WO2023030040A1 - 用于组织块培养的固定装置、组织块培养系统及培养方法 - Google Patents

用于组织块培养的固定装置、组织块培养系统及培养方法 Download PDF

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WO2023030040A1
WO2023030040A1 PCT/CN2022/113582 CN2022113582W WO2023030040A1 WO 2023030040 A1 WO2023030040 A1 WO 2023030040A1 CN 2022113582 W CN2022113582 W CN 2022113582W WO 2023030040 A1 WO2023030040 A1 WO 2023030040A1
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Prior art keywords
culture
tissue block
fixing
culture tank
connecting arm
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PCT/CN2022/113582
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English (en)
French (fr)
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李玉霞
凌焱
孙琦
谢利莹
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艾一生命科技(广东)有限公司
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Publication of WO2023030040A1 publication Critical patent/WO2023030040A1/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
    • C12M23/10—Petri dish
    • 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
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • 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
    • C12M23/48—Holding appliances; Racks; Supports
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources

Definitions

  • the invention relates to the technical field of tissue culture, in particular to a fixation device for tissue block culture, a tissue block culture system and a culture method.
  • MSCs Mesenchymal Stem Cells
  • tissues such as umbilical cord, bone marrow, fat and umbilical cord blood, and have strong self-renewal and multi-dermal differentiation capabilities.
  • Studies have shown that it has a significant effect on immune regulation and promotion of tissue repair, and has been widely used in clinical research and treatment of many major diseases such as rheumatoid arthritis, lupus erythematosus, graft-versus-host disease, and liver fibrosis.
  • Umbilical cord mesenchymal stem cells are extracted from the umbilical cord of newborns after full-term delivery and umbilical cord clamping. They have the advantages of rich sources, no harm to the body, strong proliferation ability, multi-germ layer differentiation ability, and low immunogenicity. The more professionals are recognized and applied.
  • Umbilical cord mesenchymal stem cells can be cultured by tissue block culture.
  • the commonly used separation method during culture is the blunt dissection of Wharton's jelly.
  • the umbilical artery and umbilical vein are removed at the same time; then the stripped Wharton's jelly is evenly cut into pieces. ; Then use the MSCs medium to resuspend the shredded Wharton's jelly, resuspend, inoculate in a culture dish, and finally place it directly in a CO 2 incubator for culture.
  • the current tissue block culture method has the following defects: the utilization rate of the tissue block is low, and the yield of primary cells is low. Specifically, in the tissue culture of umbilical cord mesenchymal stem cells, the utilization rate of Wharton's jelly is low, and the yield of primary umbilical cord mesenchymal stem cells is low.
  • one of the objectives of the present invention is to provide a fixation device for tissue block culture, which can ensure the adherent growth of the tissue block during the culture process.
  • the second object of the present invention is to provide a tissue block culture system.
  • the third object of the present invention is to provide a tissue block culture method.
  • Fixtures for tissue block culture including:
  • An adapter which is used for detachable connection with the culture tank
  • a fixing part is provided with at least a flat bottom, and the fixing part is used to fix the tissue block, so that the tissue block is clamped and fixed between the flat bottom of the fixing part and the bottom of the culture tank ;
  • a connecting piece, the head end of the connecting piece is movably connected with the connecting piece, and the tail end of the connecting piece is connected with the fixing piece.
  • the adapter is provided with a card slot, and the card slot is locked with the side wall of the culture tank.
  • the connecting piece includes a symmetrically arranged first connecting arm and a second connecting arm, and the connecting piece is arranged between the head end of the first connecting arm and the head end of the second connecting arm;
  • the head end of the first connecting arm is rotatably connected to the connecting piece, and the head end of the second connecting arm is rotatably connected to the connecting piece;
  • the fixing piece is arranged at the tail end of the first connecting arm and Between the tail ends of the second connecting arm.
  • a connecting post is provided between the head end of the first connecting arm and the head end of the second connecting arm; the connecting post is inserted into the connecting piece and rotatably connected with the connecting piece.
  • the head end of the first connecting arm is provided with a first boss
  • the head end of the second connecting arm is provided with a second boss
  • the first boss and the second boss are both connected to each other.
  • the connecting piece is rotatably connected.
  • first connecting arm includes a first inclined piece and a first flattening piece
  • second connecting arm includes a second inclined piece and a second flattening piece
  • first inclined piece and the connecting piece Rotationally connected, the second inclined piece is rotatably connected with the connecting piece
  • the fixing piece is arranged between the first horizontal piece and the second horizontal piece.
  • a tissue block culture system comprising at least one culture unit, the culture unit comprising:
  • a culture tank which is used to hold cell culture fluid and tissue pieces
  • said fixing device comprising an adapter, a fixing member and a connecting member
  • the adapter is used for detachable connection with the culture tank
  • the fixing part is provided with at least a flat bottom, and the fixing part is used to fix the tissue block, so that the tissue block is clamped and fixed between the flat bottom of the fixing part and the bottom of the culture tank;
  • the head end of the connecting piece is movably connected with the connecting piece, and the tail end of the connecting piece is connected with the fixing piece.
  • a culture container is also included, and the culture container is used for accommodating the culture unit.
  • a cover body is also included, and the cover body is used to cover the notch of the culture tank; or, the cover body is used to cover the container opening of the culture container.
  • a method for culturing a tissue block comprising the steps of:
  • the original culture solution is added to the culture tank, and then placed in an incubator for cultivation.
  • the concentration of CO 2 in the incubator is 5%-15%.
  • the culture medium needs to be replaced during the culture process, and cysteine is added to the culture tank every 45-50 h after the culture medium is replaced, and the amount of cysteine added is 0.02%-0.2% of the volume of the original culture medium. %, the stock solution of cysteine has a concentration of 1 ⁇ Mol/L.
