WO2017028715A1 - 一种组织细胞培养装置 - Google Patents

一种组织细胞培养装置 Download PDF

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WO2017028715A1
WO2017028715A1 PCT/CN2016/094180 CN2016094180W WO2017028715A1 WO 2017028715 A1 WO2017028715 A1 WO 2017028715A1 CN 2016094180 W CN2016094180 W CN 2016094180W WO 2017028715 A1 WO2017028715 A1 WO 2017028715A1
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Prior art keywords
cavity
cell culture
stage
tissue cell
pore
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PCT/CN2016/094180
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English (en)
French (fr)
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叶雷
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重庆润泽医药有限公司
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Priority to US15/752,252 priority Critical patent/US20190024031A1/en
Priority to EP16836579.9A priority patent/EP3323879A4/en
Publication of WO2017028715A1 publication Critical patent/WO2017028715A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/16—Vibrating; Shaking; Tilting

Definitions

  • the present invention relates to a tissue cell culture device, and more particularly to a tissue cell culture device which is capable of freely growing normally in a three-dimensional space in a culture environment.
  • the tissue cell culture device generally comprises a culture vessel (such as a culture flask, a culture dish, a culture tank, etc.), and when cultured, a culture medium is added to the culture vessel, and the culture medium may be a liquid (ie, a culture solution) or a solid state, when the cells are suspension cells.
  • a culture vessel such as a culture flask, a culture dish, a culture tank, etc.
  • the culture medium may be a liquid (ie, a culture solution) or a solid state, when the cells are suspension cells.
  • the culture medium may be a liquid (ie, a culture solution) or a solid state, when the cells are suspension cells.
  • the culture medium may be a liquid (ie, a culture solution) or a solid state, when the cells are suspension cells.
  • Plates such as 2, 4, 6, 24, 96-well cell culture plates, cell culture is performed on cell culture plates, but the cells culture on the container wall or culture plate are not similar to the growth of cells in the body, in real In vivo, tissue cells grow in a three-dimensional environment. Therefore, when cells are cultured in a two-dimensional planar cell culture plate, they are limited by the planar growth environment, and the activity, morphology, and growth state of the cells are compared with those in the in vivo environment. There are great changes, and the cells are easily exposed to the shear force of the culture fluid, which will cause damage to the cells. Therefore, it is important to carry out three-dimensional cell culture. Cell culture than the two-dimensional culture more closely mimic the in vivo situation, with significant advantages over two-dimensional culture, especially in animal cells is more meaningful, because most animal cells adherent growth.
  • CN 101245313 A discloses "three-dimensional cell culture inserts, methods of making the same, kits and uses". It describes a three-dimensional cell culture insert made of a non-degradable, non-cytotoxic polymeric material having a defined and regular three-dimensional porous structure and consisting of a single porous sheet member or Secured by a plurality of porous sheet members, the porous three-dimensional structure allows cells to be attached to the outer and inner surfaces of the three-dimensional structure at the same time, and the 100%-connected porous structure allows nutrients and metabolites to be easily Exchange of the ground is conducive to the cultivation of cells.
  • CN102864119B discloses "a carrier for cell culture and a method of preparing the same". It introduces the use of three-dimensional porous graphene scaffold as a cell carrier in a culture cell for cell culture.
  • the porous graphene scaffold contains metal foam, which simulates the in vivo growth environment of cells, realizes three-dimensional culture of cells, and is beneficial to maintain cell growth state. And activity to promote cell growth.
  • the object of the present invention is to overcome the deficiencies in the prior art and to provide a tissue cell culture device which is advantageous for free and normal growth of cells in a three-dimensional space.
  • the structural characteristics of the migration to ensure the normal growth conditions of the cells can overcome the deficiencies of the prior art.
  • a tissue cell culture device comprising a tissue cell culture body, characterized in that: the porous material as the tissue cell culture body is a cavity which is classified by a material pore size and a cavity wall surrounding the cavity forming each stage, and is surrounded by The lower-level small-cavity cavity is disposed on the cavity wall constituting the upper-level large-cavity cavity; the same-level cavity is mutually penetrated, and the cavity of each stage also penetrates each other.
  • the porous material cavity is classified, and a lower-level small-cavity cavity is arranged on the cavity wall surrounding the upper-stage large-cavity cavity, so that the cell pore size becomes smaller during the cell growth and reproduction process, and the tissue cell culture body becomes smaller.
  • the nutrient solution can still flow in the tissue cell culture body, and the cells can freely migrate in the tissue cell culture body, so as to facilitate the free and normal growth of the cells in the three-dimensional space.
  • the wall surrounding the cavity constituting the upper stage is formed by uniformly filling the small hole cavity of the lower stage, and is preferably filled with a dense cloth.
  • the pores which are classified as the porous material of the tissue cell culture body by the pore size of the material are at least two or more stages.
  • a lower-stage small-cavity cavity is disposed around the cavity wall constituting the upper-level large-cavity cavity; or a lower-stage is disposed around the cavity wall constituting the largest-stage one-dimensional cavity Two-stage small cavity.
  • it is divided into more holes of porous material, and the cavity wall of each upper cavity is provided with a small hole cavity of the next stage, or a lower stage is arranged around the cavity wall constituting the upper large cavity.
  • Each of the small holes of each stage or the wall of the cavity forming the upper large cavity is provided with any combination of small holes.
  • the inventors also believe that when the pores of the porous material are divided into three stages, it is preferable that the wall of the cavity constituting the upper stage cavity is uniformly filled by the lower stage small hole cavity. Such cell grading not only facilitates cell growth, but is also convenient for preparation.
  • At least the smallest pores have a smaller pore diameter than the average diameter of the cultured cells, and the remaining other pores have a pore diameter at least larger than the average diameter of the cultured cells.
  • the pore size of the larger pores in the other stage cavities is at least twice the average diameter of the cultured cells.
  • the pore size of the smallest pore is smaller than the average diameter of the cultured cells, and the pore diameter of the largest pore is preferably at least twice the average diameter of the cultured cells.
