Fluidized bed bioreactor and large-scale culture device for organoids and cells

Abstract

The invention belongs to the technical field of biology, and discloses a fluidized bed type bioreactor and an organoid and cell large-scale culture device. The fluidized bed type bioreactor comprises a bottom flow guide structure, a main reaction cavity and a top buffer structure which are coaxially connected from bottom to top; the bottom flow guide structure comprises a lower circular truncated cone cavity and a reactor inlet, the upper end cross section of the lower circular truncated cone cavity is larger than the lower end cross section of the lower circular truncated cone cavity, and the lower end cross section of the lower circular truncated cone cavity is equal to the cross section of the reactor inlet; the main reaction cavity comprises a main cylindrical cavity and a sampling port, and the cross section of the main cylindrical cavity is equal to the cross section of the upper end of the lower circular truncated cone cavity; the top buffer structure comprises an upper circular truncated cone cavity, an upper cylindrical cavity and a reactor outlet; the upper end cross section of the upper circular truncated cone cavity is larger than the lower end cross section of the upper circular truncated cone, the upper end cross section of the upper circular truncated cone cavity is equal to the lower end cross section of the upper cylindrical cavity, and the lower end cross section of the upper circular truncated cone cavity is equal to the cross section of the main cylindrical cavity. The method has high throughput and high biocompatibility, can be used for standardized and stable culture of organoid and cells, and has reliable experimental data.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a fluidized bed type bioreactor and a device for large-scale culture of organoids and cells. BACKGROUND

[0002] As a revolutionary breakthrough in the field of biomedicine, organoid technology has received high attention from major technological powers around the world in recent years. The core goal is to simulate the physiological structure and function of real organs, and to promote the development of precision medicine, drug research and development, and regenerative medicine. Although the fluidized bed technology is widely used in the fields of chemical industry and energy, its application in organoid culture is still in the exploratory stage.

[0003] The existing organoid culture is difficult to achieve large-scale culture due to the large difference in size and structure between the microspheres and real organs, and the limitation of mass transfer. The organoid culture has heterogeneity, and it is difficult to achieve standardized culture, which limits its engineering application. SUMMARY

[0004] The present application aims to at least solve one of the technical problems in the related art. To this end, one object of the present application is to provide a fluidized bed type bioreactor with high throughput and high biological compatibility, which can be used for standardized and stable culture of organoids and cells, and the experimental data is reliable.

[0005] According to the fluidized bed type bioreactor of the first aspect of the present application, the bottom flow guide structure, the main reaction cavity and the top buffer structure are coaxially connected from bottom to top; the bottom flow guide structure comprises a lower frustum cavity and a reactor inlet coaxially connected from top to bottom, the upper end cross section of the lower frustum cavity is larger than the lower end cross section of the lower frustum cavity, and the lower end cross section of the lower frustum cavity is equal to the cross section of the reactor inlet; the main reaction cavity comprises a main cylindrical cavity and a sampling port connected to the side surface of the main cylindrical cavity, and the cross section of the main cylindrical cavity is equal to the upper end cross section of the lower frustum cavity; the top buffer structure comprises an upper cylindrical cavity and an upper frustum cavity coaxially connected from top to bottom, and a reactor outlet connected to the side surface of the upper cylindrical cavity; the upper end cross section of the upper frustum cavity is larger than the lower end cross section of the upper frustum, the upper end cross section of the upper frustum cavity is equal to the cross section of the upper cylindrical cavity, and the lower end cross section of the upper frustum cavity is equal to the cross section of the main cylindrical cavity.

