A dynamic culture device and dynamic culture method for organoids

Abstract

The application relates to the technical field of cell culture, in particular to a dynamic culture device and a dynamic culture method for organoids, which comprise a culture bin and a cross-flow reaction generator, a liquid supply bin is communicated with one end of the culture bin away from the cross-flow reaction generator, the two ends of the culture bin are connected with a second driving unit through a transmission mechanism, and the cross-flow reaction generator is rotationally connected with a first driving unit; a vortex protrusion / groove is arranged in the inner wall of the culture bin. Under the driving of the second driving unit, the culture bin is driven to rotate; under the driving of the first driving unit, the cross-flow reaction generator reciprocates along the axial direction of the culture bin, and the liquid in the culture bin collides with the vortex protrusion / groove in the inner wall of the culture bin under the transverse action of the cross-flow reaction generator, so that transverse vortex is formed. The application has the advantages of simple structure, culture environment capable of providing specific mechanical stimulation for organoids, beneficial to the expansion of organoids, and improved structure and functional maturity of organoids.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a dynamic culture device and method for organoids. BACKGROUND

[0002] Organoids refer to a three-dimensional cell complex similar to a target organ or tissue in structure and function, which is self-assembled by adult stem cells or pluripotent stem cells in vitro after induced differentiation, and has stable genetic characteristics and tissue-specific morphological and physiological function characteristics. It has a huge application scenario in the fields of life sciences such as tissue organ development, disease mechanism, drug research and development, clinical drug sensitivity detection, and precision medicine.

[0003] At present, the most commonly used method for organoids is the classic embedding method, that is, embedding the organoids in Matrigel containing different materials and nutrients for 3D culture. However, due to the differences in raw materials and complex extraction process of Matrigel, batch differences will affect the repeatability of the experiment; and this method mainly provides static physical support, which cannot simulate the dynamic mechanical stimulation under physiological conditions, and the maturity of the structure and function of the cultured organoids is lacking. Bioreactor can provide precise control of nutrients, dissolved oxygen, pH value, overcome the diffusion limitation of nutrients and oxygen, and prevent local accumulation of metabolic waste, which may be beneficial to the survival and expansion of organoids. In addition, bioreactor can provide dynamic fluid mechanical stimulation, which is crucial for the structure and functional maturity of organoids of specific tissue origin.

[0004] As disclosed in Chinese patent document CN218174985U, a multifunctional vortex rotary cell culture bioreactor can be used to amplify suspended organoids on a large scale. The vortex oscillator generates vortex oscillation, reducing the shear force of traditional paddles in the disposable inner container, which is beneficial to the formation of organoids. The control computer controls the inductive electrode, air inlet valve and liquid inlet valve to adjust the pressure and dissolved oxygen, simulate the in vivo environment, improve the culture effect, and realize the automation of the device through the connected control computer. If it needs to be reused, the disposable inner container can be replaced with a reusable sterilizable inner container. The utility model adopts the vortex oscillator to generate the required transverse vortex environment in the cell culture device.

[0005] For example, Chinese patent document CN101372665B discloses a tissue engineering reactor with tension and culture cavity rotation function, which comprises a tissue culture cavity, a culture liquid perfusion circuit passing through the culture cavity, first and second holding devices arranged in the tissue culture cavity for arranging a tissue culture to be cultured therebetween, a tissue culture tension and compression driving device coupled with the first holding device for driving the first holding device to reciprocate so as to perform tension and compression on the tissue culture, and a culture cavity rotation driving device coupled with the tissue culture cavity for rotating the tissue culture cavity.

[0006] The tissue culture between both ends of the invention is provided with tension and compression devices, so that periodic axial tension and compression equal mechanical loadings are loaded on the tissue culture. However, the invention directly gives the tissue culture a force stimulation which is very easy to exceed the limit of the force required for the growth of the tissue culture, and may cause damage to the tissue culture. SUMMARY

[0007] The present application aims to provide a dynamic culture device and method for organoids, partially solve or alleviate the above-mentioned deficiencies in the prior art, provide a relatively mild specific mechanical stimulation culture environment for organoids, and thus promote the expansion of organoids.

[0008] In order to solve the above-mentioned technical problems, the present application specifically adopts the following technical solutions:

[0009] The first aspect of the present application is to provide a dynamic culture device for organoids, comprising a culture bin, one end of the culture bin being connected with a cross-flow reaction generator, the other end of the culture bin away from the cross-flow reaction generator being communicated with a liquid supply bin, the culture bin being connected with a second driving unit through a transmission mechanism, and the cross-flow reaction generator being rotationally connected with a first driving unit; the culture bin comprises a first region close to the cross-flow reaction generator and a second region close to the liquid supply bin, the inner wall of the second region being provided with a spiral vortex protrusion or a wave-shaped vortex protrusion extending in the axial direction of the culture bin, or a plurality of concentric circular vortex protrusions being arranged in the axial direction of the culture bin or a plurality of petal-shaped vortex grooves being arranged adjacent in the axial direction of the culture bin; in the first stage of culture, the transmission mechanism drives the culture bin to rotate around the axial direction under the drive of the second driving unit, so that the liquid in the culture bin forms a circular Taylor vortex; in the second stage of culture, the execution end of the cross-flow reaction generator reciprocates along the inner wall of the first region under the drive of the first driving unit, so that the liquid in the culture bin collides with the vortex protrusion or vortex groove in the inner wall of the culture bin under the action of the cross-flow reaction generator, thereby forming a vortex.

[0010] Further, the cross-flow reaction generator comprises a driving rod extending into the first area of the culture chamber, a sliding block rotationally connected with the driving rod and adhering to the inner wall of the first area, and a hemispherical tip arranged on the side of the sliding block close to the vortex protrusions; wherein the spherical surface of the hemispherical tip is spaced apart by a plurality of helical grooves extending along the axial direction of the hemispherical tip; and each helical groove corresponds to a helical vortex protrusion, so that each helical groove serves as an extension of the helical vortex protrusion; or, the spherical surface of the hemispherical tip is spaced apart by a plurality of wave-shaped grooves extending along the axial direction; and each wave-shaped groove corresponds to a wave-shaped vortex protrusion, so that each wave-shaped groove serves as an extension of the wave-shaped vortex protrusion; when the culture chamber rotates, the first area of the culture chamber drives the sliding block to rotate relative to the driving rod; that is, the sliding block rotates synchronously with the culture chamber, and at this time the sliding block rotates relative to the driving rod (for example, the connection between the sliding block and the driving rod uses a ball bearing, so that the sliding block can rotate relative to the driving rod); when the first driving unit drives the driving rod to rotate relative to the culture chamber, the sliding block as the execution end drives the hemispherical tip to reciprocate along the inner wall of the first area.

