CN115322904B

CN115322904B - Organoid culture device simulating bladder structure and application thereof

Info

Publication number: CN115322904B
Application number: CN202211244808.5A
Authority: CN
Inventors: 董寅初; 李胜
Original assignee: Guagnzhou Jingke Biotech Co ltd; Chengdu Nuoyeide Medical Laboratory Co ltd
Current assignee: Chengdu Nuoyeide Medical Laboratory Co ltd; Shenzhen Jingke Biotechnology Co ltd
Priority date: 2022-10-12
Filing date: 2022-10-12
Publication date: 2023-01-31
Anticipated expiration: 2042-10-12
Also published as: CN115322904A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an organoid culture device for simulating a bladder structure and application thereof. The organoid culture device for simulating the bladder structure comprises an upper-layer channel structure and a lower-layer channel structure, wherein a porous membrane is arranged between the upper-layer channel structure and the lower-layer channel structure for separation, and the upper side and the lower side of the upper-layer channel structure and the lower-layer channel structure are respectively provided with a cover part and a bottom part. The device of the present invention can simulate intravenous and perfusion administration of bladder cancer.

Description

Organoid culture device simulating bladder structure and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an organoid culture device for simulating a bladder structure and application thereof.

Background

Bladder cancer is a common clinical malignancy of the urinary system, and the bladder can be prevented from relapse by perfusion administration after early lesion excision of a patient. The in vitro model of the tumor provides a new method for screening the anti-tumor drugs. The organoid is used as a new platform and has wide application prospect. However, in the current in vitro administration method of bladder cancer organoids, because the common organoid model lacks the interstitial and muscular layer structure for simulating the bladder, the administration route under the normal physiological structure is difficult to simulate. Although the preparation of a bladder structure with a three-layer structure can simulate the physiological structure of the bladder has been reported, the culture time is too long, and the clinical rapid diagnosis application is difficult to realize. Therefore, the device which can simulate the bladder tissue structure and perform perfusion administration can be provided, and the defects of the prior art can be alleviated.

Disclosure of Invention

In view of the above, the present invention provides an organoid culture device simulating bladder structure and a method for simulating perfusion administration of bladder cancer. The specific technical scheme is as follows.

An organoid culture apparatus simulating a bladder structure comprises an upper-layer channel structure and a lower-layer channel structure, wherein a porous membrane is arranged between the upper-layer channel structure and the lower-layer channel structure for separation, and a cover part and a bottom part are respectively arranged on the upper side and the lower side of the upper-layer channel structure and the lower-layer channel structure; the upper layer channel structure consists of at least 2 upper layer sample adding holes, 1 upper layer connecting channel and 1 upper layer culture channel, wherein 1 of the upper layer sample adding holes is mutually communicated with the upper layer connecting channel and the upper layer culture channel; the lower-layer channel structure consists of at least 1 lower-layer sample adding hole, at least 1 lower-layer connecting channel and 1 lower-layer culture channel, wherein the number of the lower-layer sample adding holes is consistent with that of the lower-layer connecting channels and is mutually communicated with the lower-layer culture channels; the porous membrane is provided with at least 1 connecting hole which corresponds to and is communicated with at least 1 upper layer sample adding hole and at least 1 lower layer sample adding hole.

Further, the number of the sampling holes on the upper layer channel structure may be 2 or 3, the number of the connection holes on the porous membrane may be 1 or 2, and the number of the sampling holes on the lower layer channel structure may be 1 or 2.

Further, the cover part is provided with a cover part sample adding hole which corresponds to and is communicated with the sample adding hole on the upper layer channel structure; the bottom is directly connected to the lower channel structure.

Further, the thickness of the upper layer channel structure is 1-3mm, the upper layer sampling hole is a circular hole with the diameter of 1-3mm, the width of the upper layer connecting channel is 0.5-2mm, and the upper layer culture channel is a long groove structure with the length of 28-30mm and smooth two ends; the thickness of the lower-layer channel structure is 1-4mm, and the shapes and the sizes of the lower-layer sampling hole, the lower-layer connecting channel and the lower-layer culture channel are all consistent with those of the upper-layer structure.