[0006] In the fluidized bed bioreactor of the first aspect of the present application, during the fluidized culture, the culture medium flows from the reactor inlet into the fluidized bed bioreactor and flows upward, and flows out from the reactor outlet; and the microspheres as the carriers of organoids and cell culture during the fluidized culture are located in the main cylindrical cavity of the main reaction cavity, and the culture medium penetrates into the microspheres to grow cells; the microsphere samples are taken out through the sampling port to dynamically monitor the growth of organoids and cells. The size of the microspheres can be adjusted in the range of 30 microns to 2 millimeters in diameter according to the needs, and the material of the microspheres can be various materials including natural gel materials and various polymer materials; the structure of the microspheres includes core-shell structure, porous structure, multi-layer structure, hollow structure, homogeneous structure, responsive structure, etc. The size variation coefficient of the gel microspheres is within 5%, the culture environment of each microsphere is almost equal, and the uniformity is good, which is conducive to the standardized culture of organoids and cells. Due to the design of the lower circular cone cavity, the flow field distribution can be effectively improved and the fluidized dead angle of the traditional flat bottom structure can be eliminated; the top buffer structure has an upper circular cone cavity and a cylindrical cavity, which can effectively realize the smooth transition of the flow field and prevent the impact of microspheres caused by high-speed fluidization, avoid the damage or deformation of microspheres, and be conducive to the stable culture of organoids and cells; according to the size of the microsphere diameter and the number of microsphere culture, through fluidization simulation and actual experiment, the flow rate range suitable for the size and number of the microspheres is obtained, the lowest effective fluidization speed is selected under the premise of ensuring the mixing efficiency and mass transfer, so that the microspheres can almost be free from fluid shear stress damage, and the fluidized bed bioreactor of the first aspect of the present application has high biological compatibility.

[0007] The fluidized bed bioreactor of the first aspect of the present application can adopt a multi-scale design. For example, a large fluidized bed bioreactor has a main cylindrical cavity with a radial dimension of 50 mm, a height of 150 mm, an upper circular truncated cone cavity with a height of 25 mm, an upper cylindrical cavity with a height of 25 mm, a radial dimension of 100 mm, a reactor outlet with a radial dimension of 8 mm, a lower circular truncated cone cavity with a height of 50 mm, and a reactor inlet with a radial dimension of 8 mm. The circulating medium volume of the large fluidized bed bioreactor is in the order of tens of thousands of liters, and can be used for the cultivation of millions of microspheres, and one microsphere can be used for the cultivation of hundreds of cells. In actual use, the scale can be appropriately enlarged or reduced according to requirements. For another example, a small fluidized bed bioreactor has a main cylindrical cavity with a radial dimension of 10 mm, a height of 50 mm, an upper circular truncated cone cavity with a height of 10 mm, an upper cylindrical cavity with a height of 10 mm, a radial dimension of 30 mm, a reactor outlet with a radial dimension of 3 mm, a lower circular truncated cone cavity with a height of 10 mm, and a reactor inlet with a radial dimension of 3 mm. The circulating medium volume of the small fluidized bed bioreactor is in the order of tens of milliliters, and in actual use, the scale can be appropriately enlarged or reduced according to requirements. That is, the fluidized bed bioreactor of the first aspect of the present application adopts a multi-scale design, can be used for the cultivation of ultra-large-scale microspheres, and can be used for high-throughput cell cultivation.

[0008] The fluidized bed bioreactor of the first aspect of the present application can support non-destructive sampling and real-time quality monitoring during fluidized culture experiments by providing the sampling port, and ensure the reliability and repeatability of experimental data.

[0009] In summary, the fluidized bed bioreactor of the first aspect of the present application has the following advantages: through multi-scale design, it can be used for the cultivation of ultra-large-scale microspheres with tens of milliliters to tens of thousands of liters of circulating medium, high-throughput cell cultivation, a microsphere size variation coefficient within 5%, almost equal cultivation environment for each sphere, good uniformity, and high biocompatibility of the fluidized bed bioreactor, almost no fluid shear stress damage, and stable cultivation of organoids and cells. The fluidized bed bioreactor of the first aspect of the present application can be used for dynamic cultivation in different application scenarios, and the experimental data is reliable.

[0010] In some embodiments, the fluidized bed bioreactor is made of transparent glass.

[0011] In some embodiments, the bottom flow guide structure, the main reaction cavity, and the top buffer structure are independent pieces, and the bottom flow guide structure and the main reaction cavity, and the main reaction cavity and the top buffer structure are respectively detachably and sealingly connected.

[0012] In some embodiments, the angle of the side cavity surface of the upper conical cavity relative to the horizontal plane is 135 degrees.

[0013] In some embodiments, the structural dimensions of the fluidized bed bioreactor are obtained through fluidization simulation optimization calculation and experimental verification.

[0014] The second object of the present invention is to provide a large-scale organoid and cell culture device.

[0015] The large-scale organoid and cell culture device according to the second embodiment of the present invention comprises an oxygen supply device, a liquid storage device, a power pump, a dissolved oxygen detector, and a fluidized bed bioreactor according to the first embodiment of the present invention;

[0016] Wherein, the oxygen supply device is connected to the liquid storage device and is used to input oxygen into the liquid storage device;

[0017] The liquid storage device, the power pump, the dissolved oxygen detector and the fluidized bed bioreactor are connected end to end in sequence to form a closed circulation system. The liquid storage device is used to store culture medium, the power pump is used to provide power for the circulation of culture medium, and the dissolved oxygen detector is used to detect the oxygen concentration in the culture medium before entering the fluidized bed bioreactor. The fluidized bed bioreactor is used to provide space for organoid and cell culture.

