Abstract
The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.
Highlights
Macrophages are essential for both the innate and adaptive immune system, as they play key roles in different biological processes, such as antigen presentation and processing, microbial killing, cytokine production, and clearance of apoptotic cells, among others [1,2,3]
Because the dimethyl sulfoxide (DMSO) concentrations vary among the protocols for freezing immortalized cell lines, we first investigated the optimal DMSO concentration to be used in a protocol to freeze bone marrow cells, which should remain viable to allow further generation of bone marrow-derived macrophages (BMDM)
We found that BMDM obtained from fresh and frozen BM cells are able to support the intracellular replication of L. amazonensis (Fig. 8)
Summary
Macrophages are essential for both the innate and adaptive immune system, as they play key roles in different biological processes, such as antigen presentation and processing, microbial killing, cytokine production, and clearance of apoptotic cells, among others [1,2,3]. Because differentiated primary macrophages display limited multiplication, several investigators have used immortalized macrophage-like myeloid cell lines, such as J774A.1, RAW264.7 and P388D1. These cells usually differ from primary macrophages, as the selective pressure imposed by continual subculture usually results in the loss of genes that are not important for multiplication yet are key for macrophage immune functions. High numbers of BMDM can be obtained from a single mouse. These features have favored the choice of BMDM as a macrophage model in most immunological studies
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