A method for aerosol jet printing 3D organoids

Abstract

The present invention discloses a method for printing 3D organoids by aerosol jetting, comprising the following steps: Step 1: obtaining a cell precipitate of a tissue to be printed; Step 2: depositing the cell precipitate and the matrix gel suspension on a substrate by aerosol jetting to obtain a 3D organoid; The aerosol jetting method is as follows: placing the cell precipitate and the matrix gel suspension in an atomizer, generating an aerosol mist by a pneumatic atomization device; sandwiching the aerosol mist in N2, starting the nozzle deposition head to concentrate the aerosol mist; N2 and the aerosol mist generate a high-speed particle flow through the nozzle, the nozzle to the substrate is maintained at 2 to 5 μm, and the constructed organoid is obtained by inoculating it in a culture dish with 0.01 μL / droplet. The present invention can increase the proliferation rate of organoids, shorten the construction time of organoids, and improve the success rate of organoid samples.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and particularly relates to a method for aerosol jet printing 3D organoids. Background Art

[0002] Organoids are cell clusters that are cultured to form a structure similar to an organ with a certain morphological structure and function. The culturing of organoids realizes the high-information complementarity of the existing 2D culturing methods and animal model systems. The difference of organoids is that cells first grow in basement membrane gels and then develop into a few 3D-shaped cell cultures.

[0003] Human development and diseases can be simulated through organoids because organoids grow from human stem cells or induced pluripotent stem cells derived from adult cells. Their composition and structure are also similar to the primary tissues, and they are easy to operate and cryopreserve. This means that organoids can be used for the analysis of human diseases that are difficult to simulate through 2D cell culture or animal models, and researchers only need a small amount of original tissue to culture organoids. At the same time, organoids have great therapeutic potential. Organoid models with genetic similarity to patients can be constructed using surgical and biopsy techniques. Personalized drug sensitivity testing can be performed through organoid systems derived from patients to provide precise treatment for patients.

[0004] Currently, the common method for constructing organoids is to adhere 10 μL - 50 μL of gel droplets to the culture dish wall in a hemispherical shape. Due to the large volume of the microdroplets in this form of culture method, the density between cells is relatively low, resulting in a decrease in the density of organoids. The deposition of organoids on the bottom of the culture well plate cannot fully achieve a 3D structure, which hinders the growth of organoids and is not conducive to high-throughput detection, automated culture, and has poor repeatability. In order to improve the culturing technology for constructing organoids, there is an urgent need to achieve 3D culture of organoids in a new way.

[0005] Aerosol jet 3D printing utilizes the principle of aerodynamics and can achieve a thickness of nanometers and features of micrometers, and is applied in many fields such as medical devices or industrial components. Aerosol jet 3D printing realizes mass production through the coordinated operation of multiple nozzles. Currently, printing parameters such as the carrier gas flow rate, consumption gas flow rate, and sheath gas flow rate of aerosol jet printing technology affect the droplet size of the aerosol, and the matrix gel is a very fragile colloid. Too fast operation speed will affect the structure of the gel droplets. Therefore, the parameters of aerosol jet printing will affect the cell viability of organoid printing and the number of cells encapsulated by the matrix gel, cannot ensure the degree of cell dispersion, and affect the success rate of organoids and the repeat rate of samples. Summary of the Invention

[0006] The present invention provides a method for aerosol jet printing 3D organoids in view of the problems existing in the prior art.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for aerosol jet printing 3D organoids, comprising the following steps:

[0009] Step 1: Obtain the cell pellet of the tissue to be printed;

[0010] Step 2: Deposit the cell pellet and the matrix gel suspension on the substrate by aerosol jet, and 3D organoids can be obtained;

[0011] The aerosol jet method is as follows:

[0012] Place the cell pellet and the matrix gel suspension in an atomizer, and generate an aerosol mist through a pneumatic atomization device; clamp the aerosol mist in N2, and start to concentrate the aerosol mist to the nozzle deposition head; N2 and the aerosol mist generate a high-speed particle flow through the nozzle, and the distance from the nozzle to the substrate is maintained at 2-5 μm, and inoculate 0.01 μL per droplet into a culture dish to obtain the constructed organoids.

