An in vitro experimental model for studying the mechanism of tumor neurotropic invasion

Abstract

The present invention discloses an in vitro experimental model for studying the mechanism of tumor neurotropic invasion, comprising a first culture medium supply bottle, a first pump, a first culture chamber, a first waste liquid collection bottle, a second culture medium supply bottle, a second pump, a second culture chamber, and a second waste liquid collection bottle; the first culture 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, and the first culture chamber is located to the left of the second culture chamber. The present invention simulates a three-dimensional model of in vitro tissue and cell culture that is highly similar to the in vivo microenvironment, reduces and optimizes animal experiments, highly complies with the 3R principles of current animal ethics, and can improve the validity of experimental results, bridging the gap between the cellular and molecular levels in physiological, pathological, and pharmacological research and animal and clinical experiments.

Description

Technical Field

[0001] The present invention belongs to the field of life science technology, and in particular relates to an in vitro experimental model for studying the mechanism of tumor neurotropic invasion. Background Art

[0002] Peroneurotropic invasion (PNI) is a phenomenon of local tumor invasion and metastasis in which tumor cells invade the epineurium, perineurium, or endoneurium along the nerve fibers and extend along the nerves. PNI is an independent factor for poor patient prognosis, increasing the likelihood of recurrence and reducing survival rate. It is common in squamous cell carcinoma, pancreatic cancer, head and neck, colon and rectum, biliary tract and gastric malignancies. It is not a simple way of spread, but involves a complex interaction between nerves and tumors. Tumor cells can infiltrate along the nerves to distances beyond the tumor boundary, and the occurrence of neuroinvasion is also associated with inflammatory responses and neuropathic pain. The lack of effective in vivo or in vitro models in current research has led to a bottleneck in the study of the mechanism of neuroinvasion, and the main mechanism driving tumor cell invasion into nerves is still unclear.

[0003] Most of the in vitro research models that have been constructed so far use tumor cells and mouse dorsal root ganglia in matrix gel ( The basic steps are as follows:

[0004] 1) Isolation of mouse dorsal root ganglia in vitro;

[0005] 2) Place the mouse dorsal root ganglion on a glass slide and completely cover its surface with Matrigel. This process should be performed on ice.

[0006] 3) Place the glass slide containing the dorsal root ganglia and covered with Matrigel in a well plate and place it in a 37°C incubator to allow the Matrigel to solidify;

[0007] 4) Evenly inject tumor cell solution into the well plate containing dorsal root ganglia covered with matrigel for co-culture;

[0008] 5) Culture dorsal root ganglia or tumor cells alone as control group;

[0009] 6) Regularly observe the growth status of tumor cells and dorsal root ganglia.

[0010] The disadvantages of the prior art are that tumor cells and dorsal root ganglia are co-cultured in the same chamber, and some tumor cells are already in contact with the dorsal root ganglia when injected with Matrigel, failing to reflect the long-range infiltration phenomenon seen in vivo when tumors invade nerves. The co-existence of tumor cells and neural tissue in the same chamber also makes it difficult to distinguish whether certain induced changes are due to tumor cell metabolism affecting the nerves or the nerves affecting the tumor. Furthermore, culture in well plates results in brief interruptions in cell nutrition during medium replacement, failing to simulate the in vivo microenvironment of continuous blood transport through the vascular network. Deviations from the microenvironment can affect cell expression, potentially leading to the non-detection of certain relevant signaling molecules, thus affecting experimental results. To address these shortcomings, the present invention aims to create an in vitro simulation system that more closely resembles the actual in vivo microenvironment, thereby improving the accuracy and effectiveness of experimental results.

