Lung adenocarcinoma 2.5 D organ yield increase and consumption reduction production process using 96-well plate

Abstract

The invention provides a lung adenocarcinoma 2.5 D organ yield-increasing and consumption-reducing production process using a 96-well plate, the process uses a conventional 96-well plate with the diameter of each well less than 1cm to reduce the loss of raw materials, and meanwhile, two new methods suitable for the 96-well plate are provided to increase the diameter and yield of 2.5 D organs. The invention relates to the technical field of organoids, raw material loss is reduced by using a 96-well plate, and the contact opportunity between cells is increased by limiting tumor cells in a narrow space, so that the volume production of 2.5 D organoids of lung adenocarcinoma can be realized. The 96-well plate with a smaller area is used for reducing the loss of raw materials, and meanwhile, the yield and diameter of the organoid can be greatly increased.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and in particular to a production process for increasing the yield and reducing the consumption of lung adenocarcinoma 2.5D organoids using a 96-well plate. Background Art

[0002] 2.5D culture is a method in which cells grow on a 2D surface but exhibit characteristics similar to those of cells grown on a 3D surface, providing intermediate conditions between traditional 2D and 3D culture. This method uses an uneven layer of extracellular matrix (ECM) to provide a textured surface for cell growth, and is defined by researchers as a means of bridging the gap between two-dimensional and three-dimensional growth environments, which is the advantage of 2.5D culture. A variety of 2.5D culture modes have been developed to optimize culture results and reduce culture costs. For example, researchers have developed high-quality, low-cost 2.5D culture technology by combining egg white-alginate (EWA) to develop a new hydrogel as an extracellular matrix and scaffold material. The 2.5D culture method using transwell systems has also shown great potential in establishing an in vitro model of pulmonary fibrosis. As early as 2020, researchers proposed a hypothesis for the preparation of 2.5D organoids and achieved success, believing that certain culture medium components can enable patient-derived 3D organoid cells to be cultured in matrix-free 2D culture conditions without losing the characteristics of their original 3D organoids, such as marker expression, stemness, and drug sensitivity. To simplify the process, researchers have developed new culture protocols or changed the culture medium formula to achieve economical and high-yield 2.5D organoid culture.

[0003] We have also developed a lung adenocarcinoma 2.5D organoid culture process in the early stage, that is, culture in a 10cm diameter culture dish, but the number of organoids formed by this process is small, only dozens of organoids are formed in each culture dish, and the diameter is small, about 50 microns. This output is generally only enough for organoid identification testing. If it is to meet subsequent clinical applications and scientific research, more culture dishes are needed to achieve it, which is time-consuming, labor-intensive, and space-consuming, and the cost is increased accordingly. In order to improve this situation, this patent has developed a new culture process, that is, using a 96-well plate with a diameter of less than 1cm per well to constrain the range of tumor cell activity, and by increasing the cell seeding density to increase the frequency of contact between cells, and then by changing the culture fluid formula to ultimately achieve increased production and revenue, while achieving low-cost advantages. Summary of the invention

[0004] In view of this, the present invention aims to propose a production process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate, which uses a smaller 96-well plate to reduce raw material loss while significantly improving the yield and diameter of organoids.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate, comprising the following steps:

[0007] Step 1: Obtain a sample of malignant pleural effusion;

[0008] Step 2: Filter the malignant pleural effusion sample in step 1 through a 100-micron cell mesh to obtain a filtered sample;

[0009] Step 3: Centrifuge the filtered sample at 200G for 5 minutes at 4°C to obtain a centrifuged sample;

[0010] Step 4: Add red blood cell lysis solution to the centrifuged sample to remove red blood cells;

[0011] Step 5: Add PBS to the sample without red blood cells to terminate lysis and wash the cell pellet;

[0012] Step 6: If cell clusters are produced in step 5, use 0.25% trypsin to digest the cell clusters. If cell clusters are not produced, skip this step.