Furthermore, the thickness of the porous film is 10-50 μm, and the material is PET or PDMS. The pores on the porous membrane are used for the exchange of substances and information between the upper channel and the lower channel.

Further, the thickness of the cover part is 2-5mm, and the thickness of the bottom part is 0.5-2mm.

The method for simulating the administration of the bladder cancer by adopting the organoid culture device comprises the following steps:

step 1: assembling the cover, the upper channel structure, the porous membrane, the lower channel structure and the bottom of the organoid culture device together in sequence; wherein the bottom can be connected with a glass or a culture dish for observation or fixation under a microscope;

step 2: myoblast, fibroblast and HUVEC umbilical vein endothelial cells are added through a cover sample adding hole which is formed in the cover of the organoid culture device and communicated with the upper-layer channel structure, corresponding cell culture medium is added at the same time, the mixture is shaken up and then subjected to static culture, myoblast, fibroblast, HUVEC umbilical vein endothelial cells and corresponding cell culture medium are added through a cover sample adding hole which is formed in the cover and communicated with the lower-layer channel structure after the cells are attached to the wall, and the cells are dynamically cultured for 3-4 days to form a simulated bladder structure;

and step 3: adding the primary bladder cancer cells or organoids derived from the bladder cancer patient and Matrigel into an upper channel structure and a lower channel structure of the device respectively by the same sample adding method as the step 2 (namely, adding the primary bladder cancer cells or organoids derived from the bladder cancer patient and Matrigel into the upper channel structure through a cover sample adding hole on a cover part of the organoid culture device, which is communicated with the upper channel structure, and adding the primary bladder cancer cells or organoids derived from the bladder cancer patient and Matrigel into the lower channel structure through a cover sample adding hole on the cover part, which is communicated with the lower channel structure), and then performing standing culture for 3-4 days to form organoids;

and 4, step 4: an antitumor drug was added to the device (in the upper/lower channel structure) by the same loading method as in step 2.

By the above steps 2 and 3, a simulated bladder cancer structure is actually formed. The mode of administration of step 4 mimics intravenous and perfusion administration of bladder cancer. In particular, when the organoids are located in the superior channel structure, administration through veins has been simulated by the addition of anti-cancer drugs to the superior channel structure, since the drugs may act directly on the tumor organoids under such conditions. The drug is administered into the lower passage, and the drug needs to pass through a muscle layer and a fiber layer prepared in an organoid culture device, the structures simulate the wall structure of the bladder, and the drug can act on the organoid after passing through the wall structure of the bladder, so that the perfusion administration route of the bladder is simulated.

Further, the dynamic culture in step 2 comprises shaking by a shaker or perfusion culture using a constant flow pump.

Further, the shake cultivation method is that the organoid culture apparatus is placed on a shaker such that the shaking direction of the shaker is in line with the long axis of the organoid culture apparatus; the shaking frequency of the shaker was 50-300rpm.

Further, the flow velocity of the perfusion culture of the constant flow pump is set to be 20 mu l-1ml/min.

The application of the organoid culture device in preparing a bladder cancer administration model which can simulate the intravenous administration and perfusion administration of bladder cancer.

The invention may also comprise an organoid culture device with an improved structure that mimics the structure of the bladder.

The improved organoid culture device is additionally provided with a vent hole communicated with the atmosphere on the basis of the device; and the shape of the upper and lower layer culture channels is changed into a circular closed groove structure, and on the basis, the length of the lower layer culture channel is longer than that of the upper layer culture channel.

Advantageous technical effects

The invention constructs the tissue structure of the simulated bladder in vitro by the organoid chip technology, simulates the bladder structure by constructing physiological bladder epithelium, muscular layer structure and connective tissue, simulates a real bladder cancer model by the growth of surface cancer cells, further simulates the intravenous administration and perfusion administration routes of the bladder, and creates more favorable conditions and platforms for the drug screening of the bladder cancer.

The invention utilizes the pressure to cause the deformation of the porous membrane in the chip and generate mechanical stretching effect on the cells on the surface of the porous membrane, thereby promoting the differentiation of muscle cells to form muscle fibers so as to simulate a muscle layer, simulating a matrix structure by fibroblast differentiation and simulating a vascular structure by vascular endothelial cell differentiation.