[0013] Further, the aerosol mist is generated by providing pressurized N2 in the closed cavity of the atomizer to keep the gas flow rate of the atomizer at 600-640 cm 3 / min.

[0014] Further, the gas flow rate consumed during the generation of the high-speed particle flow is 580-620 scm, and N2 concentrates the aerosol mist at a flow rate of 50-60 scm.

[0015] Further, the method for preparing the cell pellet in step 1 is as follows:

[0016] S1: Place the sample in a culture dish containing a buffer solution, and retain the required tissue;

[0017] S2: Wash the tissue in S1, add a tissue digestive solution, cut it into pieces, then continue to add the tissue digestive solution, add a buffer solution after digestion is completed, and centrifuge;

[0018] S3: Discard the supernatant, add a tissue digestive solution to continue digestion, add a buffer solution after digestion is completed, and centrifuge to collect cells;

[0019] S4: Add a buffer solution to resuspend the cells, pass through a cell sieve, centrifuge, and collect the cells to obtain a cell pellet.

[0020] Further, after step S4, the following steps are further included:

[0021] Add a red blood cell lysate to the cell pellet for red blood cell lysis, and then centrifuge to obtain a cell pellet.

[0022] Furthermore, during the preparation of the cell pellet, the buffer contains 10% (by mass) of double antibodies.

[0023] Furthermore, the digestion conditions in S2 are as follows: digestion in a shaker at 37 °C with 5% CO2 for 1 h; the digestion conditions in S3 are as follows: digestion in a shaker at 37 °C with 5% CO2 for 20 min.

[0024] Furthermore, the centrifugation conditions in steps S3 and S4 are as follows: 1200 rpm, centrifugation for 3 min.

[0025] Furthermore, the centrifugation conditions are as follows: 1200 rpm, centrifugation for 3 min.

[0026] The technical solution adopted in the present invention is as follows:

[0027] (1) The present invention can improve the proliferation rate of organoids, shorten the construction time of organoids, and improve the success rate of organoid samples.

[0028] (2) The present invention can better simulate the three-dimensional structure of in vivo organs and does not require primary expansion.

[0029] (3) The method of the present invention is easy to operate, has a high degree of cell dispersion and good uniformity, enables a higher cell throughput in a single batch, and improves work efficiency. Description of the Drawings

[0030] Figure 1 It is a 3D organoid model diagram for aerosol jet printing.

[0031] Figure 2 It is a pattern diagram of the sample concentrated on the culture dish.

[0032] Figure 3 It is a culture diagram for constructing pancreatic cancer organs in Example 1 of the present invention.

[0033] Figure 4 It is a culture diagram for constructing breast cancer organoids in Example 2 of the present invention.

[0034] Figure 5 It is a culture diagram for constructing lung cancer organoids in Example 3 of the present invention.

[0035] Figure 6 It is a comparative schematic diagram of the culture diagrams of two kinds of organoids in Comparative Example 1 (b) and Example 2 (a) of the present invention.

[0036] Figure 7 It is a comparative schematic diagram of the culture diagrams of two kinds of organoids in Comparative Example 2 (b) and Example 2 (a) of the present invention. Detailed Embodiments

[0037] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0038] A method for aerosol jet printing of 3D organoids, comprising the following steps:

[0039] Step 1: Obtain the cell pellet of the tissue to be printed;

[0040] The cell pellet was prepared as follows:

[0041] S1: Depending on the sample size, transfer the sample to a 3.5 cm or 10 cm culture dish containing pre-cooled DPBS (containing 10% double antibody), remove fat tissue and blood vessels as much as possible, and only retain the required tissue.

[0042] S2: Place the tissue in S1 into a 15 mL centrifuge tube and wash it with DPBS (containing 10% double antibody). Repeat the washing until the washing solution is clear. Remove the DPBS (containing 10% double antibody), transfer the sample to a 1.5 mL EP tube, and add 200 μL digestion solution I (Master Aim TM Tissue digestion solution, 10-100-046), use sterile scissors to cut the tissue into 0.5-1mm 3 Transfer the chopped tissue from the EP tube to a 15 mL centrifuge tube and add 4 mL of digestion solution I (Master Aim TM Tissue digestion solution, 10-100-046), digested for 1 h in a shaker at 5% CO2 and 37°C. Add 8 mL DPBS (containing 10% double antibody) to stop digestion, and centrifuged at 1200 rpm for 3 min.

