function；
(2) for the poly- ball culture substrate of cell of the invention using agarose as gel rubber material, agarose has good life
Object compatibility, can preferably analogue body inner cell living environment；
(3) the poly- ball culture substrate of cell of the invention is irrigated molding using substrate mould, and the substrate mould uses
Photosensitive resin is made as raw material, and using 3D printing technique, not only reduces the manufacturing cost of substrate mould, manufactured
The service life for the substrate mould come also greatly prolongs, and is more advantageous to the cost for reducing the poly- ball culture of cell；
(4) by easy operation, liver cell is made quickly and efficiently to form poly- sphere in vitro, it can be thin with liver in analogue body
The physiological property of born of the same parents simultaneously keeps its biological activity for a long time；It can be obtained by the aperture and cell inoculation number of control protrusion simultaneously
Workflow is simplified without operations such as additional poly- sphere transfers to the different poly- spheres of uniform cell.
Detailed description of the invention
Fig. 1 is the object construction figure of the poly- ball culture substrate of cell of the invention.
Fig. 2 is the local electron-microscope scanning figure of the poly- ball culture substrate of cell of the invention, shows the structure of poly- ball.
Fig. 3 is the cross-sectional view of the poly- ball of the poly- ball culture substrate of cell of the invention.
Fig. 4 is the schematic perspective view of the substrate mould of the formation poly- ball culture substrate of cell of the invention.
Fig. 5 is the cross-sectional view of the salient point of the substrate mould of the invention.
Fig. 6 is to be cultivated human hepatoma HepG2 cell using the method for the three-dimensional poly- ball culture of animal cells in vitro of the invention
To the 7th day poly- ball status diagram of cell.
Fig. 7 be using the method cell ball obtained of the three-dimensional poly- ball culture of animal cells in vitro of the invention diameter with
The graph of relation of cultivated days.
Fig. 8 is flat using the method cell ball obtained and two dimension of the three-dimensional poly- ball culture of animal cells in vitro of the invention
Plate culture cell obtained compares ALT level and cultivated days relationship.
Fig. 9 is flat using the method cell ball obtained and two dimension of the three-dimensional poly- ball culture of animal cells in vitro of the invention
Plate culture cell obtained compares albumin secretory volume and cultivated days relationship.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, present invention is further described in detail.
Embodiment one
The poly- ball culture substrate of cell
With reference to Fig. 1, the poly- ball culture substrate 1 of cell of the invention provides the outer base of ectosome growth for animal cell culture
Matter, the poly- bulb matrix 1 of the cell include cultivation region 11 and the enclosing 12 for being connected to 11 periphery of cultivation region, 12 end face of enclosing
Higher than the end face of the cultivation region 11, to guarantee the culture solution that can accommodate certain volume in cultivation region 11.Preferably, described
Cultivation region 11 and enclosing 12 are integrally formed.In view of biocompatibility, the cell is poly-, and to seek culture substrate 1 be suitable for using agarose
Preparation.
Further, with reference to Fig. 2, the cultivation region 11 has been uniformly distributed several poly- ball 111, for guiding in the cell
The zooblast grown in poly- ball culture substrate 1 forms the poly- ball of cell, to form the three-dimensional knot in the case of being similar to tumor growth
Structure.
Specifically, with reference to Fig. 3, each poly- ball 111 includes the segment-shaped cavity 1112 and position positioned at lower half portion
In the cylindrical cavity 1111 of top half, the segment-shaped cavity 1112 and cylindrical cavity 1111 connect, and in institute
The top for stating cylindrical cavity 1111 forms opening, and the opening is concordant with the end face of the cultivation region 11.Preferably, the circle
The diameter of cylindrical cavity 1111 is identical as the diameter of segment-shaped cavity 1112, to keep smoothed inside the poly- ball 111
It crosses, here, unified indicated with diameter R；The depth H of the poly- ball 111 is greater than the diameter R of the segment-shaped cavity 1112, with
The poly- ball 111 is maintained there are enough depth；With reference to Fig. 2, the space D between the adjacent poly- ball 111 is with adjacent institute
The expression of the distance between centre of sphere of segment-shaped cavity 1112 is stated, the space D is 2 times of the diameter R, making full use of
Under the premise of the end face space for stating cultivation region 11, the poly- ball phase of cell in the adjacent poly- ball 111 can be reduced as far as possible
Mutually aggregation.
In a kind of possible embodiment, the diameter R value 400um, the depth H value 500um, institute as a result,
State space D value 800um.However, it should be appreciated by those skilled in the art that the diameter R, depth H, space D and the training
Lateral dimension, the shape in area 11 are supported, or even can be carried out according to the actual conditions of cell culture with the height of the enclosing 12
Adjustment, to adapt to existing cell culture container, such as Tissue Culture Dish, 6 orifice plates, 12 orifice plates, 24 orifice plates and 48 orifice plates.