  • the fixing device for tissue block culture provided by the present invention, when utilizing the tissue block for cell culture, the tissue block is clamped and fixed between the flat bottom of the fixture and the bottom of the culture tank, and the culture solution is replaced When the tissue block is in a fixed position, there will be no displacement, so it will not affect the cells to continue to swim out between the flat bottom of the fixture and the bottom of the culture tank, and maintain adherent growth, thereby improving the utilization of the tissue block Ratio, increased yield of primary cells.
  • the fixing device for tissue block culture provided by the present invention is an independent new device, and it and the culture tank are two independent devices, which are convenient for cleaning and storage.
  • This fixing device can be flexibly assembled with a ready-made culture tank (such as a culture dish). If a fixing device is needed during the cultivation process, the fixing device and the culture tank can be assembled; if no fixing device is needed during the cultivation process, just follow the routine
  • the culture method can be operated, and the flexibility is strong. That is to say, the user does not need to redesign the culture tank (such as the culture dish) separately, and the fixing device has strong versatility and has a good application prospect.
  • FIG. 1 is a schematic diagram of a fixing device provided by an embodiment of the present invention.
  • Fig. 2 is another schematic diagram of the fixing device provided by the embodiment of the present invention.
  • FIG. 3 is a schematic diagram of the connection between the fixing device and the culture tank provided by the embodiment of the present invention.
  • Figure 4 is a schematic diagram of a culture system provided by an embodiment of the present invention.
  • Example 5 is a schematic diagram of cell growth with and without a fixation device provided in Example 1 of the present invention.
  • Fig. 6 is a schematic diagram of cell growth under different CO2 concentration conditions provided by Example 1 of the present invention.
  • Fig. 7 is a schematic diagram of cell growth under the conditions of adding cysteine and not adding cysteine provided in Example 1 of the present invention.
  • connecting piece 11, first locking arm; 12, second locking arm; 20, connecting piece; 21, first connecting arm; 22, second connecting arm; 23, first inclined piece; 24, 25, the first flat piece; 26, the second flat piece; 30, the fixing piece; 40, the culture tank; 50, the culture container; 60, the cover.
  • one embodiment of the present invention provides a fixation device for tissue block culture, including an adapter 10 for detachable connection with a culture tank 40, and a fixer 30 for fixing a tissue block , and a connecting piece 20 for connecting the connecting piece 10 and the fixing piece 30.
  • the fixing device of the embodiment of the present invention is transparent, preferably made of transparent resin material, and the fixing device formed of transparent resin material can be sterilized by soaking in 75% ethanol, which is easy to operate.
  • the adapter 10 is used for detachable connection with the culture tank 40, that is, when the fixing device of the embodiment of the present invention needs to be used, the adapter 10 is connected with the culture tank 40, and when not in use, the adapter 10 can be removed from the culture tank. 40 dismantled.
  • detachable connection for example including but not limited to clamping, clamping, concave-convex matching connection, magnetic connection and the like.
  • the connecting piece 10 is detachably connected to the culture tank 40 by snap-fitting.
  • a slot may be provided on the connecting piece 10 to snap-fit with the side wall of the culture tank 40 through the slot.
  • a first clamping arm 11 and a second clamping arm 12 are provided on the adapter 10, and a clamping groove is formed between the first clamping arm 11 and the second clamping arm 12. Just insert the edge down.
  • the adapter 10 when the adapter 10 and the culture tank 40 are connected in a clamping manner, the adapter 10 can be set as a clip; A bump is set on the adapter 10, and a groove matching the bump is set on the culture tank 40, or a groove is set on the adapter 10, and a bump matching the groove is set on the culture tank 40;
  • the culture tank 40 adopts the magnetic connection method, it is sufficient to install corresponding magnets on the adapter 10 and the culture tank 40 respectively, but the cell culture devices are generally glass devices, resin devices, etc., and the magnetic connection method is optional, but not optimal. of.
  • the culture tank 40 is used to hold culture fluid and tissue pieces, and the material of the culture tank 40 is resin, glass or plastic, etc., and the preferred color is a transparent culture tank 40, through which the transparent culture tank 40 can visually Observe the cell culture process.
  • the shape of the culture tank 40 can have multiple options, including but not limited to square, conical, circular, etc., specifically glass culture dishes can be selected for use.
  • the bottom of the culture tank 40 is flat, and the culture tank 40 with a flat bottom is conducive to laying out the tissue block.
  • the fixing part 30 in the embodiment of the present invention is provided with at least a flat bottom, and the fixing part 30 is used to fix the tissue block, so that the tissue block is clamped and fixed between the flat bottom of the fixing part 30 and the bottom of the culture tank 40 .
  • the fixing part 30 is used to fix the tissue block, so that the tissue block is clamped and fixed between the flat bottom of the fixing part 30 and the bottom of the culture tank 40 .
  • the shape of the fixture 30 includes but not limited to cuboid, cylinder, truncated cone, cone, prism (triangular prism, pentagonal prism, hexagonal prism, etc.), and the fixture 30 can at least provide a flat bottom, tissue One side of the block is attached to the bottom of the culture tank 40, and the flat bottom of the fixing member 30 is pressed against the other side of the tissue block.
  • the size and weight of the fixing member 30 need to be reasonably designed according to the actual situation (such as the size of the culture tank 40, the type of the tissue block), etc., so as to be able to fix the tissue block without generating excessive pressure
  • the principle is to cause damage to tissue blocks and cells.
  • the tissue block is Wharton's glue
  • the pressure applied to the Wharton's glue by the fixture 30 can be set to be about 500-1000Pa.
  • the pressure of 500-1000Pa can be fixed. Hold Huatong glue, and it will not cause damage to Huatong glue and stem cells due to excessive pressure.
  • the weight of the entire fixing device can be set to about 3.71g
  • the bottom of the connecting part 20 and the bottom of the fixing part 30 are both flat bottoms, and they together form a platform with an area of about 1cm
  • the weight of the platform is about 1g
  • a force of 0.0098N is generated.
  • the area of Wharton's glue at the bottom of the platform is about 10-20mm 2
  • the cultured tissue block is not Wharton's jelly
  • the weight of the fixture, the size of the platform, and the weight of the platform need to be flexibly adjusted according to the actual situation.
