A gastric cancer organoid microarray culture medium and its application

Abstract

This invention relates to the field of gastric cancer organoid microarray technology, and more particularly to a gastric cancer organoid microarray culture medium and its applications. The invention provides a novel culture medium adapted for gastric cancer organoid microarray culture. The medium components include basal medium, penicillin / streptomycin dual antibiotics, antibacterial agents, hydroxyethylpiperazine ethanesulfonic acid, B-27 additive, Glutamax culture medium additive, R-vertebral protein 1, scutellarin B, and tetramethylpyrazine, among other components. This invention solves key problems in existing technologies such as slow growth, morphological instability, poor heterogeneity adaptation, and low long-term culture activity of gastric cancer organoids, providing an ideal experimental model for high-throughput screening and precision medicine applications on microarrays.

Description

Technical Field

[0001] This invention relates to the field of gastric cancer organoid chip technology, and more particularly to a gastric cancer organoid chip culture medium and its application. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors worldwide, with persistently high incidence and mortality rates, posing a serious threat to human health. Although some progress has been made in the diagnosis and treatment of gastric cancer in recent years, due to the complexity and heterogeneity of the disease, existing treatment methods still have many limitations, and patient prognoses are often poor. Therefore, in-depth research into the pathogenesis of gastric cancer, exploration of new therapeutic targets and drugs, and optimization of individualized treatment plans have become key issues in the current field of gastric cancer research.

[0003] The emergence of organoid technology has brought new opportunities to cancer research. Organoids are organ-like structures generated by culturing stem cells in vitro. They possess self-renewal and self-organization capabilities and are highly similar to their source tissues or organs in structure and function. As a research tool at the human tissue level, organoids have complex structures similar to real organs and can partially mimic the physiological functions of their source tissues or organs, demonstrating enormous application potential in many areas such as basic research on organ physiology and pathology, drug screening and development, gene therapy, and regenerative medicine.

[0004] Gastric cancer organoids can preserve the genetic and phenotypic heterogeneity of patient tumors, providing an ideal model for studying tumor occurrence, development, drug screening, and precision medicine. However, traditional organoid culture methods have some problems, such as difficulty in precisely controlling culture conditions, complex culture medium composition, and high cost. In addition, traditional culture methods cannot fully simulate the dynamic environment inside the human body, such as the continuous supply of nutrients and oxygen, and the timely removal of waste.

[0005] Microfluidic technology, through precise control of fluid flow at the micrometer level, can provide cells with a culture environment that more closely resembles in vivo physiological conditions. Microfluidic organoid chips combine the advantages of microfluidic technology and organoid culture, simulating the dynamic changes of the tumor microenvironment and providing more stable and controllable conditions for organoid growth and development. For example, microfluidic chips can simulate the circulatory system within tissues, providing organoids with constant and sufficient nutrients and oxygen, thereby improving organoid survival and proliferation capacity. Furthermore, microfluidic chips can also achieve high-throughput screening and analysis, improving research efficiency.

[0006] Currently, although research has been conducted on culturing gastric cancer organoids using microfluidic chips, many technical challenges remain. Existing culture media systems, mostly modified from traditional organoid culture formulations, are difficult to fully adapt to the unique culture environment of microfluidic chips. Within the microchannels of a microfluidic chip, the culture medium needs to continuously circulate in a dynamic fluid environment. The composition and ratio of existing media cannot precisely match the dynamic requirements of gastric cancer organoids for nutrients and growth factors under fluid shear forces, resulting in limited organoid proliferation rates and difficulty in maintaining long-term culture activity. Furthermore, due to the significant biological heterogeneity of gastric cancer organoids from different patients, existing general-purpose culture media lack customized designs to address this heterogeneity. This makes it difficult to achieve efficient expansion when culturing gastric cancer organoids from different genetic backgrounds, failing to meet the needs of personalized clinical diagnosis and treatment and high-throughput drug screening.