[0013] Step 7: Use a 40-micron cell sieve to filter out tumor cells;

[0014] Step 8: Count tumor cells under a microscope;

[0015] Step 9: Prepare 2.5D scaffold cell layer in a 96-well plate. Resuspend the sample cell pellet from step 1 and add 10 4 The cells were seeded in a 96-well plate and cultured in DMEM-F12 medium containing 10% fetal bovine serum in an atmosphere of 5% CO. 2 Culture in a 37°C constant temperature incubator for 1-2 days until a dense layer of adherent cells, i.e., a 2.5D scaffold cell layer, is formed at the bottom of the culture dish. After removing the suspended cells, add an equal amount of DMEM-F12 culture medium and continue culturing for later use;

[0016] Step 10: 20U tumor cell inoculation: 1U = 1.1 x 10 6 The tumor cells prepared in step 8 were inoculated on the 2.5D scaffold cell layer in a 96-well plate at 20 U per well, and the culture medium was used in a 5% CO 2 Continue culturing in a 37°C constant temperature incubator to wait for cell aggregation and differentiation, and replace the culture medium every 3 days. After 3-10 days, when the organoid particles are visible to the naked eye, i.e. 2.5D organoids, count and measure the diameter under a microscope.

[0017] Step 11: Collect the 2.5D organoids from each well of the 96-well plate from step 10 using a 40 μm cell strainer.

[0018] Furthermore, if complete digestion is not possible in step 6, 0.1% type I collagenase is added.

[0019] Furthermore, if the number of tumor cells in step eight is small, a cell screen or a TransferMan 4r micromanipulator is used for enrichment.

[0020] Furthermore, if the sample in step nine does not form a scaffold cell layer, X20009933 is used as the 2.5D scaffold cell layer;

[0021] Here’s how:

[0022] X20009933 cells were plated at 10 4 The cells were seeded in 96-well plates using DMEM-F12 medium containing 10% fetal bovine serum and incubated in 5% CO 2 Culture in a 37°C constant temperature incubator for 1-2 days until the cells adhere to the wall and grow to cover the bottom of the well plate before proceeding to the next step.

[0023] Furthermore, the X20009933 is an adherent cell derived from malignant pleural effusion of a patient with lung adenocarcinoma, has a mesenchymal-like cell morphology, and can be stably propagated.

[0024] Furthermore, the homemade nutrient solution in step 10 is a mixture of MPE and DMEM-F12 in a ratio of 1:1-1:3.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using 96-well plates described in the present invention increases the yield and diameter of organoids, effectively reduces production costs, and can produce more than a thousand organoids in each batch, a quantity that fully meets the needs of subsequent clinical applications and scientific research. This process is applicable to malignant pleural effusion samples from all patients. Since different organoids can be cultured in each well and different treatment conditions can be set, this process can provide solid technical support for the mass production and high-throughput treatment of 2.5D lung adenocarcinoma organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0028] Figure 1This is a process flow chart of increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate as described in an embodiment of the present invention;

[0029] Figure 2 It is a substitute raw material for the 2.5D scaffold cell layer of the production process for increasing the yield and reducing the consumption of 2.5D organoids of lung adenocarcinoma using a 96-well plate as described in the embodiment of the present invention;

[0030] Figure 3 The lung adenocarcinoma 2.5D organoids formed under different ratios of malignant pleural effusion and DMEM-F12 were observed under an optical microscope in the process for increasing the yield and reducing the consumption of the lung adenocarcinoma 2.5D organoids using a 96-well plate as described in the embodiment of the present invention;

[0031] Figure 4 This is a statistical graph of the number and diameter of lung adenocarcinoma 2.5D organoids formed under different ratios of malignant pleural effusion and DMEM-F12 in the lung adenocarcinoma 2.5D organoid production process for increasing production and reducing consumption using a 96-well plate as described in an embodiment of the present invention;

[0032] Figure 5 The 2.5D organoids formed under different gradient tumor cell seeding density conditions are observed under an optical microscope in the process for increasing production and reducing consumption of 2.5D organoids for lung adenocarcinoma using a 96-well plate as described in the embodiment of the present invention;

[0033] Figure 6 It is a statistical graph of the number and diameter of lung adenocarcinoma 2.5D organoids formed at different gradient tumor cell seeding densities in the lung adenocarcinoma 2.5D organoid production process for increasing production and reducing consumption using a 96-well plate as described in an embodiment of the present invention; DETAILED DESCRIPTION

[0034] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0035] In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "upper", "lower", "inner", "back", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0036] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0037] Based on the above design concept, an exemplary process flow chart of the lung adenocarcinoma 2.5D organoid production process using a 96-well plate to increase production and reduce consumption is shown in the following figure: Figure 1As shown, it mainly includes the following steps:

[0038] Step 1: Obtain a sample of malignant pleural effusion;

[0039] Step 2: Filter the malignant pleural effusion sample in step 1 through a 100-micron cell mesh to obtain a filtered sample;

[0040] Step 3: Centrifuge the filtered sample at 200G for 5 minutes at 4°C to obtain a centrifuged sample;

[0041] Step 4: Add red blood cell lysis solution to the centrifuged sample to remove red blood cells;

[0042] Step 5: Add PBS to the sample without red blood cells to terminate lysis and wash the cell pellet;

[0043] Step 6: If cell clusters are produced in step 5, use 0.25% trypsin to digest the cell clusters. If cell clusters are not produced, skip this step.