  • tissue cell culture bodies mentioned in the above tissue culture apparatus are particularly suitable for use as medical implants.
  • the tissue cell culture apparatus further includes a culture dish in which the tissue cell culture body is placed, and the tissue cell culture body is fixed in the culture dish.
  • the tissue cell culture body is preferably made into a plate structure.
  • the plate thickness of the tissue cell culture having a plate structure is not more than 20 times the average pore diameter of the largest primary cavity of the porous material.
  • the culture dish is fixed to a vibration table.
  • the present invention provides a tissue cell culture device which is favorable for free and normal growth of a three-dimensional space of a cell, and the porous material is used as a tissue cell culture body, in particular, the porous material is divided into a cavity having a pore size of a material and a cavity surrounding the cavity.
  • the wall is rationally designed, the cavities of the same level are mutually penetrated, and the cavities of the respective stages are also connected to each other, thereby creating a suitable three-dimensional free normal growth space for the cells, and the small cavities are used.
  • the interpenetrating pores on the wall of the cavity ensure that the nutrient solution or the metabolite flows through, forming a microcirculation system that satisfies the cell growth, and the large cavity is used for cell adherence.
  • the cells can travel freely in the three-dimensional space between the large and large cavities, especially when the holes are divided into three levels, the smallest cavities are used for the transport and flow of nutrient solution or metabolites, and the intermediate cavities are used for cells. Inhabited, the largest hole is used for cell migration and passage.
  • the culture provided by the invention is not easy to form a physiological dead space or a dead space in the cell culture process, and there is no cell growth disorder zone or a nutrient rich zone. Even if the cells increase, the cavity becomes small or blocked, and the nutrition is not hindered. Any infiltration or permeation of liquid or metabolites in cell culture, thus ensuring the smooth and normal growth of cells, more in line with the growth of cells.
  • the tissue cell culture device of the present invention hierarchically sets the pores of the porous material, and rationally designs the number of pore cavities and the size of the cavities to meet various growth environmental conditions of various tissue cells. .
  • the smaller pore size of the pores is smaller than the average diameter of the cultured cells, which is more conducive to the transport and flow of the nutrient solution or the metabolites.
  • the pore size of the other pores is at least larger than the average diameter of the cultured cells, further ensuring the demand for cell colonization.
  • the pore size of the larger pore cavity is at least twice the average diameter of the cultured cells, so that the cells can smoothly migrate and travel smoothly under the requirement of cell colonization, and ensure that the cells smoothly enter the culture body and migrate and grow in the culture body.
  • the smaller pore size is smaller than the average diameter of the cultured cells, and the capillary force is generated due to the small pore size, which promotes the infiltration and flow of the culture solution and the metabolite, and can inhale the cells into the cell culture and promote the tissue cells. Migration in the culture, and the growth rate of the cells is increased due to the presence of capillary forces.
  • the cell culture body adopts a plate structure to facilitate the growth of adherent cells into the interior, avoiding cell occlusion on the surface, and facilitating cell removal, so that the culture medium can pass through the cell culture body.
  • the tissue cell culture body provided by the present invention is placed in a culture dish, and an external vibration source is provided through the vibration table for the culture condition, and the vibration can be performed vertically, horizontally, and along the vertical axis, and at least two of the three vibrations.
  • the composite vibration can promote the flow of the culture solution in the culture dish, and the concentration of the tissue cells and the distribution of various components therein are uniform.
  • Figure 1 is a schematic view of a tissue cell culture device of the present invention
  • Figure 2 is a cross-sectional view of Figure 1A-A (with the petri dish cover 8 removed);
  • 3 is a schematic view showing the structure of a tissue cell culture body, 3-1 is a main view, 3-2 is a left view, and 3-3 is a top view;
  • Figure 4 is an enlarged view of a portion B of Figure 3;
  • Figure 5 is a cross-sectional view of Figure 4C-C.
  • 1 is a culture dish
  • 2 is a tissue cell culture body
  • 3 is a fixed block of a fixed tissue cell culture body, which is fixed in the culture dish 1 by welding
  • 4 is a vibration table
  • 5 is fixed.
  • 6 is a fixed tissue cell culture body 2
  • the pressure block, 7 is a screw
  • 8 is a petri dish cover.
  • the tissue cell culture body 2 is placed in the recess 5 of the fixed block 3, and the tissue cell culture body 2 is fixed to the fixed block 3 by the compact 6 and the screw 7, thereby being fixed in the culture dish 1, and the culture dish 1 can be commonly used.
  • the culture plate 1 is fixed to the vibrating table 4 by means of a platen and a bolt fixing method, and the petri dish cover 8 is placed on the petri dish 1.
  • FIG. 4 and FIG. 5 wherein 9 is the largest first-order cavity of the tissue cell culture body 2, and 10 is the cavity wall of the cavity, and the cavity is three-dimensionally penetrated.
  • the cavity wall 10 of the cavity 9 is composed of a smaller cavity 11 (lower stage hole) and a cavity wall 12 surrounding the cavity 11, and an enlarged view of the cavity wall 10 in conjunction with Fig. 4, Fig. 5C-
  • the cavity 11 is also three-dimensionally penetrated, and the two-stage cavities are also three-dimensionally connected to each other.
  • tissue cell culture body having a multistage pore structure of three or more stages can be formed.
  • the cells are placed on the tissue culture body 2, and then the culture solution is added to the culture dish 1, and the culture dish cover 8 is placed on the culture dish 1, and the vibrating table 4 is started to vibrate the culture dish 1. After the completion of the culture, the cells on the tissue cell culture body 2 were taken out.
  • a tissue cell culture apparatus comprising a tissue culture body 2, the cell culture body 2 is fixed in a culture dish 1, and the culture dish 1 is fixed on a vibration table 4.
  • the tissue cell culture body 2 is a porous tantalum material; the porous tantalum material is a cavity which is classified by a material pore size and a cavity wall surrounding the cavity forming each stage, and surrounds a cavity wall constituting the upper large cavity.
