WO2021114129A1 - 一种用于肿瘤嗜神经侵袭机制研究的体外实验模型 - Google Patents

一种用于肿瘤嗜神经侵袭机制研究的体外实验模型 Download PDF

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WO2021114129A1
WO2021114129A1 PCT/CN2019/124504 CN2019124504W WO2021114129A1 WO 2021114129 A1 WO2021114129 A1 WO 2021114129A1 CN 2019124504 W CN2019124504 W CN 2019124504W WO 2021114129 A1 WO2021114129 A1 WO 2021114129A1
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culture chamber
culture
chamber
tumor
pump
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PCT/CN2019/124504
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French (fr)
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阎梦萦
鲁艺
钟成
李梦
王璐璐
王立平
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中国科学院深圳先进技术研究院
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Publication of WO2021114129A1 publication Critical patent/WO2021114129A1/zh

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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

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  • the invention belongs to the technical field of life sciences, and specifically relates to an in vitro experimental model for the study of tumor neurophilic invasion mechanism.
  • PNI Tumor neurophilic invasion
  • squamous cell carcinoma pancreatic cancer
  • pancreatic cancer head and neck
  • colon and rectum biliary tract and stomach.
  • Tumor cells can infiltrate along nerves to a distance beyond the tumor boundary, and the occurrence of nerve invasion is also related to inflammatory reactions and neuropathic pain.
  • the lack of effective in vivo or in vitro models has caused the research on the mechanism of neural invasion to fall into a bottleneck. The main mechanism that drives tumor cells to invade the nerves is currently unclear.
  • the disadvantage of the prior art is that the tumor cells and the dorsal root ganglion are co-cultured in the same room, and some tumor cells have been in contact with the dorsal root ganglion when the matrigel is injected, and cannot reflect the long-distance infiltration of the tumor during neurophilic invasion in the body. Phenomenon, tumor cells and nerve tissues are mixed in one room, and it is difficult to distinguish whether some induced changes are nerves affected by the metabolism of tumor cells or tumors affected by nerves. Furthermore, culture in well plates, changing the medium to make the cell nutrition short-lived Intermittent, unable to simulate the microenvironment of the continuity of blood transport in the vascular network in the body.
  • the purpose of the present invention is to create an in vitro simulation system that is closer to the real microenvironment in the body, and to improve the accuracy and effectiveness of the experimental results.
  • the orifice plate culture method is modified, combined with the dual-flow bioreactor dual-chamber culture, one chamber for culturing tumor cells, one chamber for culturing Schwann cells or dorsal root ganglia or other nerve explants, in continuous culture Under the condition of base supply replacement, it can be combined with other new porous hydrogel scaffolds for three-dimensional culture, which highly simulates the real physiological microenvironment in the body.
  • the patent of the present invention is mainly to design an effective in vitro model for the study of tumor neurophilic invasion mechanism. Its design is intended to simulate a three-dimensional model of in vitro tissue and cell culture that is highly similar to the in vivo microenvironment, thereby reducing optimization of animal experiments. It is highly in line with the current 3R principles in animal ethics, and can improve the validity of experimental results, bridging the gap between the cellular and molecular level in physiological and pathological and pharmacological research to animal experiments and clinical experiments.
  • the technical solution adopted by the present invention is: an in vitro experimental model for the study of tumor neurophilic invasion mechanism, including a first medium supply bottle, a first pump, a first culture chamber, and a first waste liquid
  • the collection bottle, the second medium supply bottle, the second pump, the second culture chamber, the second waste liquid collection bottle; the first medium supply bottle, the first pump, the first culture chamber and the first waste liquid collection bottle are connected in sequence ,
  • the second culture medium supply bottle, the second pump, the second culture chamber and the second waste liquid collection bottle are connected in sequence.
  • a first valve is provided between the first culture chamber and the second culture chamber.
  • the chamber and the second culture chamber are connected together, and the first culture chamber is located on the left side of the second culture chamber.
