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Updated: Aug 10, 2026
Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture
Published on: April 8, 2016
Impact of different cell isolation techniques on lymphocyte viability and function
A B Klein1, S G Witonsky, S Ansar Ahmed
124061-0442,
Insights
Choosing the right lymphocyte isolation method is crucial for accurate immunological assays. Mechanical dissociation techniques impact lymphocyte function, with screen and plunger methods preserving T-cell proliferation better than stomacher or slide methods.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Lymphocyte isolation techniques significantly impact immunological assay outcomes.
- Comparative assessment of lymphocyte isolation methods, particularly regarding cell recovery, viability, and function, is lacking.
- Mechanical dissociation methods for lymphoid organs require thorough evaluation.
Purpose of the Study:
- To determine the effect of different mechanical tissue dissociation methods on lymphocyte viability and function.
- To compare four distinct mechanical dissociation techniques: metallic screen, sheer force slide, commercial stomacher, and plunger-screen.
- To evaluate the influence of isolation methods on cell recovery, morphology, apoptosis, necrosis, and mitogen-induced proliferation.
Main Methods:
- Lymphoid organs (spleen and thymus) were mechanically dissociated using four methods.
- Cell viability was assessed using trypan blue exclusion and 7-amino-actinomycin D (7-AAD) staining.
- Lymphocyte proliferation was measured via Alamar Blue assay, and surface antigen expression via flow cytometry.
Main Results:
- No significant differences were observed in lymphocyte viability, morphology, apoptosis, or necrosis across all tested techniques.
- Lymphocyte surface antigen expression remained comparable among the different isolation methods.
- Mitogen-induced splenic T-cell proliferation was significantly higher with metallic screen and plunger-screen methods compared to sheer force slide and commercial stomacher.
Conclusions:
- Lymphocyte recovery, morphology, and viability are generally unaffected by the mechanical dissociation techniques evaluated.
- Lymphocyte function, specifically mitogen-induced proliferation, is negatively impacted by the sheer force slide and commercial stomacher isolation methods.
- The choice of mechanical dissociation method is critical for preserving lymphocyte function in immunological studies.
Abstract:
The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte proliferation was measured using the Alamar Blue assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant differences in lymphocyte viability, morphology, or surface antigen expression were observed among the different techniques. Likewise, cellular apoptosis and necrosis were comparable across all the techniques. However, mitogen induced splenic T-cell proliferation was higher in cells collected using the metallic screen and plunger-screen isolation methods as compared to the sheer force slide or commercial stomacher procedures. These data suggest that cell recovery, morphology, and viability are not affected by isolation techniques. However, lymphocyte function, as assessed by mitogen induced proliferation, was negatively affected by the sheer force slide or commercial stomacher isolation techniques.