[0007] Therefore, developing a culture medium suitable for culturing gastric cancer organoids in microfluidic chips is imperative. This culture medium must fully consider the fluid characteristics of the microfluidic chip and the heterogeneity of gastric cancer organoids, optimizing the ratio and combination of nutrients, growth factors, cytokines, and extracellular matrix components to achieve precise regulation of the growth, differentiation, and metabolism of gastric cancer organoids. Developing such a culture medium is expected to overcome existing technological limitations, providing more efficient and reliable technical support for basic research, drug screening, and clinical diagnosis and treatment of gastric cancer, thus promoting the development of gastric cancer research and treatment. Summary of the Invention

[0008] The purpose of this invention is to provide a gastric cancer organoid microarray culture medium and its application.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a gastric cancer organoid microarray culture medium, comprising the following components:

[0011]

[0012]

[0013] The rest are basic culture media.

[0014] Preferably, the basal culture medium is advanced DMEM / F12 medium with a concentration of 1.5×; the double antibiotics are penicillin and streptomycin.

[0015] Preferably, the antibacterial agent is Primocin.

[0016] The present invention also provides the application of the aforementioned gastric cancer organoid microarray culture medium in the preparation of gastric cancer organoids.

[0017] Preferably, the method for preparing the gastric cancer organoid includes the following steps:

[0018] (1) Take the primary gastric cancer cells and mix them with matrix gel to obtain a matrix gel-cell mixture;

[0019] (2) The matrix gel cell mixture is injected into the gastric cancer organoid chip, incubated, and then injected into the culture medium for further culture.

[0020] Preferably, the concentration of the primary gastric cancer cell precipitate is 1500–2500 cells / μl.

[0021] Preferably, the matrix adhesive in step (1) is Matrigel Matrix Basement Membrane (Corning / REF356231).

[0022] Preferably, the incubation time in step (2) is 40-60 min, the temperature is 36-38 °C, and the CO2 concentration is 4-6%.

[0023] Preferably, the culture temperature in step (2) is 36–38°C and the CO2 concentration is 4–6%.

[0024] Preferably, the culture medium is changed every 2 to 3 days during the culture process described in step (2).

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

[0026] This invention discloses a novel culture medium suitable for gastric cancer organoid microarray culture. After extensive experimentation, the inventors achieved a breakthrough by screening a combination of components that selectively promote tumor cell growth, inhibit normal cell proliferation, and optimize three-dimensional microenvironment compatibility. By optimizing the concentration of key growth factors and introducing active ingredients from traditional Chinese medicine to reduce non-tumor cell contamination, the purity of tumor cells in gastric cancer organoids is improved, promoting the dominant growth of tumor cells and enabling the experimental model to more accurately reflect tumor characteristics. Furthermore, this culture medium solves common problems in microarray culture such as low spheroidization efficiency, competition from normal cells, and obstacles to material transport. Particularly innovative is the addition of Y-27632 culture medium as a ROCK inhibitor, which strengthens intercellular connections and resists structural damage caused by microfluidic shear forces. The addition of scutellarin and ligustrazine not only reduces free radical damage through antioxidant effects but also improves the material exchange efficiency within the organoids, solving the problem of core cell necrosis caused by mass transfer barriers in traditional culture media. This culture medium, through the synergistic effect of multiple components, is fully adapted to the dynamic environment of microfluidic chips, effectively solving key problems in existing technologies such as slow growth of organoids, unstable morphology, poor heterogeneity adaptation, and low activity in long-term culture, providing an ideal experimental model for high-throughput screening and precision medicine applications of microarrays.

[0027] This invention, through a technical system of "basic microenvironment maintenance - precise regulation of signaling pathways - characteristic intervention of traditional Chinese medicine components," forms a unique synergistic regulatory network, achieving a precise balance between the cross-activation and inhibition of multiple pathways. The multi-target natural properties of traditional Chinese medicine components can also specifically address issues such as stromal cell contamination and cell migration imbalance. Chemical drugs cannot achieve this dual microenvironment purification, and the removal of any core component leads to a significant decrease in tumor cell purity. Secondly, its chip culture scenario has high specificity; the component concentration is optimized through the chip microscale effect, forming an irreplaceable dose window, ensuring the structural stability and material transport efficiency of organoids within the chip. Attached Figure Description

[0028] Figure 1 The cell state on day 1 of culture in Example 1 (scale bar is 200 μm, the same below).

