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Isolation and immunophenotyping of mononuclear cells from human lung tissue
Takashi Shimizu1, Fumio Fujimori, Yuichi Shimaoka
1The Division of Respiratory Medicine, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
Insights
Researchers successfully isolated and phenotyped mononuclear cells from human lung tissue using collagenase digestion. This method allows for detailed analysis of interstitial immune cells in lung cancer patients.
Area of Science:
- Immunology
- Pulmonology
- Cell Biology
Background:
- Understanding the immune cell composition of lung tissue is crucial for diagnosing and treating lung diseases.
- Current methods for isolating lung mononuclear cells can be complex and may affect cell viability.
Purpose of the Study:
- To develop and validate a method for quantitatively isolating and immunologically phenotyping mononuclear cells from human lung tissue.
- To characterize the immune cell populations present in the lung interstitium of lung cancer patients.
Main Methods:
- Normal lung tissue from lung cancer patients was digested with collagenase to isolate mononuclear cells.
- Peripheral blood mononuclear cells (PBMNC) from healthy volunteers served as controls.
- Flow cytometry and immunohistochemistry were used to analyze cell surface markers (CD45, CD3, CD4, CD8, CD19, CD68, CD161).
Main Results:
- A high percentage (92.1%) of isolated lymphoid cells expressed CD45, similar to PBMNC.
- Interstitial mononuclear cells were successfully phenotyped using flow cytometry for T cells (CD3, CD4, CD8), B cells (CD19), and NK cells (CD161).
- Immunohistochemistry confirmed the presence of these cells in the lung interstitium and showed the removal of alveolar macrophages.
Conclusions:
- Collagenase digestion enables efficient isolation and flow cytometric analysis of interstitial mononuclear cells from fresh lung specimens.
- This technique provides a reliable method for studying lung immune cell populations without further purification.
- Immunohistochemistry validates the flow cytometry findings, confirming the location of immune cells within the lung interstitium.
Objective:
To quantitatively isolate and immunologically phenotype mononuclear cells contained in human lung tissue.
Methods:
Normal appearing lung tissue as far distal to the resected lesion as possible was obtained from lung cancer patients. Lung tissue was thoroughly washed and cut into small pieces and digested with collagenase. Peripheral blood mononuclear cells (PBMNC) were prepared from controls using Ficoll gradient. Isolated cells and PBMNC were analyzed by flow cytometry. We immunohistochemically stained snap-frozen lung tissue with anti-CD3, CD4, CD8, CD20, and CD161 antibodies.
Participants:
Nineteen patients with lung cancer who underwent lobectomy were enrolled. Twelve healthy volunteers also participated as controls for flow cytometric analysis of PBMNC.
Results:
In forward scatter vs side scatter, 92.1+/-7.8% of isolated cells in the lymphoid population expressed leukocyte common antigen, CD45. The frequency of CD45-positive cells in the lymphoid population from lung tissue was as high as that from PBMNC (p=0.118). CD45-positive cells were successfully further extended by anti-CD3, CD4, CD8, CD19, and CD161 antibodies. Monocyte-macrophages bearing CD68 were also detected. CD68-positive alveolar macrophages disappeared from alveolar spaces after thorough washing by immunohistochemical staining. Mononuclear cells in the interstitium were positively stained by anti-CD3, CD4, CD8, CD20, and CD161 monoclonal antibodies.
Conclusions:
We could isolate interstitial cells and analyze cell surface markers via flow cytometry from fresh lung specimens by collagenase digestion without further purification. Immunohistochemistry confirmed the presence of the cells detected by flow cytometry in the lung interstitium.
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