An automated transmembrane culture device for organoids

Abstract

The present invention provides an automated transmembrane organoid culture device, comprising a high-throughput automated perfusion module, a multi-channel fluid exchange module, and a movable interlayer module; the device is suitable for commercial multi-porous cell culture plates. The automation of the transmembrane culture mode is achieved. The automated culture process is suitable for organoid culture for a certain period of time, and the fluid exchange process does not involve artificial disturbance of the microenvironment and labor input.

Description

Technical Field

[0001] The invention relates to an automated transmembrane organoid culture device, belonging to the field of biomedical engineering. Background Art

[0002] Organoids are three-dimensional multicellular structures in vitro with self-organizing properties, which share a certain degree of similarity with living organs at the cell type or tissue function level. Thanks to the development of stem cell technology, researchers have been able to expand and induce differentiation of appropriate cell types, establish three-dimensional organoid models in vitro that mimic real biological processes, and apply them to topics such as development and disease. For example, Qian et al. constructed forebrain organoids and reproduced the structure of radial glial cell layers. They infected the organoids with Zika virus and observed microcephaly caused by suspected virus-specific infection. In addition, independent work by Drost et al. and Matano et al. used CRISPR / CAS9 to introduce continuous mutations in colon organoids, reproducing the process of adenoma carcinoma turning into adenocarcinoma. At present, organoid models of pancreas, kidney, liver, lung, heart, thymus, blood vessels, brain, etc. have been established in the field. Compared with model organisms, organoid in vitro systems have the potential to have higher homology and operability, which can open a more complex and flexible technical window for biomedical research. The growth of organoids is affected by exogenous and endogenous signals. How to construct a stable physical structure and chemical microenvironment in the early stages of differentiation is a core technical issue in the field. Microenvironment stability and targeting not only rely on the development of biomimetic biomaterials and the optimization of small molecule induction reagents, but also rely on the innovation of bioengineering methods. Common technical breakthroughs include three-dimensional aggregation methods that can cause cells to aggregate into three-dimensional structures of appropriate size, providing necessary structural support; micropatterning, microfluidics and other technologies guide asymmetric differentiation of tissues by precisely controlling the spatial distribution of fluids; high-throughput automated devices integrate sample induction, culture, readout and other modules to improve experimental efficiency and throughput. How to improve the output of organoid culture and analysis with an engineering mindset has always been one of the focuses of the field.

[0003] Organoid technology has problems in both cell aggregation and long-term culture. The main problems with cell aggregation are flux and parallelism. Methods such as microchambers and droplets can only achieve the aggregation of cells at the scale of hundreds of microns, which is not conducive to the generation of millimeter-scale organoids; colloidal mosaic methods are difficult to control the starting cell amount, and batch differences are large; colloidal mosaics, three-dimensional printing and other methods rely on base gel (also known as Matrigel, a commercial extracellular matrix purified from mouse sarcoma) as a substrate / ink, and its complex and uneven chemical composition may affect the repeatability and parallelism of the experiment. In addition, in terms of long-term culture, there are also major contradictions such as microenvironmental disturbances, excessive reliance on manpower, and devices that are not conducive to timing analysis. Organoid differentiation relies on the combined effects of small molecule gradients and physical environmental factors. In the current organoid culture method, its microenvironment is very easy to introduce unnecessary disturbances, such as sudden changes in temperature, humidity, nutrients, etc. during the replacement of culture medium, and changes in the position of organoids caused by fluids, which will lead to the lack of repeatability and parallelism of the final organoid culture. At the same time, high-throughput culture of organoids relies too much on manpower, especially the periodic fluid exchange process. The culture cycle of organoids varies according to the type. It can be as short as 10 days for cardiac organoids to show observable phenotypes, while it can be as long as 60 days for lung organoids and hundreds of days for brain organoids to mature. Moreover, due to the limitations of the design principles and experimental throughput of the engineering devices themselves, organoid research often lacks three-dimensional morphological characteristics and temporal gene expression profile identification. Inspired by these methodological difficulties, the present invention aims to provide a possible solution to the above problems. Summary of the invention

[0004] In response to the above problems, the present invention provides an automated transmembrane organoid culture device, which is characterized in that it includes a high-throughput automated perfusion module, a multi-channel fluid exchange module, and a movable interlayer module; wherein the high-throughput automated perfusion module includes a reagent diversion pipeline and a multi-channel valve for adding and / or removing culture fluid; the multi-channel fluid exchange module is used to connect the high-throughput automated perfusion module and a cell culture well plate; the multi-channel fluid exchange module includes a pipeline bracket and a needle-like structure; the needle-like structure extends into the culture well of the cell culture well plate; the movable interlayer module is used to fix the cell culture well plate and the transmembrane device, and control the vertical distance between the two during the fluid exchange process.

[0005] According to a preferred embodiment, it also includes an interpolation segmentation module for splitting the space inside the transmembrane device to achieve the effect of placing multiple organoid cell clusters on the semipermeable membrane at the bottom of the transmembrane component.

[0006] According to a preferred embodiment, the cell culture well plate refers to a flat-bottom tissue culture well plate produced by Corning Costar.