  • the lower-level small-cavity cavity is set; the same-level cavities are mutually penetrated and the cavities of the respective stages are also mutually penetrated.
  • the porous tantalum material has a two-stage cavity, and the cavity wall of the upper-level large-pore cavity which is evenly distributed and interpenetrated is composed of uniformly distributed, interpenetrating lower-level small-cavity cavities, and the two-stage cavities are mutually They also penetrate each other.
  • the pore size of the upper large cavity is 160 ⁇ m to 460 ⁇ m, and the pore size of the lower small cavity is 200 nm to 500 nm;
  • the tissue culture body 2 is a plate body having a thickness of 5.5 mm, which is 17 times the average pore diameter of the upper large cavity.
  • smooth muscle tissue cells were cultured.
  • smooth muscle tissue cells were placed on tissue culture cell 2, and MEM containing 12% serum was added to the culture dish 1, and the temperature was controlled at 35 ° C to 37 ° C.
  • the lid 5 is capped on the culture dish 1, and other specific operations are the same as those of the conventional cell culture technique.
  • the vibrating table 4 is activated to vibrate the petri dish 1 and the vibration is in a vertical vibration mode with an operating frequency of 80 Hz. After culturing for 3 days, the cells on the tissue cell culture body 2 were taken out and observed, and the results showed that the cultured cells were full and the growth state was good.
  • the porous material was prepared as follows:
  • urea with particle size of 300nm-600nm is used as the smallest primary pore former, and 300nm-600nm starch is used as binder, according to the volume ratio of tantalum powder: urea: starch: distilled water. 1:1.5:1:7 is formulated into a slurry.
  • the slurry is uniformly filled into a polyester foam having a rib diameter of 260 ⁇ m to 560 ⁇ m by a foam impregnation method to form a green body and dried, and then crushed to prepare a raw material containing a cerium powder having a particle diameter of 260 ⁇ m to 560 ⁇ m, and a pore forming agent. Mixed particles of polyester foam.
  • the mixed particles, methyl cellulose having a particle diameter of 260 ⁇ m to 560 ⁇ m are uniformly mixed in a volume ratio of 2:1, and placed in a sealed mold to form a dense body.
  • the compact body is vacuum sintered; the sintered body is subjected to a conventional subsequent treatment according to the coffin process to obtain a porous crucible having secondary pores.
  • the cell culture body 2 of the tissue cell culture apparatus of the present embodiment adopts a porous silica material, has a three-stage cavity, and is evenly distributed, and the cavity walls of the first-stage large-hole cavity which are mutually penetrated are connected to each other by the second stage, the first The three-stage cavity is filled, and the holes of each stage are also connected to each other.
  • the first-stage large-pore cavity has a pore diameter of 1000 ⁇ m to 1500 ⁇ m
  • the second-stage pore cavity has a pore diameter of 70 ⁇ m to 90 ⁇ m
  • the third-stage pore has a pore diameter of 400 nm to 700 nm
  • the tissue cell culture body 2 has a thickness of 24 mm, which is a first-order large.
  • the pore volume has an average pore diameter of 19 times.
  • the culture solution is EAGLE containing 20% calf serum, and the cultured cells are epithelial tissue cells. Others are the same as in the first embodiment.
  • the vibrating table adopts a vibration mode that rotates along a vertical axis, and the operating frequency is 70 Hz.
  • the culture results showed that the cell body was full and the growth state was good.
  • the porous material was prepared as follows:
  • urea having a particle diameter of 500 nm to 800 nm is used as a minimum first-order (third-order) pore-forming agent, and ethyl cellulose having a particle diameter of 100 ⁇ m to 150 ⁇ m is used as the second stage.
  • the pore-forming agent was prepared into a slurry by using silica powder of 500 nm to 800 nm as a binder in accordance with a silica powder:urea:ethylcellulose:starch:distilled water at a volume ratio of 1:1.5:1:1:12.
  • a polyester foam having a pore diameter of from 1100 ⁇ m to 1700 ⁇ m is used, and the slurry is uniformly filled therein by a foam impregnation method to form a green body and dried, followed by vacuum sintering to obtain a porous silica having a tertiary pore.
  • the tissue cell culture body 2 of the tissue cell culture apparatus of the present embodiment adopts a porous tantalum material and has a three-stage pore cavity in which the cavity wall of the largest first-order pore cavity (hereinafter referred to as a first-stage cavity) which is uniformly distributed and mutually penetrated is interposed. It is composed of a uniformly distributed and inter-connected intermediate first-stage cavity (hereinafter referred to as a second-stage cavity), and the cavity wall of the second-stage cavity is uniformly distributed and penetrated with a minimum level of the cavity (hereinafter referred to as the third stage) The cavities are filled; and the cavities of the respective stages are also connected to each other.
  • a first-stage cavity the cavity wall of the largest first-order pore cavity
  • the third stage a minimum level of the cavity
  • the first stage cavity has a pore diameter of 140 ⁇ m to 600 ⁇ m
  • the second stage cavity has a pore diameter of 50 ⁇ m to 90 ⁇ m
  • the third stage cavity has a pore diameter of 300 nm to 600 nm
  • the tissue cell culture body 2 has a thickness of 7 mm, which is a first stage cavity.
  • the average pore diameter was 18 times
  • the culture solution was BME containing 18% serum
  • the cultured cells were osteoblast tissue cells, and the same as in Example 1.
  • the culture condition is that the vibration table is alternately operated by horizontal vibration and vibration along the vertical axis, and the working frequencies are 65 Hz and 60 Hz, respectively.
  • the culture results showed that the cells grew in good condition.
  • the porous material was prepared as follows:
  • methyl cellulose with a particle diameter of 400nm-700nm is used as a minimum first-order pore-forming agent, and polystyrene having a particle diameter of 400nm-700nm is used as a binder.
  • the slurry was prepared in a volume ratio of 1:2:1:7.5 in accordance with the powder:methylcellulose:polystyrene:distilled water.
