How to maintain intestinal tissue resident cells (TRM) in culture?

Claudia Aiello, this is what i do everyday so whatever i suggest here should work for you today and for many years as longs as you are working on Memory T cells.

Maintaining tissue resident memory T cells (TRM) derived from fresh intestinal tissue in culture can be challenging due to their specialized nature and the unique environment they require. Here are several suggestions to improve the viability and maintenance of TRM in culture:

Optimization Steps:

Tissue Preparation and Digestion: Gentle Dissociation: Ensure the tissue dissociation process is gentle enough to preserve cell viability. Overly aggressive mechanical or enzymatic dissociation can damage the cells. Enzyme Cocktail: Optimize the enzyme cocktail used for tissue digestion. Some protocols use collagenase IV and DNase I, but the concentration and duration may need fine-tuning.

Culture Conditions: Oxygen Levels: Mimic the hypoxic conditions of the intestinal environment by culturing cells under low oxygen conditions (e.g., 1-5% O2). Nutrients and Cytokines: Ensure the culture media is well-supplemented. You are already using ImmunoCult T cell Expansion Media with PenStrep, FBS, IL-2, IL-7, and IL-15, which is good. Consider adding other factors like TGF-β to help maintain TRM phenotype and function. Co-Culture Systems: Consider co-culturing TRM with other cells from the intestinal microenvironment, such as stromal cells or epithelial cells, to provide necessary survival signals.

Survival Factors and Inhibitors: Inhibitors of Apoptosis: Adding inhibitors of apoptosis, such as Z-VAD-FMK, may help increase cell viability. Anti-inflammatory Agents: Using agents to reduce stress-induced inflammation, such as antioxidants or anti-inflammatory cytokines, could help.

Cell Density: Optimal Seeding Density: Ensure you are seeding cells at an optimal density. Too low a density can result in poor survival due to lack of cell-cell interactions.

Matrix and Scaffolds: Extracellular Matrix (ECM) Components: Using ECM components like Matrigel, collagen, or fibronectin can provide a more natural environment for TRM. 3D Culture Systems: Consider using 3D culture systems or organoid cultures to better mimic the tissue architecture.

Potential Adjustments in Protocol:

Modify Enzyme Digestion: Ensure a balanced concentration of collagenase and DNase to avoid over-digestion. Use lower concentrations and shorter digestion times to minimize cell stress and death.

Adjust Culture Media Composition: Continue using ImmunoCult T cell Expansion Media but consider adjusting the concentrations of IL-2, IL-7, and IL-15. Add TGF-β to help maintain TRM identity.

Incorporate Anti-Apoptotic Agents: Include Z-VAD-FMK or other caspase inhibitors to prevent apoptosis during initial culture phases.

Evaluate Oxygen Levels: Cultivate cells under hypoxic conditions (1-5% O2) to mimic the intestinal environment.

Utilize Co-Culture Systems: Include stromal or epithelial cells in your culture to provide supportive signals. Use transwell inserts to separate but allow communication between cell types.

Matrix and Scaffold Use: Utilize Matrigel or collagen-coated plates to provide an ECM-like environment. Consider 3D culture systems or organoids for a more natural environment.

Example Protocol Adjustment:

Day 0: Tissue Preparation and Initial Culture

Digest intestinal tissue gently with optimized collagenase IV and DNase I concentrations.

Perform double negative selection to remove unwanted cells and enrich for CD3+ cells.

Plate cells in ImmunoCult T cell Expansion Media supplemented with PenStrep, 10% FBS, 20 ng/mL IL-2, 10 ng/mL IL-7, 10 ng/mL IL-15, and 1 ng/mL TGF-β.

Culture under hypoxic conditions (2-5% O2).

Day 1-6: Culture Maintenance

Refresh media every 48-72 hours with fresh cytokines and supplements.

Monitor cell viability daily via flow cytometry.

Adjust cell density by splitting or concentrating cells as needed.

Optionally, incorporate ECM components or co-culture systems.

By optimizing these factors, you can enhance the viability and maintenance of intestinal TRM cells in culture.

Best of Luck!

Saif Wahid

Culturing tissue-resident memory T cells (TRM) from fresh intestinal tissue can indeed be challenging due to their unique microenvironment and specific requirements. Here are some detailed suggestions and considerations to help improve the viability and culture conditions for TRM cells:

1. Sorting and Purification

FACS Sorting: Sorting for CD3+ cells using FACS rather than bead enrichment is a good step. FACS sorting will provide a purer population of T cells and reduce the presence of dead cells and other contaminating cell types, which can contribute to the death of TRM.
Viability Dye Exclusion: During FACS sorting, use a viability dye to exclude dead cells, which will help in obtaining a healthier population of live TRM cells.

2. Culture Conditions

Culture Media Optimization: While ImmunoCult T cell Expansion Media is suitable, optimizing it further to closely mimic the intestinal microenvironment can help.
Consider the following:
Cytokine Cocktail: The combination of IL-2, IL-7, and IL-15 is good for T cell survival and expansion. Adding TGF-beta is an excellent idea, as it is crucial for maintaining TRM phenotype and function. Start with a low concentration (e.g., 1-5 ng/mL) and titrate as needed. Serum Source: Ensure the FBS used is of high quality and endotoxin-free. Some batches of FBS can be better for T cell culture than others. Additional Supplements: Consider adding other supplements like beta-mercaptoethanol (50 µM) to reduce oxidative stress and support T cell survival.

3. Microenvironment Mimicry

Extracellular Matrix (ECM) Components: TRM cells thrive in a specific tissue architecture and ECM environment. Adding ECM components such as fibronectin, collagen, or Matrigel to the culture can provide better support for TRM survival and function.
Co-culture Systems: Co-culturing TRM cells with stromal cells or intestinal epithelial cells can provide necessary survival signals and better mimic the natural microenvironment.

4. Minimizing Stress and Cell Death

Gentle Handling: Minimize mechanical stress during tissue dissociation and cell isolation. Use gentle dissociation methods and avoid prolonged enzymatic digestion.
Anti-apoptotic Agents: Consider adding anti-apoptotic agents like Z-VAD-FMK (a pan-caspase inhibitor) to prevent apoptosis during the initial culture period.

5. Additional Techniques

Cell Density: Maintain an optimal cell density in the culture. Too low a density can lead to poor survival due to lack of cell-cell interactions and necessary cytokine support.
Oxygen Levels: Maintain physiological oxygen levels (~5% O2) instead of atmospheric oxygen levels (~21% O2) to better mimic the tissue environment and reduce oxidative stress.

Example Protocol Adjustments

Cell Isolation and Sorting:After double negative selection, perform FACS sorting for CD3+ cells, excluding dead cells using a viability dye (e.g., DAPI or PI). Collect sorted cells in a buffer containing serum to minimize stress.

Culture Setup:Use ImmunoCult T cell Expansion Media supplemented with PenStrep, 10% FBS, IL-2 (50 U/mL), IL-7 (10 ng/mL), IL-15 (5 ng/mL), and TGF-beta (1-5 ng/mL). Consider adding beta-mercaptoethanol (50 µM) and anti-apoptotic agents like Z-VAD-FMK (20 µM). Plate cells on a surface coated with ECM components (e.g., fibronectin, collagen, or Matrigel).

Maintenance:Culture cells at 37°C with 5% CO2 and ~5% O2 (physiological oxygen). Monitor cell viability daily and replenish cytokines and medium as needed.

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