[0043] S3: Discard the supernatant and add 2 mL of digestion solution II (Master Aim TM Tissue digestion solution, 10-100-046), digested for 20 min in a shaker at 5% CO2 and 37°C. After digestion, 4 mL of DPBS (containing 10% double antibody) was added to terminate digestion, and the cells were collected by centrifugation at 1200 rpm for 3 min.

[0044] S4: Add 2 mL DPBS (containing 10% double antibody) to resuspend the cells, pass through a 100 μM cell sieve, centrifuge at 1200 rpm for 3 minutes, and collect the cells. Carefully discard the supernatant to avoid contact with the cells.

[0045] If the cell pellet contains a lot of red blood cells (the cell pellet appears red), add 1-2 mL of red blood cell lysis buffer to lyse the red blood cells. Use a 1 mL pipette tip to pipette up and down several times and incubate at room temperature for 2 minutes. If the cell pellet volume is less than 100 μL, use 1 mL of red blood cell lysis buffer. If it is greater than 100 μL, use 2 mL of red blood cell lysis buffer. Centrifuge at 1200 rpm for 3 minutes to collect the cells.

[0046] Step 2: Deposit the cell pellet and the Matrigel suspension on the substrate by aerosol jetting to obtain 3D organoids;

[0047] The aerosol jetting method is as follows:

[0048] Use aerodynamic focusing to precisely deposit the cell pellet and a Matrigel suspension with twice the volume on the substrate. The cell pellet and the Matrigel suspension are placed in an atomizer, and the temperature of the atomizer is kept at 4 °C all the time. A suspension aerosol mist with a diameter of 10 μM to 50 μM is generated through a pneumatic atomization device. The aerosol mist is generated by providing pressurized N2 in the closed cavity of the atomizer. Keep the gas flow rate of the atomizer at 600 - 640 standard cubic centimeters per minute (sccm) to generate droplets. Then the suspension aerosol mist is sandwiched in the supplied N2 and driven to the nozzle deposition head, and the consumption gas flow rate is controlled at 580 - 620 sccm. The sheath gas N2 focuses the aerosol mist at a flow rate of 50 - 60 sccm. When the sheath gas and the aerosol pass through the nozzle, they accelerate and are concentrated in a tight droplet flow flowing within the sheath gas, thereby generating a high-speed particle flow, and maintaining a distance of 2 - 5 μM during the movement from the nozzle to the substrate. In this way, organoids are inoculated into a culture dish at 0.01 μL / droplet by aerosol jetting, and the droplets present a 3D microsphere shape.

[0049] Use a pipette gun to add an appropriate volume of the corresponding organoid culture medium along the wall of the culture dish, and culture it in an incubator with 5% CO2 at 37 °C, and observe the culture situation.

[0050] Example 1

[0051] A method for aerosol jet printing 3D organoids, comprising the following steps:

[0052] Step 1: Obtain a cell pellet of the tissue to be printed;

[0053] The method for preparing the cell pellet is as follows:

[0054] S1: Select a pancreatic cancer surgical sample, transfer the sample to a 3.5 cm culture dish containing pre-cooled DPBS (containing 10% double antibiotics), and remove adipose tissue, blood vessels, etc. as much as possible, and only retain the required tissue.

[0055] S2: Put the tissue in S1 into a 15 mL centrifuge tube, wash it with DPBS (containing 10% double antibiotics), and rinse repeatedly until the washing solution is clear. Remove the DPBS (containing 10% double antibiotics), transfer the sample to a 1.5 mL EP tube, and add 200 μL of Digestive Solution Ⅰ (MasterAim TMTissue digestive fluid, 10 - 100 - 046), use sterilized scissors to cut the tissue into pieces, cut to 0.5 - 1 mm 3 in size until the chopped tissue pieces look relatively uniform and somewhat viscous. Transfer the chopped tissue from the EP tube to a 15 mL centrifuge tube, and add 4 mL of Digestive Fluid Ⅰ (MasterAim TM Tissue digestive fluid, 10 - 100 - 046), digest in a shaker at 5% CO₂, 37 °C for 1 h. Add 8 mL of DPBS (containing 10% double antibiotics) to terminate digestion, centrifuge at 1200 rpm for 3 min.