Embodiment two
Substrate mould
With reference to Fig. 4, substrate mould 2 of the invention should for the perfusion mold as the poly- ball culture substrate 1 of the cell
Substrate mould 2 includes the first template 21 and the second template 22 of mutually nested formation form chamber.
First template 21 includes plate body and the boss 211 that inside is arranged in and the circumferential setting of the neighbouring boss 211
Groove 212.211 surface of boss has been uniformly distributed several salient points 213, for accordingly in the poly- ball culture medium of the cell
Poly- ball 111, i.e., the interior shape of the shape and size of the outer profile of the described salient point 213 and the poly- ball 111 are formed in matter 1
It is identical with size, formpiston of the salient point 213 as the poly- ball 111.
Specifically, with reference to Fig. 5, each salient point 213 includes positioned at the spheroid 2131 of top half and positioned at lower half
Partial cylindrical body 2132, the cylindrical body 2132 and spheroid 2131 are integrally formed.Preferably, the cylindrical body 2132 is straight
Diameter is identical as the diameter of the spheroid 2131, and the diameter is also indicated with R；The height H of the salient point 213 is greater than the segment
The diameter R of body 2131；The space D of the adjacent salient point 213 is with the distance between the centre of sphere of the adjacent spheroid 2131
It indicates, the space D is 2 times of the diameter R.In a kind of possible embodiment, the diameter R value 400um is described
Depth H value 500um, as a result, the space D value 800um.
With continued reference to Fig. 4, the groove 212 in first template 21 has slot bottom and surrounds the inside of the slot bottom two sides
Cell wall and outside cell wall, the inside cell wall are played a role by the side wall of the boss 211, the part-structure of the outside cell wall
The hole wall that internal through-hole 221 is provided with by second template 22 plays a role, the cross-sectional shape of the through-hole 221 and big
The small outer profile with the outside cell wall matches, it is preferable that the cross-sectional shape of the through-hole 221 and the outside cell wall
Outer profile is identical, and selectively, the size of the cross section of the through-hole 221 is equal to or more than the outer profile of the outside cell wall
Size.
Further, mutually matched location structure is further respectively had between first template 21 and the second template 22.
As shown in figure 4, the location structure includes the location hole 214 for being positioned close to 21 edge of the first template, and setting exists
The locating notch 222 of the edge of second template 22 matched with the location hole 214.The location hole 214 can be with
Setting is one or more, and the quantity of the quantity of the locating notch 222 and the location hole 214 corresponds.
Further, the location structure can also include setting and second template 22 in first template 21
Outer profile the identical locating slot of shapes and sizes, so as to the nested arrangements between second template 22 and the first template 21
Closer, in this embodiment, the groove 212 in first template 21 is raised for the locating slot
Platform, the outside cell wall of the groove 212 is played a role by the hole wall of the through-hole 221 completely as a result,.
The form chamber is limited by the hole wall of the boss 211, groove 212 and through-hole 222 as a result,.
First template 21 and the second template 22 need to be prepared respectively, are then combined to obtain the complete base
Matter mold 2.For the design and producing cost for reducing the substrate mould 2,3D printing skill is used in a kind of possible embodiment
Art is produced, and is specially printed using the DLP3d Phoenix Touch 1080P printer that precision is 50um, and
Using photosensitive resin as printed material.The size of the pouring molding chamber determines that reverse mould is formed by that cell is poly- to seek culture substrate
1 size is preferably reference, the cell with the size of existing cell culture container according to the actual conditions of cell culture
Culture vessel is Tissue Culture Dish, 6 orifice plates, 12 orifice plates, 24 orifice plates and 48 orifice plates etc..
The step of preparing the cell poly- ball culture substrate using the substrate mould 2 is as follows:
(1) it will be heated to by autoclaved agarose after cooling in molten condition；Wherein, the agarose is dense
Degree is 1.5%~2%；The heating temperature of the agarose is 80 DEG C.
(2) agarose of molten condition is filled into the form chamber of the assembled substrate mould 2 while hot
In.
(3) it is demoulded cooling in gelatinous agarose, obtains the poly- ball culture substrate 1 of the cell；It, will when demoulding
Second template 2 is taken out, and the poly- ball culture substrate 1 of cell may be detached from second template 2, it is also possible to be stayed in
In first template 21, no matter which kind of situation can carry out the poly- ball culture substrate 1 of the cell and the disengaging of any template
Subsequent culture uses.