  • first culture chamber and the second culture chamber are the same. Both the first culture chamber and the second culture chamber are provided with a microporous membrane.
  • the microporous membrane separates the first culture chamber and the second culture chamber into the upper chamber and the chamber.
  • the upper chamber and the lower chamber are equipped with two interfaces, one inlet and one outlet, which are respectively an upper inlet, an upper outlet, a lower inlet, and a lower outlet.
  • microporous membrane is used to cover Matrigel or to place porous hydrogel scaffolds.
  • the first culture chamber is used for culturing tumor cells; the second culture chamber is used for culturing Schwann cells or dorsal root ganglia or other nerve explants.
  • the first culture chamber is used for culturing Schwann cells or dorsal root ganglia or other nerve explants; the second culture chamber is used for culturing tumor cells.
  • the model can establish three culture modes: 1) Close the first valve, the first culture room will culture tumor cells alone, and the second culture room will culture Schwann cells or dorsal root ganglia or other nerve explants.
  • the model is two completely independent three-dimensional culture systems, which can be analyzed and tested separately as a control experiment; the cultures in the first culture room and the second culture room can be exchanged;
  • the model can simulate molecular crosstalk between tissues in the body. It can observe the influence of tumor cells on nerve invasion when the tumor cells are in the upstream position, and can detect the induction of tumor cell metabolism on nerves;
  • the model can simulate the molecular crosstalk between tissues in the body.
  • the influence of tumor cells on nerve invasion can be observed when the tumor cells are in the downstream position, and the induction effect of nerve cell metabolism on tumor cells can be detected.
  • the model can be simplified to include the first culture medium supply bottle, the first pump, the first culture chamber, and the second culture chamber.
  • Room, the first waste liquid collection bottle, the first culture medium supply bottle, the first pump, the first culture chamber, the second culture chamber, and the first waste liquid collection bottle are connected in sequence; or include the second culture medium supply bottle, the second The pump, the first culture chamber, the second culture chamber, the second waste liquid collection bottle, the second medium supply bottle, the second pump, the first culture room, the second culture room, and the second waste liquid collection bottle are connected in sequence;
  • a first valve is provided between the first culture chamber and the second culture chamber, the first culture chamber and the second culture chamber are connected together by the first valve, and the first culture chamber is located on the left side of the second culture chamber .
  • Both rooms are equipped with a second valve.
  • the main shortcomings of the existing in vitro models are: First, they cannot reflect the long-distance infiltration of tumors along the nerves; Second, they cannot simulate the continuous circulation of blood in the culture microenvironment; Third, it is difficult to find the metabolites of tumors or nerve-related cells against each other. Impact.
  • the advantages of the present invention are as follows: 1. Using a dual culture chamber model, one chamber for culturing tumor cells, and the other chamber for culturing nerve Schwann cells or dorsal root ganglia or other nerve explants. Simulating the tandem between tissues in the body, tumor cells invade the nerves at a long distance; 2. The mutual position of the dual culture chambers can be adjusted to control the different effects when the tumor cells are located upstream and downstream of the nerve; 3.
  • the present invention is a continuous supply And the system for renewing the culture medium truly simulates the blood circulation in the body, and combined with the use of porous hydrogel scaffolds and other three-dimensional culture methods, it is closer to the real physiological microenvironment in the body, and the cell expression is closer to the real situation in the body. 4.
  • the model can be further used to study how to effectively block tumor invasion to nerves in time.
  • the model of the present invention simulates a three-dimensional model of in vitro tissue and cell culture that is highly similar to the in vivo microenvironment, thereby reducing optimization of animal experiments, highly conforming to the 3R principle of current animal ethics, and can improve the validity of experimental results, and bridge the physiology The gap between the cellular and molecular level in pathological and pharmacological research to animal experiments and clinical experiments.
  • Figure 1 is a schematic diagram of the structure of an in vitro experimental model used in the study of tumor neurophilic invasion mechanism of the present invention
  • FIG. 2 is a simplified structural diagram of the in vitro experimental model used for the study of tumor neurophilic invasion mechanism of the present invention
  • Fig. 3 is a schematic diagram of the structure of the first culture chamber and the second culture chamber of the present invention.
