An organoid cell culture system and method

Abstract

The application provides a high-flux organoid cell culture system, which is characterized in that the system comprises a cell culture container and a modified culture medium, wherein the cell culture container comprises a chip, a PCR tube or a common U-shaped 96-well plate; and the modified culture medium comprises mTeSR1, Y-27632 and Pluronic F-127. The cell culture system of the application realizes high-efficiency acquisition of a first-step uniform spherical cell cluster by improving the formula of the culture medium, and greatly shortens the formation time of the spherical cell cluster; meanwhile, the obtained spherical cell cluster has the potential to be cultured and differentiated in multiple different directions for a long time, and can be differentiated into liver, kidney and brain multiple types of organoids only by replacing the corresponding differentiation culture medium; and the system can be realized in various containers such as a chip, a culture dish and a PCR tube, and has universality.

Description

Technical Field

[0001] This invention relates to an organoid cell culture system and method, belonging to the field of cell biology. Background Technology

[0002] Organoids are three-dimensional cell culture models that can effectively simulate the dynamic changes and key physiological functions of related tissues in vivo, making them significant for research on organ development and clinical medicine. A common practice in culturing 3D organoids derived from embryonic stem cells or induced pluripotent stem cells is to directly seed a digested single-cell suspension into a low-adsorption U-shaped 96-well culture dish or other low-adsorption culture dishes. The cells' self-organizing ability allows them to spontaneously form cell clusters, which gradually develop into organoids after a period of development. However, this method has significant drawbacks due to the limited self-organizing ability of cells: First, cell aggregation efficiency is very low, with a large number of cells failing to aggregate effectively and ultimately undergoing apoptosis; second, cells tend to form heterogeneous cell clusters; third, the time required to form stable cell clusters is long, typically 2-3 days; fourth, organoid culture time is long, and if the entire culture process is done manually, the workload is large and the results cannot be guaranteed; fifth, the throughput of currently used culture dishes is low, which cannot meet the needs of high-throughput drug screening.

[0003] Therefore, there is a need to develop an automated organoid culture system that improves the efficiency of uniform cell cluster formation, shortens the time for stable cell cluster formation, and increases culture throughput. Summary of the Invention:

[0004] This invention discloses a high-throughput organoid cell culture system. By improving the culture medium formulation in the first step, it achieves highly efficient acquisition of uniform spherical cell clusters and significantly shortens their formation time. Simultaneously, the obtained spherical cell clusters possess the potential for long-term differentiation in multiple directions without altering the basic properties of the starting cells, enabling them to develop into various different organoids with high efficiency, high reproducibility, and high survival rate. This culture system can be implemented in various common bioreactors such as chips, culture dishes, and PCR tubes, and is also applicable to various cell types from different sources, demonstrating its versatility. The designed chip can culture multiple organoids in a high-throughput, automated manner, enabling large-scale drug screening.

[0005] This invention provides an organoid cell culture system, characterized in that the system comprises:

[0006] Cell culture containers and modified culture media, wherein the cell culture containers include chips, PCR tubes or ordinary U-shaped 96-well plates; and the modified culture media include mTeSR1, Y-27632 and Pluronic F-127.

[0007] According to a preferred embodiment of the present invention, the concentration of Pluronic F-127 used in the culture medium is 1 μM-100 μM, preferably 5 μM-80 μM, and more preferably 10 μM-50 μM.

[0008] According to a preferred embodiment of the present invention, the chip is a PDMS chip, comprising an upper channel layer and a lower organoid culture chamber.

[0009] According to a preferred embodiment of the present invention, the upper channel layer of the chip is used to deliver culture medium, which enters from one end and exits from the other end.

[0010] According to a preferred embodiment of the present invention, the lower layer of the chip is a self-made quadrangular pyramid with a bottom groove to assist cells in forming spheres.

[0011] According to a preferred embodiment of the present invention, the lower layer of the chip can be perforated by drilling holes in the grooves at the bottom of a pyramid, and each hole can culture 9-16 cell spheres at a time.

[0012] According to a preferred embodiment of the present invention, after the cell culture system is added to the culture medium, a sealing film needs to be attached to the top of the chip.

[0013] According to a preferred embodiment of the present invention, the chip culture chamber has no special surface modification.

[0014] According to a preferred embodiment of the present invention, the PDMS chip needs to be treated with Pluronic F-127 for low adsorption before the cells to be cultured are added.