  • a polyester foam having a rib diameter of 60 ⁇ m to 100 ⁇ m and a pore diameter of 200 ⁇ m to 400 ⁇ m is used, and the slurry is uniformly filled therein by a foam impregnation method to form a green body, dried, and then crushed to form a crucible having a particle diameter of 60 ⁇ m to 100 ⁇ m.
  • Mixed particles of powder raw material, pore former and polyester foam are used, and the slurry is uniformly filled therein by a foam impregnation method to form a green body, dried, and then crushed to form a crucible having a particle diameter of 60 ⁇ m to 100 ⁇ m.
  • the compact body is vacuum sintered; the sintered body is subjected to a conventional subsequent heat treatment according to the coffin process to obtain a porous crucible having a tertiary pore.
  • the tissue cell culture body 2 of the tissue cell culture apparatus of the present embodiment adopts a porous titanium material and has a four-stage cavity in which the cavity wall of the largest first-order cavity (ie, the first-stage cavity) uniformly distributed and interpenetrated is composed of
  • the second-stage cavities are evenly distributed and interpenetrated, and the cavity walls of the second-stage cavities are filled by uniformly distributed and inter-connected third-stage and fourth-stage cavities; and the cavities of the respective stages are also mutually Through.
  • the pore size of the first stage cavity is 700 ⁇ m ⁇ 1200 ⁇ m
  • the pore size of the second stage cavity is 200 ⁇ m ⁇ 300 ⁇ m
  • the pore size of the third stage cavity is 400nm ⁇ 700nm
  • the pore size of the fourth stage cavity is 10nm ⁇ 100nm
  • the tissue cell culture body 2 The thickness was 17 mm, which was 16 times the average pore diameter of the first-stage cavity.
  • the culture solution was high-sugar DMEM containing 10% fetal bovine serum and double antibody, and the cultured cells were HUVEC cells, and the same as in Example 1.
  • the culture condition is that the vibration table is alternately operated by horizontal vibration and vertical vibration, and each vibration is 2 minutes, and the working frequencies are 50 Hz and 70 Hz, respectively.
  • the culture results showed that the cells grew in good condition.
  • the porous material was prepared as follows:
  • urea with a particle size of 5 nm to 120 nm is used as the minimum first stage (fourth stage) pore former, and ethyl cellulose having a particle diameter of 500 nm to 800 nm is used as the third stage pore.
  • a cavity pore former was prepared by using a starch of 5 nm to 120 nm as a binder, and a slurry of titanium powder:urea:ethylcellulose:starch:distilled water at a volume ratio of 1:1.5:1:1:12.
  • a polyester foam having a rib diameter of 300 ⁇ m to 400 ⁇ m and a pore diameter of 400 ⁇ m to 600 ⁇ m is used, and the slurry is uniformly filled therein by a foam impregnation method to form a green body and dried, and then crushed to obtain a titanium powder having a particle diameter of 300 ⁇ m to 400 ⁇ m.
  • Mixed particles of raw materials, pore former and polyester foam are used, and the slurry is uniformly filled therein by a foam impregnation method to form a green body and dried, and then crushed to obtain a titanium powder having a particle diameter of 300 ⁇ m to 400 ⁇ m.
  • the compact body is vacuum sintered; the sintered body is subjected to a conventional subsequent heat treatment in accordance with a titanium material process to obtain porous titanium having a four-stage pore.
  • the tissue cell culture body 2 of the tissue culture apparatus of the present embodiment adopts a porous CoNiCrMo alloy material, and has a five-stage cavity in which the cavity wall of the largest first-order cavity (ie, the first-stage cavity) which is evenly distributed and mutually penetrated is interposed.
  • the second-stage and third-stage cavities are uniformly filled and inter-connected, and the cavity walls of the second-stage and third-stage cavities are filled by the fourth-stage and fifth-stage cavities which are evenly distributed and mutually penetrated; And the cavities of each stage also penetrate each other.
  • the first stage cavity diameter is 800 ⁇ m ⁇ 1300 ⁇ m
  • the second stage cavity cavity is 100 ⁇ m ⁇ 200 ⁇ m
  • the third stage cavity cavity is 30 ⁇ m ⁇ 70 ⁇ m
  • the fourth stage hole cavity The diameter is 500 nm to 700 nm
  • the pore size of the fifth stage cavity is 50 nm to 200 nm
  • the thickness of the tissue cell culture body 2 is 20 mm, which is 18 times the average pore diameter of the first stage cavity
  • the culture medium contains 10% fetal bovine serum.
  • the high-glucose DMEM of the double antibody the cultured cells were HUVEC cells, and the other was the same as in Example 1.
  • the culture condition is that the vibrating table adopts horizontal vibration mode, and the working frequency is 70 Hz.
  • the culture results showed that the cells grew in good condition.
  • the porous material was prepared as follows:
  • urea with a particle size of 60 nm to 300 nm is used as the smallest primary (fifth stage) pore former, and ethyl cellulose having a particle diameter of 600 nm to 800 nm is used as the fourth stage.
  • the pore-forming agent was prepared into a slurry by using 60 nm to 300 nm of starch as a binder in accordance with CoNiCrMo alloy powder:urea:ethylcellulose:starch:distilled water at a volume ratio of 1:1.5:1:1:12.
  • a polyester foam having a rib diameter of 40 ⁇ m to 80 ⁇ m and a pore diameter of 400 ⁇ m to 600 ⁇ m is used, and the slurry is uniformly filled therein by a foam impregnation method to form a green body and dried, and then crushed to form a CoNiCrMo-containing material having a particle diameter of 40 ⁇ m to 80 ⁇ m.
  • Mixed powder of alloy powder raw material, pore former and polyester foam is used, and the slurry is uniformly filled therein by a foam impregnation method to form a green body and dried, and then crushed to form a CoNiCrMo-containing material having a particle diameter of 40 ⁇ m to 80 ⁇ m.
  • the compact body is vacuum sintered; the sintered body is subjected to a conventional subsequent heat treatment according to the CoNiCrMo alloy process to obtain a porous CoNiCrMo alloy having five-stage pores.