[0007] According to a preferred embodiment, the transmembrane device is a hanging cup-shaped culture chamber (polytetrafluoroethylene hydrophobic membrane, diameter 12 mm, pore size 0.4 μm) from Merck Millipore. Below the flat bottom is a semipermeable membrane that can be placed in the culture well of a 24-well plate. The chamber between the culture well and the transmembrane device is called a culture chamber.

[0008] According to a preferred embodiment, the high-throughput automated perfusion module is composed of a single reagent shunt line and three multichannel valve-culture well-multichannel valve-injector parallel groups, and the liquid in the culture chamber can be added and removed by positive pressure and negative pressure drive, respectively.

[0009] According to a preferred embodiment, the multi-channel fluid exchange module includes a Y-shaped tee, a connector, a pipeline bracket and a needle-like structure thereof; wherein the Y-shaped tee pipeline is connected to the perfusion pipeline of the automated perfusion module, the connector fixes the former on the upper surface of the pipeline bracket plane, and the needle-like structure below extends from one side of the culture chamber to a position close to the bottom of the hole.

[0010] According to a preferred embodiment, the movable sandwich module includes a transmembrane device fixture, a motor gear set, left and right fan blades, a well plate base and a device base. The cell culture well plate is placed on the well plate base, and the transmembrane device fixture is used to fix multiple transmembrane devices, and moves in a vertical direction under the joint drive of the motor gear set and the left and right fan blades to achieve a change in the distance between the semipermeable membrane and the bottom of the culture well. This process contributes to the stability and repeatability of the automated perfusion process.

[0011] The device also includes several optional components. According to a preferred embodiment, the intercalation segmentation module can split the space inside the transmembrane device to achieve the effect of placing multiple organoid cell clusters on the semipermeable membrane at the bottom of the transmembrane component.

[0012] In summary, the present invention relates to an automated culture device, which is based on a transmembrane culture mode and is used for organoid culture. The device is characterized in that: by integrating independently developed devices, commercial equipment and biomedical consumables, it effectively combines U-shaped pore three-dimensional aggregation and transmembrane culture methods, overcomes the methodological difficulties encountered by several organoid experimental technologies, and realizes the automation of the transmembrane culture mode. This automated culture process is suitable for organoid culture of a certain length of time. The liquid replacement process does not involve artificial disturbance of the microenvironment and labor input, and realizes high parallelism. The batch three-dimensional aggregation, the generation of a stable microenvironment and non-human intervention semi-closed long-term culture. In view of the high degree of freedom of the transmembrane culture system, this automated device can be used for long-term experiments on a variety of organoids or biological samples, and the automated transmembrane cultured organoids have phenotypic and omics characteristics similar to those of general culture modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0014] Figure 1 .Schematic diagram of the U-shaped pore high-throughput three-dimensional aggregation and transmembrane culture device;

[0015] Figure 2 .Automated transmembrane culture device for organoids and U-shaped well clusters - automated transmembrane culture process;

[0016] Figure 3 .Schematic diagram of high-throughput automated perfusion module;

[0017] Figure 4 .Detailed assembly diagram of multi-channel fluid exchange module;

[0018] Figure 5 .Detailed assembly drawing of the movable mezzanine module;

[0019] Figure 6 .Schematic diagram of the design of the movable mezzanine module;

[0020] Figure 7 .Schematic diagram of interpolation segmentation module;

[0021] Figure 8 .High-throughput transmembrane culture device based on 6-well plates;

[0022] Fig. 9 .Automated transmembrane culture device for cardiac organoid differentiation;

[0023] Fig.10 .Biological replicates of different experimental groups of cardiac organoids;

[0024] Fig.11 .Immunofluorescence staining images of cardiac organoids.

[0025] in, Figure 1, Schematic diagram of U-shaped hole high-throughput three-dimensional clustering and transmembrane culture device. 101 is a commercial U-shaped bottom 96-well plate, the main material is polystyrene (PS). 102 is a commercial flat-bottom 24-well plate, also made of polystyrene. 103 is a commercial cell transmembrane culture plug-in, which looks like a transparent handleless water cup. There is a semi-permeable membrane at the bottom of the plug-in. The semi-permeable membrane can be adapted to different biochemical experiments depending on the pore size and material type. This device uses a hydrophilic polytetrafluoroethylene membrane with an outer diameter of 12 mm and a pore size of 0.4 um. 104 is a spherical cell cluster. (A) U-shaped hole high-throughput three-dimensional clustering. After embryonic stem cells are digested and suspended with accutase, a specified number of U-shaped holes pre-treated with PF127 are added. In this form, long-term culture is carried out. When changing the medium, the liquid around the cell cluster needs to be sucked away and then new culture medium is added. During the process, the cell cluster may be blown up, causing microenvironmental disturbance. (B) Transmembrane culture device. The transmembrane device is nested in the flat-bottom culture well, dividing the culture well into the inner side and the outer side, with the outer side being the culture chamber. The spherical cell cluster is placed on the semipermeable membrane of the transmembrane device, and the culture medium is replaced from the chamber side, and the small molecule exchange is completed by diffusion into the inner side. The initial configuration of the biochemical microenvironment inside and outside can be different; even more, the material and pretreatment process of the semipermeable membrane can also vary depending on the research topic (such as using collagen or base gel pretreatment). The proportions of the objects in the picture are not the proportions of the real objects.