[0011] Further, the culture bin extends a first opening at one end connected with the liquid supply bin, the liquid supply bin top is provided with a second opening, the first opening and the second opening are communicated through a conduit, the first opening and the second opening are provided with hydrophobic filter membrane. Further, the radius, pitch and spiral height of the spiral vortex protrusion are 8mm-12mm, 40mm-50mm and 32mm-39mm respectively. Further, the cross section of the spiral vortex protrusion perpendicular to the axial direction of the culture bin is an equilateral triangle. Further, the depth of the spiral groove gradually decreases along the axial direction of the hemispherical tip, or the radius of the wave-shaped groove gradually decreases along the axial direction of the hemispherical tip. Further, the wave-shaped vortex protrusion is at least six adjacent adjacent, and the radius of each wave-shaped vortex protrusion is a semicircle with a radius of 1.5mm-3.5mm. Further, the concentric circular ring vortex protrusion is at least six spaced along the axial direction, and the radius of the concentric circular ring vortex protrusion gradually decreases along the axial direction of the culture bin (1) and in the direction close to the cross-flow reaction generator. Further, the petal-shaped vortex groove is a plurality of arc grooves arranged continuously along the circumferential direction of the culture bin, and the sawtooth height formed between adjacent two arc grooves is 1.5mm-3mm; the radius of the arc length groove is 34mm-38mm. Further, the driving rod and the sliding block are matched by spline or guide key structure, or matched by ball bearing structure, so that the sliding block can rotate compared with the driving rod. Specifically, there are many existing structures to realize the rotation between the two, which will not be repeated here. Further, the dynamic culture device further comprises: a support shell, the culture bin is fixed on the top of the support shell through a fixing device, the fixing device comprises: a fixed part fixed on the top of the support shell and a semicircular part connected with the fixed part, part of the semicircular part is provided as an open hollow center structure, the culture bin is fixed or taken out through the open semicircular part. Further, the first driving unit comprises: a first motor, a first driving wheel connected with the output shaft of the first motor, the outer surface of the first driving wheel is provided with a thread which can be engaged with the external thread on the driving rod of the cross-flow reaction generator; when the first motor drives the first driving wheel to rotate continuously in the clockwise and counterclockwise directions, the cross-flow reaction generator makes reciprocating motion along the axial direction, so that the sliding block reciprocates along the inner wall of the first area in the culture bin. Further, the second driving unit comprises: a second motor, a second driving wheel connected with the output shaft at both ends of the second motor. Under the drive of the second motor, the two second driving wheels drive the transmission mechanism, such as the driven gear, to rotate in the same direction, thereby driving the culture bin to rotate. Further, the support shell is a rectangular parallelepiped, the first driving unit, the second driving unit and the liquid supply bin are located in the interior of the support shell.

[0012] The second aspect of the application is to provide a dynamic culture method of organoids, comprising: using the dynamic culture device for dynamic culture of organoids; periodically collecting growth images of organoids in the culture chamber by using the image acquisition module; using the host module to process the growth images of organoids in the culture chamber collected by the image acquisition module to obtain the current size of the organoids, and determining whether the current size is greater than or equal to a first preset threshold; if the current size of the organoids in the culture chamber is less than the first preset threshold, controlling the second driving unit to maintain the current first preset rotating speed; if the current size of the organoids in the culture chamber is greater than or equal to the first preset threshold but less than a second preset threshold, controlling the second driving unit to adjust the current first preset rotating speed to a second preset rotating speed; the second preset rotating speed is greater than the first preset rotating speed; and when it is determined that the current size of the organoids in the culture chamber is greater than or equal to the second preset threshold but less than a third preset threshold, controlling the second driving unit to maintain the current second preset rotating speed while controlling the first driving unit to rotate so that the execution end of the cross-flow reaction generator reciprocates along the inner wall of the culture chamber; wherein the frequency of the reciprocating motion of the cross-flow reaction generator is 10 times / min-15 times / min, and the stroke (i.e. the distance of the sliding block reciprocating along the inner wall of the first region) is 5mm-10mm.

[0013] Beneficial effects: The vortex protrusion or vortex groove is arranged in the culture chamber, so that a slight mechanical stimulation (i.e. I-level mechanical stimulation) is formed during the rotation of the culture chamber; at the same time, the cross-flow reaction generator is arranged in the culture chamber, so that when the cross-flow reaction generator reciprocates along the axis of the culture chamber, the transverse flow of the liquid and the vortex groove of the inner wall of the culture chamber interact to form a specific vortex, thereby providing a specific mechanical stimulation (i.e. II-level mechanical stimulation) for the culture environment of the organoids to promote the growth thereof. For example, in the initial stage, the culture chamber is controlled to rotate to create a suitable growth environment. At this time, since the cell size is small and fragile, only the culture chamber is driven to rotate around the axis, and the vortex protrusion or vortex groove generates a gentle axial and tangential mixing flow during rotation to ensure uniform suspension of cells, sufficient nutrition and gas exchange, while avoiding excessive shear force to damage the newly formed cells. In the later stage, the cross-flow reaction generator is controlled to reciprocate axially while the culture chamber is rotating to create an environment conducive to cell growth. Since the cell aggregates or tissues grow to a certain size, the core may face the limitation of nutrient or oxygen diffusion. At this time, the cross-flow reaction generator is driven to reciprocate left and right in the culture chamber to generate a relatively strong radial mixing flow and turbulence (more accurately, three-dimensional vortex rather than simple transverse vortex), thereby facilitating cell expansion.

[0014] This invention creates a special environment more suitable for culturing organoids based on the motion of the culture chamber and the crossflow reactor. The inner wall of the culture chamber is provided with vortex protrusions or vortex grooves to provide a gentler mechanical stimulation. In the initial stage, the culture chamber is rotated by a power device, thereby generating a gentle axial and tangential mixed flow in the first area of ​​the culture chamber, ensuring uniform cell suspension and sufficient nutrient and gas exchange, while avoiding excessive shear force that could damage the new cells. In the later stage, the crossflow reactor moves within the culture chamber while the culture chamber rotates, thereby generating a strong radial mixed flow and turbulence. This method creates an environment that is more suitable for organoid growth.

[0015] Furthermore, since organoids are easily affected by fluid shear forces in the early stages, which can damage their structure, the reciprocating rate of the crossflow reactor can be adjusted accordingly. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0017] Figure 1 A perspective view illustrating the internal structure of an embodiment of the dynamic culture device of the present invention; Figure 2 for Figure 1 A perspective view of the dynamic culture device shown from a first-person perspective; Figure 3 for Figure 1 A perspective view of the dynamic culture device shown from a second angle; Figure 4 for Figure 1 A perspective view of the dynamic culture device shown from a third-angle perspective; Figure 5A In order to test the effect of culture chambers with different tube wall structures on the expansion rate of kidney organoids, three groups of organoids were cultured for 10 days using the dynamic culture device of the present invention. Bright field images of kidney organoids under light microscopy were obtained from the substrate gel culture group, the static suspension culture group, and the dynamic suspension culture group. Figures 5B-5D The following graphs show the organoid number, organoid area, and cell number statistics of kidney organoids obtained after 10 days of organoid culture using the dynamic culture device of the present invention: in the substrate gel culture group, static suspension culture group, and dynamic suspension culture group. Figure 6AFor testing the influence of the culture bin with different tube wall structure design on the structure and function of kidney organoids, three groups of organoids are cultured in the dynamic culture device, and when it is found that the structures of the three groups of organoids are different, the staining results of the kidney organoids of the Matrigel culture group, the static suspension culture group and the dynamic suspension culture group after H&E staining are respectively obtained; Figure 6B For adding P-gp transporter inhibitor (P-gpi) and not adding P-gp transporter inhibitor (P-gpi) in the kidney organoids of the Matrigel culture group, the static suspension culture group and the dynamic suspension culture group respectively, the average fluorescence intensity statistical chart of the kidney organoids is obtained; Figure 7 For the schematic diagram of the extension direction of the semispherical screw groove on the slider in the cross-flow reaction generator in an embodiment of the dynamic culture device of the application; Figure 8 For the distribution schematic diagram of the semispherical screw groove on the slider in the cross-flow reaction generator in an embodiment of the dynamic culture device of the application; Figure 9 For the schematic diagram of the extension direction of the semispherical screw groove on the slider in the cross-flow reaction generator in an embodiment of the dynamic culture device of the application; Figure 10 For the distribution schematic diagram of the semispherical screw groove on the slider in the cross-flow reaction generator in an embodiment of the dynamic culture device of the application; Figure 11 For the connection structure schematic diagram between one end of the reaction driving rod and the slider; Figure 12 For the structure schematic diagram of the distribution of the reaction wave-shaped groove in the culture bin; Figure 13 For Figure 12 The cross-sectional view of the culture bin shown in the figure; Figure 14 For the distribution schematic diagram of the reaction wave-shaped groove in the culture bin; Figure 15 For the axial distribution schematic diagram of the reaction petal-shaped groove in the culture bin; Figure 16 For Figure 15 The cross-sectional view of the culture bin shown in the figure; Figure 17 For the structure schematic diagram of the reaction petal-shaped groove in the culture bin; Figure 18 For the schematic diagram of the extension direction of the reaction screw groove in the culture bin; Figure 19 For Figure 18 The cross-sectional view of the culture bin shown in the figure; Figure 20 For the circumferential distribution schematic diagram of the reaction multiple screw grooves in the culture bin; Figure 21 For the schematic diagram of the axial extension of the reaction concentric circular ring groove in the culture bin; Figure 22 For the schematic diagram of the axial extension of the reaction concentric circular ring groove in the culture bin; Figure 23 For the distribution schematic diagram of the reaction multiple concentric circular ring grooves with different radii in the culture bin; Figures 24A-24D Respectively, in the culture bin with wave-shaped vortex protrusions, the cell density is 0.25×10 5 / mL, 0.5×105 1x10 5 2x10 5 4x10 5 Figures of the number of organoids, the area of organoids, the number of cells and the maturity of the transport function of organoids obtained by organoid culture at 1x10 Figures 25A-25D Figures of the number of organoids, the area of organoids, the number of cells and the maturity of the transport function of organoids obtained by organoid culture at 1x10