[0029] Figure 2 The cell state on day 3 of culture in Example 1.

[0030] Figure 3 The cell state on day 5 of culture in Example 1.

[0031] Figure 4 The cell state on day 7 of culture in Example 1.

[0032] Figure 5 The cell state on day 1 of experimental group 1.

[0033] Figure 6 The cell state of experimental group 1 on day 3 of culture.

[0034] Figure 7 The cell state of experimental group 1 on day 5 of culture.

[0035] Figure 8 The cell state of experimental group 1 on day 7 of culture.

[0036] Figure 9 The cell state of control group 1 on day 1 of culture.

[0037] Figure 10 The cell state of control group 1 on day 3 of culture.

[0038] Figure 11 The cell state of control group 1 on day 5 of culture.

[0039] Figure 12 The cell state of control group 1 on day 7 of culture.

[0040] Figure 13 The cell state on day 1 of experimental group 2.

[0041] Figure 14The cell state of experimental group 2 on day 3 of culture.

[0042] Figure 15 The cell state of experimental group 2 on day 5 of culture.

[0043] Figure 16 The cell state of experimental group 2 on day 7 of culture.

[0044] Figure 17 The cell state of control group 2 on day 1 of culture.

[0045] Figure 18 The cell state of control group 2 on day 3 of culture.

[0046] Figure 19 The cell state of control group 2 on day 5 of culture.

[0047] Figure 20 The cell state of control group 2 on day 7 of culture.

[0048] Figure 21 The cell state of experimental group 3 on day 1 of culture.

[0049] Figure 22 The cell state of experimental group 3 on day 3 of culture.

[0050] Figure 23 The cell state of experimental group 3 on day 5 of culture.

[0051] Figure 24 The cell state of experimental group 3 on day 7 of culture.

[0052] Figure 25 The cell state on day 1 of the control group (3).

[0053] Figure 26 The cell state on day 3 of the control group.

[0054] Figure 27 The cell state of the control group on day 5 of culture.

[0055] Figure 28 The cell state of the control group on day 7 of culture. Detailed Implementation

[0056] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1: Primary gastric cancer organoid culture

[0058] Gastric cancer tissue samples obtained from clinical surgery or biopsy were removed from the sample collection tube and placed in a 6cm cell culture dish containing 2mL of washing solution to remove necrotic, calcified tissue, adipose tissue (for non-adipose tissue samples), blood clots, etc. The tissue samples were washed 8 times.

[0059] Sample digestion: Transfer the tissue block to a sterile 5mL centrifuge tube, add 1mL of 1×PBS (containing penicillin-streptomycin-gentamicin solution) to rinse the culture dish, and collect as many remaining tissue fragments as possible into the aforementioned 5mL centrifuge tube. Use autoclaved ophthalmic scissors to cut the tissue into 1mm pieces in the centrifuge tube. 3 Left and right size. Centrifuge at 4℃, 1500 rpm, 5 min, discard supernatant. After centrifugation, remove the centrifuge tube, add preheated tissue digestion solution at a ratio of 6 ml / g of sample to digestion solution, gently resuspend the tissue block with a sterile 5 mL pipette, and tighten the centrifuge tube cap. Incubate the centrifuge tube in a water bath at 37℃ for 5 min, then place it in a vertical shaker in a 37℃ incubator for 50 min, setting the shaker speed to 60 rpm. Observe the tissue digestion. The observation criteria for terminating digestion can be referenced as follows:

[0060] 1) The tissue mass shrinks in size and becomes looser in texture;

[0061] 2) The clarity of the tissue digestion fluid decreases and its viscosity increases due to the increased number of single cells and the release of tissue fragments;

[0062] 3) A small amount of digestive fluid can be taken and observed under a microscope to see if obvious cell clumps appear;

[0063] 4) The total digestion time should not exceed 60 minutes.

[0064] After digestion is complete, remove the centrifuge tube from the incubator and briefly centrifuge to allow any digestive fluid or residual tissue fragments adhering to the tube cap to fall to the bottom. Add an equal volume of stop solution and mix well to terminate digestion. Centrifuge at 4°C, 1500 rpm, for 5 minutes, and discard the supernatant. If a large amount of connective tissue fragments remain in the digested tissue fluid, filter through a 100 μm cell sieve to remove large connective tissue fragments and collect the filtrate. If no obvious connective tissue fragments are observed, tissue digestion is complete.