[0018] Since the large-scale organoid and cell culture device of the second embodiment of the present invention adopts the fluidized bed bioreactor of the first embodiment of the present invention, the large-scale organoid and cell culture device of the second embodiment of the present invention has basically the same technical effects as the fluidized bed bioreactor of the first embodiment of the present invention, and will not be repeated here.

[0019] In some embodiments, a dynamic monitoring system is further included, and the dynamic monitoring system is connected to the sampling port through a sampling control valve.

[0020] In some embodiments, a culture supernatant metabolite detection system is further included; an outlet control valve is provided between the reactor outlet and the liquid storage device, and the culture supernatant metabolite detection system is connected to the outlet control valve.

[0021] In some embodiments, the system further comprises a fixing bracket for fixing the fluidized bed bioreactor.

[0022] In some embodiments, the liquid storage device comprises a liquid storage bottle, the top of which is provided with a circulating medium inlet connected to the reactor outlet, a circulating medium outlet connected to the power pump, a sterile oxygen inlet connected to the oxygen supply device, and a sterile oxygen outlet for external discharge.

[0023] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the fluidized bed bioreactor of the present application;

[0025] Figure 2 is a schematic diagram of the organoid and cell large-scale culture device of the present application;

[0026] Figure 3 is a schematic diagram of the organoid and cell large-scale culture device of the present application;

[0027] Figure 4 is a partial schematic diagram of the liquid storage device; Figure 3

[0028] Figure 5 is a diagram of a comparative experiment of the organoid and cell large-scale culture device of the present application and a conventional culture dish on the mass transfer of 500-micron-diameter microspheres in a 4-kDa FITC-dextran solution as the culture medium under fluidized culture and static culture;

[0029] Figure 6 is a diagram of a comparative experiment of the organoid and cell large-scale culture device of the present application and a conventional culture dish on the mass transfer of 500-micron-diameter microspheres in a 10-kDa FITC-dextran solution as the culture medium under fluidized culture and static culture;

[0030] Figure 7 is a diagram of a comparative experiment of the organoid and cell large-scale culture device of the present application and a conventional culture dish on the mass transfer of 500-micron-diameter microspheres in a 70-kDa FITC-dextran solution as the culture medium under fluidized culture and static culture;

[0031] Figure 8 is a fluorescence imaging diagram of the organoid and cell large-scale culture device of the present application on the fluidized culture of 500-micron-diameter microspheres in a 70-kDa FITC-dextran solution as the culture medium at a specified time;

[0032] Figure 9 ​Fluorescence imaging of 500 μm diameter microspheres at the indicated times during static culture in a 70 kDa FITC-dextran solution in a conventional culture dish.

[0033] Large-scale organoid and cell culture device 1000; fluidized bed bioreactor 100; bottom guide structure 11; reactor inlet 111; lower conical cavity 112; main reaction chamber 12; main cylindrical cavity 121; sampling port 112; top buffer structure 13; upper conical cavity 131; upper cylindrical cavity 132; reactor outlet 133; oxygen supply device 200; oxygen pump 21; liquid storage device 300; liquid storage bottle 31; circulating culture medium inlet 311; circulating culture medium outlet 312; sterile oxygen inlet 313; sterile oxygen outlet 314; power pump 400; pre-pump control valve 41; dissolved oxygen detector 500; dynamic monitoring system 600; sampling control valve 61; culture supernatant metabolite detection system 700; outlet control valve 71. DETAILED DESCRIPTION

[0034] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0035] The following combination Figures 1 to 9 The fluidized bed bioreactor 100 according to the first embodiment and the large-scale organoid and cell culture device 1000 according to the second embodiment of the present invention are described below.