[0015] According to a preferred embodiment of the present invention, the obtained homogeneous cell spheroids can be used to obtain various organoids such as liver, kidney, and brain simply by changing the corresponding differentiation culture medium.

[0016] The beneficial effects of this invention are:

[0017] The organoid culture system used in this invention, by changing the formulation of the first-step cell spheroid assembly culture medium and using a high-throughput chip with a pyramidal base, can achieve the following beneficial effects:

[0018] First, the high cell aggregation rate improves organoid formation efficiency. Second, it yields a large number of uniform organoids with precisely defined sizes. Third, it significantly shortens the spheroidization time from 2-3 days to 12 hours. Fourth, it reduces culture costs by lowering the price of organoid culture media and replacing expensive low-adsorption culture dishes with cheaper, ordinary U-shaped culture dishes. Fifth, the chip of this invention greatly simplifies cell culture operations, enabling high-throughput organoid production through automated processes. Sixth, this system can be applied to the culture of various organoids without affecting cell differentiation pathways. The spheroidization effect of this culture medium in the PDMS chip makes high-throughput screening on the chip possible. Attached Figure Description

[0019] Figure 1 organoid chip schematic diagram

[0020] Figure 2 Organoids cultured in a modified medium in a standard U-shaped 96-well plate

[0021] Figure 3 Kidney organoids cultured in a modified culture medium in a standard U-shaped 96-well plate

[0022] Figure 4 Liver organoids cultured in a modified medium in a standard U-shaped 96-well plate

[0023] Figure 5 Brain organoids cultured in a standard U-shaped 96-well plate using a modified culture medium

[0024] Figure 6 External images of various organs cultured over long periods

[0025] Figure 7 Expression of genes related to organoid differentiation and maturation Detailed implementation method:

[0026] To further illustrate the core content of this invention, the following examples are provided. These examples are intended to further explain the invention and do not constitute a limitation on it.

[0027] Organoids represent a key advancement in stem cell research. As three-dimensional (3D) cell cultures, organoids can mimic some key characteristics of organs in vitro. These in vitro culture systems originate from self-renewing stem cell populations that can differentiate into multiple organ-specific cell types, reproducing the structure and some functions of the corresponding organs, thus providing a highly physiologically relevant system.

[0028] Based on the starting cells, organoid culture methods can be divided into two main categories: those derived from primary tissues and those derived from embryonic stem cells or induced pluripotent stem cells. Organoids derived from primary tissues have low differentiation potential; typically, only one type of organoid can be obtained from a single primary tissue. However, organoids derived from stem cells have multi-directional differentiation potential, allowing for the acquisition of various organoids, making them an excellent model for studying the development of different organs.

[0029] There are generally two methods for culturing stem cell-derived organoids. One method involves culturing stem cell clones that are several hundred micrometers in size, while the other method involves digesting the stem cell clones into discrete individual cells and then using the cells' self-organizing ability to aggregate them into spheres, forming organoids. Since the size of the stem cell clones is uncontrollable, the former method results in highly uneven sizes for each organoid. The latter method, however, allows for indirect control of organoid size by adjusting the number of discrete cells input, thus achieving organoids with a more uniform size.

[0030] Culture medium is not only the basic substance that provides cell nutrition and promotes cell proliferation, but also the living environment for cell growth and reproduction. The nutrients required by different cells vary during culture. In the culture of embryonic stem cells, to help cells aggregate into spheres, it is usually necessary to add substrate gel, growth factors that maintain normal growth and development, etc., to the culture medium. In addition, the culture time for organoids is usually long. Therefore, not only is the culture cost high, but the success rate cannot be guaranteed.

[0031] The present invention provides a culture medium for assembling organoid cell spheres, comprising mTeSR1, Y-27632 and Pluronic F-127, wherein the concentration of Pluronic F-127 used is 1μM-100μM, preferably 5μM-80μM, and more preferably 10μM-50μM.

[0032] Pluronic F-127 is a nonionic surfactant used in cell culture and other treatments. For example, it can be used for low-adsorption pretreatment of cells and proteins in PDMS chips; fabrication of nanocarriers for transporting small molecule drugs; construction of neural conduits; fluorescent labeling of blood vessels, astrocytes, and neurons; and is also a common material for bioprinting. This invention is the first to incorporate Pluronic F-127 into a culture medium for the culture of 3D organoids, promoting cell aggregation into uniform spherical cell clusters and differentiation into organoids. Pluronic F-127 is mild on cells and highly compatible with various cell culture media. Pluronic F-127 can rapidly and effectively aggregate cells into spherical cell clusters while preserving the cells' multidirectional differentiation potential, without affecting the subsequent development of the organoids.