  • the connecting piece 20 of the embodiment of the present invention is used to connect the connecting piece 10 and the fixing piece 30.
  • the head end of the connecting piece 20 is movably connected with the connecting piece 10. change, it is convenient to adjust the position of the connecting piece 20, and facilitate the connection of the entire fixing device with the culture tank 40; the tail end of the connecting piece 20 is connected with the fixing piece 30.
  • the connection mode between the connecting piece 20 and the fixing piece 30 can be a fixed connection or a flexible connection.
  • the connecting member 20 includes a first connecting arm 21 and a second connecting arm 22 arranged symmetrically, and the connecting member 10 is arranged at the head end of the first connecting arm 21 and at the Between the head ends; the head end of the first connecting arm 21 is rotatably connected to the connecting piece 10, and the head end of the second connecting arm 22 is rotatably connected to the connecting piece 10; the fixing member 30 is arranged on the tail end of the first connecting arm 21 and the second Between the tail ends of the two connecting arms 22 .
  • the whole device adopts a left-right symmetrical setting method, which can provide uniform force for the tissue block, and the device is elegant in appearance.
  • a connecting post is arranged between the head end of the first connecting arm 21 and the head end of the second connecting arm 22, one end of the connecting post is connected to the first connecting arm 21, and the other end of the connecting post is connected to the second connecting arm 22.
  • the connecting arm 22 is connected, and the connecting post is inserted into the connecting piece 10 to be rotatably connected to the connecting piece 10 .
  • the head end of the first connecting arm 21 is provided with a first boss
  • the head end of the second connecting arm 22 is provided with a second boss
  • the connector 10 corresponds to the first connecting arm 21
  • a first groove matching the first boss is provided on the side of the connector 10
  • a second groove matching the second boss is arranged on the side of the connector 10 corresponding to the second connecting arm 22, and the first boss matches with the second connecting arm 22.
  • the first groove is rotatably connected
  • the second protrusion is rotatably connected to the second groove, so that both the first connecting arm 21 and the second connecting arm 22 are rotatably connected to the connecting member 10 .
  • the first protrusion and the second protrusion are preferably cylindrical protrusions
  • the first groove and the second groove are preferably cylindrical grooves.
  • the first connecting arm 21 includes a first inclined part 23 and a first flat part 25, and the second connecting arm 22 includes a second inclined part 24 and a second flat part 26; the first inclined part 23 and The connecting piece 10 is rotatably connected, and the second inclined piece 24 is rotatably connected to the connecting piece 10 ; the fixing piece 30 is arranged between the first flat piece 25 and the second flat piece 26 .
  • the inclined connecting piece 20 naturally slopes toward the bottom of the culture tank 40 .
  • the first flat part 25 is provided with a flat bottom
  • the second flat part 26 is provided with a flat bottom
  • the fixing part 30 is sandwiched between the first flat part 25 and the second flat part 26;
  • the plane bottom of the first flat part 25, the plane bottom of the fixing part 30 and the plane bottom of the second flat part 26 are successively connected and on the same horizontal plane, the plane bottom of the first flat part 25, the fixing part
  • the plane bottom of 30 and the plane bottom of the second flat part 26 jointly form a platform, and the platform is pressed on the tissue block during the culture process of the tissue block.
  • the connecting member 20 may also only provide a connecting function for connecting the engaging member 10 and the fixing member 30, and only the fixing member 30 provides a plane for pressing and fixing the tissue mass.
  • the connecting member 20 may also only be provided with one connecting arm, and be connected to the fixing member 30 through one connecting arm. At this time, in order to receive more uniform force, the connecting arm is connected to the center of the top surface of the fixing member 30 , or the connecting arm is connected to the center of the opposite side of the fixing member 30 .
  • the connecting part 20 and the fixing part 30 may be two parts manufactured separately, and assembled after being manufactured; the connecting part 20 and the fixing part 30 may also be integrally formed parts.
  • the tissue block when using the tissue block for cell culture, the tissue block is clamped and fixed between the flat bottom of the fixture 30 and the bottom of the culture tank 40, and the culture When the liquid is in the liquid, the tissue block is in a fixed position and will not be displaced, so it will not affect the cells to continue to swim out between the flat bottom of the fixture 30 and the bottom of the culture tank 40, and keep growing adherently, thereby improving the tissue quality.
  • the utilization of the block is relatively high, which improves the yield of primary cells.
  • an embodiment of the present invention also provides a tissue block culture system, including at least one culture unit, and the culture unit includes:
  • a culture tank 40 the culture tank 40 is used to hold cell culture fluid and tissue pieces;
  • the fixing device includes a connecting piece 10, a fixing piece 30 and a connecting piece 20;
  • the adapter 10 is used for detachable connection with the culture tank 40;
  • the fixing part 30 is provided with at least a flat bottom, and the fixing part 30 is used to fix the tissue block, so that the tissue block is clamped and fixed between the flat bottom of the fixing part 30 and the bottom of the culture tank 40;
  • a head end of the connecting piece 20 is movably connected with the connecting piece 10 , and a tail end of the connecting piece 20 is connected with the fixing piece 30 .
  • the tissue block is Wharton's jelly.
  • a culture container 50 is also included, and the culture container 50 is used for accommodating the culture unit.
  • the culture container 50 includes multiple culture units, and the multiple culture units are arranged in the culture container 50 one by one;
  • the volume of the culture container 50 should be larger than the volume of the culture tank 40, so as to accommodate the culture unit.
  • Options for the culture container 50 include, but are not limited to, square containers, cylindrical containers, and the like.
  • a cover body 60 is also included, and the cover body 60 is used to cover the notch of the culture tank 40 ; or, the cover body 60 is used to cover the container opening of the culture container 50 .
  • One embodiment of the present invention also provides a tissue block culture method, comprising the following steps:
  • the original culture solution is added to the culture tank 40, and then placed in an incubator for culture.