[0044] Step 7: Use a 40-micron cell sieve to filter out tumor cells;

[0045] Step 8: Count tumor cells under a microscope;

[0046] Step 9: Prepare 2.5D scaffold cell layer in a 96-well plate. Resuspend the sample cell pellet from step 1 and add 10 4 The cells were seeded in a 96-well plate and cultured in DMEM-F12 medium containing 10% fetal bovine serum in an atmosphere of 5% CO. 2 Culture in a 37°C constant temperature incubator for 1-2 days until a dense layer of adherent cells, i.e., a 2.5D scaffold cell layer, is formed at the bottom of the culture dish. After removing the suspended cells, add an equal amount of DMEM-F12 culture medium and continue culturing for later use;

[0047] Step 10: 20U tumor cell inoculation: 1U = 1.1 x 10 6 The tumor cells prepared in step 8 were inoculated on the 2.5D scaffold cell layer in a 96-well plate at 20 U per well, and the culture medium was used in a 5% CO 2 Continue culturing in a 37°C constant temperature incubator to wait for cell aggregation and differentiation, and replace the culture medium every 3 days. After 3-10 days, when the organoid particles are visible to the naked eye, i.e. 2.5D organoids, count and measure the diameter under a microscope.

[0048] Step 11: Collect the 2.5D organoids from each well of the 96-well plate from step 10 using a 40 μm cell strainer.

[0049] Step 6: If complete digestion is not possible, add 0.1% collagenase type I.

[0050] If the number of tumor cells in step eight is small, use a cell screen or TransferMan 4r micromanipulator to enrich them.

[0051] If the sample in step 9 did not form a scaffold cell layer, X20009933 was used as the 2.5D scaffold cell layer;

[0052] Here’s how:

[0053] X20009933 cells were plated at 10 4 The cells were seeded in 96-well plates using DMEM-F12 medium containing 10% fetal bovine serum and incubated in 5% CO 2 Culture in a 37°C constant temperature incubator for 1-2 days until the cells adhere to the wall and grow to cover the bottom of the well plate before proceeding to the next step.

[0054] X20009933 is an adherent cell derived from malignant pleural effusion of a patient with lung adenocarcinoma. It has a mesenchymal-like cell morphology and can be stably passaged.

[0055] The homemade nutrient solution in step 10 is a mixture of MPE and DMEM-F12 in a ratio of 1:1-1:3.

[0056] Step 1: Obtain a sample of malignant pleural effusion;

[0057] Five MPE samples of patients with pathologically confirmed lung adenocarcinoma were collected. Patients with initial treatment and drug resistance can be included in this process. Patients aged 18-65 years old, with malignant pleural effusion of 100-200 ml, in which the tumor cell count must reach 10 7 Patients with multiple cancers, patients undergoing targeted chemotherapy or receiving thoracic perfusion chemotherapy, and patients with transformation of lung adenocarcinoma to squamous cell carcinoma or small cell lung cancer need to be excluded. The sample acquisition has obtained written informed consent from the patient and was approved by the Ethics Committee of Jilin Cancer Hospital (approval number: 202110-040-01);

[0058] Step 2: Under sterile conditions, the malignant pleural effusion in step 1 is filtered using a 100-micron cell mesh to remove filamentous mucus substances to obtain a filtered sample;

[0059] Step 3: The filtered sample was centrifuged at 200G for 5 min at 4°C to collect tumor cells and supernatant.

[0060] Step 4: Incubate the cell pellet with 5 ml of sterile FACS Lysing Solution (BD, USA) at room temperature for 5 min until the liquid becomes transparent to remove red blood cells;

[0061] Step 5: Add 20 ml PBS to stop lysis and wash, centrifuge at 200G for 5 min, discard the supernatant, wash once with 5 ml PBS, centrifuge at 200G for 5 min, and discard the supernatant;

[0062] Step 6: If there are cells aggregated or flocculent or micro-cancer nests in the precipitate, they need to be digested with 0.25% trypsin (Genview, USA) and / or 0.1% type I collagenase (Invitrogen, USA) to a single cell suspension. If no cell clusters are produced, skip this step;

[0063] Step 7: Use a 40-micron cell sieve to filter out tumor cells;

[0064] Step 8: Count tumor cells under a microscope. If the number of tumor cells is small, a cell screen or TransferMan 4r micromanipulator may be used for enrichment.