[0033] The reagents and consumables used in the following examples are from the following sources:

[0034] Traditional culture medium formulation: mTeSR1 + 50 μM Y-27632

[0035] Modified culture medium formula: mTeSR1 + 50 μM Y-27632 + 10 μM Pluronic F-127

[0036] Reagents: mTeSR1 (STEMCELL Technologies, 85850); Y-27632 (Selleck, S1049); Pluronic F-127 (Sigma, P2443).

[0037] Consumables: 96-well round-bottom low-adsorption culture dish (Corning, 7007); 96-well round-bottom untreated culture dish (Corning, 3788); PDMS prepolymer (MOMENTIVE, RTV615); qPCR sealing film (applied biosystems, 4311971).

[0038] All of the above-mentioned components are available for purchase, and their composition, dosage form, and specifications are known in the art and will not be elaborated here. In this embodiment, the above-mentioned components are combined to obtain a culture medium for the first step of organoid culture, cell spheroid assembly, thereby improving the success rate of organoid culture.

[0039] Example 1: Chip Design and Fabrication

[0040] like Figure 1 As shown, the PDMS chip is divided into two parts. The upper layer contains channels for transporting culture medium, which enters from one end and exits from the other. The lower layer is the bottom of a self-made square pyramid to help cells spherize.

[0041] When fabricating the PDMS chip, the upper chip is prepared using a 5:1 AB solution ratio, and the lower chip is prepared using a 10:1 AB solution ratio. The upper chip requires holes approximately 5 mm in diameter drilled at each of the eight circular areas, and smaller holes at the smaller circles on the left and right sides for inserting hollow steel needles to allow the culture medium to flow in or out. The upper and lower chips are then bonded together at 80°C for approximately 8 hours. In actual use, after adding the culture medium, a sealing film needs to be applied to the top for sealing. The chip has no special surface modification.

[0042] According to a preferred embodiment of the present invention, the chip is a PDMS chip, comprising an upper channel layer and a lower organoid culture chamber.

[0043] According to a preferred embodiment of the present invention, the upper channel layer of the chip is used to deliver culture medium, which enters from one end and exits from the other end.

[0044] According to a preferred embodiment of the present invention, the lower layer of the chip is a self-made quadrangular pyramid with a bottom groove to assist cells in forming spheres.

[0045] According to a preferred embodiment of the present invention, the lower layer of the chip can be shaped by drilling holes in the grooves at the bottom of a pyramid, and each hole can be used to culture 9-16 organoids at a time.

[0046] According to a preferred embodiment of the present invention, after the cell culture system is added to the culture medium, a sealing film needs to be attached to the top of the chip.

[0047] According to a preferred embodiment of the present invention, the chip culture chamber has no special surface modification.

[0048] Example 2: Uniform and stable organoids can be obtained by culturing them in a conventional U-shaped 96-well plate using a modified culture medium.

[0049] Human embryonic stem cells with a cell density suitable for passage were digested with Accutase enzyme in a 5% CO2 incubator at 37°C for 5-10 minutes. The cells were then gently pipetted onto mTeSR1 medium, centrifuged at 1000 rpm for 5 minutes, and the supernatant was collected to resuspend the embryonic stem cells. The cell pellet was mixed with a modified culture medium to achieve a concentration of 5000 cells / 100 μL, yielding the culture medium. This culture medium was then seeded into standard U-shaped 96-well plates (100 μL / well). During culture, an appropriate amount of differentiation medium was added at the set time intervals.

[0050] After 3 days of culture, the results are as follows Figure 2 The bottom right image shows organoids cultured on a low-adsorption plate (ULA plate), while the rest are organoids cultured on ordinary plates. After 3 days of culture, the organoids showed good growth, and it can be seen that the organoids treated with PF-127 were uniform and stable, while organoids obtained by other methods had more excess clumps, especially those on the ULA plate. These results indicate that adding PF-127 to the culture medium can improve the uniformity of organoid formation, and that combining it with ordinary U-shaped 96-well plates can achieve better results than ULA plates.

[0051] Example 3: Spheroidization experiment of different organoids in a conventional U-shaped 96-well plate

[0052] In a standard U-shaped 96-well plate, homogeneous cell spheroids were obtained using a modified culture medium. Based on this, kidney organoids could be effectively obtained by changing the culture medium to different corresponding differentiation media. Figure 3 ), liver organoids ( Figure 4 ), brain organoids ( Figure 5 ).