  • the tissue block is Wharton's jelly
  • the original culture medium is DMEM-F12 phenol red-free medium
  • the culture medium is changed every 2-3 days during the culture process, and the Huatong glue is still in a fixed position when the culture medium is changed, which does not affect the cells to continue to swim out of it and grow on the wall. More preferably, the culture medium is replaced every 3 days during the culture process.
  • the concentration of CO in the incubator is 5% to 15%, preferably 8% to 12%, specifically, it can be set to 5%, 7%, 8%, 10% %, 12% or 15%, more preferably the concentration of CO2 is 10%.
  • the culture medium needs to be replaced during the culture process, and cysteine is added to the culture tank 40 every 45-50 h after the culture medium is replaced, and the amount of cysteine added is 0.02%-0.2% of the volume of the original culture medium.
  • the stock solution of cysteine has a concentration of 1 ⁇ Mol/L.
  • cysteine is added according to the concentration of the stock solution without dilution, that is, the concentration of cysteine added is 1 ⁇ Mol/L. Adding cysteine can not only improve the utilization efficiency of Huatong glue, but also increase the proliferation and migration rate of cells.
  • cysteine (Cystenie) to the culture tank 40 every 48h after changing the culture solution
  • the amount of added cysteine is 0.1% Cysteine of the original culture solution volume
  • the stock solution of cysteine is 1 ⁇ Mol/L.
  • the tissue block culture method provided by the embodiment of the present invention has a high utilization rate of the tissue block, a high yield of primary cells, and can ensure the proliferation rate and migration rate of the cells.
  • a tissue block culture system using a fixture the specific scheme is as follows:
  • the fixing device includes an adapter 10, a fixing part 30 and a connecting part 20, and the connecting part 20 includes a first connecting arm 21 and a second connecting arm 21.
  • Arm 22, the tail end of first connecting arm 21 and the second connecting arm 22 all provide plane bottom, and fixture 30 is rectangular parallelepiped fixture 30, and the plane bottom of two connecting arms and the bottom surface of rectangular parallelepiped fixture 30 form a small joint together.
  • the platform (1cm ⁇ 1cm), the fixing device utilizes the pressure generated by its own gravity to make the plate and the bottom of the petri dish adhere tightly.
  • the small section of Huatong jelly peeled off from the umbilical cord is clamped between the flat plate and the bottom of the culture dish to form a culture unit, then the culture unit is placed in a culture dish (culture container 50) with a diameter of 10 mm to form a culture system, and then What is needed is to cover the culture system with the cover 60 .