[0056] S3: After discarding the supernatant, add 2 mL of Digestive Fluid Ⅱ (MasterAim TM Tissue digestive fluid, 10 - 100 - 046), digest in a shaker at 5% CO₂, 37 °C for 20 min. After digestion, add 4 mL of DPBS (containing 10% double antibiotics) to terminate digestion, centrifuge at 1200 rpm for 3 min, and collect the cells.

[0057] S4: Add 2 mL of DPBS (containing 10% double antibiotics) to resuspend the cells, pass through a 100 μM cell sieve, centrifuge at 1200 rpm for 3 min, and collect the cells. Carefully discard the supernatant, avoiding contact with the cells.

[0058] Add 2 mL of red blood cell lysate for red blood cell lysis. Use a 1 mL pipette tip to pipette up and down several times, incubate at room temperature for 2 min. Centrifuge at 1200 rpm for 3 min, and collect the cells.

[0059] Step 2: Deposit the cell pellet and Matrigel suspension on the substrate by aerosol jetting to obtain 3D organoids;

[0060] The aerosol jetting method is as follows:

[0061] Aerodynamic focusing was used to precisely deposit the cell pellet and 1.5 volumes of the Matrigel suspension onto the substrate. The cell pellet and Matrigel suspension were placed in the nebulizer, and the nebulizer temperature was kept at 4°C. A suspension aerosol mist with a diameter of 10 μM to 50 μM was generated by a pneumatic atomization device. The aerosol mist was produced by providing pressurized N2 in the closed chamber of the nebulizer. The nebulizer gas flow rate was maintained at 600 standard cubic centimeters per minute sccm to produce droplets. The suspension aerosol mist was then sandwiched in the supplied N2 and driven to the nozzle deposition head, so that the consumed gas flow rate was controlled at 580 sccm. The sheath gas N2 focused the aerosol mist at a flow rate of 50 sccm. When the sheath gas and aerosol pass through the nozzle, they are accelerated and concentrated in a tight droplet stream flowing in the sheath gas, thereby generating a high-speed particle stream and maintaining a distance of 2 μM during the movement from the nozzle to the substrate. This method uses aerosol spray to inoculate 0.01μL / droplet into a culture dish to construct organoids, and the droplets appear as 3D microspheres.

[0062] Use a mobile gun to add an appropriate volume of the corresponding organoid culture medium along the wall of the culture dish, culture in a 5% CO2, 37°C incubator, and observe the culture conditions.

[0063] On the 5th day of culture, 3 mL of TrypLE Express (Life Technologies, 12605-010) digestive enzyme was added to digest the organoids for 5 min, and 5 mL of DPBS (Biological Industries, 02-023-1ACS) was added to stop the digestion. The cells were centrifuged at 1200 rpm for 3 min, resuspended with 1 mL of organoid culture medium, and counted using a cell counter.

[0064] Cultivation conditions such as Figure 3 shown.

[0065] Example 2

[0066] A method for aerosol jet printing of 3D organoids, comprising the following steps:

[0067] Step 1: Obtain the cell pellet of the tissue to be printed;

[0068] The cell pellet was prepared as follows:

[0069] S1: Select breast cancer surgical specimens and transfer them to a 3.5 cm culture dish containing pre-cooled DPBS (containing 10% double antibody). Remove fat tissue and blood vessels as much as possible and retain only the required tissues.