[0018] Figures of the number of organoids, the area of organoids, the number of cells and the maturity of the transport function of organoids obtained by organoid culture at 1x10 DETAILED DESCRIPTION

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In this document, suffixes such as "module," "component," or "unit" used to represent elements are only for the purpose of illustrative purposes and have no specific meaning in themselves. Therefore, "module," "component," or "unit" can be used interchangeably. In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. In this document, unless otherwise expressly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. "And / or" in this document includes any and all combinations of one or more of the listed related items. "A plurality" in this document means two or more, i.e., it includes two, three, four, five, etc.

[0020] Example 1: As Figures 1-4 As shown, this embodiment proposes a dynamic culture device for organoids, including: a culture chamber 1, and a crossflow reaction generator 103 with one end extending into the culture chamber 1. The other end of the culture chamber 1 away from the crossflow reaction generator 103 is connected to a liquid supply chamber 5. A transmission mechanism (such as a driven gear) is fixedly connected to each end of the culture chamber 1. The crossflow reaction generator 103 is rotatably connected to a first drive unit, and the two transmission mechanisms are rotatably connected to a second drive unit.

[0021] In some embodiments, the culture bin 1 is provided as a hollow cylindrical structure, and the interior of the culture bin 1 is divided into a first region close to the cross-flow reactor 103 and a second region close to the liquid supply bin 5, and the inner wall of the second region is provided with vortex protrusions or vortex grooves extending along the axial direction of the culture bin 1. Preferably, in some embodiments, the vortex protrusions can adopt helical vortex protrusions or wave-shaped vortex protrusions extending along the axial direction of the culture bin 1, or the inner wall of the second region of the culture bin 1 is provided with a plurality of concentric circular vortex protrusions arranged along the axial direction of the culture bin 1 at intervals; or the vortex grooves can adopt a plurality of petal-shaped vortex grooves arranged adjacent to each other along the axial direction of the culture bin 1.

[0022] In particular, the cross-flow reactor 103 comprises a sliding block 105 (i.e., the execution end of the cross-flow reactor 103) attached to the inner wall of the first region of the culture bin 1, which is used to seal the second region to prevent the liquid in the second region from entering the first region, and the sliding block 105 can rotate synchronously with the culture bin 1; the cross-flow reactor 103 further comprises a drive rod having one end rotatably connected to the sliding block 105 and extending into the first region of the culture bin 1, and the other end of the drive rod extending out of the first region and connected with a first driving unit; when the culture bin 1 rotates without external force, the sliding block 105 rotates synchronously with the culture bin 1; when the first driving unit drives the drive rod to rotate, so that the drive rod reciprocates along the axial direction of the culture bin 1, the drive rod drives the sliding block 105 to move along the axial direction of the culture bin 1, i.e., the drive rod drives the sliding block 105 to reciprocate synchronously, and the sliding block 105 can also rotate relative to the drive rod. Preferably, as shown in Figure 11 the sliding block 105 and the drive rod are rotatably connected by a ball bearing.

[0023] Further, in some embodiments, in order to prevent the cross-flow reactor from reciprocating during the reciprocating process, causing a large mechanical stimulus due to the large space difference between the first region and the second region, which affects cell growth, or even destroys the cells, therefore, one end of the sliding block 105 close to the vortex protrusion / vortex groove side is provided with a hemispherical tip, and the spherical surface of the hemispherical tip is provided with a structure for extending the vortex protrusion, for example, a groove. Specifically, a plurality of helical grooves extending along the axial direction are arranged at intervals on the spherical surface of the hemispherical tip, and each helical groove corresponds to a helical vortex protrusion, so that each helical groove extends the helical vortex protrusion, see Figures 7-8The purpose of the hemispherical tip is to make the trajectory of the rotating fluid transition naturally, avoiding the strong turbulence and shear peak generated when the traditional flat tip suddenly approaches or moves away from the second region, thus causing excessive mechanical stimulation. Therefore, the helical groove of the hemispherical tip is extended to the helical vortex protrusion, converting rotational kinetic energy into axial laminar flow (rather than radial turbulent flow), significantly reducing the vortex intensity at the junction of the two-stage structure.

[0024] In other embodiments, the spherical surface of the hemispherical tip is provided with a plurality of axially extending wavy grooves; and each of the wavy grooves corresponds to a wavy vortex protrusion, so that each of the wavy grooves serves as an extension of the wavy vortex protrusion, see Figures 9-10 .

[0025] More preferably, the depth of the helical groove on the hemispherical tip gradually decreases along the axial direction of the hemispherical tip, or the radius of the wavy groove gradually decreases along the axial direction of the hemispherical tip.

[0026] Preferably, a bearing structure (e.g., a ball bearing structure) is introduced between the end of the drive rod and the slider 105 for rotational connection. Such a structure design allows the slider 105 to rotate synchronously with the culture chamber 1 when the culture chamber 1 rotates (and since the slider 105 is attached to the inner wall of the culture chamber 1, it can achieve sealing, which is similar to a sealed piston, and will not be described here), but does not affect the rotation of the drive rod of the cross-flow reactor 103. Moreover, the design allows the drive rod to move axially and reciprocally, and the groove on the spherical surface can also correspond to the vortex protrusion, see Figure 11 .

[0027] Preferably, the vortex protrusion or vortex groove is designed to form a specific vortex when the liquid in the culture chamber 1 encounters the vortex protrusion or vortex groove during lateral flow, thereby providing specific mechanical stimulation to the organoids. Different vortex groove structures (which can be determined in advance according to the characteristics of the cultured organoids) can produce different hydrodynamic effects, further amplifying the advantages of dynamic culture in promoting organoid expansion, see Figures 12-23 .

[0028] In particular, the radius, pitch, and helical height of the helical vortex protrusion are 8-12 mm, 40-50 mm, and 32-39 mm, respectively; preferably, 10 mm, 45 mm, and 36 mm.

[0029] Preferably, the cross-section of the helical vortex protrusion perpendicular to the axial direction of the culture chamber is an equilateral triangle. More preferably, the helical direction is to the right, see Figures 18-19 .

[0030] In particular, the wave-shaped vortex protrusions are arranged in multiple groups (preferably 12 groups) along the circumferential direction of the culture chamber 1, and each group comprises multiple semicircles arranged axially adjacent to each other with a radius of 1.5mm-3.5mm (preferably 3mm), as shown in Figures 12-14 .

[0031] In particular, the concentric circular protrusions comprise at least six concentric circular protrusions arranged axially adjacent to each other with a radius decreasing in turn. Preferably, the height of the radial protrusion of the concentric circular protrusions is 4mm-9mm, and the length of the concentric circular protrusions along the axial direction of the culture chamber 1 is 5mm-7mm, preferably 6mm, as shown in Figures 21-23 .