The present invention mimics bladder cancer by two routes of administration, including infusion and intravenous administration. Perfusion administration is the delivery of drugs from the urethra into the bladder and intravenous administration is the delivery of drugs from blood vessels. Administration of the device of the present invention from the superior access may simulate intravenous administration and administration from the inferior access may simulate perfusion administration.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.

FIG. 1 is an overall view of a type I bladder chip;

FIG. 2 is a top and side view of a type I bladder chip;

FIG. 3 is a schematic view of the structure of the cover portion and the cover portion sampling hole A/B of the type I bladder chip;

FIG. 4 is a schematic view of the structure of the upper channel of the type I bladder chip showing the loading wells, connecting channels and culture channels;

FIG. 5 is a schematic structural view of a porous PET film or PDMS film of a type I bladder chip;

FIG. 6 is a schematic view of the structure of the lower channel of the I-type bladder chip showing the wells, connecting channels and culture channels;

FIG. 7 is a bottom schematic view of a type I bladder chip;

FIG. 8 is a schematic diagram of cell barrier in type I bladder chip culture;

FIG. 9 is an overall view of a type II bladder chip;

FIG. 10 is a top and left view of a type II bladder chip;

FIG. 11 is a schematic view of the structure of the cover portion and the cover portion well A/B/C of the type II bladder chip;

FIG. 12 is a schematic view of the structure of the upper channel of type II bladder chips showing the wells, connecting channels and culture channels;

FIG. 13 is a schematic structural view of a II-type bladder chip porous PET membrane or PDMS membrane;

FIG. 14 is a schematic view of the structure of the lower channel of a type II bladder chip showing the wells, connecting channels and culture channels;

FIG. 15 is a bottom schematic view of a type II bladder chip;

FIG. 16 is a schematic view of cell barrier in type II bladder chip culture;

FIG. 17 is an overall view of a type III bladder chip;

FIG. 18 is a top and side view of a type III bladder chip;

FIG. 19 is a schematic view showing the structure of the cap part of the type III bladder chip, the cap part sampling hole A, the sampling hole B and the vent hole;

FIG. 20 is a schematic view of the structure of the upper channel of type III bladder chip showing the loading wells, connecting channels and culture channels;

FIG. 21 is a schematic structural view of a type III bladder chip porous PET membrane or PDMS membrane;

FIG. 22 is a schematic view of the structure of the lower channel and bottom of a type III bladder chip showing the wells, connecting channels and culture channels;

FIG. 23 is a schematic diagram of cell barriers in type III bladder chip culture.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the description of range 1-6 should be viewed as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5 and 6. The above rules apply regardless of the breadth of the range.

Summary of reference numerals in the specification

Type I chip: a cover part: 110; cap loading hole a:111; cap portion sampling hole B:112, a first electrode; upper channel structure: 120 of a solvent; upper layer sampling hole a:121; upper layer sample adding hole B:122; an upper layer connecting channel: 123; upper culture channel: 124; porous membrane: 130, 130; connection hole (1): 131; the lower layer channel structure: 140 of a solvent; lower layer sampling hole B:141, a solvent; lower layer connecting channel: 142; the lower culture channel: 143; bottom: 150.

a type II chip: a cover part: 210; cap loading hole a:211; cap portion sampling hole B:212; lid portion sampling hole C (liquid outlet hole C): 213; upper channel structure: 220, 220; upper layer sampling hole a:221; upper layer sampling hole B:222; upper layer sampling hole C:223; an upper layer connecting channel: 224; upper culture channel: 225, a step of mixing; porous membrane: 230; connection hole (1): 231; connection hole (2): 232; the lower layer channel structure: 240; lower layer sampling hole B:241, a first electrode and a second electrode; lower layer sampling hole C:242; lower layer connecting channel B: 243. lower layer connecting channel C:244; the lower culture channel: 245; bottom: 250.

type III chip: a cover part: 310; cap loading hole a:311; cap portion sampling hole B:312; lid vent: 313; upper channel structure: 320, a first step of mixing; upper layer sampling hole a:321; upper layer sampling hole B:322, respectively; an upper layer connecting channel: 323; upper culture channel: 324, respectively; an upper layer vent hole: 325; porous membrane: 330; connecting hole (1): 331; membrane vent: 332; the lower layer channel structure: 340, respectively; lower layer sampling hole B:341; lower layer connecting channel: 342; the lower culture channel: 343.

example 1

The invention designs two organoid culture devices (hereinafter referred to as bladder chips) for simulating bladder structures, which are referred to as I-type bladder chips and II-type bladder chips respectively.