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Abstract

本发明提供了一种组织培养装置，其包括细胞培养体。所述细胞培养体的多孔材料由以孔径大小分级的孔腔及孔壁构成，且上级大孔腔的腔壁上设置下级小孔腔；同级孔腔及各级孔腔间相互贯通。

Description

一种组织细胞培养装置 技术领域
本发明涉及组织细胞培养装置，特别是一种利于细胞在培养环境下，能够在三维空间自由正常生长的组织细胞培养装置。
背景技术
组织细胞培养是一种在药物开发、细胞生物学、毒理学、生物工程以及组织工程领域中非常有用并且被广泛使用的技术。组织细胞培养装置一般包括培养容器(如培养瓶、培养皿、培养罐等)，培养时，培养容器中加培养基，培养基可以是液态(即培养液)或固态，当细胞为悬浮型细胞时，一般只需培养基，但当细胞为贴附型细胞时，若培养容器中只有培养基，细胞就只能贴容器壁培养，在常规的细胞培养中，在培养容器中加入了细胞培养平板，如2，4，6，24，96孔细胞培养板，细胞培养在细胞培养平板上进行，但细胞在容器壁或培养平板上培养与细胞在体内的生长方式并不相仿，在真实的活体内，组织细胞均在三维空间环境中生长，因此在目前二维平面的细胞培养板培养细胞时，受平面生长环境的限制，细胞的活性、形貌和生长状态等与体内环境下相比有很大改变，且细胞易暴露于培养液流体剪切力下，会对细胞造成损伤，因此，进行三维细胞培养具有重要的意义，三维细胞培养比二维培养能更接近地模拟体内的情况，具有比二维培养的显著优势，特别是对动物细胞更有意义，因为大多数动物细胞贴壁生长。
为进行三维细胞培养，研究人员设计了一些装置：
CN 101245313A公开了″三维细胞培养插入件、其制造方法、成套用具及用途″。它介绍了一种三维细胞培养插入件，它由非降解性的无细胞毒性的聚合物材料制成，该插入件具有确定的和规则的三维多孔结构，并且其由单个多孔片层构件构成或由多个多孔片层构件固定在一起，该种多孔的三维结构使细胞可以同时贴附于该三维结构的外表面和内表面，其100％联通的多孔的结构能够允许营养物和代谢产物容易地进行交换，有利于细胞的培养。
CN102864119B公开了″用于细胞培养的载体及其制备方法″。它介绍了用三维多孔石墨烯支架作为细胞载体放入培养池中进行细胞培养，多孔石墨烯支架含有金属泡沫，模拟细胞的体内生长环境，实现了细胞的三维培养，有利于维持细胞的生长状态和活性，促进细胞生长。
仔细分析上述现有技术，它们存在的不足之处在于：当培养的细胞逐渐增多时，会阻塞孔隙形成生理死腔或者是营养物质富集区，致使营养物或新陈代谢物难以流动，并阻碍细胞的迁移生长，也会使细胞在培养体内生长分布不均匀，生长状况欠佳。
发明内容
本发明目的是为了克服现有技术中的不足，而提供一种有利于细胞在三维空间自由正常生长的组织细胞培养装置。
发明人认为如果提供一种细胞培养装置，即使在细胞生长繁殖过程中，随着细胞数量逐渐增多致使细胞培养体的孔隙变小或堵塞，仍具备满足营养液或新陈代谢物在多孔的培养体内自由迁移的结构特点，以保证细胞的正常生长条件，则能克服现有技术的不足。
本发明目的通过如下技术方案实现：
一种组织细胞培养装置，包括组织细胞培养体，其特征在于：作为该组织细胞培养体的多孔材料是以材料孔径大小进行分级的孔腔及围绕形成各级孔腔的腔壁构成，且围绕构成上级大孔腔的腔壁上设置下级小孔腔；同级孔腔均相互贯通，且各级孔腔相互间也彼此贯通。将多孔材料孔腔进行分级，且在围绕形成上级大孔腔的腔壁上设置下级小孔腔，使得细胞生长繁殖过程中，即使细胞数量逐渐增多使得培养体孔隙变小，该组织细胞培养体仍能使营养液在组织细胞培养体内流动，并能使细胞在组织细胞培养体内自由迁移，以利于细胞在三维空间自由正常生长。
进一步地，上述多孔材料中，围绕构成上级大孔腔的腔壁是由下级小孔腔均布填充构成，以密布充满为佳。
上述组织细胞培养装置中，作为组织细胞培养体的多孔材料以材料孔径大小进行分级的孔腔至少分为两级以上。当该多孔材料的孔腔分为三级时，围绕构成上一级大孔腔的腔壁上设置有下一级小孔腔；或者是围绕构成最大一级孔腔的腔壁上设置有下级两级小孔腔。以此类推，分为更多级的多孔材料的孔腔，其每上级孔腔的腔壁上设置有下一级的小孔腔，或者围绕构成上级大孔腔的腔壁上设置有下级的各级小孔腔或者围绕构成上级大孔腔的腔壁上设置有任意组合的各级小孔腔。
发明人还认为，当多孔材料的孔腔分为三级时，其中围绕构成上一级孔腔的腔壁是由下一级小孔腔均布填充为佳。这样的孔腔分级不仅利于细胞生长，而且也便于制备。
上述组织细胞培养装置中，较小级孔腔中，至少最小级孔腔的孔径要小于培养细胞的平均直径，剩余的其他级孔腔的孔径至少要大于培养细胞的平均直径。
上述组织细胞培养装置中，所述其他级孔腔中的较大级孔腔的孔径至少为培养细胞平均直径的两倍以上。
上述组织细胞培养装置中，最小级孔腔的孔径小于培养细胞的平均直径，最大级孔腔的孔径至少为培养细胞平均直径的两倍以上为佳。
上述组织细胞培养装置中所提及的组织细胞培养体特别适用作为医用植入体的应用。