  • the weight of the whole fixing device is 3.71g, and the weight of the 1cm 2 platform is about 1g, which generates a force of 0.0098N.
  • the fixing device in Example 1 is made of transparent resin material, which can be directly soaked in 75% ethanol for sterilization.
  • tissue block culture method of embodiment 1 comprises the following steps:
  • Figure 5a shows the cell growth state after 12 days of culture under the same culture conditions without a fixture
  • Figure 5b shows the cell growth state after 12 days of culture using the fixture of Example 1.
  • the initial medium was DMEM-F12 phenol red-free medium containing 15% serum, and the medium was replaced every 3 days during the cultivation process.
  • Adding 0.1% Cysteine (Cystenie stock solution concentration 1 ⁇ Mol/L) to the system every 48 hours after the culture medium the cell proliferation and migration rate were further improved, and the cell growth state was better.
  • Figure 7a is a graph of the cell growth state without adding Cysteine
  • Figure 7b is a graph of the cell growth state when adding 0.1% (v/v) Cysteine, it can be seen that after adding 0.1% (v/v) Cysteine, the cells The growth state is better.

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Abstract

一种用于组织块培养的固定装置和组织块培养系统，包括衔接件（10）、固定件（30）和连接件（20）；衔接件（10）用于与培养槽（40）可拆卸连接；固定件（30）至少设置有平面底部，固定件（30）用于固定组织块，以使组织块夹持固定在固定件（30）的平面底部和培养槽（40）的槽底之间；连接件（20）的首端与衔接件（10）活动连接，连接件（20）的尾端与固定件（30）连接。利用组织块培养的固定装置在更换培养液时，组织块处在固定位置，不会发生位移，因此不会影响细胞继续从固定件的底部和培养槽的槽底之间游出，并保持贴壁生长，从而提高了组织块的利用率较，提高了原代细胞的产量。提供一种组织块培养方法，组织块利用率较高，原代细胞产量较高，且能保证细胞的增殖速率和迁移速率。

Description

用于组织块培养的固定装置、组织块培养系统及培养方法 技术领域
本发明涉及组织培养技术领域，尤其涉及一种用于组织块培养的固定装置、组织块培养系统及培养方法。
背景技术
间充质干细胞(Mesenchymal Stem Cells,MSCs)是一种成体干细胞，它存在于脐带、骨髓、脂肪和脐带血等一些组织中，具有很强的自我更新及多胚层分化能力。研究表明它在免疫调节和促进组织修复方面疗效显著，已经大量用于类风湿性关节炎、红斑狼疮、移植物抗宿主病、肝纤维化等多种重大疾病的临床研究和治疗上。
脐带间充质干细胞从足月分娩断脐后的新生儿脐带中提取，具有来源丰富、不伤及身体、增殖能力强、具有多胚层分化能力、免疫原性低等优点，已经得到了越来越多专业人士的认可和应用。
脐带间充质干细胞可采取组织块培养法进行培养，培养时常用的分离方法为华通氏胶钝性剥离法，首先同时去除脐动脉和脐静脉；接着将剥离的华通氏胶均匀剪碎；然后用MSCs培养基重悬剪碎的华通氏胶，重悬后接种于培养皿中，最后直接置于CO 2培养箱培养。
但是，目前的组织块培养法存在以下缺陷：组织块利用率较低，原代细胞产量较低。具体到脐带间充质干细胞组织培养中，则为华通氏胶利用率较低，原代脐带间充质干细胞产量较低。
发明内容
为了克服现有技术的不足，本发明的目的之一在于提供一种用于组织块培养的固定装置，能够确保组织块在培养过程中贴壁生长。
本发明的目的之二在于提供一种组织块培养系统。
本发明的目的之三在于提供一种组织块培养方法。
本发明的目的之一采用如下技术方案实现：
用于组织块培养的固定装置，包括：
衔接件，所述衔接件用于与培养槽可拆卸连接；
固定件，所述固定件至少设置有平面底部，所述固定件用于固定组织块，以使所述组织块夹持固定在所述固定件的平面底部和所述培养槽的槽底之间；
连接件，所述连接件的首端与所述衔接件活动连接，所述连接件的尾端与所述固定件连接。
进一步地，所述衔接件设置有卡槽，所述卡槽与所述培养槽的侧壁卡接。
进一步地，所述连接件包括对称而设的第一连接臂和第二连接臂，所述衔接件设置在所述第一连接臂的首端和所述第二连接臂的首端之间；所述第一连接臂的首端与所述衔接件转动连接，所述第二连接臂的首端与所述衔接件转动连接；所述固定件设置在所述第一连接臂的尾端和所述第二连接臂的尾端之间。
进一步地，所述第一连接臂的首端和所述第二连接臂的首端之间设置有连接柱；所述连接柱插入所述衔接件与所述衔接件转动连接。
进一步地，所述第一连接臂的首端设置有第一凸柱，所述第二连接臂的首端设置有第二凸柱，所述第一凸柱和所述第二凸柱均与所述衔接件转动连接。
进一步地，所述第一连接臂包括第一倾斜件和第一平置件，所述第二连接臂包括第二倾斜件和第二平置件；所述第一倾斜件与所述衔接件转动连接，所述第二倾斜件与所述衔接件转动连接；所述固定件设置于所述第一平置件和所述第二平置件之间。
本发明的目的之二采用如下技术方案实现：
一种组织块培养系统，包括至少一个培养单元，所述培养单元包括：
