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J Leukoc Biol. 2015 Jan;97(1):201-9. doi: 10.1189/jlb.5TA0814-373. Epub 2014 Oct 30.

An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells.

Author information

1
Departments of *Surgery and Medicine, University of Pennsylvania School of Medicine, and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
2
Departments of *Surgery and Medicine, University of Pennsylvania School of Medicine, and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA evgeniy.eruslanov@uphs.upenn.edu.

Abstract

Careful preparation of human tissues is the cornerstone of obtaining accurate data in immunologic studies. Despite the essential importance of tissue processing in tumor immunology and clinical medicine, current methods of tissue disaggregation have not been rigorously tested for data fidelity. Thus, we critically evaluated the current techniques available in the literature that are used to prepare human lung tumors for immunologic studies. We discovered that these approaches are successful at digesting cellular attachments and ECMs; however, these methods frequently alter the immune cell composition and/or expression of surface molecules. We thus developed a novel approach to prepare human lung tumors for immunologic studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This enzymatic digestion cocktail optimized cell yield and cell viability, retrieved all major tumor-associated cell populations, and maintained the expression of cell-surface markers for lineage definition and in vivo effector functions. To our knowledge, we present the first rigorously tested disaggregation method designed for human lung tumors.

KEYWORDS:

collagenase; enzymatic digestion; tumor-infiltrating immune cells

PMID:
25359999
PMCID:
PMC4771617
DOI:
10.1189/jlb.5TA0814-373
[Indexed for MEDLINE]
Free PMC Article

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