[0053] Figure 3 The image on the left shows kidney organoids on the third day of differentiation in a 96-well culture dish with low adsorption. They are in very poor condition and have a loose morphology. Figure 3 The middle image shows a kidney organoid on the third day of differentiation after being cultured on a modified medium in a standard 96-well culture dish; the organoid has good morphology. Figure 3 The image on the right shows a kidney organoid on the sixth day of differentiation after being cultured on a modified medium in a standard 96-well culture dish. The organoid is in good growth condition.

[0054] Figure 4 The image on the left shows liver organoids on the third day of differentiation in a 96-well culture dish with low adsorption. They are in very poor condition and have a loose morphology. Figure 4 The middle image shows liver organoids differentiated on a modified culture medium on a standard 96-well plate on day three, exhibiting good morphology. Figure 4 The image on the right shows liver organoids differentiated on a modified culture medium on a standard 96-well plate on the sixth day. Although there are many dead cells, this is related to the conditions of liver differentiation. Overall, the condition is good and can be cultured continuously.

[0055] Figure 5 The figures show brain organoids at days 1, 4, 5, 7, 10, and 14. These organoids were cultured on standard 96-well plates using a modified culture medium. The brain organoids grew and developed normally.

[0056] The development of organoids during long-term culture.

[0057] Organoid formation requires a long time; therefore, it is necessary to test the feasibility of the culture system of this invention for long-term organoid development. Various organoids were cultured in ordinary U-shaped 96-well plates, and their morphological characteristics were observed. Liver organoids, kidney organoids, and brain organoids were continuously cultured in ordinary U-shaped 96-well plates and on microarrays, respectively, and the spherical state of the organoids was observed and photographed.

[0058] Figure 6 A-6C are morphological images of liver organoids on day 17, kidney organoids on day 22, and brain organoids on day 53, respectively, cultured in a standard U-shaped 96-well plate. Figure 6 D-6F are morphological images of liver organoids cultured on day 17, kidney organoids on day 23, and brain organoids on day 14 in PDMS chips. Scale bar is 500 μm.

[0059] Example 4: Organoids cultured in conventional 96-well plates and chips can differentiate and mature normally.

[0060] After homogeneous cell spheroids were formed, different types of organoids were obtained by changing the culture medium to the corresponding culture medium. At different culture time points, different types of organoids were removed from the culture dish, cellular RNA was extracted, and the expression levels of genes related to the development and maturation of different organs were detected by qRT-PCR, with human embryonic stem cells as a reference.

[0061] The results are as follows Figure 7 As shown in Figures a, b, and c, organoids cultured in a standard U-shaped 96-well plate are examples of organoids. Figure 7 a represents a liver organoid from day 14, which showed significantly increased expression levels of genes related to liver development and maturation compared to human embryonic stem cells; Figure 7 b is a kidney organoid from day 21, and its expression levels of kidney development-related genes are significantly higher compared to human embryonic stem cells; Figure 7 c represents the brain organoid at day 37. Compared with human embryonic stem cells, the expression levels of neural stem cell markers during neural development are significantly increased, and the expression levels of genes related to the cerebral cortex are significantly elevated. Figure 7 In Figure d, the liver organoids were cultured on the chip to day 17. Compared with human embryonic stem cells, the expression levels of genes related to liver development and maturation were significantly increased, indicating that the organoids can develop and mature normally in the culture system of the modified culture medium and PDMS chip.

Claims

1. A method for culturing organoids, the method comprising: digesting human embryonic stem cells with cell density meeting passage requirements with Accutase digestive enzyme in a 5% CO2 incubator at 37°C for 5-10 minutes; then gently pipetting adherent cells with mTeSR1 medium; centrifuging at 1000 rpm for 5 minutes; collecting the supernatant and resuspending the embryonic stem cells; mixing the cell pellet with a modified medium to achieve a concentration of 5000 cells / 100 μL to obtain the culture medium; seeding the culture medium into a standard U-shaped 96-well plate at 100 μL / well; during culture, supplementing an appropriate amount of differentiation medium at set intervals; the modified medium consists of mTeSR1 medium + 50 μM Y-27632 and 10 μM Pluronic F-127; the resulting homogeneous cell spheroids can differentiate into liver organoids, kidney organoids, or brain organoids simply by changing the corresponding differentiation medium.

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