[0026] Figure 2, automated transmembrane culture device for organoids and U-shaped well clustering - automated transmembrane culture process. (A) Assembly diagram of the automated transmembrane culture device for organoids. 201 is a high-throughput automated perfusion module, of which 2011 is a TecanCabro Centris syringe pump, 2012 is a 10-channel valve for moistening, and 2013 is a 50 ml centrifuge tube. 202 is a multi-channel fluid exchange module, 203 is a removable interlayer module, and the two modules are installed in a cell culture incubator at 204 (37 degrees Celsius, 5% carbon dioxide). The high-throughput automated perfusion module is placed outside the incubator and mounted on top of the optical platform shown in 2014. The perfusion pipeline enters the box from the detection port on the rear side and is connected to the Y-shaped tee of the multi-channel fluid exchange module. The wires of the removable interlayer and other soft wires can be inserted through the gap in the incubator door. (inlet) Bottom view of the culture device, in which 4 independent culture wells can be seen. The white circular area 205 is the unwetted polytetrafluoroethylene porous membrane, and the black part 206 below is the integrated needle connector of aluminum alloy. The location of the connector is the culture chamber, which is the area for changing the culture medium. (B) A partial enlarged view of the movable sandwich transmembrane device fixture and the orifice base. The rotatable fan blades on the long side of the base can be seen. (C) A partial enlarged view of the polyetheretherketone connector above the multi-channel liquid exchange module. (D) A partial enlarged view of the movable sandwich motor gear set. The protruding shafts on both sides are concentric with the rotatable fan blades, and the two can be docked together. (E) U-shaped hole cluster-automated transmembrane culture process. Compared with the typical U-shaped hole cluster + U-shaped hole culture mode, we placed the cell cluster on the semipermeable membrane of the transmembrane device for long-term culture. This new culture mode relies on chemical diffusion inside and outside the semipermeable membrane. On the one hand, it can reduce the fluid disturbance and temperature disturbance caused by the open-lid liquid exchange, and at the same time, it also liberates scientific research labor to a certain extent due to the use of automated liquid exchange.

[0027] Figure 3 , schematic diagram of the high-throughput automated perfusion module. The perfusion module consists of a single reagent diversion line and three multichannel valve-culture well-multichannel valve-syringe parallel groups. 301 is a reagent tube, 302 is a multichannel valve, 303 is a syringe pump, and 304 is a group of 8 culture wells. The perfusion module integrates 8 multichannel valves and 4 syringe pumps, which can be used for 24 culture wells. There are three modes for the flow of fluid in the perfusion line, namely adding new culture medium, removing old culture medium and cleaning the perfusion line. Precise control of liquid flow can be achieved through script programming.

[0028] Figure 4, detailed assembly diagram of the multi-channel liquid exchange module. (A) Exploded view of the module assembly. 401 is a three-way plastic pipeline, 402 is a polyetheretherketone connector, and 403 is an aluminum alloy liquid pipeline bracket. The liquid pipeline bracket has a total of 24 independent needle connectors. The needle connector is opened from top to bottom, and the length can just extend to the bottom of the 24-well plate culture well. There is a cylindrical pipe in the center of the tube for liquid to pass. Each culture well is connected by a hand-made Y-shaped three-way pipeline and two 1.6 mm outer diameter plastic pipelines, one for liquid inlet and the other for liquid extraction. This arrangement can minimize the flow of bubbles into the culture well, while maintaining the consistency of the fluid direction and avoiding contamination caused by mixing of waste liquid and new culture medium. In the lower left corner of the figure, it can be seen that there is a circular hole structure opened on the short side of the bracket. This hole is designed to allow the fixture, base and other components of the multi-channel liquid exchange module and the movable interlayer module to dock with the motor gear set after assembly. (B) Cross-sectional view of the liquid pipeline. The polyetheretherketone connector and the pipeline bracket are fixed by 10-32 threads. The hollow channel of the aluminum alloy pipeline has two sections, a 1.5 mm channel near the top and a 1.0 mm channel at the bottom. (C) The assembly relationship between the polyetheretherketone connector and the three-way plastic pipeline. Insert the plastic pipeline into the connector, and screw the connector along the thread of the aluminum alloy bracket to fix the liquid pipeline well. The three-way is handmade, specifically, two bent steel needles are placed in the plastic hose, fixed with epoxy resin adhesive, and cleaned with 75% alcohol and phosphate buffer solution in sequence before use.

[0029] Figure 5, the detailed assembly drawing of the movable sandwich module. (A) The assembly relationship of the various components of the module. 501 is the transmembrane device fixture, 502 is the motor gear set, 503 is the left and right side blades, 504 is the orifice plate base, and 505 is the device base. The orifice plate base is fixed to the device base, and the blades are engaged in the gap at the long side of the base. The transmembrane device fixture is opened up and down, and can fix 24 transmembrane culture plugs with a diameter of 12 mm. The motor gear set can move forward and backward along the slide rail on the side of the device base. (B) Assembly drawing of the motor gear set. The gear set is driven by the 28BYG two-phase hybrid stepper motor shown in 5021, the synchronous belt of 5022, the large / small synchronous wheel (48 teeth / 20 teeth) and the stepped gear (20 teeth) of 5023. The transmission chain will eventually cause the two side shafts shown in 5024 to rotate at a speed of "motor: shaft = 2.4:1". (C) Assembly drawing of the two side blades. The fan blades are divided into two parts, one is the long shaft with fan blades as shown in 5031, and the other is the short barrel docking original part 5032. The connection between the two is covered with a small plastic bearing with a diameter of 7 mm. (D) Assembly diagram of the well plate base. The well plate base is an easy-to-assemble layered structure that can fix the transmembrane device fixture, transmembrane culture plug-in, left and right fan blades and commercial 24-well plates. From top to bottom, they are the upper base of 5041, the lower base of 5042, the 24-well plate of 5043 and the 24-well plate fixing of 5044. The upper and lower bases and the fixings and the bases can be fixed by M2.5 screws. The fan blades on the left and right sides are first placed in the grooves on the long side of the lower base, and then the upper base is covered and the assembly is completed after the screws are locked.