[0070] S2: Transfer the tissue in S1 into a 15 mL centrifuge tube, wash it with DPBS (containing 10% double antibodies), and repeatedly rinse until the washing solution is clear. Remove the DPBS (containing 10% double antibodies), transfer the sample to a 1.5 mL EP tube, and add 200 μL of Digestive Solution Ⅰ (MasterAim TM Tissue Digestive Solution, 10 - 100 - 046). Use a sterilized scissors to cut the tissue into pieces, cut it to a size of 0.5 - 1 mm 3 until the chopped tissue pieces look relatively uniform and somewhat viscous. Transfer the chopped tissue from the EP tube to a 15 mL centrifuge tube, add 4 mL of Digestive Solution Ⅰ (MasterAim TM Tissue Digestive Solution, 10 - 100 - 046), and digest in a shaker at 5% CO2 and 37 °C for 1 h. Add 8 mL of DPBS (containing 10% double antibodies) to terminate the digestion, and centrifuge at 1200 rpm for 3 min.

[0071] S3: After discarding the supernatant, add 2 mL of Digestive Solution Ⅱ (MasterAim TM Tissue Digestive Solution, 10 - 100 - 046), and digest in a shaker at 5% CO2 and 37 °C for 20 min. After the digestion is completed, add 4 mL of DPBS (containing 10% double antibodies) to terminate the digestion, centrifuge at 1200 rpm for 3 min, and collect the cells.

[0072] S4: Add 2 mL of DPBS (containing 10% double antibodies) to resuspend the cells, pass them through a 100 μM cell sieve, centrifuge at 1200 rpm for 3 min, and collect the cells. Carefully discard the supernatant, avoiding contact with the cells.

[0073] Add 2 mL of red blood cell lysate for red blood cell lysis. Use a 1 mL pipette tip to pipette up and down several times, and incubate at room temperature for 2 min. Centrifuge at 1200 rpm for 3 min, and collect the cells.

[0074] Step 2: Deposit the cell pellet and the Matrigel suspension on the substrate by aerosol jetting to obtain 3D organoids;

[0075] The aerosol jetting method is as follows:

[0076] Aerodynamic focusing was used to precisely deposit the cell pellet and 1.5 volumes of the Matrigel suspension onto the substrate. The cell pellet and Matrigel suspension were placed in the nebulizer, and the nebulizer temperature was kept at 4°C. A suspension aerosol mist with a diameter of 10 μM to 50 μM was generated by a pneumatic atomization device. The aerosol mist was provided by pressurized N2 in the closed chamber of the nebulizer. The nebulizer gas flow rate was maintained at 620 standard cubic centimeters per minute sccm to produce droplets. The suspension aerosol mist was then sandwiched in the supplied N2 and driven to the nozzle deposition head, so that the consumed gas flow rate was controlled at 600 sccm. The sheath gas N2 focused the aerosol mist at a flow rate of 55 sccm. When the sheath gas and aerosol pass through the nozzle, they are accelerated and concentrated in a tight droplet stream flowing in the sheath gas, thereby generating a high-speed particle stream and maintaining a distance of 2 μM during the movement from the nozzle to the substrate. This method uses aerosol spray to inoculate 0.01μL / droplet into a culture dish to construct organoids, and the droplets appear as 3D microspheres.

[0077] Use a mobile gun to add an appropriate volume of the corresponding organoid culture medium along the wall of the culture dish, culture in a 5% CO2, 37°C incubator, and observe the culture conditions.

[0078] On the 5th day of culture, 3 mL of TrypLE Express (Life Technologies, 12605-010) digestive enzyme was added to digest the organoids for 5 min, and 5 mL of DPBS (Biological Industries, 02-023-1ACS) was added to stop the digestion. The cells were centrifuged at 1200 rpm for 3 min, resuspended with 1 mL of organoid culture medium, and counted using a cell counter.

[0079] Cultivation conditions such as Figure 4 shown.

[0080] Example 3

[0081] A method for aerosol jet printing of 3D organoids, comprising the following steps:

[0082] Step 1: Obtain the cell pellet of the tissue to be printed;

[0083] The cell pellet was prepared as follows:

[0084] S1: Select lung cancer surgical specimens and transfer them to a 3.5 cm culture dish containing pre-cooled DPBS (containing 10% double antibody). Remove fat tissue and blood vessels as much as possible and retain only the required tissues.