[0032] In particular, three petal-shaped vortex grooves are arranged along the axial direction of the culture chamber 1, as shown in Figure 15 and Figure 16 Each petal-shaped vortex groove comprises multiple (preferably 36) arc-shaped grooves arranged along the circumferential direction of the culture chamber 1 and extending along the axial direction of the culture chamber 1, and the sawtooth formed between two adjacent arc-shaped grooves has a height of 1.5mm-3mm (preferably 2mm), and the radius of the arc-shaped groove is 34mm-38mm (preferably 36mm), as shown in Figures 15-17 . Further, the sawtooth between two adjacent petal-shaped vortex grooves is staggered.

[0033] In some embodiments, a first transmission mechanism 101 and a second transmission mechanism 102 are fixedly installed at the two ends of the culture chamber 1, and preferably the two transmission mechanisms are driven gears, and the drive gear in the second drive unit is engaged with the driven gears and provides power to them, so that the two transmission mechanisms can drive the culture chamber 1 to rotate (i.e. the culture chamber rotates around its axial direction).

[0034] As shown in Figure 1 and Figure 2 , in some embodiments, a support shell 3 is further included, which is a cuboid, and the culture chamber 1 is fixed on the top of the support shell 3, and the first drive unit, the second drive unit and the liquid supply chamber 5 are arranged inside the support shell 3.

[0035] In some embodiments, a first opening 104 is arranged on one side (as shown in Figure 1 ) of the culture chamber 1, preferably the left side. The liquid supply chamber 5 is a cuboid container with culture medium inside, and the top of the liquid supply chamber 5 is provided with a second opening 501. The two openings are connected by a conduit 6, so that the culture chamber 1 and the liquid supply chamber 5 are connected.

[0036] Preferably, the first opening 104 and the second opening 501 are both provided with a layer of 0.22μm hydrophobic filter membrane to prevent the liquid in the culture chamber 1 from flowing into the liquid supply chamber 5 when the organoids are carried into the liquid supply chamber 5.

[0037] In some embodiments, the culture bin 1 is fixed on the top of the bracket shell 3 by a fixer, which includes, from left to right, a first fixer 301, a second fixer 302, a third fixer 303, and a fourth fixer 304. The first fixer 301 and the second fixer 302 are fixed on the left end part of the culture bin 1, and the third fixer 303 and the fourth fixer 304 are fixed on the right end part of the culture bin 1. The structure of the four fixers includes a fixed part at the bottom fixed to the bracket shell 3, and a semicircular part at the upper part in the form of an open hollow circle center, which can be opened or closed to take out or fix the culture bin 1.

[0038] In particular, the specifications of the four fixers are adaptively adjusted according to the different connection parts of the culture bin 1. The second fixer and the third fixer are fixed on the surface of the culture bin, so the hollow circle center (the part for placing the culture bin when opened) is adapted to the size specifications of the culture bin. The first fixer is fixed on the first opening, and the fourth fixer is fixed on the cross-flow reaction generator, so the specifications are relatively small.

[0039] In some embodiments, the first driving unit is arranged inside the bracket shell 3 and includes a first motor 2 and a first driving wheel 201 arranged on the output shaft of the first motor 2. The first driving wheel 201 is provided with external threads to be threadedly connected with the external threads on the driving rod of the cross-flow reaction generator 103. Under the driving of the first motor 2, the first driving wheel 201 continuously changes the direction of rotation in clockwise and counterclockwise directions, so that the driving rod rotates relative to the culture bin 1 (for example, the culture bin 1 is provided with an opening at one end close to the driving rod for the rotation of the driving rod, and the size of the opening is larger than that of the driving rod; or for example, the culture bin has a tubular structure with one end closed and the other end open, wherein the closed end is connected to the liquid supply bin through a conduit, and the opening end is sized to facilitate the sliding block to enter and exit), so as to make reciprocating motion along the axial direction of the culture bin 1. Preferably, the sliding block 105 is provided with an elastic sealing ring on the surface of the inner wall of the culture bin.

[0040] In some embodiments, the second driving unit is arranged inside the bracket shell 3 opposite to the culture bin and includes a second motor 4, two second rotating shafts respectively connected to the two ends of the second motor 4, and a second driving wheel 401 connected to the two second rotating shafts. The two second driving wheels are located opposite to the second transmission mechanisms of the culture bin and are respectively engaged with the two transmission mechanisms. Under the driving of the second motor 4, the two second driving wheels 401 drive the two transmission mechanisms to rotate in the same direction. In turn, the culture bin is rotated, and the centrifugal force of the rotating culture bin resists gravity, so that the organoids are in a suspended state.

[0041] In some embodiments, the device further comprises a main control module (or host computer) electrically connected with the first driving unit and the second driving unit. The main control module controls the reciprocating frequency and amplitude of the cross-flow reactor 103 to adjust the shear force. In particular, the main control module controls the reciprocating frequency of the cross-flow reactor 103, and the lateral shear force received by the organoids will not be damaged. Preferably, the image acquisition module periodically acquires the growth image of the organoids in the culture chamber 1; and sends it to the host computer to determine whether the current size of the cells in the culture chamber is greater than or equal to the first preset threshold value. If the current size is less than the first preset threshold value (for example, less than 20 pm), the corresponding control instruction is sent to the main control module to control the second driving unit to keep the current first preset rotating speed; if the current size is greater than or equal to the first preset threshold value, the corresponding control instruction is sent to the main control module to control the second driving unit to adjust its current first preset rotating speed to a second preset rotating speed. This is repeated until a new current size greater than or equal to a second preset threshold value (for example, 35 pm-50 pm; preferably, the value for kidney organoids is 40 pm) is obtained, and the corresponding control instruction is sent to the main control module to control the first driving unit to rotate, so that the slider 105 of the cross-flow reactor 103 reciprocates along the inner wall of the culture chamber 1; wherein the frequency of the reciprocating motion of the cross-flow reactor 103 is 10 times / min-15 times / min (preferably, the frequency for kidney organoids is 12 times), and the stroke is 5 mm-10 mm (preferably, the stroke for kidney organoids is 5 mm). Specifically, the second preset rotating speed is greater than the first preset rotating speed. Further, when the host computer determines that the current size of the organoids in the culture chamber 1 is greater than or equal to a third preset threshold value (for example, greater than or equal to 50 pm), the corresponding control instruction is sent to the main control module to control the second driving unit to adjust its current second preset rotating speed to a third preset rotating speed. Of course, in some embodiments, the main control module can also be integrated into the host computer. Of course, the main control module can also be integrated into the image acquisition module, that is, the image acquisition module performs image analysis and controls the first driving unit and the second driving unit in the device to work.

[0042] The working principle of the device is as follows: initial stage: the first transmission mechanism 101 and the second transmission mechanism 102 are driven by the second driving unit to drive the culture chamber 1 to rotate, at this time, the cross-flow reaction generator 103 does not rotate (i.e., the culture chamber rotates around the driving rod of the cross-flow reaction generator); the organoid in the initial stage is in a suspended state. Later stage: after the organoid forms a certain volume and structure, when it is necessary to strengthen the lateral stimulation, the cross-flow reaction generator 103 is driven by the first driving unit to reciprocate along the axis direction of the culture chamber 1, and the liquid in the culture chamber 1 collides more strongly with the vortex protrusion / vortex groove in the inner wall of the culture chamber 1 under the lateral action of the cross-flow reaction generator 103, so as to form a lateral vortex to mechanically stimulate the organoid.