该组织细胞培养装置还包括放置组织细胞培养体的培养皿，所述组织细胞培养体固定于所述培养皿中。
上述组织细胞培养装置中，所述组织细胞培养体制成板块结构为佳。
上述组织细胞培养装置中，呈板块结构的组织细胞培养体的板厚不大于所述多孔材料最大一级孔腔的平均孔径的20倍。
上述组织细胞培养装置中，所述培养皿固定于振动工作台上。
本发明的有益效果：
1、本发明提供了有利于细胞三维空间自由正常生长的组织细胞培养装置，将多孔材料作为组织细胞培养体，特别是该多孔材料以材料孔径大小进行分级的孔腔及围绕形成孔腔的腔壁进行合理设计，同级孔腔均相互贯通且各级孔腔相互间也彼此贯通，给细胞创造了适宜的三维自由正常生长的空间，小孔腔用 于营养液或新陈代谢物的传输、流动，腔壁上的互为贯通的孔腔可保证营养液或新陈代谢物流动通过，构成满足细胞生长的微循环体系，大孔腔用于细胞贴壁寄居，细胞能在大孔腔及大孔腔之间的三维空间自由穿行，特别是当孔分成三级时，最小级孔腔用于营养液或新陈代谢物的传输、流动，中间级孔腔用于细胞寄居，最大级孔用于细胞迁移、穿行。本发明提供的培养体在细胞培养过程中不易形成生理死腔或浓度死腔，不会出现细胞生长障碍区或者是营养物质富集区，即使细胞增多，孔腔变小或堵塞也不阻碍营养液或新陈代谢物在细胞培养体内的任意浸润或称渗透，从而保障细胞的顺利正常生长，更加符合细胞的生长规律。
2、本发明所述的组织细胞培养装置，对多孔材料的孔腔进行分级设置，而且合理设计各级孔腔的级数及孔腔的大小，以满足各类组织细胞的各种生长环境条件。特别是较小级的孔腔孔径小于培养细胞的平均直径，更利于充满构成营养液或新陈代谢物的传输、流动，其他级孔腔的孔径至少大于培养细胞的平均直径进一步保证了细胞寄居的需求，较大级孔腔的孔径至少为培养细胞平均直径的两倍以上则进一步在满足细胞寄居的需求下使细胞能顺利地自由迁移、穿行，并保障细胞顺利进入培养体内，在培养体内迁移生长；而较小级孔腔孔径小于培养细胞的平均直径，由于孔径小还会产生毛细力，能促进培养液、新陈代谢物的浸润流动，并能将细胞吸入细胞培养体内，并促进其在组织细胞培养体中的迁移，而由于毛细力的存在还会提高细胞的生长速度。
3、细胞培养体采用板块结构便于贴壁细胞长入内部，避免细胞在表面阻塞，也便于细胞取出，便于培养液穿过细胞培养体。
4、将本发明提供的组织细胞培养体置于培养皿，对培养条件通过振动工作台提供一种外部振动源，可以进行垂直、水平、沿垂直轴转动的振动，以及三种振动中至少两个的复合振动，可促使培养皿中的培养液的流动，使其中的组织细胞的浓度、及各种成分分布均匀。
附图说明
下面将结合附图与实施例对本发明作进一步阐述。
图1为本发明组织细胞培养装置示意图；
图2为图1A-A截面图(去掉培养皿盖8)；
图3为组织细胞培养体结构示意图，3-1为主视图，3-2为左视图，3-3为俯视图；
图4为图3局部B放大图；
图5为图4C-C截面图。
具体实施方式
下面结合附图对本发明的具体实施方式作说明，实施方式以本发明技术方案为前提，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不仅限于下述的实施方式。
在图1、图2中，1为培养皿，2为组织细胞培养体，3为固定组织细胞培养体的固定块，它通过焊接固定在培养皿1内，4为振动工作台，5为固定块3开的凹槽，用于使组织细胞培养体2放入固定，6为固定组织细胞培养体2的 压块，7为螺钉，8为培养皿盖。组织细胞培养体2放入固定块3的凹槽5中，通过压块6、螺钉7使组织细胞培养体2固定于固定块3上，从而在培养皿1中固定，培养皿1可通过常用的压板、螺栓固定方式使培养皿1固定在振动工作台4上，培养皿盖8盖在培养皿1上。
如图3、图4、图5所示，其中，9为组织细胞培养体2的最大一级孔腔，10为该孔腔的腔壁，该孔腔是三维贯通的。从图4可知，孔腔9的腔壁10由更小的孔腔11(下一级孔)及围绕孔腔11的腔壁12构成，结合图4对腔壁10的放大图、图5C-C截面图可知，孔腔11也是三维贯通的，且两级孔腔彼此也三维贯通。
进一步类推，可以形成三级以上的多级孔结构的组织细胞培养体。
培养细胞时，将细胞置于组织细胞培养体2上，然后将培养液加入培养皿1中，将培养皿盖8盖在培养皿1上，启动振动工作台4，使培养皿1振动。培养完成后，将组织细胞培养体2上的细胞取出。
以下详细给出本发明的实施例：
实施例1：
参见附图，一种组织细胞培养装置，它包括组织细胞培养体2，细胞培养体2固定于培养皿1中，培养皿1固定在振动工作台4上。该组织细胞培养体2为一多孔钽材料；该多孔钽材料是以材料孔径大小进行分级的孔腔及围绕形成各级孔腔的腔壁构成，且围绕构成上级大孔腔的腔壁上设置下级小孔腔；同级孔腔均相互贯通且各级孔腔相互间也彼此贯通。
具体在本例，该多孔钽材料，它具有两级孔腔，均匀分布、相互贯通的上级大孔腔的腔壁由均匀分布、相互贯通的下级小孔腔填充构成，且两级孔腔相互间也彼此贯通。上级大孔腔的孔径为160μm～460μm，下级小孔腔孔径为200nm～500nm；组织细胞培养体2为一板块体材，其厚度为5.5mm，为上级大孔腔的平均孔径的17倍。
