Comprehensive Guide: Culture and Multiplex Fluorescence IHC Identification of Lung Cancer Organoids

1. Concept: Lung Cancer Organoids and Their Research Significance

Lung cancer organoids are three-dimensional (3D) in vitro culture models derived from lung cancer tissues or cells, which can recapitulate the histological features, genetic characteristics, and tumor microenvironment of the original tumor. As a cutting-edge tool in precision oncology research, lung cancer organoids overcome the limitations of traditional 2D cell cultures that fail to simulate the complex spatial structure and intercellular interactions of tumors. They have been widely applied in drug sensitivity testing, tumor heterogeneity research, mechanism exploration of tumor occurrence and development, and personalized treatment strategy formulation.

Multiplex fluorescence immunohistochemistry (mIHC) is an essential technique for the identification and functional characterization of lung cancer organoids. By simultaneously detecting multiple specific markers, this technology enables accurate verification of the authenticity of organoids and in-depth analysis of the expression and distribution of key molecules, providing reliable experimental basis for subsequent research applications.

2. Research Frontiers

In recent years, lung cancer organoids have become a research hotspot in the field of oncology. The establishment of stable and reproducible organoid culture systems, coupled with high-throughput detection technologies such as multiplex fluorescence IHC, has accelerated the translation of basic research to clinical practice. Current frontiers include the construction of patient-derived lung cancer organoids (PDOs) for personalized medicine, the simulation of tumor-immune cell interactions using organoid models, and the development of organoid-based drug screening platforms. The combination of lung cancer organoid culture and multiplex fluorescence IHC identification is crucial for promoting these frontier research directions and improving the efficiency and accuracy of research.

3. Research Significance

The standardized culture and accurate identification of lung cancer organoids are the prerequisites for their effective application in scientific research and clinical practice. A well-established culture system ensures the stability and reproducibility of organoids, while multiplex fluorescence IHC identification confirms the authenticity and functional characteristics of organoids, avoiding experimental deviations caused by impure or non-representative models.

This not only improves the reliability of research results but also lays a foundation for the development of personalized treatment plans for lung cancer patients, which is of great significance for advancing the field of precision oncology and improving the prognosis of lung cancer patients.

4. Related Mechanisms, Research Methods and Product Applications

4.1 Standard Protocol for Lung Cancer Organoid Culture

The culture of lung cancer organoids relies on the Organotial Human Lung Cancer Organoid Medium Kit (Catalog No.: abs9443) from the Absin product line of ANT BIO PTE. LTD., which provides a comprehensive and optimized culture environment for the growth and differentiation of lung cancer organoids. The detailed protocol is as follows:

1.       Tissue Transportation: Place the collected tissue in a pre-cooled (2-8°C) tissue preservation solution vial, transport it to a clean laboratory quickly for tissue processing and cell isolation, take photos and register information.

2.       Preparation of Culture Dishes: Prepare several culture dishes, add pre-cooled (4°C) primary culture buffer for later use.

3.       Tissue Processing: Disinfect the sampling vial, transfer the tissue to the culture dish, wash it three times with primary culture buffer to remove impurities, and cut the tissue into small pieces of approximately 1-3mm³ using ophthalmic scissors or a scalpel.

4.       Digestion: Digest the tissue with human lung cancer primary tissue digestion solution, shake and digest at 37°C for 10-20min (observe the digestion status at any time during the digestion process).

5.       Termination of Digestion: Take a small amount of liquid for microscopic observation. After observing a large number of single cells or cell clusters below 70um under the microscope, add three times the volume of primary culture buffer to terminate the digestion.

6.       Filtration and Centrifugation: Filter using a 100um pore size sieve (Catalog No.: abs7009), collect the filtrate, centrifuge at 300g for 5min to enrich the cells, remove the supernatant, add primary culture buffer to resuspend and centrifuge again.

7.       Matrigel Calculation: After step 6, observe the volume of the collected tissue, add 25 times the tissue volume of Matrigel (Catalog No.: abs9495) to resuspend and plate.

8.       Plating: Taking a 24-well cell culture plate as an example, spot 25ul of the tissue-Matrigel mixture into each well for plating (operate at 4°C).

9.       Gel Formation and Culture: Place the plated culture plate in a 37°C incubator for 10-15min to form a gel, then add human lung cancer organoid medium (restored to room temperature) for culture.