[0043] The purpose of the device for optimizing the culture environment in stages is as follows: the environment required in the initial stage is the environment created by only the rotation of the culture chamber, when the cell size is small and fragile, only the culture chamber is driven to rotate around the shaft. The vortex protrusion or vortex groove generates a mild axial and tangential mixing flow when rotating, which ensures that the cells are uniformly suspended, the nutrition and gas exchange are sufficient, and at the same time, the newly born cells are prevented from being damaged by excessive shear force. In the later stage, the culture chamber is rotated, and the cross-flow reaction generator is controlled to reciprocate axially, so as to create an environment conducive to cell growth, when the cell aggregate or tissue grows to a certain size, the core thereof can face the limitation of nutrition or oxygen diffusion. At this time, the slider is started to reciprocate left and right in the smooth first area. The reciprocating motion generates a strong radial mixing flow and turbulence (more accurately, a three-dimensional vortex rather than a simple lateral vortex). The dynamic flow field generated by the reciprocating motion can exert a stronger shear force on the cells than the simple rotation. Such shear force has the following advantages: stimulating cell function: for certain cell types (such as endothelial cells, osteoblasts, and chondrocytes), appropriate fluid shear force is an important physiological signal that can promote their differentiation, extracellular matrix secretion, or expression of specific functional proteins. Controlling the size and morphology of aggregates: preventing excessive aggregation of cells to form a large, easily necrotic mass, which helps to form a more uniform and healthier tissue-like structure. Simulating the physiological microenvironment: closer to the dynamic mechanical stimulation experienced by in-vivo tissues (such as blood vessels and bones).

[0044] Based on the culture device described above, the application further provides a dynamic culture method of an organoid, which comprises:

[0045] S101, dynamic culture of organoids is performed using the dynamic culture device described above; specifically, the culture conditions can refer to those in Embodiment Two or Embodiment Three, which are not repeated here; S102, the growth images of the organoids in the culture bin 1 in the dynamic culture device are periodically collected using an image collection module; specifically, the image collection module can use existing high-definition camera equipment; S103, the current size of the organoids is obtained by image processing of the growth images of the organoids in the culture bin collected by the image collection module using a host control module (or a host computer); S104, it is determined whether the current size is greater than or equal to a first preset threshold value; if the current size of the organoids in the culture bin 1 is less than the first preset threshold value (for example, less than 20 μm), step S105 is performed; if the current size of the organoids in the culture bin 1 is greater than or equal to the first preset threshold value, step S106 is performed; S105, the current first preset rotating speed of the second driving unit is maintained, and step S102 is performed; S106, the current first preset rotating speed of the second driving unit is adjusted to a second preset rotating speed, steps S102-S103 are performed, and when a new current size is obtained, step S107 is performed; specifically, the second preset rotating speed is greater than the first preset rotating speed; S107, it is determined whether the current size of the organoids in the culture bin 1 is greater than or equal to a preset second preset threshold value (for example, 35 μm-50 μm; preferably, the value for kidney organoids is 40 μm); if yes, step S108 is performed, otherwise step S102 is performed; S108, while the second driving unit maintains the second preset rotating speed, the first driving unit is controlled to rotate so that the slider 105 of the cross-flow reactor 103 reciprocates along the inner wall of the culture bin 1; wherein the frequency of the reciprocating motion of the cross-flow reactor 103 is 10 times / min-15 times / min (preferably, the frequency for kidney organoids is 12 times), and the stroke is 5 mm-10 mm (preferably, the stroke for kidney organoids is 5 mm).

[0046] Further, the dynamic culture method further comprises the step of: S109, when it is determined that the current size of the organoids in the culture bin 1 is greater than or equal to a third preset threshold value (for example, greater than or equal to 50 μm), the current second preset rotating speed of the second driving unit is adjusted to a third preset rotating speed.

[0047] In this embodiment, the rotating speed of the culture bin is dynamically adjusted according to the different size of the organoids, and when the size of the organoids reaches a condition, the reciprocating motion of the cross-flow reactor is controlled to generate strong radial mixing flow and turbulence, thereby more favoring cell expansion.

[0048] Embodiment Two: This embodiment is to test the influence of culture bins with different tube wall structure designs on the expansion rate of kidney organoids.

[0049] 1. Take different tube wall structure design of culture warehouse (including spiral vortex convex, that is, dynamic suspension group 2, wave vortex convex, that is, dynamic suspension group 3, concentric ring vortex convex, that is, dynamic suspension group 4, and petal-shaped vortex groove, that is, dynamic suspension group 5) of 5mL specification and control culture warehouse without groove structure, remove the cross-flow reaction generator, add 5mL of 1% FBS DPBS solution with a pipette, remove the air bubbles, and place at room temperature for a few minutes to wet the tube wall, prevent the organoids from adhering to the tube wall.

[0050] 2. Take an appropriate amount of tissue sample or organoid, add an appropriate amount of TrypLE digestion solution, and digest at 37℃ for 3-8 minutes. Observe the digestion under a microscope every 2 minutes during the period. When more than 90% of the tissue or organoid is digested into single cells, add 4 times the volume of DPBS to terminate digestion.

[0051] 3. 600g, 4℃, centrifuge for 5 minutes, remove the supernatant, add an appropriate amount of DPBS, mix well with a pipette gun, and count. Take an appropriate amount of cell suspension, 600g, 4℃, centrifuge for 5 minutes.

[0052] 4. Remove the supernatant, add an appropriate amount of culture medium, and adjust the cell density to 1×10 5 / mL. Matrigel culture group: take 2×10 5 cells embedded in 100μL matrigel, inoculated in a conventional 12-well culture plate, and after solidification, add 2mL of culture medium; static suspension culture group: transfer 2mL of cell suspension to a low adsorption 12-well culture plate. The tube wall without groove structure (that is, dynamic suspension group 1) and the culture warehouse with spiral vortex convex, wave vortex convex, concentric ring vortex convex or petal-shaped vortex groove are set as dynamic suspension culture groups 1-5 respectively, and 2mL of cell suspension is added to each culture warehouse. Load the cross-flow reaction generator back into the culture warehouse and adjust the position of the cross-flow reaction generator to exclude excess gas in the culture warehouse.

[0053] 5. Place each culture warehouse on the shell, connect one end with the conduit of the liquid supply warehouse, adjust the rotation speed of the culture warehouse to 10-20rpm on the main control panel, and culture at 37℃, 5% CO2.

[0054] 6. After 2-3 days of culture, the cell aggregates and self-assemble into a certain volume of organoids, adjust the rotation speed according to the size change of the organoids.

[0055] 7. When the size of the organoids reaches about 40μm, drive the cross-flow reaction generator to reciprocate, the frequency of reciprocation is 10-15 times / min, the stroke is 5mm, and culture at 37℃, 5% CO2.

[0056] 8. During the culture period, the growth status of organoids should be observed regularly. The rotation speed needs to be adjusted according to the size changes of the organoids to maintain their suspension (Table 1). On the 10th day, photographs were taken under a microscope to count the number and area of ​​organoids. After photographing, the organoids were digested into single cells with an appropriate amount of TrypLE digestion solution and counted. The differences in number, size, and absolute cell number among the different groups of organoids were compared.

[0057] Table 1. Rotation speed required to maintain suspension of organoids of different sizes throughout the entire culture cycle.

[0058]

[0059] Table 2. Effects of different tube wall structure designs on the number of kidney organoids.

[0060]

[0061] Table 3. Effects of different tube wall structure designs on the area of ​​kidney organoids

[0062]

[0063] Table 4. Effects of different tube wall structure designs on the number of kidney organoid cells.

[0064]

[0065] like Figures 5A-5D The results showed that after 10 days of culture, different tube wall structure designs affected the number of organoids and the total area of ​​organoids (1×10⁻⁶). 5 μm 2 ) and cell number (1×10 5 The effects of these effects are shown in Tables 2, 3, and 4: The number of kidney organoids in the matrix gel group was 33.2 ± 5.4, and the total organoid area was (3.2 ± 0.3) × 10⁻⁶. 5 μm 2 The cell count was (2.7±0.2)×10⁻⁶. 5 The number of kidney organoids in the statically suspended group was 45.3±6.2, and the total organoid area was (4.7±0.4)×10⁻⁶. 5 μm 2 The cell count was (3.4±0.2)×10⁻⁶. 5 The number of kidney organoids in the dynamically suspended group 1 was 56.1±4.2, and the total organoid area was (6.4±0.8)×10⁻⁶. 5 μm 2 The cell count was (4.1±0.2)×10⁻⁶. 5 The number of kidney organoids in the dynamically suspended group 2 was 72.8±7.7, and the total organoid area was (9.4±1.0)×10⁻⁶.5 μm 2 , cell number was (5.3 ± 0.3) x 10 5 . The kidney organoids in dynamic suspension group 3 had 68.7 ± 10.8 organoids, total organoid area was (8.5 ± 0.9) x 10 5 μm 2 , cell number was (5.0 ± 0.3) x 10 5 . The kidney organoids in dynamic suspension group 4 had 58.2 ± 5.3 organoids, total organoid area was (6.6 ± 0.6) x 10 5 μm 2 , cell number was (4.2 ± 0.3) x 10 5 . The kidney organoids in dynamic suspension group 5 had 66.9 ± 4.3 organoids, total organoid area was (7.7 ± 0.8) x 10 5 μm 2 , cell number was (4.9 ± 0.2) x 10 5 .