[0011] The present invention changes the well plate culture method and combines the use of a dual-chamber culture in a dual-flow bioreactor, with one chamber culturing tumor cells and one chamber culturing Schwann cells or dorsal root ganglia or other neural explants. Under the condition of continuous culture medium supply and replacement, other novel porous hydrogel scaffolds can be used for three-dimensional culture, highly simulating the real physiological microenvironment in the body. Summary of the Invention

[0012] The patent of this invention mainly designs an effective in vitro model for studying the mechanism of tumor neurotropic invasion. 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 and optimizing animal experiments. It is highly consistent with the 3R principle in current animal ethics, and can improve the validity of experimental results, bridging the gap from the cellular and molecular level in physiological, pathological and pharmacological research to animal experiments and clinical experiments.

[0013] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is: an in vitro experimental model for studying the mechanism of tumor neurotropic invasion, comprising a first culture medium supply bottle, a first pump, a first culture chamber, a first waste liquid collection bottle, a second culture medium supply bottle, a second pump, a second culture chamber, and a second waste liquid collection bottle; the first culture 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 first valve connects the first culture chamber and the second culture chamber together, and the first culture chamber is located on the left side of the second culture chamber.

[0014] Furthermore, the first culture chamber and the second culture chamber have the same structure, and are both provided with microporous membranes, which separate the first culture chamber and the second culture chamber into an upper layer and a lower layer. The upper layer and the lower layer are both provided with two interfaces, one inlet and one outlet, namely, an upper inlet, an upper outlet, a lower inlet, and a lower outlet.

[0015] Furthermore, the microporous membrane is used to cover the matrix gel or place the porous hydrogel scaffold.

[0016] Furthermore, a second valve is provided between the first pump and the first culture chamber, between the first culture chamber and the first waste liquid collection bottle, between the second pump and the second culture chamber, and between the second culture chamber and the second waste liquid collection bottle.

[0017] Furthermore, the first culture chamber is used to culture tumor cells; and the second culture chamber is used to culture Schwann cells or dorsal root ganglia or other neural explants.

[0018] Furthermore, the first culture chamber is used to culture Schwann cells or dorsal root ganglia or other neural explants; and the second culture chamber is used to culture tumor cells.

[0019] Furthermore, the model can establish three culture modes: 1) With the first valve closed, tumor cells are cultured in the first culture chamber alone, while Schwann cells, dorsal root ganglia, or other neural explants are cultured in the second culture chamber alone. In this case, the model becomes two completely independent three-dimensional culture systems that can be analyzed and tested separately as control experiments. The cultures in the first and second culture chambers can be swapped.

[0020] 2) Open the first valve, culture tumor cells in the first culture chamber alone, and culture Schwann cells or dorsal root ganglia or other neural explants in the second culture chamber alone. This model can simulate molecular crosstalk between tissues in vivo, allowing observation of the impact of tumor cells on neural invasion when located upstream, and detection of the induction of tumor cell metabolism on nerves.

[0021] 3) Open the first valve, culture Schwann cells or dorsal root ganglia or other neural explants in the first culture chamber alone, and culture tumor cells in the second culture chamber alone. At this time, the model can simulate the molecular crosstalk between tissues in the body, observe the impact of tumor cells on neural invasion at downstream locations, and detect the induction of neural cell metabolism on tumor cells.

[0022] Furthermore, when it is not necessary to distinguish and collect metabolites from the first culture chamber and the second culture chamber for analysis, the model can be simplified as follows: comprising a first culture medium supply bottle, a first pump, a first culture chamber, a second culture chamber, a 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 comprising a second culture medium supply bottle, a second pump, the first culture chamber, the second culture chamber, and the second waste liquid collection bottle, the second culture medium supply bottle, the second pump, the first culture chamber, the second culture chamber, and the second waste liquid collection bottle are connected in sequence;

[0023] 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 through the first valve. The first culture chamber is located on the left side of the second culture chamber.

[0024] Furthermore, a second valve is provided between the first pump and the first culture chamber, between the second culture chamber and the first waste liquid collection bottle, or between the second pump and the first culture chamber, between the second culture chamber and the second waste liquid collection bottle.