[0085] S2: Put the tissue in S1 into a 15 mL centrifuge tube, wash it with DPBS (containing 10% double antibodies), and rinse repeatedly until the washing solution is clear. Remove the DPBS (containing 10% double antibodies), transfer the sample to a 1.5 mL EP tube, and add 200 μL of Digestive Solution Ⅰ (MasterAim TM Tissue Digestive Solution, 10-100-046). Use a sterilized scissors to cut the tissue into pieces until it reaches a size of 0.5 - 1 mm 3 until the chopped tissue pieces look relatively uniform and somewhat viscous. Transfer the chopped tissue from the EP tube to a 15 mL centrifuge tube, add 4 mL of Digestive Solution Ⅰ (MasterAim TM Tissue Digestive Solution, 10-100-046), and digest it in a shaker at 5% CO2 and 37 °C for 1 h. Add 8 mL of DPBS (containing 10% double antibodies) to terminate the digestion, and centrifuge at 1200 rpm for 3 min.

[0086] S3: After discarding the supernatant, add 2 mL of Digestive Solution Ⅱ (MasterAim TM Tissue Digestive Solution, 10-100-046), and digest it in a shaker at 5% CO2 and 37 °C for 20 min. After the digestion is completed, add 4 mL of DPBS (containing 10% double antibodies) to terminate the digestion, and centrifuge at 1200 rpm for 3 min to collect the cells.

[0087] S4: Add 2 mL of DPBS (containing 10% double antibodies) to resuspend the cells, pass them through a 100 μM cell sieve, centrifuge at 1200 rpm for 3 min, and collect the cells. Carefully discard the supernatant, avoiding contact with the cell pellet.

[0088] Add 2 mL of red blood cell lysate for red blood cell lysis. Use a 1 mL pipette tip to pipette up and down several times, and incubate at room temperature for 2 min. Centrifuge at 1200 rpm for 3 min to collect the cells.

[0089] Step 2: Deposit the cell pellet and the Matrigel suspension on the substrate by aerosol jetting to obtain 3D organoids;

[0090] The aerosol jetting method is as follows:

[0091] Use aerodynamic focusing to precisely deposit the cell pellet and a 1.5-fold volume of Matrigel suspension on the substrate. The cell pellet and Matrigel suspension are placed in an atomizer, while maintaining the atomizer temperature at 4°C all the time. A suspension aerosol mist with a diameter of 10 μM to 50 μM is generated by a pneumatic atomization device. The aerosol mist is provided with pressurized N2 in the closed cavity of the atomizer. Keep the gas flow rate of the atomizer at 640 standard cubic centimeters per minute (sccm) to generate droplets. Then the suspension aerosol mist is sandwiched in the supplied N2 and driven to the nozzle deposition head, and the consumption gas flow rate is controlled at 620 sccm. The sheath gas N2 focuses the aerosol mist at a flow rate of 60 sccm. When the sheath gas and the aerosol pass through the nozzle, they are accelerated and concentrated in a tight droplet flow flowing within the sheath gas, thereby generating a high-speed particle flow and maintaining a distance of 2 μM during the movement from the nozzle to the substrate. In this way, the organoids are inoculated in a culture dish at 0.01 μL / droplet by aerosol spraying, and the droplets present 3D microspheres.

[0092] Use a movable pipette gun to add an appropriate volume of the corresponding organoid medium along the wall of the culture dish, and culture it in a 5% CO2, 37°C incubator, and observe the culture situation.

[0093] On the 5th day of culture, add 3 mL of TrypLE Express (Life Technologies, 12605-010) digestive enzyme, digest the organoids for 5 minutes, add 5 mL of DPBS (Biological Industries, 02-023-1ACS) to terminate the digestion, centrifuge at 1200 rpm for 3 minutes, resuspend with 1 mL of organoid medium, and count with a cell counter.

[0094] The culture situation is as Figure 5 shown.

[0095] Comparative Example 1

[0096] This comparative example provides a comparison of two methods for constructing organoids. One is the culture method used in Example 2, and the other is the culture method for constructing droplet organoids.

[0097] The method of Example 2 will not be elaborated.