In the embodiment of the present invention, the two bladder chips are made of PDMS and each include 5 parts of structures, which are, from top to bottom, a cover, an upper channel structure, a porous membrane structure, a lower channel structure, and a bottom.

The structure of the type I bladder chip is shown in figures 1-7.

The cover portion 110: the thickness is 2-5mm, and the sampling device comprises cover part sampling holes A111 and B112. Each cover part sample adding hole is a circular opening with the diameter of 1-3 mm.

Upper-layer channel structure 120: the thickness is 1-3mm, and the device is composed of three parts, namely upper layer sample adding holes A121 and B122, an upper layer connecting channel 123 formed between the upper layer sample adding holes and the channels and an upper layer culture channel 124, wherein the upper layer sample adding holes are circular openings with the diameter of 1-3mm, a hole is respectively arranged at the 1-5mm positions of the left side and the right side of the upper layer culture channel 124, the hole connected with the channel structure is the upper layer sample adding hole A121, and the hole is also corresponding to and communicated with the cover part sample adding hole A111; another independent hole is an upper sample addition hole B122, which is in communication with the lower channel structure 140; the upper layer connecting channel 123 is 0.5-2mm wide, one end of the upper layer connecting channel is connected with the upper layer sample adding hole A121, and the other end of the upper layer connecting channel is connected with the upper layer culture channel 124; the upper culture channel 124 is composed of a long groove with two smooth ends, 28-30 mm.

Porous membrane 130: the material is porous PET film or PDMS film with thickness of 10-50 μm. In the type I bladder chip, a circular connecting hole (1) 131 is formed in the membrane to communicate with the upper/lower sample application holes B122/141. The layer can separate the upper channel and the lower channel of the chip to form a double-channel structure of the chip. Wherein the polyethylene terephthalate (PET) film has pores of 0.4-8 μm; the PDMS membrane has pores of around 9 μm.

Lower channel structure 140: the thickness is 1-4mm, and the device is composed of three parts of a lower layer sample adding hole B141, a lower layer connecting channel 142 formed between the lower layer sample adding hole B141 and the channel and a lower layer culture channel 143. And the sizes of the lower well B141, the connecting channel 142 and the culture channel 143 are the same as those of the upper channel structure.

Bottom 150: the thickness is 0.5-2mm, the upper part is connected with the lower layer channel structure, and the lower part can be connected with glass or a culture dish through plasma treatment, so that the chip is fixed.

Type II bladder chip:

the type II chip is slightly different from the type I chip in the design of the sample addition hole, and the structure of the type II chip is shown in figures 9-15.

The lid 210 of the type II chip is provided with a211, B212, and C213 sample wells, wherein the sample well C213 may also be referred to as a liquid outlet C. The cap addition hole a211 corresponds to and communicates with the upper addition hole 221 of the upper channel structure 220, and the cap addition hole B212 and the cap addition hole C213 communicate with the lower addition hole B241 and the lower addition hole C242 of the lower channel structure 240, respectively, through the connection hole (1) 231 and the connection hole (2) 232 provided in the porous membrane 230.

The upper layer channel structure 220 is provided with 3 sample adding holes, wherein the upper layer sample adding hole A221 is communicated with the upper layer connecting channel 224 and the upper layer culture channel 225; the upper sample addition holes B222 and C223 communicate with the holes B212 and C213 of the lid section and the holes B241 and C242 of the lower layer, respectively.

It will be appreciated that since the lower channel structure 240 is provided with two wells which are connected to the channels, respectively, there are two lower connecting

channels

243 and 244 in the lower channel structure, one of which is connected to wells B241 and C242, respectively, and the other to a common lower culture channel 245.