  • the first medium supply bottle 2. The first pump, 3. The first culture chamber, 4. The first waste liquid collection bottle, 5. The second medium supply bottle, 6. The second pump, 7. The second Cultivation chamber, 8. Second waste liquid collection bottle, 9. First valve, 10. Microporous membrane, 11. Upper inlet, 12. Upper outlet, 13. Lower inlet, 14. Lower outlet, 15. Upper chamber, 16 .The lower part of the room, 17. The second valve.
  • the in vitro culture model system of the present invention is not limited to the study of the pathological mechanism of tumor neurophilic invasion, but can also be extended to the study of other pathological and pharmacological mechanisms.
  • An in vitro experimental model for the study of tumor neurophilic invasion mechanism includes a first medium supply bottle 1, a first pump 2, a first culture chamber 3, a first waste liquid collection bottle 4, and a The second medium supply bottle 5, the second pump 6, the second culture chamber 7, the second waste liquid collection bottle 8; the first medium supply bottle 1, the first pump 2, the first culture chamber 3 and the first waste liquid collection
  • the bottle 4 is connected in sequence, the second culture medium supply bottle 5, the second pump 6, the second culture chamber 7 and the second waste liquid collection bottle 8 are connected in sequence, and the first culture chamber 3 and the second culture chamber 7 are arranged between the first culture chamber 3 and the second culture chamber 7.
  • a valve 9 connects the first culturing chamber 3 and the second culturing chamber 7 together, and the first culturing chamber 3 is located on the left side of the second culturing chamber 7.
  • the first pump 2 transports the culture medium in the first culture medium supply bottle 1 into the first culture chamber 3, the metabolized waste liquid in the first culture chamber 3 is discharged into the first waste liquid collection bottle 4, and the first valve 9 is opened At this time, a part of the culture medium flows through the first culture chamber 3 and then enters the second culture chamber 7.
  • the second pump 6 transports the fresh culture medium in the second culture medium supply bottle 5 into the second culture chamber 7.
  • the waste liquid metabolized in 7 is discharged into the second waste liquid collection bottle 8.
  • the speed of the liquid can be controlled by adjusting the speed of the gear driven by the voltage of the pump.
  • the first culturing chamber 3 and the second culturing chamber 8 have the same structure. As shown in FIG. 3, the first culturing chamber 3 and the second culturing chamber 8 are both provided with a microporous membrane 10, which connects the first culturing chamber 3 Separate the upper 15 and lower 16 chambers from the second culture chamber 8.
  • the upper 15 and lower 16 chambers are each provided with one inlet and one outlet, which are an upper inlet 11, an upper outlet 12, a lower inlet 13, and a lower outlet 14, respectively.
  • the microporous membrane 10 is used to cover the matrigel or to place the porous hydrogel scaffold. In this way, after the first culture chamber 9 and the second culture chamber 7 are connected, the migration of the cells in the vertical direction and the horizontal direction can be simulated at the same time.
  • the first culture chamber 3 is used for culturing tumor cells; the second culture chamber 7 is used for culturing Schwann cells or dorsal root ganglia or other nerve explants. Or the first culture chamber 3 is used for culturing Schwann cells or dorsal root ganglia or other nerve explants; the second culture chamber 7 is used for culturing tumor cells.
  • Three culture modes can be established for the model: 1) Close the first valve 9, the first culture chamber 3 cultures tumor cells alone, and the second culture chamber 7 cultures Schwann cells or dorsal root ganglia or other nerve explants alone.
  • the model is two completely independent three-dimensional culture systems, which can be analyzed and tested separately as a control experiment; the cultures in the first culture chamber 3 and the second culture chamber 7 can be exchanged;
  • the model can simulate the molecules between the tissues in the body Crosstalk, you can observe the influence of tumor cells on nerve invasion when the tumor cells are in the upstream position, and detect the induction of tumor cell metabolism on nerves;
  • the model can simulate the molecules between the tissues in the body Crosstalk can be used to observe the influence of tumor cells on nerve invasion when the tumor cells are in the downstream position, and to detect the inducing effect of nerve cell metabolism on tumor cells.