[0065] (If a large number of red blood cells are observed in the cell pellet, red blood cell lysis should be performed. Specific procedure: Add 1 mL of red blood cell lysis buffer to the cell pellet, mix well by pipetting, incubate on ice for 2 min, then add 2 mL of stop solution to neutralize. Centrifuge at 4°C, 1500 rpm, for 5 min, and discard the supernatant.)

[0066] Wash the cell pellet: Resuspend the cell pellet thoroughly in an appropriate volume of 1×PBS (penicillin-streptomycin-gentamicin solution), centrifuge at 4°C, 1500 rpm for 5 min, and discard the supernatant. Repeat this operation 3 times. Before the last wash, take a small amount of cell suspension and an equal volume of 0.4% trypan blue solution for staining and counting, and record the cell viability and number.

[0067] Cell-carrying gel preparation: Divide the cell suspension into equal portions according to the number of microarray cells to be inoculated. Transfer to 1.5 mL microcentrifuge tubes and centrifuge. Mix the matrix gel (Matrigel Matrix Basement Membrane (Corning / REF356231)) with the prepared primary gastric cancer cell pellet (2000 cells / μl). Prepare Xellar OC-Plex 32 microarrays in advance for inoculation; the components are shown in Table 1.

[0068] Table 1: Composition of Culture Medium for Gastric Cancer Organoid Microarrays

[0069]

[0070]

[0071] Xellar OC-Plex 32 microarray seeding: Aspirate 1.5 μl of the MTG-cell mixture using a 2 μl pipette. Holding the pipette, place the tip in the microarray channel port and gently suspend the tip in the well, slowly injecting the MTG-cell mixture. Using a pipette to aspirate 50 μl of 1×PBS and inject it into the rib area of ​​the microarray can reduce gel deformation caused by evaporation. Incubate the device at 37°C, 5% CO2 for 1 hour to allow the gel to solidify. Add 60 μl of culture medium to the perfusion channel + port using a 200 μl pipette, then add 20 μl of culture medium to each gel port. (Note the presence of air bubbles in the perfusion channel; if large air bubbles are present, they must be removed. To remove air bubbles: After the gel solidifies, inject the corresponding amount of culture medium into the perfusion channel and quickly observe under a microscope for air bubbles. If present, use a 200 μl pipette tip to aspirate three times in the perfusion channel to remove the liquid. Use an empty pipette tip to remove any remaining air from the perfusion channel before adding fresh liquid. Incubate overnight in an incubator. The next day, quickly examine the gel under a microscope; if air bubbles are present, repeat the process, adding a reservoir layer – subsequent steps.)

[0072] The total volume of culture medium for each chip was 100 μl (60 μl + 20 μl + 20 μl = 100 μl). The chips were placed in a 37°C, 5% CO2 incubator and cultured. The culture medium was changed every 3 days, and the culture status of gastric cancer organoids was observed.

[0073] The result is shown in the figure: Figure 1 To show the cell state on day 1 of culture, Figure 2 To show the cell state on day 3 of culture. Figure 3 To show the cell state on day 5 of culture. Figure 4 The cell state on day 7 of culture.

[0074] Gastric cancer organoids were continuously cultured for 7 days using the gastric cancer organoid microarray-specific culture medium described in this invention. The organoids showed a significant increase in volume, a dense structure, and clear edges, exhibiting a highly refractive and translucent state. Cell activity was good, and no obvious signs of apoptosis or necrosis were observed, suggesting that this culture system can efficiently maintain the proliferative potential and biological characteristics of gastric cancer organoids.

[0075] Example 2: Culture of gastric cancer organoid cell lines

[0076] AGS Cell Resuscitation: Prepare 15ml centrifuge tubes in advance, and preheat the culture media (Tables 2 and 3) to 37°C. Remove the cryovials from the liquid nitrogen container and quickly place them in a 37°C water bath to thaw. Gently shake the cryovials until the organoids inside are completely thawed (approximately 2 minutes). Once thawed, immediately remove the cryovials and transfer the cell suspension to a centrifuge tube containing 5ml of culture medium. Centrifuge at 1000 rpm for 5 minutes at 4°C. Discard the supernatant, resuspend the cell pellet in 1×PBS, and centrifuge again to wash the cells.