[0066] Compared with the Matrigel group, the kidney organoids in static suspension group had significantly increased organoid number, total organoid area and cell number; compared with the static suspension group, the kidney organoids in dynamic suspension group 1 had significantly increased organoid number, total organoid area and cell number; and compared with dynamic suspension group 1, the kidney organoids in dynamic suspension groups 2, 3 and 5 had further increased organoid number, total organoid area and cell number.

[0067] This shows that the suspension culture method is more conducive to the expansion of kidney organoids than embedding culture in Matrigel, and the dynamic suspension method can further improve the expansion rate of kidney organoids. In addition, the design of spiral vortex protrusions, wave vortex protrusions, concentric circular vortex protrusions or petal-shaped vortex grooves on the wall of the culture tank can produce special fluid dynamics effects, further amplifying the beneficial effects of dynamic culture in promoting organoid expansion.

[0068] Example Three: This example tests the effects of culture tanks with different wall structure designs on the structure and function of kidney organoids:

[0069] 1. Take 5mL size culture tanks with different wall structure designs (including spiral, wave, concentric circular and petal-shaped groove structures) and a control culture tank without a groove structure, remove the cross-flow reaction generator, use a pipette to add 5mL of 1% FBS DPBS solution, tap the injector wall with your fingers to remove air bubbles, and place it at room temperature for a few minutes to wet the wall and prevent the organoids from sticking to the wall.

[0070] 2. Take an appropriate amount of tissue sample or organoid, add an appropriate amount of TrypLE digestion solution, digest at 37°C for 3-8 minutes, observe the digestion under a microscope every 2 minutes during the period, and when more than 90% of the tissue or organoid is digested into single cells, add 4 times the volume of DPBS to terminate the digestion.

[0071] 3. 600g, 4°C, centrifuge for 5 minutes, remove the supernatant, add an appropriate amount of DPBS, mix well with a pipette gun, and then count. Take an appropriate amount of cell suspension, 600g, 4°C, centrifuge for 5 minutes.

[0072] 4. Remove the supernatant, add an appropriate amount of culture medium, and adjust the cell density to 1x10 5 / mL. Matrigel culture group: take 2x10 5 cells embedded in 100 μL of matrigel, inoculated in a conventional 12-well culture plate, after solidification, add 2 mL of culture medium; static suspension culture group: transfer 2 mL of cell suspension to a low adsorption 12-well culture plate; the culture chambers with no groove structure and the groove structures of spiral, wave, concentric ring or petal shape are sequentially set as dynamic suspension culture groups 1-5, 2 mL of cell suspension is added to each culture chamber, and the cross-flow reactor is loaded back into the culture chamber, and the position of the cross-flow reactor is adjusted to exclude excess gas in the culture chamber.

[0073] 5. Place each culture chamber on the rotating device, connect the head opening end with the conduit of the liquid supply chamber, adjust the rotation speed of the culture chamber to 10-20 rpm on the main control panel, and culture at 37°C, 5% CO2.

[0074] 6. After 2-3 days of culture, when the cells aggregate and self-assemble into a certain volume of organoids, adjust the rotation speed according to the size change of the organoids.

[0075] 7. When the size of the organoids reaches about 40 μm, drive the cross-flow reactor to reciprocate, with a frequency of 10-15 times / min and a stroke of 5 mm, and culture at 37°C, 5% CO2.

[0076] 8. During the culture period, observe the growth state and morphological structure of the organoids regularly. When differences in the structure of the organoids are found among the groups, half of the organoids are subjected to H&E staining; the remaining half is used for functional experiment determination to compare the differences in tissue structure complexity and functional maturity of the organoids among the groups.

[0077] Table 5 Influence of different tube wall structure designs on the transport function of kidney organoids

[0078]

[0079] For example, Figure 6BThe effects of different tube wall structure designs shown in Table 5 on the transport function of the kidney organoids were as follows: the average fluorescence intensity of the Matrigel group without the addition of P-gp inhibitor (P-gpi) was 52.6±5.4, and the average fluorescence intensity of the Matrigel group with the addition of P-gp inhibitor (P-gpi) was 56.7±6.1. The average fluorescence intensity of the static suspension group without the addition of P-gp inhibitor (P-gpi) was 44.3±4.8, and the average fluorescence intensity of the static suspension group with the addition of P-gp inhibitor (P-gpi) was 54.6±5.2. The average fluorescence intensity of the dynamic suspension group 1 without the addition of P-gp inhibitor (P-gpi) was 31.3±2.9, and the average fluorescence intensity of the dynamic suspension group 1 with the addition of P-gp inhibitor (P-gpi) was 53.8±5.8. The average fluorescence intensity of the dynamic suspension group 2 without the addition of P-gp inhibitor (P-gpi) was 30.2±3.2, and the average fluorescence intensity of the dynamic suspension group 2 with the addition of P-gp inhibitor (P-gpi) was 54.7±5.2. The average fluorescence intensity of the dynamic suspension group 3 without the addition of P-gp inhibitor (P-gpi) was 14.8±1.8, and the average fluorescence intensity of the dynamic suspension group 3 with the addition of P-gp inhibitor (P-gpi) was 55.7±6.2. The average fluorescence intensity of the dynamic suspension group 4 without the addition of P-gp inhibitor (P-gpi) was 17.4±1.9, and the average fluorescence intensity of the dynamic suspension group 4 with the addition of P-gp inhibitor (P-gpi) was 57.5±6.5. The average fluorescence intensity of the dynamic suspension group 5 without the addition of P-gp inhibitor (P-gpi) was 29.5±2.6, and the average fluorescence intensity of the dynamic suspension group 5 with the addition of P-gp inhibitor (P-gpi) was 58.3±6.7.

[0080] As shown in the H&E staining results of FIG. 6, the kidney organoids of the Matrigel group were small in size and had relatively simple tissue structures. The kidney organoids of the static suspension group were larger in size and showed no significant changes in structural characteristics. The kidney organoids of the dynamic suspension group 1 were further increased in size and had relatively complex structures similar to kidney tissues. Compared with the dynamic suspension group 1, the kidney organoids of the dynamic suspension groups 3 and 4 showed chamber-like structures, further increasing the complexity of the tissue structures. Figure 6A Figure 6B ​The results of P-gp transporter function test showed that in dynamic suspension group 1, without adding P-gp transporter inhibitor (P-gpi), the calcein dye in the kidney organoids was transported to the outside of the cells as a heterologous substance, and after adding the inhibitor, the efflux of calcein was significantly blocked, indicating that the kidney organoids in the dynamic suspension culture group had mature excretion function; in contrast, the kidney organoids in the matrigel group did not have efflux of calcein with or without the addition of P-gpi, and there was no significant difference in fluorescence intensity, indicating that the kidney organoids in the matrigel group lacked mature excretion function; the kidney organoids in the static suspension group had moderate excretion function, between the matrigel group and the dynamic suspension group. The kidney organoids in dynamic suspension groups 2-5 also had obvious excretion function, and dynamic suspension groups 3 and 4 had further improved function maturity compared to dynamic suspension group 1.