Embodiment three
The physical signs of poly- ball incubation liver cell
(1) human liver cancer cell HepG2 is taken, is incubated at containing 10% fetal calf serum, penicillin (100u/ml), streptomysin
In (100u/ml) dual anti-DMEM high glucose medium, 37 DEG C, 5%CO2, relative humidity 90% incubator in cultivate, periodically
Observe cell growth status.It is digested with 0.25% pancreatin within every 2~3 days, carries out secondary culture.In 5-6 generation to be reached, take 24 orifice plates
Mold size, with 500, every hole of mold cell inoculation, continuous culture 7 days measures cell dia and cultivated days relationship.
As shown in fig. 7, scattering upon starting first day cell of cell inoculation, not poly- ball measures it using Image J software
Diameter 75um or so.As number of days increases, cell is slowly drawn close by the state of scattering, and sphere diameter first increases and then decreases, cell exists
Proliferation, cell bulb diameter are continuing to increase, while statistics indicate that: probably at the 3-4 days or so, cell forms sphere, and Fig. 6 is
HepG2 is cultivated to the 7th day cell in poly- ball growth conditions.
(2) the poly- glomus cell cell ALT of measurement three-dimensional cell is horizontal, using two-dimensional flat plate culture as control, by HepG2 cell
It is incubated at and contains in the dual anti-DMEM high glucose medium of 10% fetal calf serum, penicillin (100u/ml), streptomysin (100u/ml),
37 DEG C of volume fractions are 5%CO2, cultivate in the incubator of relative humidity 90%, routine observation cell growth status.Every 2~3 days
It is digested with 0.25% pancreatin, carries out secondary culture.In 5-6 generation to be reached, takes 24 hole board mold sizes, with 1560, the every hole of mold
Cell inoculation, continuous culture 6 days, takes cells and supernatant, measures cell ALT level compared with two-dimensional flat plate.ALT (alanine
Aminopherase) horizontal reverse answers meronecrosis horizontal.
As shown in figure 8, cell ALT (alanine aminotransferase) the value level and two dimension ALT of three-dimensional poly- ball culture are horizontal
Value it was found that, three-dimensional poly- ball culture ALT maintains always reduced levels, and two-dimensional flat plate culture increases with number of days, and ALT is horizontal
It significantly rises, shows that meronecrosis increases.
(3) the poly- glomus cell albumin secretory volume of three-dimensional cell is measured, it is using two-dimensional flat plate culture as control, HepG2 is thin
Born of the same parents are incubated at containing in the dual anti-DMEM high glucose medium of 10% fetal calf serum, penicillin (100u/ml), streptomysin (100u/ml),
37 DEG C, 5%CO2, relative humidity 90% incubator in cultivate, routine observation cell growth status.Every 2~3 days use
0.25% pancreatin digestion, carries out secondary culture.In 5-6 generation to be reached, takes 24 hole board mold sizes, 1560 thin with the every hole of mold
Born of the same parents' inoculation, 24 orifice plate plates of cells cultures are inoculated with the same cell number 1560 in every hole, and continuous culture 4 days takes cell conditioned medium to measure
Cell Albumin Secretion is horizontal, using the complete DMEM high glucose medium of same batch as comparing.
As shown in figure 9, increasing with cultivated days, three-dimensional poly- ball culture HepG2 cell albumin level is apparently higher than two dimension
Plate culture.
In conclusion cost is relatively low, scale is controllable, process is simple for the external three-dimensional culture method of liver cell of the present invention, training
Feeding effect is significantly better than two-dimensional flat plate culture.
The above embodiment is a preferred embodiment of the present invention, but is not merely restricted to the described embodiments, other
Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, should be equivalent
Substitute mode is all included in the scope of protection of the present invention.
Claims (25)
1. a kind of poly- ball culture substrate of cell, which is characterized in that the Ago-Gel body being integrally formed comprising cultivation region and
It is connected to the enclosing of the cultivation region periphery；The cultivation region is equipped with the poly- ball that several lower half portion are segment-shaped cavity, institute
Top half to the opening for stating poly- ball is cylindrical cavity, and the cylindrical cavity and segment-shaped cavity connect, the circle
The diameter of cylindrical cavity is identical as the diameter of the segment-shaped cavity.
2. the poly- ball culture substrate of cell as described in claim 1, which is characterized in that the depth of the poly- ball is greater than the ball
Lack the diameter of shape cavity.
3. the poly- ball culture substrate of cell as claimed in claim 2, which is characterized in that the diameter of the segment-shaped cavity is
400um, the depth of the poly- ball are 500um.
4. the poly- ball culture substrate of cell as described in claim 1, which is characterized in that the centre of sphere of the adjacent segment-shaped cavity
The distance between be 2 times of the segment-shaped cavity diameter.
5. the poly- ball culture substrate of cell as described in claim 1, which is characterized in that the poly- ball is evenly distributed on the culture
In area.
6. the poly- ball culture substrate of cell as described in claim 1, which is characterized in that the end face of the enclosing is higher than the culture
The end face in area.
