In Vitro Resident Memory CD8 T Cell Differentiation Using Epithelial Organoid-T Cell Co-culture System
February 3rd, 2026
To study CD8 T cell differentiation and function in vitro, CD8 T cells can be isolated from the mouse to be co-cultured long-term with preformed infected murine vaginal epithelial organoids. Here, we describe this process and assess the acquisition of resident memory T cell markers upon co-culture.
Our research investigates CD8 resident memory T-cells in the female reproductive tract, examining how infections and diseases influence their residency, function, maintenance, and therapeutic potential. Existing methods limit survival and phenotypic stability of resident memory T-cells. This protocol enables scalable in vitro study by inducing tissue residency through organoid T-cell cult culture.
To begin, plate vaginal epithelial organoids or VEOs at a density of 10, 000 cells in 20 microliters of basement membrane extract per well of a 24-well plate. Place the plate in the incubator and maintain the cultures for seven to 10 days at 37 degrees Celsius. One day before co-culture, aspirate the cell culture media from the designated wells and wash the wells with DPBS.
Aspirate the DPBS, then dispense 600 microliters of pre-warmed 0.25%trypsin EDTA into the well. Using a BSA coated P1000 pipette, resuspend thoroughly to mechanically disrupt the basement membrane extract and transfer the contents of the well into a BSA coated sterile 15 milliliter conical tube. Now place the conical tube in a 37 degrees Celsius water bath.
After five minutes, add 10 milliliters of 10%FBS in RPMI to quench the trypsinization. Then place the tube in the centrifuge and spin at 500 G for five minutes at four degrees Celsius and discard the supernatant. Using a BSA coated pipette tip, resuspend the pellet in 100 to 300 microliters of T-cell organoid culture medium or plain DMEMF 12.
Count the cells using a hemocytometer. Calculate the volume of virus required to achieve a multiplicity of infection of 0.1 using the total cell number and the known viral titer. Aspirate the cell culture media from the VEO wells selected for infection.
Wash each well with 500 microliters of pre-warmed DPBS and place the plate in a 37 degrees Celsius incubator for five minutes. After five minutes, discard the DPBS. Add 500 microliters of organoid media containing sufficient viral particles to achieve a multiplicity of infection of 0.1.
Place the plate in a 37 degrees Celsius incubator with 5%carbon dioxide for one hour, gently swirling the plate by hand every 20 to 30 minutes. Transfer the appropriate amount of basement membrane extract required for plating from minus 80 degrees Celsius storage to four degrees Celsius and let it thaw for eight hours. Check the VEOs under a light microscope to assess size and overall health.
Confirm that the VEOs are approximately 100 micrometers in diameter with a uniform round morphology and minimal single cells. Aspirate the cell culture media from the VEO wells and wash each well with 500 microliters of cold sterile DPBS maintained at two to eight degrees Celsius. After removing the DPBS, add 200 to 500 microliters of cold organoid harvesting solution to each well, ensuring at least a 10X volume relative to the basement membrane extract droplet.
Then using a BSA coated P200 pipette, mechanically disrupt the basement membrane extract. Now place the plate on an orbital shaker set to approximately 100 revolutions per minute at four degrees Celsius for 20 minutes. After incubation, use a BSA coated pipette tip to mix and transfer the contents of each well into a BSA coated sterile 15 milliliter conical tube.
Place the tube in the centrifuge and spin at 500 G for five minutes at four degrees Celsius. Then discard the supernatant. Wash the VEO pellet with five to 10 milliliters of cold DPBS and centrifuge it again at 500 G for five minutes at four degrees Celsius.
Aspirate the DPBS, then using a P1000 pipette tip, resuspend the pellet in one milliliter of T-cell organoid culture medium and place the VEO suspension on ice until the T-cells are ready for co-culture. For collecting T-cells, check the resting T-cells under a light microscope to assess size and overall health. Confirm that the T-cell show some clustering, enlarged morphology, and no visible cell debris or fragments.