  • the model can be simplified as shown in Figure 2: including the first medium supply bottle 1, the first pump 2, the first Culture room 3, second culture room 7, first waste liquid collection bottle 4
  • the first medium supply bottle 1, first pump 2, first culture room 3, second culture room 7, first waste liquid collection bottle 4 are connected in sequence; or include the second culture medium supply bottle 5, the second pump 6, the first culture chamber 3, the second culture chamber 7, the second waste liquid collection bottle 8, the second culture medium supply bottle 5, the first
  • the two pumps 6, the first culturing chamber 3, the second culturing chamber 7, and the second waste liquid collection bottle 8 are connected in sequence; a first valve 9 is provided between the first culturing chamber 3 and the second culturing chamber 7, and the first culturing chamber 3 and the second culture chamber 7 are connected together by a first valve 9, and the first culture chamber 3 is located on the left side of the second culture chamber 7.
  • the first pump 2 or the second pump 6 transports the culture medium in the first culture medium supply bottle 1 or the second culture medium supply bottle 5 into the first culture chamber 3 and the second culture chamber 7, and finally the waste liquid flows into the first waste Liquid collection bottle 4 or second waste liquid collection bottle 8.

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Abstract

一种用于肿瘤嗜神经侵袭机制研究的体外实验模型，包括第一培养基供应瓶、第一泵、第一培养室、第一废液收集瓶、第二培养基供应瓶、第二泵、第二培养室、第二废液收集瓶；第一培养基供应瓶、第一泵、第一培养室和第一废液收集瓶依次连接，第二培养基供应瓶、第二泵、第二培养室和第二废液收集瓶依次连接，第一培养室和第二培养室之间设有第一阀门，第一培养室位于第二培养室的左侧。

Description

一种用于肿瘤嗜神经侵袭机制研究的体外实验模型 技术领域
本发明属于生命科学技术领域，具体涉及一种用于肿瘤嗜神经侵袭机制研究的体外实验模型。
背景技术
