Method for culturing cells or organoids

Abstract

The invention provides a method for culturing cells or organoids. The method comprises the following steps: mixing cells or organoids to be cultured with a hydrogel precursor solution, then curing the mixed product, and culturing the cured product, before the curing treatment, the method further comprises the step of spraying the mixed treatment product into a collection solution through an electrostatic spraying method. The method can be used for large-scale culture and amplification of cells or organoids, for example, the culture and amplification can reach a million-level high-throughput level, oil phase residues are avoided, the size uniformity is high, and the application requirements of cell therapy, establishment of an organoid biological sample library, high-throughput drug screening and the like are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for culturing cells or organoids, more particularly to a cell or organoid, a hydrogel microsphere and a culture system. BACKGROUND

[0002] Three-dimensional cell or organoid culture technology significantly improves the authenticity of cell function expression and drug response by simulating the in vivo microenvironment. With the increasing demand for scale and consistency of in vitro three-dimensional cell / organoid culture in cell therapy, regenerative medicine and high-throughput drug screening, microcarrier technology has become one of the mainstream solutions due to its ability to significantly increase cell attachment area and efficient material exchange.

[0003] The most commonly used microsphere generation techniques currently include microfluidic chip technology and coaxial droplet generation technology, which have high operating costs and low throughput, making it difficult to expand to a million-level throughput in a single run, resulting in long cell / organoid expansion cycles and high costs. In addition, the residual oil phase solvent can damage the activity of cells or biological molecules.

[0004] Therefore, there is an urgent need for a method for high-throughput culturing of cells or organoids by microcarriers to meet the comprehensive needs of high-throughput, low-cost, operability and function preservation for future cell therapy, high-throughput drug screening and large-scale functional screening. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a high-throughput three-dimensional cell / organoid culture method, which can be applied to large-scale culturing and expansion of cells or organoids.

[0006] The present application is based on the following findings of the inventors:

[0007] The principle of the most commonly used microsphere generation technique is to prepare monodisperse hydrogel microspheres at the oil / water interface, each microsphere serving as an independent culture unit and being compatible with fully automated liquid handling and high-content imaging platforms, enabling large-scale parallel culturing and real-time phenotype analysis. However, the current microsphere generation technique has high operating costs and low throughput, making it difficult to expand to a million-level throughput in a single run, resulting in long cell / organoid expansion cycles and high costs. To overcome this problem, the inventors provide a microsphere preparation and a million-level high-throughput three-dimensional cell / organoid culture method using the microspheres as carriers, which can be applied to large-scale culturing and expansion of cells or organoids, thereby meeting the requirements of cell therapy, high-throughput drug screening and other applications.

[0008] In a first aspect, the present application provides a method for culturing cells or organoids. According to embodiments of the present application, the method comprises: mixing cells or organoids to be cultured with a hydrogel precursor solution, then solidifying the mixture, and culturing the solidified product; before the solidification, further comprising spraying the mixture into a collection solution by electrostatic spraying. According to the method of embodiments of the present application, cells or organoids can be cultured and expanded at a large scale, for example, up to a high-throughput level of millions, without oil phase residues, with high size uniformity, thereby meeting the requirements of applications such as cell therapy and high-throughput drug screening.

[0009] In a second aspect, the present application provides cells or organoids. According to embodiments of the present application, the cells or organoids are obtained by the method of the first aspect of the present application. According to the cells or organoids of embodiments of the present application, the proliferation rate and functional activity are good, and can meet the requirements of applications such as cell therapy and high-throughput drug screening.

[0010] In a third aspect, the present application provides a hydrogel microsphere. According to embodiments of the present application, the hydrogel microsphere comprises: a hydrogel matrix formed by a hydrogel material with a mass fraction of 1-10%; and cells or organoids encapsulated in the hydrogel matrix; wherein the cell concentration is 10 6 -10 7 μm, and the size variation coefficient of the hydrogel microsphere is ≤10%. According to the hydrogel microsphere of embodiments of the present application, cells or organoids can be cultured and expanded at a high-throughput level of millions, improve the survival rate of cells or organoids, have good monodispersity, and have wide size adaptability, thereby meeting the requirements of applications such as cell therapy and high-throughput drug screening.

[0011] In a fourth aspect, the present application provides a culture system for preparing the aforementioned hydrogel microsphere. According to embodiments of the present application, the culture system further comprises a special medium for cells or organoids, and is suitable for a culture environment of 37°C and 5% CO2. According to the culture system of embodiments of the present application, the aforementioned hydrogel microsphere can be stably cultured for a long time.