Using a P1000 pipette, resuspend the T-cells by pipetting up and down several times in each corner of the well. Collect an conical tube and count the total number of live CD8 T-cells. Next, place the T-cell suspension into the centrifuge and spin at 584 G for five minutes at four degrees Celsius.
Discard the supernatant, and resuspend the T-cell pellet in 10 milliliters of T-cell organoid culture medium. Then transfer the appropriate number of T-cells into the conical tube containing the previously prepared VEOs to combine the cells. Place the combined VEO and T-cell suspension into the centrifuge and spin again at 584 G for five minutes at four degrees Celsius.
After discarding the supernatant, resuspend the VEOs and T cells in the appropriate volume of basement membrane extract. Then using another pipette tip, plate the suspension onto the pre-warmed tissue culture plate, dispensing 20 microliters per well for a 24-well plate. Now invert the tissue culture plate and place it upside down in a 37 degrees Celsius incubator.
Allow 30 minutes for complete basement membrane extract adhesion and solidification. After the incubation period, carefully add the supplemented complete T-cell organoid culture medium to each well. Dispense 500 microliters per well for a 24-well plate or 250 microliters per well for a 96-well plate, adding the media against the side of the well to avoid disturbing the basement membrane extract droplet.
Replace the culture medium every two days until the samples are harvested for flow cytometry analysis. Seven day old mouse VEOs infected with lymphocytic choriomeningitis virus expressing yellow fluorescent protein showed abundant viral replication as early as four hours after co-culture, visible as the fluorescent signals within the organoids. Accumulation of CD8 T-cells near infected organoids became prominent at later time points during co-culture.
Progressive aggregation of CD8 T-cells was associated with a visible reduction in yellow fluorescent protein signal in infected organoids at later time points. CD8 T-cells co-cultured with infected VEOs showed increased expression of CD 69 and CD 103 over time, indicating acquisition of a tissue-resident memory phenotype. Antigen-stimulated CD69 positive, CD103 positive, CD8 T-cells showed a significant increase in intracellular interferon gamma and interleukin 2 production compared to unstimulated controls.
This protocol enables in vitro study of CD8 resident memory T-cells, allowing efficient manipulation of epithelial and T-cells without reliance on animal models. The most important challenge in this protocol is maintaining healthy, viable T-cells and vaginal epithelial organoids as poor viability or infection compromises co-culture survival and experimental outcomes. We focused on flow cytometric analysis, but co-cultures can be collected for staining, imaging and sequencing.
The safe media enables downstream ELISA.
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Overview
This protocol demonstrates a method for studying CD8 resident memory T-cell differentiation using an organoid-T-cell co-culture system. It allows for the investigation of T-cell responses in a controlled in vitro environment, which is crucial for understanding immune responses in the female reproductive tract.
Key Study Components
Area of Science
- Immunology
- Infection Biology
- Cell Culture Techniques
Background
- CD8 T cells play a critical role in immune responses.
- Resident memory T cells are essential for long-term immunity.
- Existing methods for studying these cells have limitations.
- Organoid models provide a more physiologically relevant environment.
Purpose of Study
- To develop a scalable in vitro system for studying CD8 T cell differentiation.
- To assess the impact of infections on T cell residency and function.
- To evaluate the acquisition of resident memory T cell markers.
Methods Used
- Isolation of CD8 T cells from mice.
- Co-culture of T cells with infected vaginal epithelial organoids.
- Flow cytometric analysis to assess T cell markers.
- Monitoring of viral replication and T cell responses over time.
Main Results
- Infection of organoids leads to T cell accumulation.
- CD8 T cells exhibit increased expression of memory markers.
- Significant production of cytokines in stimulated T cells.
- This method allows for detailed study of T cell behavior in vitro.
Conclusions
- The protocol enables efficient study of CD8 resident memory T cells.
- It provides insights into T cell responses in the context of infections.
- This approach may reduce reliance on animal models for T cell research.