[0098] The method for constructing droplet organoids is as follows:

[0099] Step 1 is the same as in Example 2,

[0100] Step 2: Resuspend the obtained cell pellet in 1.5-fold volume of Matrigel equal to that in Example 2, inoculate it in a culture plate at 50 μL / droplet. After inoculation, let the culture plate stand at 37°C for 30 minutes to wait for the Matrigel to completely solidify.

[0101] Use a pipette to gently add 500 μL of lung cancer organoid culture medium preheated to 37 °C along the side wall of each well. Add DPBS (containing 10% double antibiotics) to the other wells without inoculated gel droplets for edge sealing.

[0102] Change the culture medium every 2 - 4 days and passage every 7 - 14 days, and observe the culture conditions.

[0103] On the 5th day of culture, add 3 mL of TrypLE Express (Life Technologies, 12605 - 010) digestive enzyme, digest the organoids for 5 min, add 5 mL of DPBS (Biological Industries, 02 - 023 - 1ACS) to terminate digestion, centrifuge at 1200 rpm for 3 min, resuspend with 1 mL of organoid culture medium, and count using a cell counter.

[0104] The culture conditions are as Figure 6 shown

[0105] The counting results are shown in Table 1

[0106] Culture method version Cell count (W) Viability rate (%) Culture method of Example 2 524.00 88 Culture method of Comparative Example 1 106.00 86

[0107] The observation results of the two groups are shown in Table 2

[0108]

[0109] From Figure 6 and Table 1 and Table 2, it can be seen that the aerosol jet printing 3D organoid culture method improves the proliferation rate of organoids and shortens the time for organoid construction compared with the traditional organoid construction culture method. At the same time, it improves the cell throughput, eliminates the need for primary expansion culture, facilitates subsequent experiments, and improves work efficiency.

[0110] Comparative Example 2

[0111] This comparative example provides a comparison of two organoid construction culture methods. One is the culture method used in Example 2, and the other is the gel droplet organoid construction culture method.

[0112] This comparative example provides a comparison of two organoid construction culture methods. One is the culture method used in Example 2, and the other is the microfluidic droplet 3D printing organoid culture method.

[0113] The method of Example 2 will not be elaborated here.

[0114] The microfluidic droplet 3D printing organoid culture method is as follows:

[0115] Step 1 is the same as in Example 2,

[0116] Step 2: In the microfluidic droplet printing organoid device, the cell pellet is assembled with a two-fold volume of matrix gel suspension in a cryostat (cooler), and the suspension is maintained at 4 °C to prevent its gelation. Two polytetrafluoroethylene (PTFE) injection tubes for the suspension and the volatile cell-compatible oil (HFE7000, 3M) are connected to a third tube through a three-way polydimethylsiloxane (PDMS) connector, where the cell-loaded growth factor makes the mixed solution into monodisperse droplets of 500 μM size. The solidification of the suspension droplets is accelerated by incubation through the tube in a water bath heated to 37 °C outside the cooler. The suspension droplets are equally spaced by the cell-compatible oil, undergo a sol-gel transition in the same pipeline, and are transformed into microdroplet spheres before approaching the pipeline outlet. The droplet printer discretizes the microdroplet spheres at the pipeline outlet into a 96-well microplate.

[0117] Use a pipette to add an appropriate volume of lung cancer organoid medium along the wall of the culture well plate, and culture it in an incubator at 5% CO2 and 37 °C, and observe the culture condition.

[0118] Use a pipette to add an appropriate volume of lung cancer organoid medium along the wall of the culture well plate, and culture it in an incubator at 5% CO2 and 37 °C, and observe the culture condition.

[0119] On the 5th day of culture, add 3 mL of TrypLE Express (Life Technologies, 12605-010) digestive enzyme, digest the organoids for 5 min, add 5 mL of DPBS (Biological Industries, 02-023-1ACS) to terminate the digestion, centrifuge at 1200 rpm for 3 min, resuspend with 1 mL of organoid medium, and count with a cell counter.

[0120] The culture conditions are as Figure 7 shown.