[0036] like Figure 1 As shown, the fluidized bed bioreactor 100 according to the first embodiment of the present invention includes a bottom guide structure 11, a main reaction chamber 12 and a top buffer structure 13 coaxially connected from bottom to top. The bottom guide structure 11 includes a lower conical cavity 112 and a reactor inlet 111 coaxially connected above and below. The upper cross-section of the lower conical cavity 112 is larger than the lower cross-section of the lower conical cavity 112, and the lower cross-section of the lower conical cavity 112 is equal to the cross-section of the reactor inlet 111; the main reaction cavity 12 includes a main cylindrical cavity 121 and a sampling port 112 connected to the side of the main cylindrical cavity 121, and the cross-section of the main cylindrical cavity 121 is equal to the upper cross-section of the lower conical cavity 112; the top buffer structure 13 includes an upper cylindrical cavity 132 and an upper conical cavity 131 coaxially connected above and below, and a reactor outlet 133 connected to the side of the upper cylindrical cavity 132; the upper cross-section of the upper conical cavity 131 is equal to the cross-section of the upper cylindrical cavity 132, and the lower cross-section of the upper conical cavity 131 is equal to the cross-section of the main cylindrical cavity 121.

[0037] The fluidized bed bioreactor 100 of the first aspect embodiment of the present application, in the fluidized culture, the culture medium enters the fluidized bed bioreactor 100 from the reactor inlet 111 and flows upward, and flows out from the reactor outlet 133; and the microspheres in the fluidized culture process are used as carriers for organoid and cell culture, and are located in the main cylindrical cavity 121 of the main reaction cavity 12, the culture medium penetrates into the microspheres to grow cells; the microsphere samples are taken out through the sampling port 112, so as to dynamically monitor the growth of the organoid and cells. Wherein, the size of the microspheres can be adjusted in the range of 30 microns to 2 millimeters in diameter according to the needs, and the material of the microspheres can be various materials, including natural gel materials and various polymer materials; the structure of the microspheres includes core-shell structure, porous structure, multi-layer structure, hollow structure, homogeneous structure, responsive structure, etc. The size variation coefficient of the gel microspheres is within 5%, the culture environment of each microsphere is almost equal, and the uniformity is good, which is beneficial to realize the standardized culture of the organoid and cells. Due to the design of the lower circular cone cavity, the flow field distribution can be effectively improved and the fluidized dead angle of the traditional flat bottom structure can be eliminated; the top buffer structure 13 has an upper circular cone cavity and a cylindrical cavity, which can effectively realize the smooth transition of the flow field and prevent the microspheres from being impacted due to high-speed fluidization, avoid the damage or deformation of the microspheres, and be beneficial to realize the stable culture of the organoid and cells; according to the size of the microspheres and the number of the microspheres, through fluidization simulation and actual experiment, the flow rate range suitable for the size and number of the microspheres is obtained, under the premise of ensuring the mixing efficiency and mass transfer, the lowest effective fluidization speed is selected, so that the microspheres can almost avoid the damage of fluid shear stress, so that the fluidized bed bioreactor 100 of the first aspect embodiment of the present application has high biological compatibility.

[0038] The fluidized bed bioreactor 100 of the first aspect of the present application can adopt a multi-scale design. For example, a large fluidized bed bioreactor 100, the radial dimension of the main cylindrical cavity 121 is 50 mm, the height of the main cylindrical cavity 121 is 150 mm, the height of the upper circular table cavity 131 is 25 mm, the height of the upper cylindrical cavity 132 is 25 mm, the radial dimension of the upper cylindrical cavity 132 is 100 mm, the radial dimension of the reactor outlet 133 is 8 mm, the height of the lower circular table cavity 112 is 50 mm, and the radial dimension of the reactor inlet 111 is 8 mm. The circulating medium volume of the large fluidized bed bioreactor 100 is in the order of tens of thousands of liters, and can be used for the cultivation of millions of microspheres, and one microsphere can be used for the cultivation of hundreds of cells. In actual use, appropriate magnification or reduction can be made according to the needs. For another example, a small fluidized bed bioreactor 100, the radial dimension of the main cylindrical cavity 121 is 10 mm, the height of the main cylindrical cavity 121 is 50 mm, the height of the upper circular table cavity 131 is 10 mm, the height of the upper cylindrical cavity 132 is 10 mm, the radial dimension of the upper cylindrical cavity 132 is 30 mm, the radial dimension of the reactor outlet 133 is 3 mm, the height of the lower circular table cavity 112 is 10 mm, and the radial dimension of the reactor inlet 111 is 3 mm. The circulating medium volume of the small fluidized bed bioreactor 100 is in the order of tens of milliliters, and appropriate magnification or reduction can be made according to the needs in actual use. That is, the fluidized bed bioreactor 100 of the first aspect of the present application adopts a multi-scale design, can be used for super-large-scale microsphere cultivation, and can be used for high-throughput cell cultivation.