培养槽，所述培养槽用于承装细胞培养液和组织块；
至少一个所述固定装置，所述固定装置包括衔接件、固定件和连接件；
所述衔接件用于与所述培养槽可拆卸连接；
所述固定件至少设置有平面底部，所述固定件用于固定所述组织块，以使所述组织块夹持固定在所述固定件的平面底部和所述培养槽的槽底之间；
所述连接件的首端与所述衔接件活动连接，所述连接件的尾端与所述固定件连接。
进一步地，还包括培养容器，所述培养容器用于收容所述培养单元。
进一步地，还包括盖体，所述盖体用于遮蔽所述培养槽的槽口；或者，所述盖体用于遮蔽所述培养容器的容器口。
本发明的目的之三采用如下技术方案实现：
一种组织块培养方法，包括以下步骤：
将组织块接种在培养槽中，并使所述组织块的一面贴附于所述培养槽的槽底；
将所述固定装置与所述培养槽进行组装，其中所述衔接件与所述培养槽进行可拆卸连接，所述固定件压在所述组织块上；
向所述培养槽中添加原始培养液，然后置于培养箱中培养。
进一步地，所述培养箱中CO 2的浓度为5％～15％。
进一步地，培养过程中需要更换培养液，更换培养液后每45-50h向所述培养槽中加入半胱氨酸，半胱氨酸的加入量为所述原始培养液体积的0.02％-0.2％，半胱氨酸的储液为浓度1μMol/L。
相比现有技术，本发明的有益效果在于：
(1)本发明所提供的用于组织块培养的固定装置，利用组织块进行细胞培养时，组织块是夹持固定在固定件的平面底部和培养槽的槽底之间的，更换培养液时，组织块处在固定位置，不会发生位移，因此不会影响细胞继续从固定件的平面底部和培养槽的槽底之间游出，并保持贴壁生长，从而提高了组织块的利用率较，提高了原代细胞的产量。
(2)本发明所提供的用于组织块培养的固定装置，是独立的新型装置，其与培养槽是两个独立的器件，方便清洗和存放。此固定装置可与现成的培养槽(例如培养皿)灵活组装，如果培养过程中需要固定装置时，将固定装置和培养槽进行组装即可；如果培养过程中不需要固定装置时，就按常规培养法操作即可，灵活性强。也就是说，使用者不需要另行对培养槽(例如培养皿)进行重新设计，此固定装置的通用性强，具有很好的应用前景。
附图说明
图1为本发明实施例所提供的固定装置的示意图；
图2本本发明实施例所提供的固定装置的另一示意图；
图3为本发明实施例所提供的固定装置与培养槽的连接示意图；
图4为本发明实施例所提供的培养系统的示意图；
图5为本发明实施例1所提供的有固定装置和无固定装置时的细胞生长情况示意图；
图6为本发明实施例1所提供的在不同CO 2浓度条件下细胞生长情况示意图；
图7为本发明实施例1所提供的在补加半胱氨酸和不加半胱氨酸条件下的细胞生长情况示意图。
图中：10、衔接件；11、第一卡臂；12、第二卡臂；20、连接件；21、第一连接臂；22、第二连接臂；23、第一倾斜件；24、第二倾斜件；25、第一平置件；26、第二平置件；30、固定件；40、培养槽；50、培养容器；60、盖体。
具体实施方式
下面，结合附图以及具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。在下述实施例中所采用的原材料、设备等除特殊限定外均可以通过商业渠道购买获得。
如图1-2所示，本发明一实施方式提供了一种用于组织块培养的固定装置，包括用于与培养槽40可拆卸连接的衔接件10、用于固定组织块的固定件30、以及用于连接衔接件10和固定件30的连接件20。
在其中的一个实施例中，本发明实施例的固定装置是透明的，优选采用透明树脂材料制作，透明树脂材料形成的固定装置可用75％乙醇浸泡除菌，操作方便。
具体地，衔接件10用于与培养槽40可拆卸连接，即需要使用本发明实施例的固定装置时，将衔接件10与培养槽40连接，不需要使用时，衔接件10可以从培养槽40中拆除。可拆卸连接的方式可以包括多种，例如包括但不限于卡接、夹设、凹凸匹配连接、磁性连接等。
作为优选的实施方式，衔接件10与培养槽40通过卡接的方式进行可拆卸连接，例如可以在衔接件10上设置卡槽，通过卡槽与培养槽40的侧壁卡接。具体地，衔接件10上设置有第一卡臂11和第二卡臂12，第一卡臂11和第二卡臂12之间形成卡槽，连接时，卡槽从培养槽40的侧壁边缘往下插入即可。
作为可选的实施方式，当衔接件10与培养槽40采用夹设的连接方式时，可以将衔接件10设置为夹子；当衔接件10与培养槽40采用凹凸匹配的连接方式时，可以在衔接件10上设置凸块，培养槽40上设置与凸块匹配的凹槽，或者在衔接件10上设置凹槽，在培养槽40上设置与凹槽匹配的凸块；当衔接件10与培养槽40采用磁性连接方式时，分别在衔接件10和培养槽40上设置相应的磁铁即可，但细胞培养器件一般为玻璃器件、树脂器件等，磁性连接的方式可选，但不是最优的。
在其中的一个实施例中，培养槽40用于承装培养液和组织块，培养槽40的材料为树脂、玻璃或塑料等，优选颜色为透明的培养槽40，通过透明培养槽40可以直观观察细胞培养过程。培养槽40的形状可以有多种选择，包括但不限于方形、锥形、圆形等，具体地可以选 用玻璃培养皿。
在其中的一个实施例中，培养槽40的槽底为平底，设有平底的培养槽40有利于组织块铺平。
本发明实施例的固定件30至少设置有平面底部，固定件30用于固定组织块，以使组织块夹持固定在固定件30的平面底部和培养槽40的槽底之间。采取这种方式，在组织块培养过程中，能够保证细胞贴壁生长并向外迁移；并在更换培养液时，组织块的位置不易发生变化，细胞的贴壁情况和迁移速率不会受到影响，能够提供组织块的利用效率，提高原代细胞的产率。
在其中的一个实施例中，固定件30的形状包括但不限于为长方体、圆柱体、圆台、圆锥、棱柱(三棱柱、五棱柱、六棱柱等)，固定件30能够至少提供平面底部，组织块的一面贴附于培养槽40的槽底，固定件30的平面底部则压在组织块的另一面上。
在其中的一个实施例中，固定件30的大小、重量需要根据实际情况(例如培养槽40大小、组织块的类型)等进行合理设计，以能够固定住组织块，又不至于产生过大压强造成组织块和细胞的损坏为原则。例如，当组织块为华通氏胶时，可以设置固定件30施加给华通氏胶的压强约为500-1000Pa，根据华通氏胶的特性和承受能力计算，500-1000Pa压强既可固定住华通胶，又不至于压强过大造成华通胶和干细胞的损坏。
在其中的一个实施例中，可以将整个固定装置的重量设置为约3.71g，连接件20的底部和固定件30的底部均为平面底部，它们共同组成一个平台，平台的面积约为1cm 2，平台重量约1g，产生0.0098N的力，平台底部华通氏胶的面积约10-20mm 2，承受压强P＝F/S，约为500-1000Pa，根据华通氏胶的特性和承受能力计算，500-1000Pa压强既可固定住华通胶，又不至于压强过大造成华通氏胶和干细胞的损坏。而当培养的组织块并非华通氏胶时，固定装置的重量、平台的大小、平台的重量等则需要根据实际的情况进行灵活调整。