[0081] The above results show that the suspension culture method is more conducive to the formation of complex tissue structure and mature function of kidney organoids than embedding culture in matrigel, and the dynamic suspension method can further improve the complexity of the tissue structure and the maturity of the function of the kidney organoids. In addition, the design of wave-shaped vortex protrusions or concentric vortex protrusions or vortex grooves on the wall of the culture chamber can produce special hydrodynamic effects, further amplifying the beneficial effects of dynamic culture in improving the complexity of the organoid structure and the maturity of the function.

[0082] According to the results of Examples 2 and 3, although different vortex protrusions or grooves designed on the inner wall of the culture chamber can promote organoid expansion, the wave-shaped vortex protrusion structure is the best for promoting the expansion rate of organoids, and it also improves the complexity of the tissue structure and the maturity of the function of the organoids, so the culture chamber containing the wave-shaped vortex protrusion structure is selected as an example for testing to explore the optimal seeding cell density and the effect of mechanical stimulation intensity on the expansion efficiency and functional maturity of organoids.

[0083] Example Four: Finding the seeding cell density conducive to organoid expansion and functional maturity.

[0084] 1. Take a plurality of 5mL specification culture chambers (with wave-shaped vortex protrusion structure on the inner wall), use a pipette to add 5mL of DPBS solution containing 1% FBS, flick the wall with fingers to remove bubbles, and place at room temperature for a few minutes to wet the wall to prevent organoids from adhering to the wall.

[0085] 2. Take an appropriate amount of tissue sample or organoid, add an appropriate amount of TrypLE digestion solution, and digest at 37°C for 3-8 minutes, observing the digestion under a microscope every 2 minutes during the period, and when more than 90% of the tissue or organoid is digested into single cells, add 4 times the volume of DPBS to terminate digestion.

[0086] 3.600g, 4°C, centrifuge for 5 minutes, remove supernatant, add appropriate amount of DPBS, mix well with pipette gun, count, take appropriate amount of cell suspension, 600g, 4°C, centrifuge for 5 minutes.

[0087] 4. Remove the supernatant, add an appropriate amount of medium, adjust the cell density to 0.25x10 5 / mL, 0.5x10 5 / mL, 1x10 5 / mL, 2x10 5 / mL and 4x10 5 / mL, respectively, transfer 2mL cell suspension to the culture chamber, load the cross-flow reactor back into the culture chamber, and adjust the position of the slider to exclude excess gas in the culture chamber.

[0088] 5. Place each culture chamber on the corresponding rotating device, connect the open end to the conduit of the liquid supply chamber, adjust the rotation speed of the culture chamber to 10-20rpm, and culture at 37°C, 5% CO2.

[0089] 6. After 2-3 days of culture, when the cells aggregate and self-assemble into a certain volume of organoids, adjust the rotation speed according to the size change of the organoids. The rotation speed adjustment strategy is shown in Table 1 above.

[0090] 7. Adjust the frequency of reciprocating movement of the slider of the cross-flow reactor to 10-15 times / min, and the stroke to 5mm, and culture at 37°C, 5% CO2.

[0091] 8. During the culture period, observe the growth state of the organoids regularly, take photos under a microscope on the 10th day, count the number and area of the organoids; after taking photos, half of the organoids are digested into single cells with an appropriate amount of TrypLE digestion solution, and then counted, to compare the differences in number, size, and absolute cell number of the organoids in each group; the remaining half is used for functional experiment determination to compare the differences in functional maturity of the organoids in each group. Thus, the optimal cell seeding density for organoid expansion and functional maturation is determined.

[0092] As shown in Figures 24A-24D , when the cell seeding density is in the range of 0.25x10 5 / mL to 2x10 5 / mL, the number of organoids, the total area of organoids, the number of cells, and the transport function of the kidney organoids all increase in a cell density-dependent manner (i.e., the greater the cell seeding density, the greater the number of organoids, the total area, and the transport function); when the cell seeding density exceeds 2x10 5The number of kidney organoids, total area, cell number and transport function showed a significant downward trend after 2 weeks.

[0093] This shows that when the inoculation cell density is low, increasing the cell density is beneficial to the formation of 3D structure of organoids, the expansion of cell number and the maturation of transport function, and when the inoculation density is too large, it may hinder the expansion efficiency and functional maturation of organoids due to the competition of nutrients and space.

[0094] Example Five: Further based on the above-mentioned culture tank with wave-shaped vortex protruding structure, the influence of mechanical stimulation intensity on the expansion efficiency and functional maturation of organoids is tested.

[0095] 1. Take a 5 mL size culture tank (with wave-shaped vortex protruding structure), remove the cross-flow reaction generator, use a pipette to add 5 mL of DPBS solution containing 1% FBS, gently shake the tube wall to remove bubbles, and place it at room temperature for a few minutes to wet the tube wall and prevent organoids from adhering to the tube wall;

[0096] 2. Take an appropriate amount of tissue sample or organoid, add an appropriate amount of TrypLE digestion solution, and digest at 37°C for 3-8 minutes. During the digestion, observe the digestion under a microscope every 2 minutes. When more than 90% of the tissue or organoid is digested into single cells, add 4 times the volume of DPBS to terminate the digestion;

[0097] 3. 600g, 4°C, centrifuge for 5 minutes, remove the supernatant, add an appropriate amount of DPBS, mix well with a pipette gun, and then count, take an appropriate amount of cell suspension, 600g, 4°C, centrifuge for 5 minutes.

[0098] 4. Remove the supernatant, add an appropriate amount of culture medium, adjust the cell density to 2x10 5 / mL, transfer 2 mL of cell suspension to each culture tank, load the cross-flow reaction generator back into the culture tank, and adjust the position of the slide to remove excess gas in the culture tank.

[0099] 5. Place each culture tank on the corresponding rotating device, connect the open end to the conduit of the liquid supply tank, adjust the rotation speed of the culture tank to 10rpm -20rpm, and culture at 37°C, 5% CO2.

[0100] 6. After 2-3 days of culture, the cell aggregates and self-assemble into a certain volume of organoids, adjust the rotation speed according to the size change of the organoids.

[0101] 7. Adjust the frequency and stroke of the piston propulsion device, and set up 6 groups of mechanical stimulation conditions with different intensities, in the following order: Group 1 (frequency 10 times / min, stroke 5 mm), Group 2 (frequency 20 times / min, stroke 5 mm), Group 3 (frequency 30 times / min, stroke 5 mm), Group 4 (frequency 10 times / min, stroke 10 mm), Group 5 (frequency 20 times / min, stroke 10 mm), and Group 6 (frequency 30 times / min, stroke 10 mm). Incubate at 37℃ and 5% CO2.

[0102] 8. During the culture period, the growth status of the organoids was observed regularly. On the 10th day, photographs were taken under a microscope to count the number and area of ​​organoids. After photographing, half of the organoids were digested into single cells using an appropriate amount of TrypLE digestion solution and counted. Differences in number, size, and absolute cell count among the groups of organoids were compared. The remaining half was used for functional experiments to compare differences in functional maturity among the groups of organoids. This was to determine the mechanical stimuli that were conducive to organoid proliferation and functional maturation.

[0103] like Figures 25A-25D As shown, compared with other groups, group 5 (frequency, stroke 10 mm) showed a significant improvement in the number of organoids, total organoid area, cell number, and maturity of transport function in kidney organoids. This indicates that setting the frequency of the crossflow reactor to 20 times / min and the stroke to 10 mm, combined with the design of the wavy convex structure on the inner wall of the culture chamber, can provide specific mechanical stimulation that is conducive to organoid expansion and the maturation of transport function, significantly amplifying the beneficial effects of dynamic culture. However, when the mechanical intensity is further increased, such as in group 6 (frequency 30 times / min, stroke 10 mm), the high-intensity, high-frequency fluid motion may cause high shear force and excessive mechanical stimulation, affecting the cell viability of organoids, causing cell detachment and dissociation of organoid structures, thereby hindering organoid expansion and functional maturation.