The invention actually comprises an improved chip structure, namely a type III bladder chip, on the basis of the type I and type II chips.

Type III bladder chip, structure is shown in FIGS. 17-22.

The whole chip structure also comprises 5 parts, namely a cover part, an upper layer channel structure, a porous membrane, a lower layer channel structure and a bottom part from top to bottom.

The cover portion 310: two cap loading holes a311 and B312 are provided, and in addition, a plurality of vent holes 313, including but not limited to 4, are provided, which are circular openings of 0.5-1 mm. The cap loading aperture a311 is connected to the upper channel structure, the cap loading aperture B312 is connected to the lower channel structure, the cap vent 313 is connected to the upper channel structure 320 and the lower channel structure 340, and the other end is open to the atmosphere.

Upper-layer channel structure 320: the thickness is 1-3mm, and the device is composed of an upper layer sample adding hole A321/B322, an upper layer connecting channel 323 and an upper layer culture channel 324 formed between the upper layer sample adding hole A321 and the channel. Wherein, the upper culture channel 324 is a circular closed groove structure. An upper layer vent 325 is also provided.

Porous membrane 330: the material is PET film or PDMS film. The membrane is provided with 1 connecting hole (1) 331 communicating with the upper/lower sample addition holes B322/341, and two membrane vent holes 332.

Lower-layer channel structure 340: consists of three parts, namely a lower layer sample adding hole B341, a lower layer connecting channel 342 formed between the lower layer sample adding hole B341 and the channel and a lower layer culture channel 343. Wherein the lower culture channel 343 is slightly longer than the upper culture channel 324, and the part thereof extending out of the culture channels is connected to the atmosphere through the membrane vent holes 332.

It is understood that a type III chip may also include a bottom structure similar to the type I or type

II chip bottom

150 or 250 structure.

Example 2

The invention relates to a preparation method of a chip.

1. Design and preparation 3D prints organoid chip mould

The printing mold is prepared by using a method of LCD photocuring printing by using water-washed photosensitive resin as printing ink of the mold. After printing, the mold needs to be subjected to post-treatment by the processes of washing, dehydration, secondary curing, secondary dehydration, high-temperature baking at 90 ℃ and release agent spraying.

2. Preparation of the chip

Mixing the PDMS matrix material with a curing agent, putting the mixture into a mold, discharging air bubbles in vacuum, and heating and curing at 90 ℃. And (3) treating the cured PDMS chip by using plasma.

3. Cutting of microporous films

A porous polyester (polyethylene terephthalate [ PET ]) film (having 0.4 to 8 μm pores) or a PDMS film (10 to 50 μm thick; having about 9 μm or so pores) was cut into a rectangular shape having a length of 30 to 50mm and a width of 20 to 30mm, and 1 or 2 circular holes having a diameter of 1 to 3mm were punched at a position of 1 to 5mm on the left or right side of the center thereof. Then plasma treatment is adopted, and collagen coating treatment is carried out after ultraviolet sterilization.

It is understood that the shape of the chip and the shape of the film of the present invention are not limited to a square or a rectangle.

Example 3

The method for simulating the administration of the bladder cancer (the use method of the chip) is carried out by adopting the type I and type II organ-like culture devices of the invention.

1. C2C12 myoblasts, 3T3 fibroblasts, and HUVEC umbilical vein endothelial cells were fed in cell culture dishes (flasks). After the cells were grown to the bottom of the dish (flask), the cells were digested to prepare a cell suspension, and the cell amount was counted.

2. Sample adding and organoid culturing.

And (3) connecting a capillary silicone tube with a syringe, adding the mixed myoblast, fibroblast and HUVEC umbilical vein endothelial cells from a cover sample adding hole A111 of the I-type bladder chip, shaking uniformly, then performing static culture, adding the myoblast, the fibroblast, the HUVEC umbilical vein endothelial cells and a culture medium into a lower channel device from a cover sample adding hole B112 after the cells adhere to the wall, and putting the mixture into a shaking table for dynamic culture for 3-4 days to form a simulated bladder structure. When the device is put into a shaking table, the shaking direction of the shaking table is consistent with the long axis of the organoid culture device; the shaking frequency of the shaker was set to 50-300rpm.