[0030] Figure 6 , schematic diagram of the design of the movable interlayer module. (A) Simplified diagram of the device. The movable interlayer module consists of three parts marked 602, 603 and 604, which are located below the liquid pipeline bracket 601. In order to facilitate the explanation of the design ideas, the specific structure of the transmembrane device fixture is simplified here, and the motor gear set, left and right fan blades, orifice plate base, device base and other components are hidden. (B) The motor gear set controls the relative distance between the transmembrane device and the bottom of the orifice plate so that it can selectively infiltrate the culture medium. During the non-page change period, the transmembrane culture plug is fully immersed in the large volume of culture medium on the outside for chemical diffusion to maintain the nutrient supply of the cell cluster on the inside; when changing the liquid, in order to avoid waste liquid diffusion, liquid residue and bubble blockage, the motor gear unit drives the transmembrane device fixture connected to the transmembrane culture plug to move up and away from the outer culture medium, and reset it after the liquid in the culture chamber is replaced.

[0031] Figure 7 Schematic diagram of the inlet partition module. (A) The inlet polyetheretherketone partition can divide the inner side of the semipermeable membrane into 7 small culture units with a diameter of 2 mm. (inlet) The stem cell cluster is placed in one of the culture units. (B) Side view of the partition insert placed in the transmembrane device. Scale bar = 500 μm.

[0032] Figure 8 , High-throughput transmembrane culture device based on 6-well plate. (A) Exploded view. (B) Cross-sectional view of the perfusion path. (C) Different configuration options between the detachable transmembrane device clamp and the liquid pipeline.

[0033] Fig. 9 , automated transmembrane culture device for cardiac organoid differentiation. (A) Flow chart of U-shaped well cluster-automated transmembrane culture of cardiac organoids. After obtaining embryonic stem cell clusters, embryonic stem cells can be induced to differentiate into cardiomyocytes by sequentially activating and inhibiting the Wnt / β-catenin signaling pathway. (B) Cell clusters are placed on a semipermeable membrane on day 0; scale bar = 500 μm. (C) U-shaped well cluster-automated transmembrane culture of cardiac organoids. The first image on the left is an in situ image, and the second image on the left is the appearance of the cell cluster after being transferred to the U-shaped well and washed with phosphate buffer solution. The third image on the left is an in situ image of the multi-cell cluster cultured with an intercalated partition on day 8. Scale bar = 500 μm.

[0034] Fig.10 , biological replicates of different experimental groups of cardiac organoids. (A) Day 0, border-washed embryonic stem cell clusters. (B) U-well clusters-U-well cultured day 8 cardiac organoids. (C)(D) U-well clusters-automated transmembrane cultured day 8 cardiac organoids, single cell cluster differentiation and multi-cell cluster differentiation, respectively. The black shadow centered at the center of the image is the 2 mm diameter chamber outline of the semipermeable membrane interpolation divider. Scale bar = 500 μm.

[0035] Fig.11 , Immunofluorescence staining images of cardiac organoids. The co-localization of vesicular structures and cardiac-specific proteins cTnT and NKX2-5 indicates that the automated transmembrane cultured cardiac organoids can reflect the heart development process. DETAILED DESCRIPTION

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0037] In the present invention, the position-related words, such as "upper", "lower", "left", "right", "vertical", "horizontal", "X direction", "Y direction", "front", "back", "high", "low", "inside", and "outside", express relative position relationships and do not represent specific positions. It can be understood by those skilled in the art that when the direction of the entire device is adjusted, all position relationships will change accordingly.

[0038] The present invention has developed an automated transmembrane organoid culture device, and based on the characteristics of the device, designed a "U-shaped hole clustering-automated transmembrane culture" culture process. Figure 1As shown in (A), U-shaped well high-throughput three-dimensional clustering is a standardized high-throughput cell cluster generation method that can be used to generate a large number of three-dimensional cell clusters with consistent sizes at one time. Figure 1 As shown in (B), the transmembrane culture device is a suspended cell experimental system. The cup-shaped transmembrane device (transwell insert) nested inside the culture well can divide the culture well into inner and outer environments. With the semipermeable membrane below the transmembrane device as the boundary, researchers can generate different culture microenvironments on the inside and outside of the membrane respectively. Depending on the different membrane materials and pore sizes, biological samples are cultured inside the transmembrane device, and the fluid is replaced from one side of the culture chamber, which can reduce the external disturbance of the inner sample. The above two culture systems are generally open-lid culture, that is, when changing the culture medium, it is necessary to use manpower to take the well plate out of the incubator, open the lid to remove the old culture medium, and add fresh culture medium.