本例培养平滑肌组织细胞，培养时，将平滑肌组织细胞置于组织细胞培养体2上，在培养皿1内加入含有12％血清的MEM，将其温度控制在35℃～37℃，将培养皿盖5盖在培养皿1上，其他具体操作与常规细胞培养技术相同。启动振动工作台4，使培养皿1振动，振动采用垂直振动方式，工作频率为80Hz。培养3天，然后将组织细胞培养体2上的细胞取出观察，结果表明，培养的细胞胞体丰满，生长状态良好。
本例中，该种多孔材料是按以下方法制得的：
(1)材料准备
采用1-10μm的钽粉作为原料，粒径为300nm-600nm的尿素作为最小一级孔造孔剂，并用300nm-600nm的淀粉作为粘合剂，按照钽粉∶尿素∶淀粉∶蒸馏水按体积比1∶1.5∶1∶7配制成浆料。
将所述浆料用泡沫浸渍法均匀填充入棱直径为260μm-560μm的聚酯泡沫中，形成坯体并干燥，然后破碎制成粒径为260μm-560μm的含有钽粉原料、造孔剂与聚酯泡沫的混合颗粒。
(2)将混合颗粒、粒径为260μm-560μm的甲基纤维素按体积比2∶1均匀混合放入密闭模具压制成致密坯体。
(3)将致密坯体真空烧结；烧结后的坯体按照钽材工艺进行常规后续处理得到具有二级孔的多孔钽。
实施例2：
本实施例的组织细胞培养装置的细胞培养体2采用多孔二氧化硅材料，具有三级孔腔，均匀分布、相互贯通的第一级大孔腔的腔壁由相互贯通的第二级、第三级孔腔填充构成，且各级孔腔相互间也彼此贯通。第一级大孔腔孔径为1000μm～1500μm，第二级孔腔孔径为70μm～90μm，第三级孔腔的孔径为400nm～700nm，组织细胞培养体2的厚度为24mm，为第一级大孔腔的平均孔径的19倍，培养液为含有20％小牛血清的EAGLE，培养细胞为上皮组织细胞。其他同实施例1。
振动工作台采用沿垂直轴转动的振动方式，工作频率为70Hz。培养结果表明，细胞胞体丰满，生长状态良好。
本例中，该种多孔材料是按以下方法制得的：
(1)材料准备
采用1-10μm的二氧化硅粉作为原料，粒径为500nm～800nm的尿素作为最小一级(第三级)孔造孔剂，用粒径为100μm～150μm的乙基纤维素作为第二级孔造孔剂，并用500nm～800nm的淀粉作为粘合剂，按照二氧化硅粉∶尿素∶乙基纤维素∶淀粉∶蒸馏水按体积比1∶1.5∶1∶1∶12配制成浆料。
(2)采用孔径为1100μm～1700μm的聚酯泡沫，将所述浆料用泡沫浸渍法均匀填充其中，形成坯体并干燥，然后真空烧结得到具有三级孔的多孔二氧化硅。
实施例3：
本实施例的组织细胞培养装置的组织细胞培养体2采用多孔铌材料，具有三级孔腔，其中，均匀分布、相互贯通的最大一级孔腔(以下称第一级孔腔)的腔壁由均匀分布、相互贯通的中间一级孔腔(以下称第二级孔腔)填充构成，第二级孔腔的腔壁由均匀分布、相互贯通的最小一级孔腔(以下称第三级孔腔)填充构成；且各级孔腔相互间也彼此贯通。第一级孔腔孔径为140μm～600μm，第二级孔腔孔径为50μm～90μm，第三级孔腔孔径为300nm～600nm，组织细胞培养体2的厚度为7mm，为第一级孔腔的平均孔径的18倍，培养液为含有18％血清的BME，培养细胞为成骨组织细胞，其他同实施例1。
培养条件是振动工作台采用水平振动、沿垂直轴转动的振动方式交替进行，工作频率分别为65Hz、60Hz。培养结果表明，细胞生长状态良好。
本例中，该种多孔材料是按以下方法制得的：
(1)材料准备
采用粒径为1-10μm的铌粉为原料，粒径为400nm-700nm的甲基纤维素做为最小一级孔造孔剂，用粒径为400nm-700nm的聚苯乙烯作为粘合剂，按照铌粉∶甲基纤维素∶聚苯乙烯∶蒸馏水按体积比1∶2∶1∶7.5配制成浆料。
采用棱直径为60μm～100μm、孔径为200μm～400μm的聚酯泡沫，将所述浆料用泡沫浸渍法均匀填充其中，形成坯体并干燥，然后破碎制成粒径为60μm～100μm的含有铌粉原料、造孔剂与聚酯泡沫的混合颗粒。
(2)将混合颗粒、粒径为60μm～100μm的乙基纤维素按体积比2∶1均匀混合后均匀地灌入棱直径为250μm-750μm、孔径为400μm-600μm的三维贯通的聚酯泡沫中，然后将聚酯泡沫放入密闭模具压制成致密坯体。
(3)将致密坯体真空烧结；烧结后的坯体按照铌材工艺进行常规后续热处理得到具有三级孔的多孔铌。
实施例4：
本实施例的组织细胞培养装置的组织细胞培养体2采用多孔钛材料，具有四级孔腔，其中，均匀分布、相互贯通的最大一级孔腔(即第一级孔腔)的腔壁由均匀分布、相互贯通的第二级孔腔填充构成，第二级孔腔的腔壁由均匀分布、相互贯通的第三级、第四级孔腔填充构成；且各级孔腔相互间也彼此贯通。第一级孔腔孔径为700μm～1200μm，第二级孔腔孔径为200μm～300μm，第三级孔腔孔径为400nm～700nm，第四级孔腔孔径为10nm～100nm，组织细胞培养体2的厚度为17mm，为第一级孔腔的平均孔径的16倍，培养液为含有10％的胎牛血清和双抗的高糖DMEM，培养细胞为HUVEC细胞，其他同实施例1。
培养条件是振动工作台采用水平振动、垂直振动方式交替进行，各振动2分钟，工作频率分别为50Hz、70Hz。培养结果表明，细胞生长状态良好。