The structure of the bladder wall is divided into structures such as endothelium, submucosa, muscular layer and connective tissue, myoblast, fibroblast and vascular endothelial cells are added into the bladder chip, the cells on the surface of the bladder chip are mechanically stretched by the deformation of a membrane in the chip, the differentiation of the muscle cells is promoted to form muscle fibers so as to simulate the muscular layer, the differentiation of the fibroblast simulates the connective tissue structure, and the differentiation of the vascular endothelial cells simulates the vascular structure.

3. Bladder cancer cells were added.

Adding bladder cancer cells or organoids from a patient and Matrigel into the device through the sampling holes A111 and B112 on the cover part, putting the device into a shaking table to continue dynamic culture, and removing the waste culture medium in the upper/lower layer channels from the sampling holes A111 and B112 on the cover part when the tumor cells are observed to be adhered to the porous membrane.

4. Simulating the administration of the drug.

The antitumor drugs are added into the upper/lower channel structures 120/140 from the cap sample adding holes A111 or B112 respectively, and then the mixture is placed in a shaking table for continuous culture, so that the killing effect of the antitumor drugs on tumors can be observed by observing the growth state of bladder cancer organoids in the chip, the clinical drug screening approach is optimized, and the drug sensitivity of patients is observed.

In the case of the type I bladder chip, it should be noted that the amount of the medium in the lower channel is not so large that the rhythmic elevation of the porous membrane 130 in the upper channel is observed during dynamic culture.

The type I chip was cultured using a shaker, and the chip bottom 150 was fixed to the bottom of a petri dish and placed in a 37 ℃ incubator. The placing direction is consistent with the shaking direction of the shaking table and the long axis of the chip so as to meet the liquid flowing requirement in the chip. Schematic cell barrier for type I bladder chip culture is shown in figure 8.

It is understood that the type II chip can also be used to simulate the administration of a bladder cancer infusion.

1. C2C12 myoblasts, 3T3 fibroblasts and HUVEC umbilical vein endothelial cells were cultured.

2. Sample adding and organoid culturing.

The cell suspension or organoid is added to the upper channel structure 220 of the chip from the cap well A211 and to the lower channel structure 240 of the chip from the cap wells B212 and C213 using a syringe connected capillary silicone tubing. The hole B of the II-type chip is a lower-layer channel culture medium sample adding hole, and the sample adding work of the lower-layer culture medium can be completed through the hole. The hole C is a culture medium outlet hole of the lower channel, the culture medium of the lower channel can be sucked out from the hole, and the effect of mechanical force on the organoid cells can be researched by changing the pressure in the lower channel.

The type II chips were slightly different from the type I chips in the dynamic culture.

The II-type chip can be connected with a constant flow pump for perfusion culture, and the flow rate of the constant flow pump for perfusion culture is set to be 20 mul-1 ml/min.

The sample adding hole B212 and the sample adding hole C213 on the cover part of the II-type chip can be also connected with an external pipeline to control the liquid flow, the flow rate and the time in the chip, so that the pressure in the lower channel is changed to cause the form change of the porous membrane, thereby generating the mechanical force action on cells and organoids, simulating the rhythmic filling and contraction of a real bladder, and restoring the physiological bladder cancer growth environment.

When the liquid is added to the lower layer of the II-type bladder chip, the liquid should be filled into the lower layer channel by the aid of the II-type bladder chip, the filling amount is only required when the porous membrane 230 deforms, and the porous membrane deforms rhythmically by controlling liquid flow. Schematic cell barrier for type II bladder chip culture is shown in figure 16.