[0039] like Figure 2 As shown, the new device and process of the present invention organically integrate the above two modes. The specific experimental steps are: U-shaped well high-throughput three-dimensional aggregation: After the embryonic stem cells are suspended and counted, they are aggregated in the U-shaped well. The inside of the U-shaped well is treated with Pluronic F-127 (PF127) overnight in advance. PF127, as a long-chain polar molecule, can effectively prevent cells from adhering to the wall and promote three-dimensional aggregation during the cell aggregation process.

[0040] Automated transmembrane culture: After embryonic stem cells are aggregated, the cell clusters are transferred to the inner side of the polytetrafluoroethylene semipermeable membrane, and the culture medium on the outer side of the membrane is replaced through the automated perfusion module. The small molecule nutrients required for differentiation rely on chemical diffusion inside and outside, thereby avoiding the destruction of the culture microenvironment and improving the stability, parallelism and repeatability of the culture experiment. In addition, the new automated culture mode can reduce labor input during the long-term organoid culture process, and can be further used to systematically explore the robustness of culture experiments and their potential benefits to downstream analysis processes.

[0041] (I) Main components

[0042] like Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown in Figure 1, the automated transmembrane culture device for organoids is a detachable device adapted for commercial 24-well plates. The experimental device consists of three parts: a high-throughput automated perfusion module, a multi-channel fluid exchange module, and a removable interlayer module. For the code of each component in the attached figure, see Table 4; for the models of commercial components and the engineering drawings of independently developed and designed components, refer to Tables 2 and 3.

[0043] 1. High-throughput automated perfusion module

[0044] This experimental device can automatically perfuse the culture wells of a commercial 24-well plate. We integrated multiple syringe pumps and commercial multi-channel valves to build a high-throughput perfusion module. The perfusion module consists of a single reagent dispenser (RD) plus three multi-channel valve-culture well-multi-channel valve-syringe pump (switch-well-switch-pump, SWSP) parallel groups, such as Figure 3 As shown. One parallel group can perfuse 8 independent culture chambers. For each chamber outside the culture well, the perfusion system pushes the reagent from one side through positive pressure drive to achieve the addition of culture medium. Considering that the culture well is not a closed system, the fluid in the chamber to be transferred will not spontaneously leave the culture well, so we add a set of negative pressure pipette lines at the other end. The lines for adding and withdrawing liquid are connected together through a Y-shaped tee. In addition to the bidirectional flow of the fluid at the multi-channel fluid exchange module when entering the culture well, most of the time it maintains a unidirectional flow, which reduces contamination and improves the efficiency of fluid exchange. Three parallel groups can just achieve liquid replacement of 24 culture wells. The reagent diversion module can select up to ten experimental reagents including water, phosphate-buffered saline (PBS), culture medium, etc. The used reagents will eventually be transferred to the waste tank.

[0045] In terms of hardware communication, thanks to the complete RS485 / 232 serial communication instructions of commercial components, we have completed the automatic control of individual hardware through the NI-VISA application programming interface (API) built into LabVIEW. We adopted the automation method of script programming and developed a set of real-time control panels that can be read and written at any time. Users only need to write the standardized code below according to their needs to achieve long-term, fully automatic high-throughput perfusion.

[0046] 2. Multi-channel fluid exchange module

[0047] Inheriting the design of the high-throughput automated perfusion module, we developed a multi-channel liquid exchange module based on a 24-well plate. This module is located in the incubator, above the movable sandwich module and below the pipeline of the high-throughput automated perfusion module. Its main function is to connect the pipeline and the well plate. Figure 4 As shown in the figure, the Y-type tee merges two plastic pipes into one culture well, and the polyetheretherketone connector fixes the pipes on the aluminum alloy bracket. Fresh culture medium enters the transmembrane culture chamber from one of the pipes, and the old culture medium leaves from the other pipe. The needle connector of the aluminum alloy liquid pipe bracket can be extended to the bottom side of the culture well, so that the perfusion module can be fully replaced.

[0048] 3. Removable mezzanine module

[0049] like Figure 5 As shown in the figure, the movable sandwich module includes several major parts, including the transmembrane device fixture, motor gear set, left and right fan blades, orifice plate base and device base. The whole is an easy-to-assemble layered structure. Its main function is to carry a commercial 24-well plate and multiple transmembrane devices and control the relative distance between the two. The following introduces these hardware structures respectively.

[0050] (1) Transmembrane device fixture: a plate-like structure that can hold up to 24 transmembrane devices with a diameter of 12 mm using double-sided tape. Each culture well is connected from top to bottom, allowing samples to be processed and imaged from above. Grooves complementary to the fan blades are designed on both sides of the long side of the fixture, allowing the fixture to sit better on the fan blades and move up and down with them.