肿瘤嗜神经侵袭(PNI)是肿瘤细胞在神经纤维周围沿着神经侵入神经外膜、神经束膜或神经内膜并延伸的肿瘤局部浸润转移现象。PNI是患者预后不良的独立因素，增加了复发的可能性和降低了存活率，常见于鳞状细胞癌，胰腺癌，头颈，结肠和直肠，胆道和胃的恶性肿瘤。它不是一种简单的扩散方式，涉及了神经和肿瘤之间复杂的相互作用，肿瘤细胞可以沿着神经浸润到肿瘤边界之外的距离，并且神经侵袭的发生也与炎性反应和神经性疼痛相关。目前的研究中因为缺少有效的体内或体外模型，使关于神经侵袭机制的研究陷入了瓶颈，驱动肿瘤细胞向神经侵袭的主要机制目前尚不明确。
目前已构建的体外研究模型大多是采用肿瘤细胞与小鼠背根神经节在基质胶(
Figure PCTCN2019124504-appb-000001
Basement Membrane Matrix)中共培养的方式。基本步骤如下：
1)体外分离小鼠背根神经节；
2)将小鼠背根神经节置于玻片上，并用基质胶将其表面完全覆盖，此过程冰上操作；
3)将含有背根神经节并覆盖了基质胶的玻片置于孔板中，放于37℃培养箱，使基质胶固化；
4)均匀注射肿瘤细胞液于含有基质胶覆盖的背根神经节的孔板中进行共同培养；
5)单独培养背根神经节或单独培养肿瘤细胞作为对照组；
6)定期观察肿瘤细胞和背根神经节的生长状态。
现有技术的缺点是肿瘤细胞和背根神经节处于同一室中共同培养，某些肿瘤细胞在注射进基质胶时已与背根神经节接触，不能反应体内肿瘤嗜神经侵袭时的远距离浸润现象，肿瘤细胞和神经组织共混一室，也很难分辨某些诱导变化是肿瘤细胞的代谢影响的神经还是神经影响的肿瘤，再者孔板中培养，更换培养基使细胞营养有短暂的间断性，不能模拟体内脉管网络血液运输连续性的微环境，微环境的偏差会影响细胞的表达，可能导致某些相关的信号分子不被发现，进而影响实验效果。针对这些缺点本发明的目的即创造更加接近体内真实微环境的体外模拟系统，提高实验结果的准确性和有效性。
本发明中更改了孔板培养方式，结合使用双流式生物反应器双室培养，一室培养肿瘤细 胞，一室培养施万细胞或背根神经节或其它神经外植体，在连续性的培养基供应更换条件下并可结合使用其它新型多孔水凝胶支架进行三维培养，高度模拟体内真实的生理微环境。
发明内容
本发明专利主要是设计一种有效的用于肿瘤嗜神经侵袭机制研究的体外模型，其设计意在模拟一个与体内微环境高度相似的体外组织及细胞培养的三维模型，从而减少优化动物实验，高度符合当下动物伦理中的3R原则，并且可以提高实验结果的有效性，弥合了生理病理及药理研究中细胞分子水平到动物实验及临床实验的鸿沟。
为了达到上述目的，本发明所采用的技术方案为：一种用于肿瘤嗜神经侵袭机制研究的体外实验模型，包括第一培养基供应瓶、第一泵、第一培养室、第一废液收集瓶、第二培养基供应瓶、第二泵、第二培养室、第二废液收集瓶；第一培养基供应瓶、第一泵、第一培养室和第一废液收集瓶依次连接，第二培养基供应瓶、第二泵、第二培养室和第二废液收集瓶依次连接，第一培养室和第二培养室之间设有第一阀门，第一阀门将第一培养室和第二培养室连接在一起，第一培养室位于第二培养室的左侧。
进一步地，第一培养室和第二培养室结构相同，第一培养室和第二培养室中均设有微孔膜，微孔膜将第一培养室和第二培养室分隔为室上层和室下层，室上层和室下层均设有一进一出两个接口，分别为上进口、上出口、下进口、下出口。
进一步地，微孔膜用于覆盖基质胶或者放置多孔水凝胶支架。
进一步地，第一泵与第一培养室之间、第一培养室与第一废液收集瓶之间、第二泵与第二培养室之间以及第二培养室与第二废液收集瓶之间均设有第二阀门。
进一步地，第一培养室用于培养肿瘤细胞；第二培养室用于培养施万细胞或背根神经节或其他神经外植体。
进一步地，第一培养室用于培养施万细胞或背根神经节或其他神经外植体；第二培养室用于培养肿瘤细胞。
进一步地，模型可建立三种培养模式：1)关闭第一阀门，第一培养室单独培养肿瘤细胞，第二培养室单独培养施万细胞或背根神经节或其他神经外植体，此时模型为两个完全独立的三维培养系统，可单独进行分析检测，作为对照组实验；其中第一培养室和第二培养室内的培养物可调换；