[0012] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0013] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0014] Figure 1 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 1 at day 1 of culture.

[0015] Figure 2 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 1 at day 4 of culture.

[0016] Figure 3 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 1 at day 8 of culture.

[0017] Figure 4 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 1 at day 12 of culture.

[0018] Figure 5 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 2 at day 1 of culture.

[0019] Figure 6 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 2 at day 4 of culture.

[0020] Figure 7 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 2 at day 8 of culture.

[0021] Figure 8 Optical micrograph of the cell or organoid loaded microspheres obtained in Example 2 at day 12 of culture. DETAILED DESCRIPTION

[0022] Embodiments of the present application are described in detail below. The embodiments described below are examples only and are not to be construed as limiting the present application.

[0023] It should be noted that the terms "first", "second" and the like in the description do not necessarily connote an absolute order or quantity, but are used to modify an element or add, specify or to distinguish a specific element from another, unless otherwise specifically indicated. Thus, a feature with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0024] In this document, the terms "comprises" or "comprising" are used in the sense of open-ended expressions, i.e., in the sense of "including, but not limited to", so as to encompass the items listed thereafter, but not excluding other items.

[0025] In this document, the terms "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs, and instances where it does not.

[0026] In this document, "A and / or B" can include a case where one of A or B alone exists, a case where both of A and B exist, and a case where A and B exist together. A, B are merely examples, and any technical features used in this application can be connected using "and / or".

[0027] The present application provides a method for culturing cells or organoids, cells or organoids, hydrogel microspheres, or a culture system for preparing hydrogel microspheres, which are described in detail below.

[0028] Method for culturing cells or organoids

[0029] In a first aspect of the present application, a method for culturing cells or organoids is provided. According to an embodiment of the present application, the method comprises mixing cells or organoids to be cultured with a hydrogel precursor solution, then solidifying the mixture, and culturing the solidified product. Before the solidification, the mixture is further sprayed into a collection solution by an electrostatic spraying method. According to the method of the present application, cells or organoids can be cultured and expanded at a large scale, such as up to a million high-throughput level, without oil phase residues, with high size uniformity, thereby meeting the requirements of cell therapy, high-throughput drug screening, and other applications.

[0030] According to an embodiment of the present application, the solidification is performed under the condition of standing for 1-10 minutes, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes, or 405 nm ultraviolet light irradiation for 1-10 minutes, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes.

[0031] According to an embodiment of the present application, the hydrogel precursor solution comprises 1-10% by mass of a hydrogel material, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range of 2-10%, 1-9% between any two values.

[0032] According to an embodiment of the present application, the hydrogel material is selected from at least one of sodium alginate, methacrylated gelatin, methacrylated hyaluronic acid, polyethylene glycol, and collagen.

[0033] According to an embodiment of the present application, the hydrogel precursor solution is subjected to a first bacteria filtration treatment by a 0.20-0.25 μm filter membrane, such as a 0.20 μm filter membrane, a 0.21 μm filter membrane, a 0.22 μm filter membrane, a 0.23 μm filter membrane, a 0.24 μm filter membrane, or a 0.25 μm filter membrane before being mixed with the cells or organoids.

[0034] According to an embodiment of the present application, the spraying the mixed processing product into the collection liquid by the electrostatic spraying method is achieved under at least one of the following conditions: a flow rate of 15-500 μL / min, a voltage of 3-20 kV, an inner diameter of the nozzle of 60-500 μm, and a collection distance of 1-5 cm.

[0035] According to an embodiment of the present application, the cell concentration is 10 6 -10 7 cells / mL, for example, the cell concentration can be 10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, 10 7 cells / mL, or a range value between 2×10 6 cells / mL-10 7 cells / mL, 3×10 6 cells / mL-10 7 cells / mL.

[0036] According to an embodiment of the present application, the collection liquid is selected from at least one of a calcium chloride solution and a mineral oil.

[0037] According to an embodiment of the present application, the collection liquid is filtered through a 0.20-0.25 μm filter membrane, for example, can be filtered through a 0.20 μm filter membrane, a 0.21 μm filter membrane, a 0.22 μm filter membrane, a 0.23 μm filter membrane, a 0.24 μm filter membrane, or a 0.25 μm filter membrane for a second bacteria filtration treatment.

[0038] According to an embodiment of the present application, the calcium chloride solution has a mass fraction of 1%-5%, for example, can be 1%, 2%, 3%, 4%, or 5%.