[0077] Counting: After resuspending the cell pellet with an appropriate amount of 1×PBS, take a small amount of cell suspension and stain with an equal volume of 0.4% trypan blue solution and count the cells. Record the cell viability and number based on the cell count.

[0078] Cell-carrying gel preparation: Divide the cell suspension into equal portions according to the number of chips to be inoculated. Transfer to 1.5 mL microcentrifuge tubes and centrifuge. Mix the matrix gel (Matrigel Matrix Basement Membrane (Corning / REF356231)) with the prepared primary gastric cancer cell pellet (2000 cells / μl).

[0079] Prepare the Xellar OC-Plex 32 chip in advance for inoculation. The culture medium composition is shown in Table 2.

[0080] Table 2: Composition of Gastric Cancer Organoid Microarray Culture Medium

[0081] Culture medium components concentration advanced DMEM / F12 culture medium 1.5× Bispecific antibodies (penicillin / streptomycin) 1.5× Antibacterial agent (Primocin) 1.5× Hydroxyethylpiperazine ethanesulfonic acid 1.5× B-27 Additive 1.5× Glutamax culture medium supplement 1.5× R-Vertrin 1 200ng / ml Noggin culture medium additives 150ng / ml N-acetylcysteine 1.25mM Epidermal growth factor (EGF) 100ng / ml Fibroblast growth factor 7 (FGF7) 50ng / ml Fibroblast growth factor 10 (FGF10) 200ng / ml Gastrin I 1nM A83-01 Culture Medium Additives 500nM SB202190 Culture Medium Additives 1μM Nicotinamide 10mM Y-27632 culture medium additives 10μM Lampflower B 10μM Ligustrazine 10μM

[0082] Xellar OC-Plex 32 microarray seeding: Use a 2μl pipette to aspirate 1.5μl of the MTG-cell mixture. Holding the pipette, place the tip of the pipette into the microarray channel port, gently suspend the tip in the well, and slowly inject the MTG-cell mixture. Use a pipette to aspirate 50μl of 1×PBS and inject it into the rib area of ​​the microarray to reduce gel deformation caused by evaporation. Incubate the device at 37°C, 5% CO2 for 1 hour to allow the gel to solidify. Use a 200μl pipette to add 60μl of culture medium to the perfusion channel + port, and then add 20μl of culture medium to each gel port (check for air bubbles in the perfusion channels; if large air bubbles are present, remove them).

[0083] Removing air bubbles: After the gel solidifies, inject the appropriate culture medium into the perfusion channel. Quickly observe under a microscope for air bubbles. If present, use a 200μl pipette tip to aspirate three times through the perfusion channel to remove the liquid. Use an empty pipette tip to remove any remaining air from the perfusion channel before adding fresh liquid. Incubate overnight in an incubator. The next day, quickly examine the sample under a microscope. If air bubbles are present, repeat the process, adding a reservoir layer (following subsequent steps).

[0084] The total volume of culture medium for each chip was 100 μl (60 μl + 20 μl + 20 μl = 100 μl). The chips were placed in a 37°C, 5% CO2 incubator and cultured. The culture medium was changed every 3 days, and the culture status of gastric cancer organoids was observed.

[0085] Experimental Example 1: Comparison of Primary Gastric Cancer Organoid Culture Methods

[0086] Experimental group 1 used the gastric cancer organoid microarray culture medium and method described in Example 1 to culture gastric cancer organoids.

[0087] Control group 1: The rest of the methods were the same as in Example 1, except that ordinary gastric cancer organoid culture medium (manufacturer: Moji Bio, catalog number: MA-0807T008LP) was used instead of the gastric cancer organoid microarray culture medium in Example 1.

[0088] The experimental results of control group 1 are as follows Figures 5-8 As shown. The experimental results of experimental group 1 are as follows. Figures 9-12 As shown.