2)打开第一阀门，第一培养室单独培养肿瘤细胞，第二培养室单独培养施万细胞或背根神经节或其他神经外植体，此时模型可模拟体内组织间的分子串音，可以观察肿瘤细胞在上游位 置时对神经侵袭的影响，可以检测肿瘤细胞代谢对神经的诱导作用；
3)打开第一阀门，第一培养室单独培养施万细胞或背根神经节或其他神经外植体，第二培养室单独培养肿瘤细胞，此时模型可模拟体内组织间的分子串音，可以观察肿瘤细胞在下游位置时对神经侵袭的影响，可以检测神经细胞代谢对肿瘤细胞的诱导作用。
进一步地，当不需要区分收集第一培养室和第二培养室的代谢产物进行分析时，该模型可以简化为：包括第一培养基供应瓶、第一泵、第一培养室、第二培养室、第一废液收集瓶，第一培养基供应瓶、第一泵、第一培养室、第二培养室、第一废液收集瓶依次连接；或包括第二培养基供应瓶、第二泵、第一培养室、第二培养室、第二废液收集瓶，第二培养基供应瓶、第二泵、第一培养室、第二培养室、第二废液收集瓶依次连接；
第一培养室和第二培养室之间设有第一阀门，所述第一培养室和第二培养室通过第一阀门连接在一起，所述第一培养室位于第二培养室的左侧。
进一步地，第一泵与第一培养室之间、第二培养室与第一废液收集瓶之间或第二泵与第一培养室之间、第二培养室与第二废液收集瓶之间均设有第二阀门。
现有的体外模型主要的缺点是：一、不能反应肿瘤沿神经的远距离浸润现象；二、不能模拟血液连续流通的培养微环境；三、很难探寻肿瘤或神经相关细胞的代谢产物对彼此的影响。本发明的优点在于：一、采用双培养室的模型，一室培养肿瘤细胞，另一室培养神经施万细胞或背根神经节或其它神经外植体，拉开了两者的距离，可以模拟体内组织间的串联，肿瘤细胞远距离向神经侵袭；二、可以调整双培养室的相互位置，对照肿瘤细胞位于神经上游和下游时产生的不同影响；三、本发明是一个连续性地供应和更新培养基的系统，很真实的模拟了体内血液循环，再结合使用多孔水凝胶支架等三维培养方式，更加接近体内真实的生理微环境，使细胞表达更接近体内真实情况。四、该模型还可以进一步用于如何可以及时有效阻断肿瘤向神经侵袭的研究。本发明模型模拟了一个与体内微环境高度相似的体外组织及细胞培养的三维模型，从而减少优化动物实验，高度符合当下动物伦理中的3R原则，并且可以提高实验结果的有效性，弥合了生理病理及药理研究中细胞分子水平到动物实验及临床实验的鸿沟。
附图说明
图1为本发明用于肿瘤嗜神经侵袭机制研究的体外实验模型的结构示意图；
图2为本发明用于肿瘤嗜神经侵袭机制研究的体外实验模型简化后的结构示意图；
图3为本发明第一培养室和第二培养室的结构示意图。
附图标记说明：
1.第一培养基供应瓶，2.第一泵，3.第一培养室，4.第一废液收集瓶，5.第二培养基供应瓶，6.第二泵，7.第二培养室，8.第二废液收集瓶，9.第一阀门，10.微孔膜，11.上进口，12.上出口，13.下进口，14.下出口，15.室上层，16.室下层，17.第二阀门。
具体实施方式
下面结合附图，对本发明的一个具体实施方式进行详细说明，但应当理解本发明的保护范围并不受具体实施方式的限制。本发明的体外培养模型系统不局限于研究肿瘤嗜神经侵袭这一种病理机制，还可拓展应用到其他病理药理机制的研究。
一种用于肿瘤嗜神经侵袭机制研究的体外实验模型，如图1所示，包括第一培养基供应瓶1、第一泵2、第一培养室3、第一废液收集瓶4、第二培养基供应瓶5、第二泵6、第二培养室7、第二废液收集瓶8；第一培养基供应瓶1、第一泵2、第一培养室3和第一废液收集瓶4依次连接，第二培养基供应瓶5、第二泵6、第二培养室7和第二废液收集瓶8依次连接，第一培养室3和第二培养室7之间设有第一阀门9，第一阀门9将第一培养室3和第二培养室7连接在一起，第一培养室3位于第二培养室7的左侧。
第一泵2将第一培养基供应瓶1内的培养基输送进第一培养室3，第一培养室3内代谢后的废液排入第一废液收集瓶4，第一阀门9打开时，也有一部分培养基流经第一培养室3再进入第二培养室7，第二泵6将第二培养基供应瓶5内的新鲜培养基输送进第二培养室7，第二培养室7内代谢的废液排入第二废液收集瓶8。其中可通过调节泵的电压带动的齿轮转速来控制液体流速。