[0039] According to an embodiment of the present application, the solidified processing product comprises hydrogel microspheres.

[0040] According to an embodiment of the present application, the hydrogel microspheres have a diameter of 50-1000 μm, for example, can be 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, or a range value between 100-1000 μm, 50-900 μm.

[0041] According to an embodiment of the present application, the generation rate of the hydrogel microspheres is 20-500 per second, for example, can be 20, 50, 70, 90, 100, 200, 300, 400, 450, 500 or a range value of 50-500 per second, 20-450 per second, at a common speed of 500 per second, a million-level microspheres can be produced per hour, so as to achieve the purpose of high-throughput amplification of three-dimensional culture organoids.

[0042] According to an embodiment of the present application, the culturing of the hydrogel microspheres is carried out at 37°C and 5% CO2.

[0043] According to an embodiment of the present application, the culturing of the hydrogel microspheres is carried out under the condition that the culture medium is replaced every 3 days.

[0044] According to a specific embodiment of the present application, the steps of the method of the present application are as follows: (i) culturing a cell line or an organoid in a culture medium, and after digestion, resuspending in a hydrogel precursor solution of a hydrogel material having at least one of a mass fraction of 1%-10% sodium alginate, methacrylated gelatin, methacrylated hyaluronic acid, polyethylene glycol, collagen, to obtain a hydrogel precursor solution mixed with a concentration of 10 6 -10 7 10 cells or organoids per mL, wherein all the hydrogel precursor solutions are filtered through a 0.22 μm filter before mixing with the cells or organoids, the culture temperature is 37°C, and the carbon dioxide concentration is 5%; (ii) using an electrostatic spraying method, spraying the hydrogel precursor solution mixed with the cells or organoids into a collection solution in the form of a spray, and then solidifying, filtering and washing, and collecting, to obtain hydrogel microspheres loaded with the cells or organoids, wherein the collection solution is a calcium chloride solution with a mass fraction of 1.5% or mineral oil, all the collection solutions are filtered through a 0.22 μm filter before use, the instrument and equipment of the electrostatic spraying method include a syringe pump, a high-pressure generating device, a liquid guide pipe, a support, a nozzle, a receiving device, the flow rate used is 15-500 μL / min, the voltage is 3-20 kV, the collection distance is 1-5 cm, the inner diameter of the nozzle is 60-500 μm, and the filtering and washing method is: filtering through a screen, the hydrogel microspheres loaded with the cells or organoids are left on the screen, and then the microspheres are washed with the culture medium; (iii) culturing the obtained hydrogel microspheres loaded with the cells or organoids, after a period of time, the cells or organoids inside proliferate to form a mass, the microspheres are digested, and the mass of the cells or organoids inside is taken out.

[0045] Cells or organoids

[0046] In a second aspect, the present application provides a cell or organoid. According to embodiments of the present application, the cell or organoid is obtained by the method of the first aspect of the present application. According to embodiments of the present application, the cell or organoid has a good proliferation rate and functional activity, and can meet the requirements of cell therapy, high-throughput drug screening and other applications.

[0047] Hydrogel microspheres

[0048] In a third aspect, the present application provides a hydrogel microsphere. According to embodiments of the present application, the hydrogel microsphere comprises: a hydrogel matrix formed by a hydrogel material with a mass fraction of 1-10%; and a cell or organoid encapsulated in the hydrogel matrix; wherein the cell concentration is 10 6 -10 7 μm, and the size variation coefficient of the hydrogel microsphere is ≤10%. According to embodiments of the present application, the hydrogel microsphere can achieve a high-throughput level of culture and expansion, improve the survival rate of the cell or organoid, has good monodispersity, and has wide size adaptability, thereby meeting the requirements of cell therapy, high-throughput drug screening and other applications.

[0049] According to embodiments of the present application, the hydrogel material is selected from at least one of sodium alginate, methacrylated gelatin, methacrylated hyaluronic acid, polyethylene glycol, and collagen.

[0050] According to embodiments of the present application, the hydrogel microsphere is prepared by electrostatic spraying under the following conditions: a flow rate of 15-500 μL / min, an electrostatic voltage of 3-20 kV, an inner diameter of the nozzle of 60-500 μm, and a collection distance of 1-5 cm.

[0051] According to embodiments of the present application, the cross-linking method of the hydrogel microsphere is one of: ion cross-linking solidification by a 1.5% calcium chloride solution and photo-crosslinking solidification by irradiation with 405 nm ultraviolet light.