[0025] The main disadvantages of existing in vitro models are: 1. They cannot reflect the long-distance infiltration of tumors along nerves; 2. They cannot simulate the culture microenvironment with continuous blood circulation; 3. It is difficult to explore the effects of the metabolic products of tumor or nerve-related cells on each other. The advantages of the present invention are: 1. It adopts a dual-culture chamber model, with one chamber culturing tumor cells and the other chamber culturing neural Schwann cells or dorsal root ganglia or other nerve explants, which increases the distance between the two and can simulate the series connection between tissues in the body and the long-distance invasion of tumor cells into nerves; 2. The relative positions of the dual culture chambers can be adjusted to control the different effects produced when tumor cells are located upstream and downstream of the nerves; 3. The present invention is a system that continuously supplies and renews culture medium, which realistically simulates the blood circulation in the body. Combined with the use of three-dimensional culture methods such as porous hydrogel scaffolds, it is closer to the real physiological microenvironment in the body, making cell expression closer to the real situation in the body. 4. The model can also be further used to study how to effectively and timely block the invasion of tumors into nerves. 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 and optimizing animal experiments, highly consistent with the 3R principles in current animal ethics, and can improve the validity of experimental results, bridging the gap from the cellular and molecular levels in physiological, pathological and pharmacological research to animal experiments and clinical experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the structure of the in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to the present invention;

[0027] Figure 2 This is a simplified schematic diagram of the in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to the present invention;

[0028] Figure 3 Schematic diagram of the structure of the first culture chamber and the second culture chamber of the present invention.

[0029] Description of reference numerals:

[0030] 1. First culture medium supply bottle, 2. First pump, 3. First culture chamber, 4. First waste liquid collection bottle, 5. Second culture medium supply bottle, 6. Second pump, 7. Second culture 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 layer of the chamber, 16. Lower layer of the chamber, 17. Second valve. DETAILED DESCRIPTION

[0031] A specific embodiment of the present invention is described in detail below with reference to the accompanying drawings. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiment. The in vitro culture model system of the present invention is not limited to studying the pathological mechanism of tumor neurotropic invasion, but can also be expanded to study other pathological and pharmacological mechanisms.

[0032] An in vitro experimental model for studying the mechanism of tumor neurotropic invasion, such as Figure 1 As shown, it includes a first culture medium supply bottle 1, a first pump 2, a first culture chamber 3, a first waste liquid collection bottle 4, a second culture medium supply bottle 5, a second pump 6, a second culture chamber 7, and a second waste liquid collection bottle 8; the first culture medium supply bottle 1, the first pump 2, the first culture chamber 3 and the first waste liquid collection bottle 4 are connected in sequence, and 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. A first valve 9 is provided between the first culture chamber 3 and the second culture chamber 7. The first valve 9 connects the first culture chamber 3 and the second culture chamber 7 together, and the first culture chamber 3 is located on the left side of the second culture chamber 7.

[0033] First pump 2 delivers culture medium from first culture medium supply bottle 1 into first culture chamber 3. Metabolized waste liquid in first culture chamber 3 is discharged into first waste liquid collection bottle 4. When first valve 9 is open, some culture medium also flows through first culture chamber 3 and into second culture chamber 7. Second pump 6 delivers fresh culture medium from second culture medium supply bottle 5 into second culture chamber 7. Metabolized waste liquid in second culture chamber 7 is discharged into second waste liquid collection bottle 8. The liquid flow rate can be controlled by adjusting the gear speed driven by the pump voltage.

[0034] The first culture chamber 3 and the second culture chamber 8 have the same structure. Figure 3 As shown, a microporous membrane 10 is provided in each of the first and second culture chambers 3, 8. The microporous membrane 10 separates the first and second culture chambers 3, 8 from an upper layer 15 and a lower layer 16. Each of the upper and lower layers 15, 16 has two inlet and outlet ports, namely an upper inlet 11, an upper outlet 12, a lower inlet 13, and a lower outlet 14. The microporous membrane 10 is used to cover the matrigel or to place a porous hydrogel scaffold. This allows the first and second culture chambers 9, 7 to be connected to simulate both vertical and horizontal cell migration.