4.2 Protocol for Multiplex Fluorescence IHC Identification of Lung Cancer Organoids

The multiplex fluorescence IHC identification of lung cancer organoids adopts the 6-Color Multiplex Fluorescence IHC Staining Kit (Mouse/Rabbit Universal Secondary Antibody, Catalog No.: abs50014) from the Absin product line of ANT BIO PTE. LTD., which enables simultaneous detection of multiple target markers and accurate characterization of organoid properties. The detailed protocol is as follows:

10.    Sample Preparation: Prepare organoid samples with reference to "Organoid Practical Camp - Fixation and Embedding".

11.    Baking Slices: Bake the slices at 60°C for 30min.

12.    Deparaffinization and Hydration: Xylene (5min) → Xylene (5min) → Xylene (5min) → 100% Ethanol (5min) → 100% Ethanol (5min) → 95% Ethanol (3min) → 85% Ethanol (3min) → 75% Ethanol (3min) → Deionized Water (5min).

13.    Antigen Retrieval: Take 200ml of antigen retrieval solution, add it to the staining box, place the tissue sections on a heat-resistant plastic slide rack in the staining box, heat in a pressure cooker until saturated pressure is reached, then continue heating for 5min, turn off the power, take out the staining box after 10min, cool to room temperature for 30min, draw a water-resistant circle, and immerse in PBST for 3 washes, 3min each time.

14.    Blocking Endogenous Peroxidase: Add 100μL of 3% H2O2 to each section, incubate at room temperature for 10min, and wash with PBST 3 times for 3min each.

15.    Blocking: Remove the PBST solution, add 100μL of 5% goat serum blocking solution to each section, incubate at room temperature for 30min, and remove the blocking solution.

16.    Primary Antibody Incubation: Add 100μL of primary antibody working solution to each section, incubate at room temperature in a humid environment for 1h, and wash with PBST 3 times for 3min each.

17.    Secondary Antibody Incubation: Remove PBST, add 100μL of secondary antibody to each section, incubate at room temperature in a humid environment for 15min, and wash with PBST 3 times for 3min each.

18.    Dye Incubation: Remove PBST, add 100μL of freshly prepared 1* dye working solution (diluted 1:100 with signal amplification solution) to each section, incubate for 10min, then wash in PBST, soak and wash at room temperature 3 times for 3min each.

19.    Antibody Elution: Preheat the antibody elution buffer (Catalog No.: abs994) at 37°C, add a small amount of antibody elution buffer to cover the sample, and let stand at room temperature for 3-5min; discard the elution buffer, add a sufficient amount of elution buffer to cover the sample again, and incubate at 37°C for 20min; wash with PBST 3 times for 3min each.

20.    Repeat steps (6)-(10) until all target antibody stainings are completed.

21.    Nuclear Staining: Add 1*DAPI working solution to the sample to immerse the sample area, incubate at room temperature for 10min, then wash the slides with 1*PBST 3 times for 2min each.

22.    Mounting: Add anti-fluorescence quenching mounting medium, mount with a coverslip, and avoid air bubbles.

23.    Scanning Imaging and Data Analysis.

4.3 Product Support from ANT BIO PTE. LTD.

The standardized culture and accurate identification of lung cancer organoids are highly dependent on high-quality experimental reagents and kits. ANT BIO PTE. LTD.'s Absin sub-brand provides a full set of solutions for lung cancer organoid research, including core products such as organoid culture medium kits, Matrigel, cell filters, and multiplex fluorescence IHC staining kits. These products have the advantages of high stability, good reproducibility, and strong compatibility, which can effectively meet the needs of lung cancer organoid culture and identification.

In addition, ANT BIO PTE. LTD. also provides supporting reagents such as antigen retrieval solutions, peroxidase blocking solutions, blocking sera, and antibody diluents, forming a one-stop supply chain for lung cancer organoid research. The professional technical support team can also provide personalized guidance for experimental operation problems, helping researchers smoothly complete the entire experimental process from organoid culture to identification.

5. Brand Mission

As a professional supplier of life science reagents, ANT BIO PTE. LTD. is dedicated to providing high-quality, reliable products and comprehensive solutions to empower global life science research. The company's three specialized sub-brands cover the full spectrum of research needs in the life science field: Absin focuses on general reagents and kits, Starter specializes in antibodies, and UA is dedicated to recombinant proteins. Our core mission is to bridge the gap between cutting-edge scientific research and practical applications, accelerate the pace of scientific discovery, and contribute to the advancement of human health and regenerative medicine.

6. Related Product List

7. Disclaimer

This article is AI-compiled and interpreted based on the original work in DOI: 10.1002/advs.202413562. All intellectual property (e.g., images, data) of the original publication shall belong to the journal and the research team. For any infringement, please contact us promptly and we will take immediate action.

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