[0051] (2) Motor gear set and left and right blades: A simple gear structure driven by a 28BYG two-phase hybrid stepper motor. The motor drive chain will eventually cause the two shafts to rotate at the same speed and different directions. Through the short barrel docking components, the gear set will drive the left and right blades in the interlayer to rotate. When the blades change from the "__" state to the " / \" state, the transmembrane device fixture will be lifted up and away from the bottom of the culture well. Figure 6 As described in the following three points, this mechanical process ensures that the culture microenvironment inside the transmembrane device can maintain stability and be fully infiltrated during the culture process without diffusion from the outside during the culture medium replacement process. Effectively avoid the diffusion of perfusion waste liquid: The device is a semi-closed system with one-way flow. All liquids leading to the culture wells must flow through the outside of the transmembrane device before flowing to the waste tank with the perfusion cycle. During this period, hundreds of microliters of liquid in the perfusion plastic tube chamber (ie, a 10 cm long, 1.6 mm outer diameter tube can hold about 30 microliters of liquid) will flow through the outside of the transmembrane device, causing unexpected diffusion. This type of diffusion can be avoided by moving the transmembrane device up and away from the outside fluid when changing the medium.

[0052] · Effectively avoid residual liquid on the outside: The plastic wall of the orifice plate and the plastic outer ring of the transmembrane device are both hydrophilic materials. Liquid is easily attached between the two layers of plastic structure under the action of surface tension, resulting in more residual liquid in the outer chamber. Lifting the transmembrane device off the bottom of the orifice plate helps to better remove the liquid. Taking pure water as an example, the average liquid replacement amount is 188 microliters when the transmembrane device is not moved up, and 88.3 microliters when the transmembrane device is moved up.

[0053] · Effectively avoid the formation of bubbles at the bottom of the polytetrafluoroethylene semipermeable membrane: Similarly, affected by the surface tension of the plastic, when the fluid enters the outer chamber, it is easy to infiltrate along the wall gap, which eventually indirectly leads to the formation of large bubbles under the transmembrane device. Large bubbles will hinder the infiltration of the membrane and cut off the diffusion process, causing the semipermeable membrane to evaporate within 24-48 hours, and the sample on the inside of the membrane will then be dehydrated and necrotic. Treating the transmembrane device with 1 mmol PF127 in advance can change the contact angle between the liquid and the plastic to reduce the probability of bubble generation. On this basis, quickly inserting the transmembrane device into the liquid surface after replacing the outer culture medium can completely prevent the formation of bubbles.

[0054] (3) Orifice plate base: frame-type structure, used to cover and fix the commercial 24-orifice plate. The long sides on both sides can be used to install the left and right fan blades, and there are three rectangular air holes on the short sides of both sides.

[0055] (4) Device base: a plate-like structure used to support other components of the movable module and the multi-channel fluid exchange module.

[0056] (II) Selection of parts

[0057] In addition to the above main modules, we also developed the following components according to individual experimental requirements:

[0058] 1. Insertion partition of transmembrane device

[0059] The main purpose of the insert partition is to divide the inner side of the polytetrafluoroethylene semipermeable membrane into 7 small compartments with a diameter of 2 mm and a height of 2 mm by means of physical barrier, so as to achieve the effect of culturing multiple organoid samples in one transmembrane device. Figure 7 As shown, the interposer partition of polyetheretherketone material can be inserted from the top of the transmembrane device fixture and seated on the polytetrafluoroethylene membrane.

[0060] 2. Transmembrane automated culture module based on 6-well plate

[0061] like Figure 8 As shown in the figure, we followed the same hardware design ideas mentioned above and conceived a set of culture modules based on 6-well plates. Only the design drawings of the multi-channel liquid exchange module and the movable interlayer module are shown here. The 6-well plate version of the device has a more simplified liquid pipeline bracket, which allows us to simplify the culture wells (every four 12 mm diameter transmembrane devices share one culture well) while also freeing up the upper space of the device to achieve further modularization of the transmembrane device fixed layer. This version of the transmembrane device fixture can be disassembled from the open space above the liquid pipeline bracket, avoiding the original cumbersome layered assembly steps. In addition, as Figure 8As shown in (C), we have also added a variety of transmembrane device fixtures. In addition to the current 12 mm outer diameter transmembrane devices, they can also be adapted to 24 mm outer diameter styles. These transmembrane devices each correspond to two unique multi-channel fluid exchange configurations, which can achieve fluid exchange inside the transmembrane device through additional perfusion lines. This hardware has the potential to be applied to the research of larger biological samples such as brain slices and fusion-like bodies, which is a further optimization direction for this automated transmembrane culture device.

[0062] IV. Conclusion

[0063] The present invention has developed an automated fluid exchange device based on a transmembrane culture mode for organoid culture. The new experimental process effectively combines U-shaped pore three-dimensional aggregation and transmembrane culture methods, overcoming the methodological difficulties encountered by several organoid experimental technologies, and achieving high parallelism of batch three-dimensional aggregation, the generation of a stable microenvironment, and semi-enclosed long-term culture without human intervention. In view of the high degree of freedom of the transmembrane culture system, this automated device can be used for long-term experiments on a variety of organoids or biological samples. Several possible research directions are listed here.

[0064] 1. Optimize the induction concentration of small molecules: Through a relatively stable medium exchange process (fixed outer culture medium residue, stable chemical diffusion), the effects of small molecules / drugs on biological samples can be systematically studied, the experimental conditions can be optimized, and the effective concentration of small molecules can be evaluated.

[0065] 2. Air-liquid interface (ALI): Study the long-term development and metabolism of intestinal, brain, kidney and other organoids under the experimental conditions of embedded colloid / unembedded colloid. Study the differentiation trajectory and morphological changes of organoids under different oxygen concentrations. Bioprinting can also be used to generate complex three-dimensional biological structures on semipermeable membranes.