第一培养室3和第二培养室8结构相同，如图3所示，第一培养室3和第二培养室8中均设有微孔膜10，微孔膜10将第一培养室3和第二培养室8分隔室上层15和室下层16，室上层15和室下层16均设有一进一出两个接口，分别为上进口11、上出口12、下进口13、下出口14。微孔膜10用于覆盖基质胶或者放置多孔水凝胶支架。这样第一培养室9和第二培养室7连接后可以同时模拟细胞垂直方向以及水平方向的迁移。
第一泵2与第一培养室3之间、第一培养室3与第一废液收集瓶4之间、第二泵6与第二培养室7之间以及第二培养室7与第二废液收集瓶8之间均设有第二阀门17。这样可以根据实验条件进行开关，来选择控制培养基的流向。
第一培养室3用于培养肿瘤细胞；第二培养室7用于培养施万细胞或背根神经节或其他神经外植体。或第一培养室3用于培养施万细胞或背根神经节或其他神经外植体；第二培养室7用于培养肿瘤细胞。
模型可建立三种培养模式：1)关闭第一阀门9，第一培养室3单独培养肿瘤细胞，第二培 养室7单独培养施万细胞或背根神经节或其他神经外植体，此时模型为两个完全独立的三维培养系统，可单独进行分析检测，作为对照组实验；其中第一培养室3和第二培养室7内的培养物可调换；
2)打开第一阀门9，第一培养室3单独培养肿瘤细胞，第二培养室7单独培养施万细胞或背根神经节或其他神经外植体，此时模型可模拟体内组织间的分子串音，可以观察肿瘤细胞在上游位置时对神经侵袭的影响，可以检测肿瘤细胞代谢对神经的诱导作用；
3)打开第一阀门9，第一培养室3单独培养施万细胞或背根神经节或其他神经外植体，第二培养室7单独培养肿瘤细胞，此时模型可模拟体内组织间的分子串音，可以观察肿瘤细胞在下游位置时对神经侵袭的影响，可以检测神经细胞代谢对肿瘤细胞的诱导作用。
当不需要区分收集第一培养室3和第二培养室7的代谢产物进行分析时，该模型可以简化为如图2所示：包括第一培养基供应瓶1、第一泵2、第一培养室3、第二培养室7、第一废液收集瓶4所述第一培养基供应瓶1、第一泵2、第一培养室3、第二培养室7、第一废液收集瓶4依次连接；或包括第二培养基供应瓶5、第二泵6、第一培养室3、第二培养室7、第二废液收集瓶8，所述第二培养基供应瓶5、第二泵6、第一培养室3、第二培养室7、第二废液收集瓶8依次连接；第一培养室3和第二培养室7之间设有第一阀门9，第一培养室3和第二培养室7通过第一阀门9连接在一起，第一培养室3位于第二培养室7的左侧。第一泵2与第一培养室3之间、第二培养室7与第一废液收集瓶4之间或第二泵6与第一培养室3之间、第二培养室7与第二废液收集瓶8之间均设有第二阀门17。第一泵2或第二泵6将第一培养基供应瓶1或第二培养基供应瓶5内的培养基输送进第一培养室3和第二培养室7，最后废液流入第一废液收集瓶4或第二废液收集瓶8。
本发明的具体方案阐述中仅具体地表达了本发明的几种实施方式，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，该实验模型并不局限使用于肿瘤嗜神经侵袭机制的研究，还可以用于看肿瘤药物效用的研究以及其它一些涉及多器官组织相互作用的病理机制研究，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (9)

  1. 一种用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，包括第一培养基供应瓶(1)、第一泵(2)、第一培养室(3)、第一废液收集瓶(4)、第二培养基供应瓶(5)、第二泵(6)、第二培养室(7)、第二废液收集瓶(8)；所述第一培养基供应瓶(1)、第一泵(2)、第一培养室(3)和第一废液收集瓶(4)依次连接，所述第二培养基供应瓶(5)、第二泵(6)、第二培养室(7)和第二废液收集瓶(8)依次连接，所述第一培养室(3)和第二培养室(7)之间设有第一阀门(9)，所述第一培养室(3)和第二培养室(7)通过第一阀门(9)连接在一起，所述第一培养室(3)位于第二培养室(7)的左侧。