[0035] Second valves 17 are provided between the first pump 2 and the first culture chamber 3, between the first culture chamber 3 and the first waste liquid collection bottle 4, between the second pump 6 and the second culture chamber 7, and between the second culture chamber 7 and the second waste liquid collection bottle 8. These valves can be opened and closed according to experimental conditions to selectively control the flow direction of the culture medium.

[0036] The first culture chamber 3 is used to culture tumor cells; the second culture chamber 7 is used to culture Schwann cells or dorsal root ganglia or other neural explants. Or the first culture chamber 3 is used to culture Schwann cells or dorsal root ganglia or other neural explants; the second culture chamber 7 is used to culture tumor cells.

[0037] The model can establish three culture modes: 1) Close the first valve 9, and culture tumor cells in the first culture chamber 3 alone, and culture Schwann cells or dorsal root ganglia or other neural explants in the second culture chamber 7 alone. In this case, the model is two completely independent three-dimensional culture systems, which can be analyzed and tested separately as a control group experiment; the cultures in the first culture chamber 3 and the second culture chamber 7 can be swapped;

[0038] 2) Open the first valve 9, and culture tumor cells in the first culture chamber 3 alone, and culture Schwann cells or dorsal root ganglia or other neural explants in the second culture chamber 7 alone. In this case, the model can simulate molecular crosstalk between tissues in the body, observe the effect of tumor cells on neural invasion when they are in an upstream position, and detect the induction effect of tumor cell metabolism on nerves;

[0039] 3) Open the first valve 9, and culture Schwann cells or dorsal root ganglia or other neural explants in the first culture chamber 3 alone, and culture tumor cells in the second culture chamber 7 alone. At this time, the model can simulate the molecular crosstalk between tissues in the body, observe the impact of tumor cells on nerve invasion when they are in a downstream position, and detect the induction effect of nerve cell metabolism on tumor cells.

[0040] When there is no need to distinguish and collect the metabolites from the first culture chamber 3 and the second culture chamber 7 for analysis, the model can be simplified as follows: Figure 2 As shown, the system includes a first culture medium supply bottle 1, a first pump 2, a first culture chamber 3, a second culture chamber 7, and a first waste liquid collection bottle 4, which are sequentially connected; or includes a second culture medium supply bottle 5, a second pump 6, the first culture chamber 3, the second culture chamber 7, and a second waste liquid collection bottle 8, which are sequentially connected; a first valve 9 is provided between the first culture chamber 3 and the second culture chamber 7, and the first culture chamber 3 and the second culture chamber 7 are connected together through the first valve 9, and the first culture chamber 3 is located on the left side of the second culture chamber 7. A second valve 17 is provided between the first pump 2 and the first culture chamber 3, between the second culture chamber 7 and the first waste liquid collection bottle 4, or between the second pump 6 and the first culture chamber 3, and between the second culture chamber 7 and the second waste liquid collection bottle 8. 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 the second waste liquid collection bottle 8.

[0041] The detailed description of the present invention only specifically describes several embodiments of the present invention, but this should not be construed as limiting the scope of the present invention. It should be noted that this experimental model is not limited to the study of the mechanisms of tumor neurotropic invasion; it can also be used to investigate the efficacy of anticancer drugs and other pathological mechanisms involving multi-organ tissue interactions, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the appended claims.