本例中，该种多孔材料是按以下方法制得的：
采用1-10μm的钛粉作为原料，粒径为5nm～120nm的尿素作为最小一级(第四级)孔腔造孔剂，用粒径为500nm～800nm的乙基纤维素作为第三级孔腔造孔剂，并用5nm～120nm的淀粉作为粘合剂，按照钛粉∶尿素∶乙基纤维素∶淀粉∶蒸馏水按体积比1∶1.5∶1∶1∶12配制成浆料。
采用棱直径为300μm～400μm、孔径为400μm～600μm的聚酯泡沫，将所述浆料用泡沫浸渍法均匀填充其中，形成坯体并干燥，然后破碎得到粒径为300μm～400μm的含有钛粉原料、造孔剂与聚酯泡沫的混合颗粒。
(2)将混合颗粒、粒径为300μm～400μm的甲基纤维素按体积比2∶1均匀混合后均匀地灌入棱直径为800μm～1300μm、孔径为1500μm-2000μm的三维贯通的聚酯泡沫中，然后将聚酯泡沫放入密闭模具压制成致密坯体。
(3)将致密坯体真空烧结；烧结后的坯体按照钛材工艺进行常规后续热处理得到具有四级孔的多孔钛。
实施例5：
本实施例的组织细胞培养装置的组织细胞培养体2采用多孔CoNiCrMo合金材料，具有五级孔腔，其中，均匀分布、相互贯通的最大一级孔腔(即第一级孔腔)的腔壁由均匀分布、相互贯通的第二级、第三级孔腔填充构成，第二级、第三级孔腔的腔壁由均匀分布、相互贯通的第四级、第五级孔腔填充构成；且各级孔腔相互间也彼此贯通。第一级孔腔孔径为800μm～1300μm，第二级孔腔孔径为100μm～200μm，第三级孔腔孔径为30μm～70μm，第四级孔腔孔 径为500nm～700nm，第五级孔腔孔径为50nm～200nm，组织细胞培养体2的厚度为20mm，为第一级孔腔的平均孔径的18倍，培养液为含有10％的胎牛血清和双抗的高糖DMEM，培养细胞为HUVEC细胞，其他同实施例1。
培养条件是振动工作台采用水平振动方式，工作频率为70Hz。培养结果表明，细胞生长状态良好。
本例中，该种多孔材料是按以下方法制得的：
采用1-10μm的CoNiCrMo合金粉作为原料，粒径为60nm～300nm的尿素作为最小一级(第五级)孔腔造孔剂，用粒径为600nm～800nm的乙基纤维素作为第四级孔腔造孔剂，并用60nm～300nm的淀粉作为粘合剂，按照CoNiCrMo合金粉∶尿素∶乙基纤维素∶淀粉∶蒸馏水按体积比1∶1.5∶1∶1∶12配制成浆料。
采用棱直径为40μm～80μm、孔径为400μm～600μm的聚酯泡沫，将所述浆料用泡沫浸渍法均匀填充其中，形成坯体并干燥，然后破碎制成粒径为40μm～80μm的含有CoNiCrMo合金粉原料、造孔剂与聚酯泡沫的混合颗粒。
(2)将混合颗粒、粒径为200μm～300μm的甲基纤维素按体积比1∶1均匀混合后均匀地灌入棱直径为900μm～1400μm、孔径为1500μm-2000μm的三维贯通的聚酯泡沫中，然后将聚酯泡沫放入密闭模具压制成致密坯体。
(3)将致密坯体真空烧结；烧结后的坯体按照CoNiCrMo合金工艺进行常规后续热处理得到具有五级孔的多孔CoNiCrMo合金。

Claims (10)

  1. 一种组织细胞培养装置，包括组织细胞培养体，其特征在于：作为该组织细胞培养体的多孔材料是以材料孔径大小进行分级的孔腔及围绕形成各级孔腔的腔壁构成，且围绕构成上级大孔腔的腔壁上设置下级小孔腔；同级孔腔均相互贯通，且各级孔腔相互间也彼此贯通。
  2. 如权利要求1所述的组织细胞培养装置，其特征在于：所述多孔材料中，每上级孔腔的腔壁上设置有下一级的小孔腔，或者围绕构成上级大孔腔的腔壁上设置有下级的各级小孔腔或者围绕构成上级大孔腔的腔壁上设置有任意组合的各级小孔腔。
  3. 如权利要求1所述的组织细胞培养装置，其特征在于：所述多孔材料的孔腔分为三级时，围绕构成上一级大孔腔的腔壁上设置有下一级小孔腔；或者是围绕构成最大一级孔腔的腔壁上设置有下级两级小孔腔。
  4. 如权利要求1或2或3所述的组织细胞培养装置，其特征在于：所述多孔材料中，围绕构成上级大孔腔的腔壁是由下级小孔腔均布填充构成的。
  5. 如权利要求1-4任一权利要求所述的组织细胞培养装置，其特征在于：较小级孔腔的孔径小于培养细胞的平均直径，其他级孔腔的孔径至少大于培养细胞的平均直径。
  6. 如权利要求5所述的组织细胞培养装置，其特征在于：所述其他级中的较大级孔腔的孔径至少为培养细胞平均直径的两倍以上。
  7. 如权利要求1-6任一权利要求所述的组织细胞培养装置，其特征在于：该组织细胞培养装置还包括放置组织细胞培养体的培养皿，所述组织细胞培养体固定于所述培养皿中。
  8. 如权利要求7所述的组织细胞培养装置，其特征在于：所述组织细胞培养体制成板块结构。
  9. 如权利要求8所述的组织细胞培养装置，其特征在于：呈板块结构的组织细胞培养体的板厚不大于所述多孔材料最大一级孔腔的平均孔径的20倍。
  10. 如权利要求7-9所述的组织细胞培养装置，其特征在于：所述培养皿固定于振动工作台上。
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