[0065] Step 9: The patient's MPE often contains multiple types of cells, such as fibroblasts, immune cells, mesenchymal cells, etc. These cells are used to make a scaffold cell layer to replace the matrix gel as a cell growth scaffold. Resuspend the MPE cell pellet and place 10 4 The cells were seeded in 96-well plates (Fisher, USA) and cultured in DMEM-F12 medium (Hyclone, USA) containing 10% fetal bovine serum (Life, USA) in an atmosphere of 5% CO. 2 Culture in a 37°C constant temperature incubator (Thermo Scientific, USA) for 1-2 days. When a dense layer of adherent cells is formed at the bottom of the culture dish, remove the suspended cells and add an equal amount of DMEM-F12 culture medium to continue culturing for later use. This layer of adherent cells is called a 2.5D scaffold cell layer in our previous process.

[0066] For the MPE samples that cannot produce the scaffold cell layer in step 9, a 2.5D scaffold cell layer is used as a substitute raw material, such as Figure 2 This raw material is derived from adherent cells derived from MPE of other lung adenocarcinoma patients, has a mesenchymal-like cell morphology, and can be stably passaged. We named this alternative raw material X20009933. X20009933 cells were cultured at 10 4 The cells were seeded in 96-well plates (Fisher, USA) using DMEM-F12 medium (Hyclone, USA) containing 10% fetal bovine serum (Life, USA) and cultured in an atmosphere of 5% CO. 2 The cells were cultured in a 37°C constant temperature incubator (Thermo Scientific, USA) for 1-2 days. The next step was performed when the cells adhered to the wall and grew to cover the bottom of the well plate.

[0067] Step 10: 20U tumor cell inoculation: 1U = 1.1 x 10 6 Cells were inoculated in step eight on the 2.5D scaffold cell layer in a 96-well plate at 20 U per well, and the culture medium was used to continue culturing in a 37°C constant temperature incubator with 5% CO2 to wait for cell aggregation and differentiation. The culture medium was replaced every 3 days. After 3-10 days, the organoid particles were visible to the naked eye, i.e., 2.5D organoids, and the numbers and diameters were measured under a microscope.

[0068] Step 11: Use a 40-micron cell mesh to filter and collect the 2.5D organoids in each well of the 96-well plate in step 10;

[0069] In this process, the culture medium formula was changed, and the ratio of MPE in DMEM-F12 culture medium was set to 0%, 25%, 50%, 75% and 100% in five gradients. 4 Pieces / cm 2 Tumor cells were inoculated on the scaffold cell layer, and the cells were resuspended in the mixture of the five gradient MPE supernatants and DMEM-F12 (Hyclone, USA) as the culture medium and then placed in 5% CO 2 The cells were cultured in a 37°C constant temperature incubator (Thermo Scientific, USA) and the culture medium was changed every 3 days until the cells aggregated and differentiated. The culture medium was cultured for about 3-10 days. When the organoid particles were visible to the naked eye, they were counted and their diameters were measured under a microscope. The yield was determined by the count of organoids, and the diameter was determined by the measurement under a microscope. The final ratio with the highest yield and the largest diameter was used as the new formula parameter of the culture medium for this process.

[0070] Adjust the ratio of MPE supernatant, the result is as follows Figure 3 and Figure 4 As shown. Organoids began to be significantly generated on the third day of culture (p<0.001), and there were significant differences in the number and diameter of organoids between different concentration groups (p<0.001). The number began to decrease significantly on the seventh day (p<0.05), and there was no significant difference in diameter change (p>0.05). The number and diameter of the 50% MPE group were the highest among all groups, with a maximum of 40 organoids per well; while the number of the 25% group was slightly lower than that of the 50% group, there was no statistically significant difference between the two groups (p>0.05). In addition, compared with the third day, although the number (65 vs. 54) and diameter (94.53±28.39 vs.86.01±26.67) on the seventh day decreased, there was no statistical difference (p=0.081). These data show that the ratio of MPE supernatant to DMEM-F12 is 1:1~1:3, which can achieve an optimal yield.