[0052] According to embodiments of the present application, the generation rate of the hydrogel microsphere is 20-500 per second.

[0053] Culture system

[0054] In a fourth aspect, the present application provides a culture system for preparing the aforementioned hydrogel microsphere. According to embodiments of the present application, the culture system further comprises a cell or organoid-specific culture medium, and is suitable for a culture environment of 37°C and 5% CO2. According to embodiments of the present application, the culture system can stably culture the aforementioned hydrogel microsphere for a long time.

[0055] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0056] Example 1:

[0057] This embodiment provides a method for culturing cells or organoids, using a hydrogel material based on sodium alginate.

[0058] 1. Preparation of hydrogel precursor solution: Weigh 1.5g of sodium alginate powder into a 100mL beaker, place a magnetic rotor in the beaker, add deionized water to 100mL, stir at 60℃ for 5h, and then filter through a 0.22μm filter membrane for sterilization to obtain a 1.5wt% sodium alginate solution.

[0059] 2. Preparation of hydrogel precursor solution containing cells or organoids: After digesting the cells or organoids, centrifuge, aspirate the supernatant, resuspend in PBS buffer, centrifuge again, aspirate the supernatant, and then resuspend in a certain amount of the hydrogel precursor solution obtained in step 1 to achieve a cell concentration of 10. 6 -10 7 A hydrogel precursor solution containing cells or organoids was obtained at a concentration of cells / mL.

[0060] 3. Preparation of the collection solution: Weigh 15g of CaCl2, add deionized water to dissolve and make up to 1L. After filtration through a 0.22μm filter membrane for sterilization, a 1.5wt% CaCl2 solution is obtained as the collection solution.

[0061] 4. Electrospray preparation of hydrogel microspheres loaded with cells or organoids: The hydrogel precursor solution mixed with cells or organoids obtained in step 2 is propelled by an injection pump through a pipeline to the nozzle. Under the action of electrostatic force, spherical droplets are formed. The droplets are sprayed into a flowing crosslinking pool containing a collection solution and allowed to stand and solidify for 2 minutes to obtain hydrogel microspheres loaded with cells or organoids. The electrospray parameters are: injection flow rate 100 μL / min, voltage 11 kV, collection distance 3.5 cm, and nozzle inner diameter 160 μm.

[0062] 5. Filtration and washing: The collection solution containing microspheres obtained in step 4 is filtered through a 70μm sieve to remove the collection solution. The hydrogel microspheres carrying cells or organoids remain on the sieve. The microspheres are then washed with culture medium.

[0063] 6. Cultivation of hydrogel microspheres loaded with cells or organoids: Transfer the microspheres obtained after washing in step 5 to a culture dish, immerse the microspheres in a cell or organoid culture medium, and observe them using an optical microscope. Figure 1 Cultured at 37℃ and 5% carbon dioxide, with the culture medium changed every 3 days. Observed using an optical microscope on days 4, 8, and 12. Figures 2-4 Clear cell proliferation can be observed.

[0064] Example 2:

[0065] This embodiment provides a method for culturing cells or organoids, wherein the hydrogel material is based on a blend of sodium alginate and matrix gel.

[0066] The difference between this embodiment and Embodiment 1 is as follows: In step 1, after obtaining the 1.5 wt% sodium alginate solution, the 1.5 wt% sodium alginate solution and matrix gel are mixed at a volume ratio of 10:1 to obtain the sodium alginate-matrix gel mixed hydrogel precursor solution, which is then placed on ice for later use; in step 2, the cells or organoids are resuspended in the hydrogel precursor solution on ice. In step 4, the liquid delivery tubing is buried under ice, and the other steps are the same as in Embodiment 1. During the culture process, observations are made using an optical microscope as follows. Figures 5-8 As shown, significant cell proliferation can be observed.

[0067] Example 3:

[0068] This embodiment provides a method for culturing cells or organoids, using a hydrogel material based on methacrylamide hyaluronic acid. The difference from Example 1 is as follows: In step 1, 5g of methacrylamide hyaluronic acid is weighed and placed in a 100mL beaker, and a magnetic rotor is placed in the beaker. A standard LAP initiator solution is added to 100mL, and the mixture is stirred at 60°C for 5 hours. Subsequently, it is filtered through a 0.22μm filter membrane for sterilization to obtain a 5wt% methacrylamide hyaluronic acid solution. In step 3, mineral oil is used to collect the solution. In step 4, the curing method involves irradiation with 405nm ultraviolet light for 5 minutes. Other steps are the same as in Example 1.