本发明实施例的连接件20用于连接衔接件10和固定件30，连接件20的首端与衔接件10活动连接，活动连接的方式使得衔接件10与连接件20之间的角度可以发生变化，便于调整连接件20的位置，便于整个固定装置与培养槽40的连接；连接件20的尾端与固定件30连接。连接件20与固定件30的连接方式可以为固定连接、也可以为活动连接。
在本发明的其中一个实施方式中，连接件20包括对称而设的第一连接臂21和第二连接臂22，衔接件10设置在第一连接臂21的首端和第二连接臂22的首端之间；第一连接臂21的首端与衔接件10转动连接，第二连接臂22的首端与衔接件10转动连接；固定件30设置在第一连接臂21的尾端和第二连接臂22的尾端之间。整个装置采取左右对称的设置方式，能够为组织块提供均匀受力，且装置美观大方。
作为优选的实施方式，第一连接臂21的首端和第二连接臂22的首端之间设置有连接柱，连接柱的一端与第一连接臂21连接，连接柱的另一端与第二连接臂22连接，连接柱插入衔接件10与衔接件10转动连接。
又或者，作为优选的实施方式，第一连接臂21的首端设置有第一凸柱，第二连接臂22的首端设置有第二凸柱，衔接件10与第一连接臂21相对应的侧面上设置有与第一凸柱匹配的第一凹槽，衔接件10与第二连接臂22相对应的侧面上设置有与第二凸柱匹配的第二凹槽，第一凸柱与第一凹槽转动连接，第二凸柱与第二凹槽转动连接，从而使得第一连接臂21和第二连接臂22均与衔接件10转动连接。第一凸柱和第二凸柱优选为圆柱形凸柱，第一凹 槽和第二凹槽优选为圆柱形凹槽。
作为优选的实施方式，第一连接臂21包括第一倾斜件23和第一平置件25，第二连接臂22包括第二倾斜件24和第二平置件26；第一倾斜件23与衔接件10转动连接，第二倾斜件24与衔接件10转动连接；固定件30设置于第一平置件25和第二平置件26之间。衔接件10与培养槽40的侧壁连接后，倾斜设置的连接件20自然向培养槽40的底部倾斜。
作为优选的实施方式，第一平置件25设置有平面底部，第二平置件26设置有平面底部，固定件30夹设于第一平置件25和第二平置件26之间；第一平置件25的平面底部、固定件30的平面底部和第二平置件26的平面底部三者依次相接且在同一个水平面上，第一平置件25的平面底部、固定件30的平面底部和第二平置件26的平面底部共同组成一个平台，此组织块培养过程中，此平台压在组织块上。而在另外的实施例当中，连接件20也可以仅提供连接作用，用于连接衔接件10和固定件30，仅由固定件30提供用于压设固定组织块的平面。
在本发明的其中一个实施方式中，连接件20也可以只设置一个连接臂，通过一个连接臂与固定件30连接。此时，为了受力更加均匀，连接臂与固定件30的顶面中央连接，或者连接臂与固定件30相对的侧面的中央连接。
在本发明的其中一个实施方式中，连接件20和固定件30可以是分开制作的两个部件，制作完成后进行组装；连接件20与固定件30也可以是一体成型的部件。
本发明实施例所提供的用于组织块培养的固定装置，利用组织块进行细胞培养时，组织块是夹持固定在固定件30的平面底部和培养槽40的槽底之间的，更换培养液时，组织块处在固定位置，不会发生位移，因此不会影响细胞继续从固定件30的平面底部和培养槽40的槽底之间游出，并保持贴壁生长，从而提高了组织块的利用率较，提高了原代细胞的产量。
如图3-4所示，本发明一实施方式还提供了一种组织块培养系统，包括至少一个培养单元，培养单元包括：
培养槽40，培养槽40用于承装细胞培养液和组织块；
至少一个固定装置，固定装置包括衔接件10、固定件30和连接件20；
衔接件10用于与培养槽40可拆卸连接；
固定件30至少设置有平面底部，固定件30用于固定组织块，以使组织块夹持固定在固定件30的平面底部和培养槽40的槽底之间；
连接件20的首端与衔接件10活动连接，连接件20的尾端与固定件30连接。
在本发明的其中一个实施方式中，组织块为华通氏胶。
作为优选的实施方式，还包括培养容器50，培养容器50用于收容培养单元。例如，包括多个培养单元，多个培养单元一一排列设置在培养容器50内；具体地，也可以仅设置一个培养单元，一个培养单元设置在一个培养容器50内。培养容器50的体积应当大于培养槽40的体积，以便于收容培养单元。培养容器50的选择包括但不限于方形容器、圆柱形容器等。
作为优选的实施方式，还包括盖体60，盖体60用于遮蔽培养槽40的槽口；或者，盖体60用于遮蔽培养容器50的容器口。
本发明一实施方式还提供了一种组织块培养方法，包括以下步骤：
将组织块接种在培养槽40中，并使组织块的一面贴附于培养槽40的槽底；
将固定装置与培养槽40进行组装，其中衔接件10与培养槽40进行可拆卸连接，固定件30压在组织块上；
向培养槽40中添加原始培养液，然后置于培养箱中培养。
在本发明的其中一个实施方式当中，组织块为华通氏胶，原始培养液为DMEM-F12无酚红培养基。
在本发明的其中一个实施方式当中，培养过程中2-3天更换一次培养液，更换培养液时华通胶仍然处在固定位置，不影响细胞继续从中游出并贴壁生长。更加优选地，培养过程中每3天更换一次培养液。
作为优选的实施方式，在培养过程中，设置培养箱中CO 2的浓度为5％～15％，优选地为8％～12％，具体地可以设置为5％、7％、8％、10％、12％或15％，更加优选地CO 2的浓度为10％。
作为优选的实施方式，培养过程中需要更换培养液，更换培养液后每45-50h向培养槽40中加入半胱氨酸，半胱氨酸的加入量为原始培养液体积的0.02％-0.2％，半胱氨酸的储液为浓度1μMol/L。在添加半胱氨酸时，半胱氨酸按照储液浓度加入，不需要经过稀释，即半胱氨酸的加入浓度为1μMol/L。加入半胱氨酸既能提高华通胶的利用效率，又提高了细胞的增殖和迁移速率。
更加优选地，更换培养液后每48h向培养槽40中加入半胱氨酸(Cystenie)，补加半胱氨酸量为原始培养液体积的0.1％Cysteine，半胱氨酸的储液为浓度1μMol/L。
本发明实施例所提供的组织块培养方法，组织块利用率较高，原代细胞产量较高，且能够保证细胞细胞的增殖速率和迁移速率。
实施例1
一种采用固定装置的组织块培养系统，具体方案如下：
取直径为6mm培养皿，在培养皿的侧面卡上本发明实施例的固定装置，固定装置包括衔接件10、固定件30和连接件20，连接件20包括第一连接臂21和第二连接臂22，第一连接臂21和第二连接臂22的尾端均提供了平面底部，固定件30为长方体固定件30，两个连接臂的平面底部和长方体固定件30的底面共同形成一个小的平台(1cm×1cm)，固定装置利用自身重力产生的压力，使平板和培养皿底部贴紧。将脐带中剥离的华通胶小段夹在平板和培养皿底部之间，形成了一个培养单元，然后将培养单元放在直径为10mm的培养皿(培养容器50)中，形成一个培养系统，然后将培养系统用盖体60覆盖住即可。