  2. 根据权利要求1所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，所述第一培养室(3)和第二培养室(8)结构相同，所述第一培养室(3)和第二培养室(8)中均设有微孔膜(10)，所述微孔膜(10)将第一培养室(3)和第二培养室(8)分隔为室上层(15)和室下层(16)，所述室上层(15)和室下层(16)均设有一进一出两个接口，分别为上进口(11)、上出口(12)、下进口(13)、下出口(14)。
  3. 根据权利要求2所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，所述微孔膜(10)用于覆盖基质胶或者放置多孔水凝胶支架。
  4. 根据权利要求1所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，所述第一泵(2)与第一培养室(3)之间、第一培养室(3)与第一废液收集瓶(4)之间、第二泵(6)与第二培养室(7)之间以及第二培养室(7)与第二废液收集瓶(8)之间均设有第二阀门(17)。
  5. 根据权利要求1-4任一所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，所述第一培养室(3)用于培养肿瘤细胞；所述第二培养室(7)用于培养施万细胞或背根神经节或其他神经外植体。
  6. 根据权利要求1-4任一所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，所述第一培养室(3)用于培养施万细胞或背根神经节或其他神经外植体；所述第二培养室(7)用于培养肿瘤细胞。
  7. 根据权利要求1-4任一所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，模型可建立三种培养模式：1)关闭第一阀门(9)，第一培养室(3)单独培养肿瘤细胞，第二培养室(7)单独培养施万细胞或背根神经节或其他神经外植体，此时模型为两个完全独立的三维培养系统，可单独进行分析检测，作为对照组实验；其中第一培养室(3)和第二培养室(7)内的培养物可调换；
    2)打开第一阀门(9)，第一培养室(3)单独培养肿瘤细胞，第二培养室(7)单独培养施 万细胞或背根神经节或其他神经外植体，此时模型可模拟体内组织间的分子串音，可以观察肿瘤细胞在上游位置时对神经侵袭的影响，可以检测肿瘤细胞代谢对神经的诱导作用；
    3)打开第一阀门(9)，第一培养室(3)单独培养施万细胞或背根神经节或其他神经外植体，第二培养室(7)单独培养肿瘤细胞，此时模型可模拟体内组织间的分子串音，可以观察肿瘤细胞在下游位置时对神经侵袭的影响，可以检测神经细胞代谢对肿瘤细胞的诱导作用。
  8. 根据权利要求1所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，当不需要区分收集第一培养室(3)和第二培养室(7)的代谢产物进行分析时，该模型可以简化为：包括第一培养基供应瓶(1)、第一泵(2)、第一培养室(3)、第二培养室(7)、第一废液收集瓶(4)，所述第一培养基供应瓶(1)、第一泵(2)、第一培养室(3)、第二培养室(7)、第一废液收集瓶(4)依次连接；或包括第二培养基供应瓶(5)、第二泵(6)、第一培养室(3)、第二培养室(7)、第二废液收集瓶(8)，所述第二培养基供应瓶(5)、第二泵(6)、第一培养室(3)、第二培养室(7)、第二废液收集瓶(8)依次连接；
    所述第一培养室(3)和第二培养室(7)之间设有第一阀门(9)，所述第一培养室(3)和第二培养室(7)通过第一阀门(9)连接在一起，所述第一培养室(3)位于第二培养室(7)的左侧。
  9. 根据权利要求8所述的用于肿瘤嗜神经侵袭机制研究的体外实验模型，其特征在于，所述第一泵(2)与第一培养室(3)之间、第二培养室(7)与第一废液收集瓶(4)之间或第二泵(6)与第一培养室(3)之间、第二培养室(7)与第二废液收集瓶(8)之间均设有第二阀门(17)。
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