[0039] The fluidized bed bioreactor 100 of the first aspect of the present application can support non-destructive sampling and real-time quality monitoring during fluidized culture experiments by providing the sampling port 112, and ensure the reliability and repeatability of experimental data.

[0040] In summary, the fluidized bed bioreactor 100 of the first aspect of the present application has the following advantages: through multi-scale design, super-large-scale microsphere cultivation can be carried out with tens of milliliters to tens of thousands of liters of circulating medium, high-throughput cell cultivation can be carried out, the size variation coefficient of the microsphere is within 5%, the cultivation environment of each sphere is almost equal, the uniformity is good, and the fluidized bed bioreactor 100 has high biocompatibility, almost eliminates fluid shear stress damage, and can realize stable cultivation of organoids and cells. The fluidized bed bioreactor 100 of the first aspect of the present application can be used for dynamic cultivation in different application scenarios, and the experimental data is reliable.

[0041] In some embodiments, the fluidized bed bioreactor 100 is made of transparent glass. Specifically, the fluidized bed bioreactor 100 is precisely machined from a high-transmittance glass material, has good chemical stability and mechanical strength, and is conducive to ensuring the overall stability of the system during dynamic fluidization of cells; the fluidized bed bioreactor 100 has transparency, realizes visual monitoring of the organoid and cell culture experiment process, and facilitates real-time observation of the fluidization state of cells and timely optimization of experimental parameters.

[0042] In some embodiments, the bottom flow guide structure 11, the main reaction cavity 12, and the top buffer structure 13 are independent pieces, and the bottom flow guide structure 11 and the main reaction cavity 12 and the main reaction cavity 12 and the top buffer structure 13 are respectively detachably and sealingly connected. In this way, the bottom flow guide structure 11, the main reaction cavity 12, and the top buffer structure 13 are convenient to process, and the bottom flow guide structure 11 and the main reaction cavity 12 and the main reaction cavity 12 and the top buffer structure 13 are respectively detachably and sealingly connected, which can ensure the airtightness of the fluidized bed bioreactor 100 and prevent leakage. Specifically, the bottom flow guide structure 11 and the main reaction cavity 12 and the main reaction cavity 12 and the top buffer structure 13 are all bolted after being sealingly clamped by flanges.

[0043] In some embodiments, the angle between the side cavity surface of the upper circular truncated cone cavity 131 and the lower end surface of the upper circular truncated cone cavity 131 is 135 degrees. In this way, the stable transition of the flow field can be effectively realized, and the impact of microspheres caused by high-speed fluidization can be prevented, the microspheres can be prevented from being damaged or deformed, and stable culture of organoids and cells can be facilitated.

[0044] In some embodiments, the structure size of the fluidized bed bioreactor 100 is obtained through fluidization simulation optimization calculation and experimental verification. The design of the fluidized bed bioreactor 100 fully considers the fluid mechanics characteristics and operational convenience, and each structural parameter is optimized and verified through calculation and experiment, which can provide a stable and reliable fluidization environment for biological processes such as cell culture.

[0045] As Figures 1 to 4As shown, the organoid and cell large-scale culture device 1000 according to the second aspect of the present application comprises an oxygen supply device 200, a liquid storage device 300, a power pump 400, a dissolved oxygen detector 500, the fluidized bed bioreactor 100 according to the first aspect of the present application; wherein the oxygen supply device 200 is connected to the liquid storage device 300, for inputting oxygen into the liquid storage device 300, and the oxygen is high-purity (99.999%) oxygen; the liquid storage device 300, the power pump 400, the dissolved oxygen detector 500 and the fluidized bed bioreactor 100 are sequentially connected end to end to form a closed circulation system, the liquid storage device 300 is used for storing culture medium, the power pump 400 is used for providing power for the circulation flow of the culture medium, the dissolved oxygen detector 500 is used for detecting the oxygen concentration in the culture medium before entering the fluidized bed bioreactor 100, and the fluidized bed bioreactor 100 is used for providing a space for organoid and cell culture.

[0046] It should be noted that the connections among the oxygen supply device 200, the liquid storage device 300, the power pump 400, the dissolved oxygen detector 500 and the fluidized bed bioreactor 100 in the organoid and cell large-scale culture device 1000 according to the second aspect of the present application are all connected through pipelines, such as silica gel pipelines.