It can be understood that the type III bladder chip of the invention can meet the culture requirements of type I and type II bladder chips at the same time, after the cells are cultured according to the culture method of the type I chip, the cells can be continuously cultured by a shaking table after being adhered to the surface of a PET film, and the cells can also be perfused and cultured by connecting the air vents of the lower layer channel with a constant flow pump. A schematic cell barrier representation of type III bladder chip culture is shown in FIG. 23.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for simulating bladder cancer drug delivery by an organoid culture device of a bladder structure is characterized in that the organoid culture device comprises an upper channel structure and a lower channel structure, a porous membrane is arranged between the upper channel structure and the lower channel structure for separation, and a cover part and a bottom part are respectively arranged at the upper side and the lower side of the upper channel structure and the lower channel structure; the upper layer channel structure consists of at least 2 upper layer sample adding holes, 1 upper layer connecting channel and 1 upper layer culture channel, wherein 1 of the upper layer sample adding holes is mutually communicated with the upper layer connecting channel and the upper layer culture channel; the lower-layer channel structure consists of at least 1 lower-layer sample adding hole, at least 1 lower-layer connecting channel and 1 lower-layer culture channel, wherein the number of the lower-layer sample adding holes is consistent with that of the lower-layer connecting channels and is mutually communicated with the lower-layer culture channels; the porous membrane is provided with at least 1 connecting hole which corresponds to and is communicated with at least 1 upper layer sample adding hole and at least 1 lower layer sample adding hole; the upper surface of the cover part is provided with a cover part sample adding hole which corresponds to and is communicated with the sample adding hole on the upper-layer channel structure; the bottom is directly connected with the lower channel structure; the method for simulating the administration of bladder cancer comprises the following steps:

step 1: assembling the cover, the upper channel structure, the porous membrane, the lower channel structure and the bottom of the organoid culture device together in sequence;

step 2: adding myoblast, fibroblast and HUVEC umbilical vein endothelial cells through a cover sample adding hole communicated with an upper-layer channel structure on a cover of the organoid culture device, simultaneously adding a corresponding cell culture medium, shaking uniformly, then performing static culture, adding myoblast, fibroblast, HUVEC umbilical vein endothelial cells and a corresponding cell culture medium through a cover sample adding hole communicated with a lower-layer channel structure on the cover after the cells are attached to the wall, and dynamically culturing for 3-4 days to form a simulated bladder structure, wherein the dynamic culture comprises shaking by a shaking table or perfusion culture by using a constant flow pump, the shaking table is placed on the shaking table, the shaking direction of the shaking table is consistent with the long axis of the organoid culture device, and the rhythmic protrusion of a porous membrane in the organoid culture device to the upper-layer channel structure is observed; or, the liquid flow is controlled to enable the porous membrane to generate rhythmical deformation during perfusion culture by using the constant flow pump;

and step 3: adding primary bladder cancer cells or organoids derived from bladder cancer patients and Matrigel into an upper-layer channel structure and a lower-layer channel structure of the device respectively by the same sample adding method as the step 2, and performing static culture for 3-4 days to form organoids after the primary bladder cancer cells derived from bladder cancer patients are added;

and 4, step 4: the antitumor drug was added to the device by the same loading method as in step 2.

2. The method of claim 1, wherein the number of wells on the upper channel structure is 2 or 3, the number of connecting holes on the porous membrane is 1 or 2, and the number of wells on the lower channel structure is 1 or 2.

3. The method of claim 1, wherein the upper layer channel structure has a thickness of 1-3mm, the upper layer sample application hole is a circular hole with a diameter of 1-3mm, the upper layer connecting channel has a width of 0.5-2mm, and the upper layer culture channel has a long groove structure with a length of 28-30mm and two rounded ends; the thickness of the lower-layer channel structure is 1-4mm, and the shapes and the sizes of the lower-layer sampling hole, the lower-layer connecting channel and the lower-layer culture channel are all consistent with those of the upper-layer structure.

4. The method of claim 1, wherein the porous film has a thickness of 10-50 μm and is made of PET or PDMS.

5. The method of claim 1, wherein the cover portion has a thickness of 2 to 5mm and the base portion has a thickness of 0.5 to 2mm.

6. The method of claim 1, wherein the frequency of shaking of the shaker is 50-300rpm; or the flow velocity of the perfusion culture of the constant flow pump is set to be 20 mu l-1ml/min.

7. The method of claim 1, wherein the simulated bladder cancer administration comprises simulated bladder cancer intravenous administration and perfusion administration.