整个固定装置的重量为3.71g，1cm 2平台的重量约1g，产生0.0098N的力，平台底部华通氏胶面积约10-20mm 2，承受压强P＝F/S，约500-1000Pa，根据华通胶的特性和承受能力计算，500-1000Pa压强既可固定住华通氏胶，又不至于压强过大造成华通氏胶和干细胞的损坏。实施例1的固定装置由透明树脂材料制作而成，可直接用75％乙醇浸泡除菌。
实施例1的组织块培养方法包括以下步骤：
同时去除脐动脉和脐静脉；将剥离的华通氏胶均匀剪碎；用MSCs培养基(含15％血清的DMEM-F12无酚红培养基)重悬剪碎的华通氏胶，然后接种于6cm培养皿中，MSCs培养基深度超过华通胶即可，接着放入10cm培养皿中并盖上盖体，最后将整个培养系统直接放于CO 2培养箱中静置培养，细胞在培养皿底部和平台底部皆可贴壁生长，并向外迁移，待细胞生长融合至80％-90％时进行消化传代。整个培养系统是透明的，由透明树脂材料制成，操 作人员可直接在显微镜下观察细胞生长情况。
华通氏胶固定后，培养过程中2-3d更换一次培养基，更换培养基时华通氏胶仍旧处在固定位置，不影响细胞继续从中游出并贴壁生长。但由于固定位置通气性较差，细胞迁移和培养速率较正常培养生长缓慢。如图5所示，图5a为无固定装置相同培养条件下，培养12天后细胞生长状态；图5b为使用实施例1固定装置培养12天后细胞生长状态。
因此，我们对在使用实施例1固定装置的情况下对细胞培养条件进行优化，改变培养时CO 2浓度，探索CO 2浓度为5％，10％和15％时，细胞的生长情况，培养时间为12天。结果如图6所示，图6a为5％CO 2条件下细胞生长状态；图6b为10％CO 2条件下细胞生长状态；图6c为15％CO 2条件下细胞生长状态。可见，在CO 2浓度为10％时，细胞迁移和生长速度较快。
接着，我们在10％CO 2培养条件的基础上对培养基成分进行优化，初始培养基为含15％血清的DMEM-F12无酚红培养基，培养过程中每3天更换一次培养基，更换培养基后每48h向体系中加入初始培养基体积0.1％的Cysteine(Cystenie储液浓度1μMol/L)，细胞增殖和迁移速率进一步提高，细胞生长状态较佳。如图7所示，图7a为不添加Cysteine的时细胞生长状态图，图7b为添加0.1％(v/v)Cysteine时细胞生长状态图，可见，添加0.1％(v/v)Cysteine后细胞生长状态较佳。
综上，利用华通氏胶进行组织块培养时，加入本发明实施例的固定装置，且优化培养条件，设置培养箱CO 2浓度为10％，并在培养时每48h加入原始培养液体积0.1％的Cysteine(Cystenie储液浓度1μMol/L)，既能提高华通氏胶的利用效率，又提高了细胞的增殖和迁移速率。使用了本发明实施例的固定装置，华通氏胶得到固定，大大方便了换液时操作。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。

Claims (10)

  1. 用于组织块培养的固定装置，其特征在于，包括：
    衔接件，所述衔接件用于与培养槽可拆卸连接；
    固定件，所述固定件至少设置有平面底部，所述固定件用于固定组织块，以使所述组织块夹持固定在所述固定件的平面底部和所述培养槽的槽底之间；
    连接件，所述连接件的首端与所述衔接件活动连接，所述连接件的尾端与所述固定件连接。
  2. 如权利要求1所述的用于组织块培养的固定装置，其特征在于，所述衔接件设置有卡槽，所述卡槽与所述培养槽的侧壁卡接。
  3. 如权利要求1所述的用于组织块培养的固定装置，其特征在于，所述连接件包括对称而设的第一连接臂和第二连接臂，所述衔接件设置在所述第一连接臂的首端和所述第二连接臂的首端之间；所述第一连接臂的首端与所述衔接件转动连接，所述第二连接臂的首端与所述衔接件转动连接；所述固定件设置在所述第一连接臂的尾端和所述第二连接臂的尾端之间。
  4. 如权利要求3所述的用于组织块培养的固定装置，其特征在于，所述第一连接臂的首端和所述第二连接臂的首端之间设置有连接柱；所述连接柱插入所述衔接件与所述衔接件转动连接；
    或者，所述第一连接臂的首端设置有第一凸柱，所述第二连接臂的首端设置有第二凸柱，所述第一凸柱和所述第二凸柱均与所述衔接件转动连接。
  5. 如权利要求3所述的用于组织块培养的固定装置，其特征在于，所述第一连接臂包括第一倾斜件和第一平置件，所述第二连接臂包括第二倾斜件和第二平置件；所述第一倾斜件与所述衔接件转动连接，所述第二倾斜件与所述衔接件转动连接；所述固定件设置于所述第一平置件和所述第二平置件之间。
  6. 一种采用权利要求1-5任一项所述的固定装置的组织块培养系统，其特征在于，包括至少一个培养单元，所述培养单元包括：
    培养槽，所述培养槽用于承装细胞培养液和组织块；
    至少一个所述固定装置，所述固定装置包括衔接件、固定件和连接件；
    所述衔接件用于与所述培养槽可拆卸连接；
    所述固定件至少设置有平面底部，所述固定件用于固定所述组织块，以使所述组织块夹持固定在所述固定件的平面底部和所述培养槽的槽底之间；
    所述连接件的首端与所述衔接件活动连接，所述连接件的尾端与所述固定件连接。
  7. 如权利要求6所述的组织块培养系统，其特征在于，还包括培养容器，所述培养容器用于收容所述培养单元。
  8. 如权利要求7所述的组织块培养系统，其特征在于，还包括盖体，所述盖体用于遮蔽所述培养槽的槽口；或者，所述盖体用于遮蔽所述培养容器的容器口。
  9. 一种组织块培养方法，其特征在于，采用如权利要求1-5任一项所述的用于组织块培养的固定装置，包括以下步骤：
    将组织块接种在培养槽中，并使所述组织块的一面贴附于所述培养槽的槽底；
    将所述固定装置与所述培养槽进行组装，其中所述衔接件与所述培养槽进行可拆卸连接，所述固定件压在所述组织块上；
    向所述培养槽中添加原始培养液，然后置于培养箱中培养。
  10. 如权利要求9所述的组织块培养方法，其特征在于，所述培养箱中CO 2的浓度为5％～15％；和/或
    培养过程中需要更换培养液，更换培养液后每45-50h向所述培养槽中加入半胱氨酸，半胱氨酸的加入量为所述原始培养液体积的0.02％-0.2％，半胱氨酸的储液为浓度1μMol/L。
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