[0066] 3. Complex / organoid fusion (assembloid): Under the experimental conditions of embedded colloid / unembedded colloid, multiple three-dimensional cell clusters of the same / different tissue sources are placed together for fusion culture to observe the long-term occurrence process of organoid fusion.

[0067] 4. Co-culture: There are two options. One is to culture multiple three-dimensional cell clusters of the same / different tissue sources in a degenerate cell culture unit (i.e., a 6-well plate transmembrane culture module) under the conditions of embedded colloid / unembedded colloid, and study whether different tissues have long-range interaction-mediated signal / metabolic phenomena under the condition of sharing culture medium. The second is to culture appropriate cell clusters on the outside of the transmembrane device as a secretion source of paracrine or cytokine, and investigate more complex long-range interaction mechanisms, such as short-chain fatty acid signaling molecules.

[0068] 5. Automated Culture of Cardiac Organoids Using human cardiac organoids (HCOs) as an example, we demonstrate the application of automated transmembrane culture devices and experimental processes in organoid culture. Functional cardiomyocyte differentiation can be achieved by regulating the Wnt / β-catenin signaling pathway with small chemical molecules, and a beating phenotype may be seen within 10 days. Here, we attempted to differentiate a spherical cell mass of 50,000 starting embryonic stem cells (ESCs) into sub-millimeter-scale cardiac organoids.

[0069] (I) Embryonic stem cell U-shaped pore clusters

[0070] 1. Treat a flat-bottomed six-well plate with DMEM + Matrigel solution overnight.

[0071] 2. After 4-5 days, the embryonic stem cells fill the wells of the plate. Wash with phosphate buffered saline to remove dead cells, and then digest with 600 μl of accutase.

[0072] 3. After centrifugation and cell counting, ESCs were passaged at a density of 200,000 cells / well. On day 0, 10 mM Y27632 was added.

[0073] 4. Replace 2 ml of mTeSR medium every 24 hours. The digested embryonic stem cells can be used for aggregation: a given number of embryonic stem cells are suspended with mTeSR + 50 mM Y27632, and then added to the U-shaped wells of a 96-well plate treated with 1 mM PF127 phosphate buffer solution overnight.

[0074] 2. Automated transmembrane differentiation of cardiac organoids

[0075] The culture protocol of cardiac organoids was modified from the method of Hoang, P. et al. to differentiate human induced pluripotent stem cells (hiPSCs) into three-dimensional cardiac microchambers. The culture reagents are shown in Table 1 below, where n is a non-negative integer.

[0076] Table 1. HCO culture reagents. (n=0,1,2…)

[0077] Fluid change time Culture medium Day 0 RPMI1640+50X B27 supplement (without insulin)+10 micromolar CHIR99021 Day 1 & 4 RPMI1640+50X B27 Supplement (without insulin) Day 2 RPMI1640+50X B27 supplement (without insulin)+5 micromolar IWP4 Day 6, 7+2n RPMI1640+50X B27

[0078] The experimental process is divided into pre-preparation, hardware assembly and sample preparation, in order:

[0079] 1. Preparation

[0080] (1) Use an automatic paper cutting machine to make a double-sided tape that fits the upper bottom surface of the transmembrane device cylinder. Use tweezers to stick the double-sided tape to the upper bottom surface of the transmembrane device.

[0081] (2) (Optional) Place the split plug-in into the transmembrane device to obtain a transmembrane device complex.

[0082] (3) The transmembrane device complex was immersed in 1 mM PF127 phosphate buffer solution overnight.

[0083] (4) Use 75% ethanol solution to pre-clean the high-throughput automated perfusion module.

[0084] (5) Pre-clean the automated perfusion module using phosphate buffered saline with Antibiotic-Antimycotic and Gentamicin.

[0085] 2. Hardware Assembly

[0086] (1) Install a new commercial 24-well plate into the removable well plate base and connect the Y-type tee with the plastic pipe for liquid exchange to the aluminum alloy pipe bracket. Both parts are placed in the incubator.

[0087] (2) Use a cut pipette tip to transfer the cell clusters in the 96-well plate to the inner side of the transmembrane device complex membrane. After removing the excess mTesR culture medium, add 100 μl of day 1 culture medium to the inner side of the membrane and 600 μl of day 0 culture medium to the outer side. (3) Glue the transmembrane device complex with the cell clusters to the fixture. After assembly, a complete transmembrane device fixed layer (fixture + multiple transmembrane device complexes) is obtained.

[0088] (4) Fix the transmembrane device to the left and right fan blades of the orifice plate base in the incubator. Close the liquid pipeline bracket and insert the rotating shafts on both sides of the motor gear set from the opened circular hole structure in the direction parallel to the long side.

[0089] (5) Set up the LabVIEW automatic perfusion program. Manually enter the command line, or select the script file that reads the perfusion program. (6) Start running the program. The complete automated fluid replacement process has the following steps: First, the motor gear unit drives the fan blades to rotate and move the interlayer upward, and the perfusion pipeline removes 600 microliters of old culture medium from each culture well. Secondly, after removing the culture medium, 1000 microliters of phosphate buffer solution and new culture medium are perfused in turn to rinse the universal pipeline. Furthermore, 600 microliters of new culture medium are perfused into each culture well. The above perfusion steps will be performed twice to reduce liquid residue. After completing the above cycle, the system perfuses phosphate buffer solution to clean the universal pipeline. Finally, the motor gear unit controls the interlayer to fall and infiltrate the new culture medium on the outside.