7. a kind of substrate mould, which is characterized in that including mutually nested to form the poly- ball culture substrate form chamber of cell
The first template and the second template of plate；The groove being circumferentially arranged in first template equipped with boss and the neighbouring boss,
The boss surface is equipped with the salient point that several top halfs are spheroid；Second template offers and the groove outer side slot
The through-hole of the outer profile cooperation of wall.
8. substrate mould as claimed in claim 7, which is characterized in that the lower half portion of the salient point is cylindrical body, the circle
Cylinder and the segment body by integral forming.
9. substrate mould as claimed in claim 8, which is characterized in that the diameter of the diameter of the cylindrical body and the spheroid
It is identical.
10. substrate mould as claimed in claim 7 or 8, which is characterized in that the height of the salient point is greater than the spheroid
Diameter.
11. substrate mould as claimed in claim 10, which is characterized in that the diameter of the spheroid is 400um, the salient point
Height be 500um.
12. substrate mould as claimed in claim 7, which is characterized in that the distance between the centre of sphere of the adjacent spheroid
It is 2 times of the spheroid diameter.
13. substrate mould as claimed in claim 7, which is characterized in that the salient point is evenly distributed on the boss.
14. substrate mould as claimed in claim 7, which is characterized in that the shape of the through-hole of second template with it is described recessed
The shape of the outer profile of slot lateral wall is identical.
15. substrate mould as claimed in claim 14, which is characterized in that the size of the through-hole of second template is equal to or greatly
In the size of the outer profile of the groove lateral wall.
16. substrate mould as claimed in claim 7, which is characterized in that first template and the second template are respectively equipped with use
In the mutually nested location structure with cooperation.
17. a kind of method of the three-dimensional poly- ball culture of animal cells in vitro, which is characterized in that it includes the following steps:
Preparation is used to form the substrate mould of the poly- ball culture substrate of cell；
The poly- ball culture substrate of the cell is prepared using the substrate mould；
The poly- ball culture substrate of the cell is placed in cell culture container and carries out the poly- nodularization culture of zooblast；
Wherein, the poly- ball culture substrate of the cell is the poly- ball culture substrate of cell as described in claim 1~6 any one,
Or
The substrate mould is the substrate mould as described in claim 7~16 any one.
18. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 17, which is characterized in that the matrix mould
Tool is obtained using 3D printing technique.
19. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 17, which is characterized in that the matrix mould
Tool is made using photosensitive resin.
20. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 17, which is characterized in that the cell is poly-
Ball culture substrate is made by agarose.
21. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 20, which is characterized in that the cell is poly-
Ball culture substrate the preparation method comprises the following steps:
It will be heated to by autoclaved agarose after cooling in molten condition；
The agarose of molten condition is filled into while hot in the assembled substrate mould；
It is demoulded cooling in gelatinous agarose, obtains the poly- ball culture substrate of the cell.
22. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 20, which is characterized in that the agarose
Concentration be 1.5%~2%.
23. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 20, which is characterized in that the agarose
Heating temperature be 80 DEG C.
24. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 17, which is characterized in that the poly- nodularization
The process of culture are as follows:
Cell with the density inoculation of each 500 cells of poly- ball by passage；
Continuous stationary culture extremely forms the poly- ball of cell.
25. the method for the three-dimensional poly- ball culture of animal cells in vitro as claimed in claim 17, which is characterized in that the cell training
Feeding container includes Tissue Culture Dish, 6 orifice plates, 12 orifice plates, 24 orifice plates and 48 orifice plates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610951267.8A CN106554937B (en) | 2016-10-26 | 2016-10-26 | A kind of external three-dimensional culture method of liver cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610951267.8A CN106554937B (en) | 2016-10-26 | 2016-10-26 | A kind of external three-dimensional culture method of liver cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106554937A CN106554937A (en) | 2017-04-05 |
| CN106554937B true CN106554937B (en) | 2019-08-27 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101904775A (en) * | 2010-06-25 | 2010-12-08 | 西安交通大学 | Tissue-like manufacture die based on spheroid elementary unit and preparation process |
| CN202492524U (en) * | 2012-02-17 | 2012-10-17 | 福建医科大学 | Microfluidic chip for unit type cell culture |
| CN203429183U (en) * | 2013-08-22 | 2014-02-12 | 广东顺德西安交通大学研究院 | Three-dimensional cell culture plate |
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| CN101904775A (en) * | 2010-06-25 | 2010-12-08 | 西安交通大学 | Tissue-like manufacture die based on spheroid elementary unit and preparation process |
| CN202492524U (en) * | 2012-02-17 | 2012-10-17 | 福建医科大学 | Microfluidic chip for unit type cell culture |
| CN203429183U (en) * | 2013-08-22 | 2014-02-12 | 广东顺德西安交通大学研究院 | Three-dimensional cell culture plate |
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