[0069] Example 4:

[0070] This embodiment provides a method for culturing cells or organoids, wherein the hydrogel material is based on methacrylated gelatin.

[0071] The difference from Example 1 is as follows: In step 1, 5g of methacrylamide gelatin was weighed and placed in a 100mL beaker, and a magnetic rotor was placed in the beaker. The LAP initiator standard solution was added to 100mL, and the mixture was stirred at 60℃ for 5h. Then, it was filtered through a 0.22μm filter membrane for sterilization to obtain a 5wt% methacrylamide gelatin solution; In step 3, mineral oil was used to collect the solution; In step 4, the curing method was irradiation with 405nm ultraviolet light for 5 minutes. The other steps were the same as in Example 1.

[0072] Cells or organoids cultured according to the methods described in Examples 1-4 were observed to show significant cell proliferation during the culture process using an optical microscope. This demonstrates that the method of this application is applicable to a variety of hydrogel materials. Cells or organoids can be cultured using the method of this application with different hydrogel materials.

[0073] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0074] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for culturing cells or organoids, characterized in that, include: The cells or organoids to be cultured are mixed with a hydrogel precursor solution, the mixture is then solidified, and the solidified product is cultured; prior to the solidification process, the mixture is further sprayed into a collection liquid by electrostatic spraying.

2. The method according to claim 1, characterized in that, The curing process is carried out under the conditions of standing for 1 to 10 minutes or irradiation with 405nm ultraviolet light for 1 to 10 minutes; Optionally, the hydrogel precursor solution comprises 1%-10% hydrogel material by mass. Optionally, the hydrogel material is selected from at least one of sodium alginate, methacrylamide gelatin, methacrylamide hyaluronic acid, polyethylene glycol, and collagen. Optionally, the hydrogel precursor solution is subjected to a first bacterial filtration treatment through a 0.20–0.25 μm filter membrane before being mixed with cells or organoids.

3. The method according to claim 1, characterized in that, The electrostatic spraying method is used to spray the mixed treatment product into the collection liquid under at least one of the following conditions: flow rate of 15-500 μL / min, voltage of 3-20 kV, nozzle inner diameter of 60-500 μm, and collection distance of 1-5 cm.

4. The method according to claim 1, characterized in that, The cell concentration was 10. 6 -10 7 per mL.

5. The method according to claim 2 or 3, characterized in that, The collection solution is selected from at least one of calcium chloride solution and mineral oil; And / or, the collected liquid undergoes a second bacterial filtration treatment through a 0.20–0.25 μm filter membrane; And / or, the calcium chloride solution has a mass fraction of 1% to 5%.

6. The method according to claim 1, characterized in that, The solidification product includes hydrogel microspheres; Optionally, the diameter of the hydrogel microspheres is 50-1000 μm; Optionally, the generation rate of the hydrogel microspheres is 20-500 per second.

7. The method according to claim 6, characterized in that, The hydrogel microspheres were cultured at 37°C and 5% CO2. And / or, the hydrogel microspheres are cultured under conditions where the culture medium is changed every 3 days.

8. A cell or organoid, characterized in that, Obtained by the method described in any one of claims 1 to 7.

9. A hydrogel microsphere, characterized in that, include: A hydrogel matrix, wherein the hydrogel matrix is ​​formed of a hydrogel material with a mass fraction of 1%-10%; Cells or organoids, wherein the cells or organoids are encapsulated in the hydrogel matrix; The cell concentration is 10 6 -10 7 The number of microspheres per mL is 50-1000 μm in diameter, and the coefficient of variation of the size of the microspheres is ≤10%. Optionally, the hydrogel material is selected from at least one of sodium alginate, methacrylamide gelatin, methacrylamide hyaluronic acid, polyethylene glycol, and collagen; Optionally, the hydrogel microspheres are prepared by electrostatic spraying under the conditions of a flow rate of 15-500 μL / min, an electrostatic voltage of 3-20 kV, a nozzle inner diameter of 60-500 μm, and a collection distance of 1-5 cm. Optionally, the crosslinking method of the hydrogel microspheres is one of the following: ionic crosslinking and curing by 1.5wt% calcium chloride solution and photocrosslinking and curing initiated by 405nm ultraviolet light irradiation; Optionally, the generation rate of the hydrogel microspheres is 20-500 per second.

10. A culture system for preparing the hydrogel microspheres of claim 9, characterized in that, The culture system also includes a special culture medium for cells or organoids, and is suitable for a culture environment of 37°C and 5% CO2.

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