[0047] Since the organoid and cell large-scale culture device 1000 according to the second aspect of the present application adopts the fluidized bed bioreactor 100 according to the first aspect of the present application, the organoid and cell large-scale culture device 1000 according to the second aspect of the present application has basically the same technical effects as the fluidized bed bioreactor 100 according to the first aspect of the present application, and thus will not be described here.

[0048] In some embodiments, a dynamic monitoring system 600 is further included, which is connected to the sampling port 112 through a sampling control valve 61, and the dynamic monitoring system 600 is used for monitoring the cell growth in the microspheres. By connecting the sampling control valve 61 to the dynamic monitoring system 600, the lossless sampling and real-time quality monitoring can be supported according to the needs during the fluidized culture experiment, so as to ensure the reliability and repeatability of the experimental data.

[0049] In some embodiments, a culture supernatant metabolite detection system 700 is further included; an outlet control valve 71 is arranged between the reactor outlet 133 and the liquid storage device 300, and the culture supernatant metabolite detection system 700 is connected to the outlet control valve 71. By connecting the outlet control valve 71 to the culture supernatant metabolite detection system 700, the culture supernatant sample can be taken out according to the needs during the fluidized culture experiment, so as to analyze the substances secreted by the cells into the culture medium.

[0050] In some embodiments, a fixing support (not shown in the figure) is further included to fix the fluidized bed bioreactor 100, so as to ensure the overall stability of the organoid and cell large-scale culture device 1000 of the second aspect of the present application during the dynamic fluidization of the cells. Specifically, a metal ring, which can be a stainless steel ring, is arranged on the fixing support, and the fluidized bed bioreactor 100 is fixed in the metal ring.

[0051] In some embodiments, the power pump 400 is a high-precision peristaltic pump, which can provide power for the circulating flow of the culture medium and accurately control the flow rate. However, the power pump 400 is not limited to the high-precision peristaltic pump, and can also be other types of power pumps 400. The dissolved oxygen detector 500 is a dissolved oxygen sensor, which can accurately measure the oxygen concentration in the culture medium.

[0052] In some embodiments, the oxygen supply device 200 includes an oxygen pump 21 for pumping oxygen into the liquid storage device 300.

[0053] In some embodiments, the liquid storage device 300 includes a liquid storage bottle 31, the top of which is provided with a circulating medium inlet 311 connected to the reactor outlet 133, a circulating medium outlet 312 connected to the power pump 400, a sterile oxygen outlet 314 connected to the oxygen supply device 200, and a sterile oxygen outlet for external discharge. It should be noted that the sterile oxygen inlet 313 and the sterile oxygen outlet 314 arranged at the liquid storage bottle 31 can construct a fluidized bed dynamic culture system integrated with the oxygen supply device 200. The flow rate of oxygen can be adjusted according to the high-precision rotor flow valve.

[0054] In some embodiments, a pre-pump control valve 41 is arranged between the liquid storage device 300 and the power pump 400.

[0055] The organoid and cell large-scale culture device 1000 of the second aspect of the present application can visually observe the gel microspheres during the fluidized culture process, detect the mass transfer and diffusion process, optimize the fluidized bed culture conditions and other parameters based on the detection results of the equilibrium time of the fluidized bed and the recovery efficiency of the microspheres, and preliminarily realize the stable culture of the cell lines and organoids.

[0056] The following gives some comparative experiments of the fluidized culture experiment of the organoid and cell large-scale culture device 1000 of the second aspect of the present application and the conventional culture, so as to further illustrate the advantages of the organoid and cell large-scale culture device 1000 of the second aspect of the present application and the fluidized bed bioreactor 100.

[0057] Example 1: Fluidized culture / static culture mass transfer of microspheres with a diameter of 500 microns

[0058] Experimental purpose: Through different molecular weight FITC-dextran labeling, real-time fluorescence microscopy imaging, analysis of real-time permeability quantitative data, comparison of mass transfer differences between microsphere fluidization / static culture.

[0059] The experimental operation steps are as follows:

[0060] S1: Dissolve the labeled FITC-dextran in DPBS to prepare a 10 μg / mL solution as the culture medium. Take out part of the culture medium and inject it into the organoid and cell large-scale culture device 1000 of the second aspect of the application. Cultivate cells in the microspheres in the fluidized bed bioreactor 100 by circulating the culture medium; take out part of the culture medium and inject it into the culture dish containing the microspheres, and soak the microspheres thoroughly.