[0090] (7) If imaging observation of the sample is required or the liquid replacement process has been completed, the liquid pipeline bracket can be removed and the interlayer together with the well plate and base can be taken out.

[0091] (III) Experimental results and analysis

[0092] Fig. 9 (A) The whole process of U-shaped well cluster-automated transmembrane culture of cardiac organoids is shown. After completing the hardware assembly, we use a pipette to transfer the cell clusters with clear boundaries to the semipermeable membrane inside the transmembrane device of the fixed layer of the transmembrane device (single cell cluster culture) or to the compartmentalized plug-in chamber (multiple cell cluster culture). Starting from day 0, the cell clusters will be placed in the device for automated medium replacement culture until the 8th day to open the lid for imaging observation and sample processing. In order to evaluate the phenotypic and gene expression characteristics of automated-cultured HCOs (aHCOs), we also prepared U-shaped well cluster-U-shaped well cultured cardiac organoids (U-bottom-manually-cultured HCOs, uHCOs) as a reference. Fig. 9 (B) and Fig. 9 (C) shows the morphological characteristics of cell clusters on day 0 and day 8, respectively. Fig.10 Images of multiple biological replicates are shown. From the bright field images, it can be noted that aHCOs have a fairly consistent phenotype. There are detached cell fragments around aHCOs, indicating that the transmembrane culture mode avoids the phenomenon of fluid washing the cell clusters and reduces the physical stimulation of the cell clusters. aHCOs have a larger cross-sectional area than uHCOs, and both have a vesicular structure. The vesicular structure of uHCOs can be observed to have a beating phenotype on days 8 to 10, while this phenotype cannot be observed in aHCOs on day 8. Immunofluorescence staining further revealed the myocardial components of the cardiac organoids on day 8. Fig.11 As shown in the figure, the signature transcription factor NKX2-5 and cardiac troponin cTnt of cardiac development are significantly expressed in both aHCOs and uHCOs. This result shows that aHCOs and uHCOs are very similar, and both can characterize the cardiac development process to a certain extent; the automated transmembrane culture device successfully realizes the non-human intervention, semi-closed, low microenvironment disturbance organoid culture. More detailed analysis depends on subsequent longer-term culture and omics analysis.

[0093] 6. Hardware parameters and drawing numbers of the experimental device

[0094] Table 2. Commercial component models of automated transmembrane organoid culture devices.

[0095]

[0096] Table 3. Engineering drawing codes for CNC machined parts of the automated transmembrane culture device for organoids.

[0097]

[0098] Table 4. Module detailed assembly code of the organoid automated transmembrane culture device.

[0099]

[0100] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. An automated transmembrane organoid culture device, characterized in that: It comprises a high-throughput automated perfusion module, a multi-channel fluid exchange module, and a movable interlayer module; wherein the high-throughput automated perfusion module comprises a reagent shunt pipeline and a multi-channel valve for adding and / or removing culture fluid; the multi-channel fluid exchange module is used to connect the high-throughput automated perfusion module and a cell culture well plate; the multi-channel fluid exchange module comprises a pipeline support and a needle-like structure; the needle-like structure extends into the culture well of the cell culture well plate; the movable interlayer module is used to fix the cell culture well plate and the transmembrane device, and control the vertical distance between the two during the fluid exchange process; The movable sandwich module includes a transmembrane device fixture, a motor gear set, left and right fan blades, a well plate base and a device base; the cell culture well plate is placed on the well plate base, and the transmembrane device fixture is used to fix multiple transmembrane devices, and moves in a vertical direction under the joint drive of the motor gear set and the left and right fan blades to achieve a change in the distance between the semipermeable membrane and the bottom of the culture well; The high-throughput automated perfusion module consists of a single reagent shunt line and three multichannel valve-culture well-multichannel valve-injector parallel groups, which can add and remove liquid from the culture chamber by positive pressure and negative pressure drive respectively; Among them, the multi-channel fluid exchange module includes a Y-shaped tee, a connector, a pipeline bracket and its needle-like structure; wherein the Y-shaped tee pipeline is connected to the perfusion pipeline of the automated perfusion module, the connector fixes the former on the upper surface of the pipeline bracket plane, and the needle-like structure below extends from one side of the culture chamber to a position close to the bottom of the hole.

2. The culture device according to claim 1, characterized in that: It also includes an interpolation segmentation module for splitting the space inside the transmembrane device to achieve the effect of placing multiple organoid cell clusters on the semipermeable membrane at the bottom of the transmembrane component.

3. The culture device according to claim 1, characterized in that: The cell culture well plate mentioned herein mostly refers to Corning Costar's flat-bottom tissue culture well plate.

4. The culture device according to claim 1, characterized in that: The transmembrane device is mostly a hanging cup-shaped culture chamber produced by Merck Millipore.

5. The culture device according to claim 1, characterized in that: Also included is an interposer partitioning module that can split the space inside the transmembrane device.

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