[0104] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0105] Those skilled in the art can clearly understand the above-mentioned embodiment method can be realized by means of software and necessary general hardware platform, of course, also can be realized by hardware, but in many cases, the former is a better embodiment. Based on such understanding, the technical solutions of the present application essentially or say the part of contribution to the prior art can be embodied in the form of software product, the computer software product is stored in a storage medium (such as ROM / RAM, disk, optical disk), including a plurality of instructions to make a computer terminal (may be a mobile phone, computer, server, or network equipment, etc.) executes the method described in various embodiments of the present application.

[0106] The embodiments of the present application are described above in combination with the drawings, but the present application is not limited to the above-mentioned specific embodiments, the above-mentioned specific embodiments are only illustrative, but not limited, those skilled in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, which all belong to the protection of the present application.

Claims

1. A dynamic culture device for organoids, characterized in that, include: A culture chamber (1) is connected to a cross-flow reaction generator (103) at one end and to a liquid supply chamber (5) at the other end of the culture chamber (1) away from the cross-flow reaction generator (103). The culture chamber (1) has a first opening (104) at the end connected to the liquid supply chamber (5). The liquid supply chamber (5) has a second opening (501) at the top. The first opening (104) and the second opening (501) are connected through a conduit (6). The first opening (104) and the second opening (501) are provided with hydrophobic filter membranes. The culture chamber (1) is connected to a second drive unit through a transmission mechanism. The cross-flow reaction generator (103) is rotatably connected to the first drive unit. The culture chamber (1) includes a first region near the crossflow reactor (103) and a second region near the liquid supply chamber (5). The inner wall of the second region is provided with spiral vortex protrusions or wave-shaped vortex protrusions extending along the axial direction of the culture chamber (1). Alternatively, the inner wall of the second region is provided with a plurality of concentric annular vortex protrusions spaced apart along the axial direction of the culture chamber (1) or a plurality of petal-shaped vortex grooves adjacent to each other along the axial direction of the culture chamber (1). In the first stage of cultivation, driven by the second driving unit, the transmission mechanism drives the cultivation chamber (1) to rotate, causing the liquid in the cultivation chamber (1) to form an annular Taylor vortex; In the second stage of cultivation, driven by the first driving unit, the execution end of the cross-flow reaction generator (103) reciprocates along the inner wall of the first region, causing the liquid in the cultivation chamber (1) to collide with the vortex protrusions or vortex grooves in the inner wall of the cultivation chamber (1) under the action of the cross-flow reaction generator (103), thereby forming a vortex.

2. The dynamic culture device for organoids according to claim 1, characterized in that, The crossflow reaction generator (103) includes a drive rod with one end extending into a first region of the culture chamber (1), a slider (105) rotatably connected to the drive rod and conforming to the inner wall of the first region, and a hemispherical tip disposed on the slider (105) near the vortex protrusion. The hemispherical tip is provided with a plurality of spiral grooves at intervals along the axial direction of the hemispherical tip; and each spiral groove corresponds to a spiral vortex protrusion, such that each spiral groove is an extension of the spiral vortex protrusion. Alternatively, the spherical tip of the hemispherical shape is provided with a plurality of wavy grooves that extend axially along the tip of the hemispherical shape at intervals; and each wavy groove corresponds to a wavy vortex protrusion, such that each wavy groove is an extension of the wavy vortex protrusion. When the culture chamber (1) rotates, the culture chamber (1) drives the slider (105) to rotate relative to the drive rod; When the first driving unit drives the driving rod to rotate relative to the culture chamber (1), the slider (105), which is the execution end, rotates synchronously with the culture chamber (1) and drives the hemispherical tip to reciprocate along the inner wall of the first region.

3. The dynamic culture device for organoids according to claim 2, characterized in that, The depth of the spiral groove gradually decreases along the axial direction of the hemispherical tip and away from the spiral vortex protrusion, or the radius of the wavy groove gradually decreases along the axial direction of the hemispherical tip and away from the wavy vortex protrusion.

4. The dynamic culture device for organoids according to claim 1, characterized in that, The radius, pitch, and helical height of the spiral vortex protrusion are 8mm-12mm, 40mm-50mm, and 32mm-39mm, respectively. Alternatively, the wave-shaped vortex protrusions are at least six adjacent to each other along the axial direction, and each wave-shaped vortex protrusion has a radius of 1.5mm-3.5mm as a semicircle. Alternatively, the concentric annular vortex protrusions are at least six spaced apart along the axial direction, and the radius of the concentric annular vortex protrusions decreases sequentially along the axial direction of the culture chamber (1) and in the direction close to the crossflow reactor. Alternatively, the petal-shaped vortex groove is a plurality of arc-shaped grooves continuously arranged along the circumference of the culture chamber (1), and the serration height between two adjacent arc-shaped grooves is 1.5mm-3mm, and the radius of the arc-shaped groove is 34mm-38mm.

5. The dynamic culture device for organoids according to any one of claims 1 to 4, characterized in that, The cross section of the spiral vortex protrusion perpendicular to the axial direction of the culture chamber (1) is an equilateral triangle.

6. The dynamic culture device for organoids according to claim 1, characterized in that, Also includes: The support shell (3) is used to fix the culture chamber (1) on the top of the support shell (3) by means of a fixing device. The fixing device includes a fixing part fixed to the top of the support shell (3) and a semi-circular part connected to the fixing part. The semi-circular part is configured as an openable hollow center structure, and the culture chamber (1) is fixed or removed by means of the openable semi-circular part.

7. The dynamic culture device for organoids according to claim 2, characterized in that, The first drive unit includes: a first motor (2), a first drive wheel (201) connected to the output shaft of the first motor (2), and the outer surface of the first drive wheel (201) is provided with a thread that can engage with the external thread on the drive rod in the cross-flow reaction generator (103); When the first motor (2) drives the first drive wheel (201) to rotate continuously in clockwise and counterclockwise directions, the crossflow reaction generator (103) reciprocates along the axial direction, thereby causing the slider (105) to reciprocate along the inner wall of the first region in the culture chamber (1).

8. The dynamic culture device for organoids according to claim 1, characterized in that, The second drive unit includes: a second motor (4) and a second drive wheel (401) connected to the output shafts at both ends of the second motor (4); Driven by the second motor (4), the two second drive wheels (401) drive the driven gears set at both ends of the culture chamber (1) to rotate, thereby driving the culture chamber (1) to rotate.

9. The dynamic culture device for organoids according to claim 6, characterized in that, The bracket housing (3) is a cuboid, and the first drive unit, the second drive unit and the liquid supply tank (5) are located inside the bracket housing (3).

10. A method for dynamic culture of organoids, characterized in that, include: Dynamic culture of organoids is performed using the dynamic culture apparatus for organoids according to any one of claims 1 to 9; The growth images of organoids in the culture chamber (1) of the dynamic culture device are periodically acquired using the image acquisition module; The main control module performs image processing on the growth images of organoids in the culture chamber (1) acquired by the image acquisition module to obtain the current size of the organoids, and determines whether the current size is greater than or equal to a first preset threshold. If the current size of the organoid in the culture chamber (1) is less than the first preset threshold, the second drive unit is controlled to maintain the current first preset rotation speed; If the current size of the organoid in the culture chamber (1) is greater than or equal to the first preset threshold, but less than the second preset threshold, the second drive unit is controlled to adjust its current first preset speed to the second preset speed; the second preset speed is greater than the first preset speed. When it is determined that the current size of the organoid in the culture chamber (1) is greater than or equal to the second preset threshold, but less than the preset third preset threshold, the second drive unit is controlled to maintain the current second preset speed while the first drive unit is controlled to rotate, so that the execution end of the crossflow reaction generator (103) reciprocates along the inner wall of the culture chamber (1); wherein the frequency of the reciprocating motion of the crossflow reaction generator (103) is 10 times / min-15 times / min, and the stroke is 5mm-10mm.

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