Claims

1. An in vitro experimental model for studying the mechanism of tumor neurotropic invasion, characterized in that: The invention comprises a first culture medium supply bottle (1), a first pump (2), a first culture chamber (3), a first waste liquid collection bottle (4), a second culture medium supply bottle (5), a second pump (6), a second culture chamber (7), and a second waste liquid collection bottle (8); the first culture medium supply bottle (1), the first pump (2), the first culture chamber (3), and the first waste liquid collection bottle (4) are 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, a first valve (9) is provided between the first culture chamber (3) and the second culture chamber (7), the first culture chamber (3) and the second culture chamber (7) are connected together through the first valve (9), and the first culture chamber (3) is located on the left side of the second culture chamber (7); The first culture chamber (3) and the second culture chamber (8) have the same structure. A microporous membrane (10) is provided in each of the first culture chamber (3) and the second culture chamber (8). The microporous membrane (10) separates the first culture chamber (3) and the second culture chamber (7) into an upper layer (15) and a lower layer (16). The upper layer (15) and the lower layer (16) are each provided with two interfaces, one inlet and one outlet, namely an upper inlet (11), an upper outlet (12), a lower inlet (13), and a lower outlet (14). A second valve (17) is provided between the first pump (2) and the first culture chamber (3), between the first culture chamber (3) and the first waste liquid collection bottle (4), between the second pump (6) and the second culture chamber (7), and between the second culture chamber (7) and the second waste liquid collection bottle (8).

2. The in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to claim 1, characterized in that: The microporous membrane (10) is used to cover the matrix glue or place the porous hydrogel scaffold.

3. The in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to claim 1 or 2, characterized in that: 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 neural explants.

4. The in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to claim 1 or 2, characterized in that: The first culture chamber (3) is used for culturing Schwann cells or dorsal root ganglia or other neural explants; the second culture chamber (7) is used for culturing tumor cells.

5. The in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to claim 1 or 2, characterized in that: The model can establish three culture modes: 1) the first valve (9) is closed, the first culture chamber (3) is used to culture tumor cells alone, and the second culture chamber (7) is used to culture Schwann cells or dorsal root ganglia or other neural explants alone. At this time, the model is two completely independent three-dimensional culture systems, which can be analyzed and tested separately as a control group experiment; the cultures in the first culture chamber (3) and the second culture chamber (7) can be exchanged; 2) Opening the first valve (9), culturing tumor cells alone in the first culture chamber (3), and culturing Schwann cells or dorsal root ganglia or other neural explants alone in the second culture chamber (7). At this time, the model can simulate molecular crosstalk between tissues in the body, observe the effect of tumor cells on nerve invasion when they are in the upstream position, and detect the induction effect of tumor cell metabolism on nerves; 3) Open the first valve (9), culture Schwann cells or dorsal root ganglia or other neural explants in the first culture chamber (3) alone, and culture tumor cells in the second culture chamber (7) alone. At this time, the model can simulate the molecular crosstalk between tissues in the body, observe the effect of tumor cells on nerve invasion when they are in the downstream position, and detect the induction effect of nerve cell metabolism on tumor cells.

6. The in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to claim 1, characterized in that: When there is no need to distinguish and collect metabolites from the first culture chamber (3) and the second culture chamber (7) for analysis, the model can be simplified as follows: comprising a first culture medium supply bottle (1), a first pump (2), a first culture chamber (3), a second culture chamber (7), and a first waste liquid collection bottle (4), wherein the first culture medium supply bottle (1), the first pump (2), the first culture chamber (3), the second culture chamber (7), and the first waste liquid collection bottle (4) are connected in sequence; or comprising a second culture medium supply bottle (5), a second pump (6), the first culture chamber (3), the second culture chamber (7), and a second waste liquid collection bottle (8), wherein the second culture medium supply bottle (5), the second pump (6), the first culture chamber (3), the second culture chamber (7), and the second waste liquid collection bottle (8) are connected in sequence; A first valve (9) is provided between the first culture chamber (3) and the second culture chamber (7). The first culture chamber (3) and the second culture chamber (7) are connected together through the first valve (9). The first culture chamber (3) is located on the left side of the second culture chamber (7).

7. The in vitro experimental model for studying the mechanism of tumor neurotropic invasion according to claim 6, characterized in that: A second valve (17) is provided between the first pump (2) and the first culture chamber (3), between the second culture chamber (7) and the first waste liquid collection bottle (4), or between the second pump (6) and the first culture chamber (3), between the second culture chamber (7) and the second waste liquid collection bottle (8).

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