[0121] The counting results are shown in Table 3:

[0122] Culture method version Cell count (W) Viability rate (%) Culture method of Example 2 498.00 89 Culture method of Comparative Example 2 346.00 89

[0123] The two groups of observation results are shown in Table 4:

[0124]

[0125]

[0126] From Figure 7 Table 3 and Table 4, it can be seen that the aerosol jet printing 3D organoid culture method shortens the organoid construction time more than the microfluidic 3D printing-based organoid culture method, and improves the cell throughput and working efficiency.

[0127] The method of 3D organoid printing by aerosol jet can improve the proliferation rate of organoids, shorten the organoid modeling time, and eliminate the need for primary amplification, thereby increasing the cell throughput, facilitating subsequent experiments, and improving work efficiency.

[0128] The present invention can increase the proliferation rate of organoids, shorten the construction time of organoids, and improve the survival rate of organoid samples. When used in subsequent experiments, there is no need for primary expansion, which can better simulate the three-dimensional structure of in vivo organs. It is convenient to operate, has a high degree of cell dispersion and good uniformity, resulting in a higher cell throughput in a single batch and improving work efficiency.

Claims

1. A method for constructing 3D organoids using aerosol jet printing, characterized in that, It includes the following steps: Step 1: Obtain the cell pellet of the tissue to be printed; Step 2: Deposit the cell pellet and the Matrigel suspension on the substrate by aerosol jetting to construct 3D organoids; The aerosol jetting method is as follows: Place the cell pellet and the Matrigel suspension in an atomizer to generate an aerosol mist through a pneumatic atomization device; clamp the aerosol mist in N2 and start to concentrate the aerosol mist to the nozzle deposition head; N2 and the aerosol mist generate a high-speed particle flow through the nozzle, keep the distance from the nozzle to the substrate at 2 - 5 μm, and inoculate it into the culture dish at 0.01 μL / droplet by aerosol jetting to construct 3D organoids.

2. The method for constructing 3D organoids using aerosol jet printing according to claim 1, characterized in that, The aerosol mist is generated by providing pressurized N2 in the closed cavity of the atomizer to maintain the gas flow rate of the atomizer at 600 - 640 cm 3 / min, producing droplets.

3. The method for constructing 3D organoids by aerosol jet printing according to claim 1, wherein During the process of generating the high-speed particle flow, the gas flow rate consumed is 580 - 620 sccm, and N2 concentrates the aerosol mist at a flow rate of 50 - 60 sccm.

4. A method for constructing 3D organoids using aerosol jet printing according to claim 1, characterized in that, The method for preparing the cell pellet in Step 1 is as follows: S1: Place the sample in a culture dish containing buffer and retain the required tissue; S2: Wash the tissue in S1, add tissue digestive solution, cut it into pieces and then continue to add tissue digestive solution. After digestion is completed, add buffer and centrifuge; S3: Discard the supernatant and then add tissue digestive solution to continue digestion. After digestion is ended, add buffer and centrifuge to collect cells; S4: Add buffer to resuspend the cells, pass through a cell sieve, centrifuge, and collect the cells to obtain the cell pellet.

5. A method for constructing 3D organoids using aerosol jet printing according to claim 4, characterized in that, After Step S4, the following steps are further included: Add red blood cell lysate to the cell pellet for red blood cell lysis, and then centrifuge to obtain the cell pellet.

6. A method for constructing 3D organoids using aerosol jet printing according to claim 4, characterized in that, During the preparation process of the cell pellet, the buffer contains 10% (mass fraction) of double antibodies.

7. A method for constructing 3D organoids using aerosol jet printing according to claim 4, characterized in that The digestion conditions in S2 are digestion in a 5% CO2, 37 °C shaker for 1 h; the digestion conditions in S3 are digestion in a 5% CO2, 37 °C shaker for 20 min.

8. A method for constructing 3D organoids using aerosol jet printing according to claim 4, characterized in that The centrifugation conditions in Steps S3 and S4 are as follows: 1200 rpm, centrifuge for 3 min.

9. A method for constructing 3D organoids using aerosol jet printing according to claim 5, characterized in that, The centrifugation conditions are as follows: 1200 rpm, centrifuge for 3 min.

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