[0061] S2: Real-time fluorescence microscopy imaging of the microspheres in the fluidized bed bioreactor 100 and the microspheres in the culture dish, respectively; for 4kDa FITC-dextran, record the fluorescence image within 0-5 minutes, take a picture every 1 minute; for 10kDa FITC-dextran, record the fluorescence image within 0-30 minutes, take a picture every 5 minutes; for 70kDa FITC-dextran, as shown in Figure 8 and Figure 9 , record the fluorescence image within 0-120 minutes, and 12 hours, 24 hours, record the fluorescence image at 0, 5, 10, 20, 30, 60, 90, 120 minutes within 0-120 minutes.

[0062] S3: Fluorescence intensity of fluorescence image. Specifically, the fluorescence intensity of the fluorescence image is obtained by ImageJ software. For a single-channel (monochrome) fluorescence picture, the gray value of each pixel represents the fluorescence intensity of that point. The fluorescence intensity formula of a specific area: average fluorescence intensity = total fluorescence intensity of the area / area of the area.

[0063] Experimental conclusion: From Figures 5 to 7 It can be seen that the microspheres have appropriate permeability and can exchange nutrients with the external microenvironment, and FITC-dextran within a certain mass range (4kDa-70kDa) can realize effective diffusion; low molecular weight substances diffuse rapidly in the microspheres, and high molecular weight substances diffuse slowly in the microspheres; fluidized culture can accelerate the diffusion process.

[0064] Example 2: Fluidization / static culture of calcium alginate microspheres with a diameter of 500 microns.

[0065] The experimental operation steps are as follows: microspheres wrap THLE cells. The circulating medium volume is 30 mL.

[0066] S1: 2000 microspheres with a diameter of 500 microns were added to the fluidized bed bioreactor 100, and the organoid and cell large-scale culture device 1000 of the second aspect of the application was assembled in the clean bench. The organoid and cell large-scale culture device 1000 of the second aspect of the application was placed in the incubator for fluidization, the power pump 400 was turned on, the flow rate of the power pump 400 was adjusted to 5 mL / min, and the fluidization process of the microspheres was observed. Another part of the microspheres with a diameter of 500 microns was placed in a culture dish for static culture.

[0067] S2: A portion of the microspheres was taken out from the fluidized bed bioreactor 100 and the culture dish respectively, and day 0 (L fluidization day) DAPI staining was performed. Z-axis layer scanning was performed under confocal, and the number of cells in the microspheres was counted by multi-dimensional microscopic image data visualization software IMARIS Viewer.

[0068] S3: On fluidization day 3 (three days), sampling and observation were performed. A portion of the supernatant sample was taken out from the fluidized bed bioreactor 100 and the culture dish respectively for analysis of the substances secreted by the cells into the culture medium, and the microspheres fluidized for three days were taken out respectively for counting of live cells, dead cells and DAPI in the microspheres.

[0069] S4: On fluidization day 6 (six days), day 9 (nine days), and day 12 (twelve days), the sampling and recording operations of step S3 were repeated.

[0070] Based on the above data, the cell survival rate and the proliferation rate of the fluidized and static cultures were compared.

[0071] Example 3: Fluidization / static culture of calcium alginate microspheres with a diameter of 200 microns.

[0072] The experimental operation steps are as follows: the microspheres were wrapped with THLE cells. The circulating medium volume was 30 mL.

[0073] S1: 20000 microspheres with a diameter of 200 microns were added to the fluidized bed bioreactor 100, and the organoid and cell large-scale culture device 1000 of the second aspect of the application was assembled in the clean bench. The organoid and cell large-scale culture device 1000 of the second aspect of the application was placed in the incubator for fluidization, the power pump 400 was turned on, the flow rate of the power pump 400 was adjusted to 3.2 mL / min, and the fluidization process of the microspheres was observed. Another part of the microspheres with a diameter of 200 microns was placed in a culture dish for static culture.

[0074] S2: A portion of the microspheres were removed from the fluidized bed bioreactor 100 and the culture dish, respectively, and DAPI staining was performed on day 0 (the day of fluidization). The z-axis layer scanning was performed under confocal microscopy, and the number of cells in the microspheres was counted using the multidimensional microscopic image data visualization software IMARIS Viewer.