[0089] Both groups used gastric cancer organoids from the same patient, with identical seeding density, culture period (7 days), and microarray microenvironment parameters. It can be seen that conventional culture media were significantly inferior to the microarray-specific culture medium of this invention in terms of gastric cancer organoid expansion efficiency, structural integrity, and cell viability, suggesting that the latter is better able to maintain the proliferative potential and biological function of tumor organoids.

[0090] Experimental Example 2: Comparison of Primary Gastric Cancer Organoid Culture Methods

[0091] Experimental group 2 used the gastric cancer organoid microarray culture medium and method described in Example 2 to culture gastric cancer organoids.

[0092] Control group 2: The rest of the methods were the same as in Example 2, except that ordinary gastric cancer organoid culture medium (manufacturer: Moji Bio, catalog number: MA-0807T008LP) was used instead of the gastric cancer organoid microarray culture medium in Example 2.

[0093] The experimental results of control group 2 are as follows Figures 13-16 As shown. The experimental results of experimental group 2 are as follows. Figures 17-20 As shown.

[0094] The results showed that, under the same AGS gastric cancer organoid model, seeding density, 7-day culture period, and chip microenvironment conditions, compared with conventional culture medium, the chip-specific culture medium of the present invention significantly improved the organoid expansion efficiency, structural integrity, and cell activity, further confirming its superior ability to maintain the proliferation potential and biological function of tumor organoids.

[0095] Experimental Example 3: Comparison of Primary Gastric Cancer Organoid Culture Methods

[0096] Experimental group 3 used the gastric cancer organoid microarray culture medium and method described in Example 2 to culture gastric cancer organoids.

[0097] Control group 3: The remaining methods were the same as in Example 2, except that no ligustrazine and tetramethylpyrazine were added to the gastric cancer organoid microarray culture medium.

[0098] The experimental results of control group 3 are as follows Figures 21-24 As shown. The experimental results of experimental group 3 are as follows. Figures 25-28 As shown.

[0099] The results showed that under the same AGS gastric cancer organoid model, seeding density, 7-day culture period, and microarray microenvironment conditions, the only difference was the culture medium formulation. The control group used the microarray culture medium of this invention, but with the removal of scutellarin (0 μM) and tetramethylpyrazine (0 μM). The experimental group used the complete formulation, with scutellarin 15 μM + tetramethylpyrazine 10 μM. In the microarray microenvironment, the combined addition of scutellarin and tetramethylpyrazine significantly promoted the proliferation, structural maintenance, and cell survival of AGS gastric cancer organoids. Its mechanism of action was closely related to its antioxidant and mitochondrial protective effects.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gastric cancer organoid microarray culture medium, characterized in that, It includes the following components: The rest are basic culture media.

2. The gastric cancer organoid microarray culture medium according to claim 1, characterized in that, The basal culture medium is advanced DMEM / F12 medium with a concentration of 1.5×; the antibiotics are penicillin and streptomycin.

3. The gastric cancer organoid microarray culture medium according to claim 1, characterized in that, The antibacterial agent is Primocin.

4. The application of the gastric cancer organoid microarray culture medium according to any one of claims 1 to 3 in the culture of gastric cancer organoids.

5. The application according to claim 4, characterized in that, The method for culturing gastric cancer organoids includes the following steps: (1) Take the primary gastric cancer cells and mix them with matrix gel to obtain a matrix gel-cell mixture; (2) The matrix gel cell mixture is injected into a gastric cancer organoid chip, incubated, and then injected with the culture medium described in any one of claims 1 to 3 for culturing.

6. The application according to claim 5, characterized in that, The concentration of the primary gastric cancer cells precipitated in step (1) is 1500-2500 cells / μl.

7. The application according to claim 5, characterized in that, The type of matrix adhesive mentioned in step (1) is Matrigel Matrix Basement Membrane.

8. The application according to claim 5, characterized in that, The incubation time in step (2) is 40-60 min, the temperature is 36-38℃, and the CO2 concentration is 4-6%.

9. The application according to claim 5, characterized in that, The culture temperature in step (2) is 36-38℃, and the CO2 concentration is 4-6%.

10. The application according to claim 5, characterized in that, In step (2), the culture medium should be changed every 2 to 3 days during the culture process.

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