[0071] This process uses unit (U) as the unit of tumor cell concentration, and defines 1U unit = 1.1 x 10 6 The cells were seeded into 96-well plates at a cell concentration gradient of 1U, 2.5U, 5U, 7.5U, 10U, and 20U, respectively. The cells were resuspended in a 1:1 mixture of MPE supernatant and DMEM-F12 (Hyclone, USA) as the culture medium and then placed in a 5% CO 2 Continue to culture in a 37°C constant temperature incubator (Thermo Scientific, USA) to wait for cell aggregation and differentiation, and replace the culture medium every 3 days. After about 3-10 days, when the organoid particles are visible to the naked eye, count and measure the diameter under a microscope. The yield is determined by the count of organoids, and the diameter is determined by the measurement under a microscope. Finally, the cell inoculation condition with the highest yield and the largest diameter is used as the execution parameter of this process.

[0072] Determine the tumor cell seeding density. Based on the above exploration results, the ratio of MPE supernatant to DMEM-F12 was 1:1. The results are as follows Figure 5 and Figure 6 As shown in the figure, with the increase of cell seeding density, the diameter and number of organoids also increased significantly. When the density reached 20U, the organoid yield increased significantly (p<0.001). Among them, the organoids with a diameter ≥100 microns showed significant differences among the groups (p<0.05), and the number of organoids in the 20U group could reach up to 42, with an average diameter of 139.74±29.05 microns. These results show that tumor cell seeding density is a key factor affecting organoid generation and diameter. In this process, a seeding density of 20U is recommended for organoid culture.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate, characterized in that: The following steps are involved: Step 1: Obtain a sample of malignant pleural effusion; Step 2: Filter the malignant pleural effusion sample in step 1 through a 100-micron cell mesh to obtain a filtered sample; Step 3: Centrifuge the filtered sample at 200G for 5 minutes at 4°C to obtain a centrifuged sample; Step 4: Add red blood cell lysis solution to the centrifuged sample to remove red blood cells; Step 5: Add PBS to the sample without red blood cells to terminate lysis and wash the cell pellet; Step 6: If cell clusters are produced in step 5, use 0.25% trypsin to digest the cell clusters. If cell clusters are not produced, skip this step. Step 7: Use a 40-micron cell sieve to filter out tumor cells; Step 8: Count tumor cells under a microscope; Step 9: Prepare 2.5D scaffold cell layer in a 96-well plate. Resuspend the sample cell pellet from step 1 and add 10 4 The cells were inoculated into a 96-well plate and cultured in a 37°C constant temperature incubator with 5% CO2 for 1-2 days using DMEM-F12 culture medium containing 10% fetal bovine serum. When a dense layer of adherent cells, i.e., a 2.5D scaffold cell layer, was formed at the bottom of the culture dish, the suspended cells were removed and an equal amount of DMEM-F12 culture medium was added to continue culturing for later use; Step 10: 20U tumor cell inoculation: 1U = 1.1 x 10 6 Cells were inoculated in step eight on the 2.5D scaffold cell layer in a 96-well plate at 20 U per well, and the culture medium was used to continue culturing in a 37°C constant temperature incubator with 5% CO2 to wait for cell aggregation and differentiation. The culture medium was replaced every 3 days. After 3-10 days, the organoid particles were visible to the naked eye, i.e., 2.5D organoids, and the numbers and diameters were measured under a microscope. Step 11: Collect the 2.5D organoids from each well of the 96-well plate from step 10 using a 40 μm cell strainer.

2. The process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate according to claim 1, characterized in that: If complete digestion is not possible in step 6, 0.1% type I collagenase was added.

3. The process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate according to claim 1, characterized in that: If the number of tumor cells in step eight is small, a cell screen or TransferMan 4r micromanipulator is used for enrichment.

4. The process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate according to claim 1, characterized in that: If the sample in step nine does not form a scaffold cell layer, X20009933 is used as the 2.5D scaffold cell layer; Here’s how: X20009933 cells were plated at 10 4 The cells were inoculated into a 96-well plate using DMEM-F12 medium containing 10% fetal bovine serum and cultured in a 37°C constant temperature incubator with 5% CO2 for 1-2 days. The next step was performed when the cells adhered to the wall and grew to cover the bottom of the well plate.

5. The process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate according to claim 4, characterized in that: The X20009933 cells are adherent cells derived from malignant pleural effusion of patients with lung adenocarcinoma, have a mesenchymal-like cell morphology, and can be stably propagated.

6. The process for increasing the yield and reducing the consumption of 2.5D lung adenocarcinoma organoids using a 96-well plate according to claim 1, characterized in that: The homemade nutrient solution in step 10 is a mixture of MPE and DMEM-F12 in a ratio of 1:1-1:3.

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