[0075] S3: On day 3 of fluidization, sampling and observation were performed. A portion of the supernatant was removed from the fluidized bed bioreactor 100 and the culture dish to analyze substances secreted into the culture medium by the cells. The microspheres, which had been fluidized for three days, were also removed to count live and dead cells and DAPI within the microspheres.

[0076] S4: Fluidization day 6 (six days), day 9 (nine days), day 12 (twelve days), repeat step S3 sampling and recording operations.

[0077] Based on the above data, the cell survival rate and proliferation rate of fluidized and static cultures were compared.

[0078] It should be noted that the English terms appearing in this specification have the following meanings: DPBS: Dulbecco's phosphate buffer solution; DAPI: 4',6-diamidino-2-phenylindole; FITC-dextran: fluorescein isothiocyanate-dextran; kDa: kilodaltons; 4kDa: molecule with a relative molecular mass of 4000; 10kDa: molecule with a relative molecular mass of 10000; 70kDa: molecule with a relative molecular mass of 70000.

[0079] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A fluidized bed bioreactor, characterized in that: It includes a bottom guide structure, a main reaction chamber and a top buffer structure coaxially connected from bottom to top; the bottom guide structure includes a lower conical cavity and a reactor inlet coaxially connected from top to bottom, the upper cross-section of the lower conical cavity is larger than the lower cross-section of the lower conical cavity, and the lower cross-section of the lower conical cavity is equal to the cross-section of the reactor inlet; the main reaction chamber includes a main cylindrical cavity and a sampling port connected to the side of the main cylindrical cavity, the cross-section of the main cylindrical cavity is equal to the upper cross-section of the lower conical cavity; the top buffer structure includes an upper cylindrical cavity and an upper conical cavity coaxially connected from top to bottom, and a reactor outlet connected to the side of the upper cylindrical cavity; the upper cross-section of the upper conical cavity is larger than the lower cross-section of the upper cone, the upper cross-section of the upper conical cavity is equal to the cross-section of the upper cylindrical cavity, and the lower cross-section of the upper conical cavity is equal to the cross-section of the main cylindrical cavity.

2. The fluidized bed bioreactor according to claim 1, characterized in that The fluidized bed bioreactor is made of transparent glass.

3. The fluidized bed bioreactor according to claim 1, characterized in that The bottom flow guide structure, the main reaction chamber and the top buffer structure are independent parts respectively. The bottom flow guide structure and the main reaction chamber, and the main reaction chamber and the top buffer structure are detachably and sealedly connected.

4. The fluidized bed bioreactor according to claim 1, characterized in that The angle between the side cavity surface of the upper frustum cavity and the horizontal plane is 135 degrees.

5. The fluidized bed bioreactor according to claim 1, characterized in that: The structural dimensions of the fluidized bed bioreactor are obtained through fluidization simulation optimization calculation and experimental verification.

6. A large-scale organoid and cell culture device, characterized in that: The fluidized bed bioreactor comprises an oxygen supply device, a liquid storage device, a power pump, a dissolved oxygen detector, and the fluidized bed bioreactor according to any one of claims 1 to 5; Wherein, the oxygen supply device is connected to the liquid storage device and is used to input oxygen into the liquid storage device; The liquid storage device, the power pump, the dissolved oxygen detector and the fluidized bed bioreactor are connected end to end in sequence to form a closed circulation system. The liquid storage device is used to store culture medium, the power pump is used to provide power for the circulation of culture medium, and the dissolved oxygen detector is used to detect the oxygen concentration in the culture medium before entering the fluidized bed bioreactor. The fluidized bed bioreactor is used to provide space for organoid and cell culture.

7. The large-scale organoid and cell culture device according to claim 6, characterized in that: It also includes a dynamic monitoring system, which is connected to the sampling port through a sampling control valve.

8. The large-scale organoid and cell culture device according to claim 6, characterized in that: It also includes a culture supernatant metabolite detection system; an outlet control valve is provided between the reactor outlet and the liquid storage device, and the culture supernatant metabolite detection system is connected to the outlet control valve.

9. The large-scale organoid and cell culture device according to claim 6, characterized in that: It also includes a fixing bracket, which fixes the fluidized bed bioreactor.

10. The large-scale organoid and cell culture device according to claim 6, characterized in that: The liquid storage device includes a liquid storage bottle, the top of which is provided with a circulating medium inlet connected to the reactor outlet, a circulating medium outlet connected to the power pump, a sterile oxygen inlet connected to the oxygen supply device, and a sterile oxygen outlet for discharging outward.

Images