Monocyte proliferation in a cytokine-freem serum-free system

S. Bennett, S. B. Por, E. R. Stanley, S. N. Breit

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

The aim of this study was to establish a cytokine-free, serum-free system which would enable the long-term survival and proliferation of human peripheral blood monocytes. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by adherence to untreated plastic petri dishes and maintained up to 6 weeks in serum-free medium (SFM) consisting of IMEM, insulin, transferrin, sodium selenite and BSA. Maximal cell proliferation occurred during the first 2 weeks of culture and corresponded to the appearance of large numbers of pure, nonadherent culture-derived macrophages. Monocyte maturation was characterised by the modulation of specific cell surface antigens. The percentage of cells staining for the transferrin receptor increased with time, whereas the percentages of cells expressing CD11b, CD11c and HLA-DR remained greater than 60% for the 15 days studied. The mean fluorescent intensities (MFI) of all these antibodies increased significantly with time. The only differences found between the adherent and nonadherent cells, using the above antibodies, were with the MFI for CD11b and CD11c. In both cases, the intensity of staining was significantly greater in the adherent cells. Estimation of cytokine production by cells maintained for 5 weeks in SFM found that they constitutively produced large amounts of macrophage colony-stimulating factor (M-CSF) in the absence of any exogenous stimuli. These cells were also found to secrete high levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during the 1st week and granulocyte macrophage colony-stimulating factor (GM-CSF) during the 3rd week. However, the addition of exogenous GM-CSF (5 U/ml, S5) was found to significantly inhibit monocyte proliferation up to 17 days. This is the first report of proliferation associated with long-term survival of culture derived macrophages in a serum-free, cytokine-free system.

Original languageEnglish (US)
Pages (from-to)201-212
Number of pages12
JournalJournal of Immunological Methods
Volume153
Issue number1-2
DOIs
StatePublished - Aug 30 1992

Fingerprint

Monocytes
Cytokines
Serum
Serum-Free Culture Media
Granulocyte-Macrophage Colony-Stimulating Factor
Macrophages
Staining and Labeling
Sodium Selenite
Transferrin Receptors
Macrophage Colony-Stimulating Factor
Antibodies
HLA-DR Antigens
Surface Antigens
Transferrin
Plastics
Interleukin-6
Blood Cells
Tumor Necrosis Factor-alpha
Cell Proliferation
Insulin

Keywords

  • Defined medium
  • Differentiation
  • Monocytes
  • Monokine
  • Surface antigen

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Monocyte proliferation in a cytokine-freem serum-free system. / Bennett, S.; Por, S. B.; Stanley, E. R.; Breit, S. N.

In: Journal of Immunological Methods, Vol. 153, No. 1-2, 30.08.1992, p. 201-212.

Research output: Contribution to journalArticle

Bennett, S. ; Por, S. B. ; Stanley, E. R. ; Breit, S. N. / Monocyte proliferation in a cytokine-freem serum-free system. In: Journal of Immunological Methods. 1992 ; Vol. 153, No. 1-2. pp. 201-212.
@article{a920cb95217a47c49be0b23d05215c29,
title = "Monocyte proliferation in a cytokine-freem serum-free system",
abstract = "The aim of this study was to establish a cytokine-free, serum-free system which would enable the long-term survival and proliferation of human peripheral blood monocytes. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by adherence to untreated plastic petri dishes and maintained up to 6 weeks in serum-free medium (SFM) consisting of IMEM, insulin, transferrin, sodium selenite and BSA. Maximal cell proliferation occurred during the first 2 weeks of culture and corresponded to the appearance of large numbers of pure, nonadherent culture-derived macrophages. Monocyte maturation was characterised by the modulation of specific cell surface antigens. The percentage of cells staining for the transferrin receptor increased with time, whereas the percentages of cells expressing CD11b, CD11c and HLA-DR remained greater than 60{\%} for the 15 days studied. The mean fluorescent intensities (MFI) of all these antibodies increased significantly with time. The only differences found between the adherent and nonadherent cells, using the above antibodies, were with the MFI for CD11b and CD11c. In both cases, the intensity of staining was significantly greater in the adherent cells. Estimation of cytokine production by cells maintained for 5 weeks in SFM found that they constitutively produced large amounts of macrophage colony-stimulating factor (M-CSF) in the absence of any exogenous stimuli. These cells were also found to secrete high levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during the 1st week and granulocyte macrophage colony-stimulating factor (GM-CSF) during the 3rd week. However, the addition of exogenous GM-CSF (5 U/ml, S5) was found to significantly inhibit monocyte proliferation up to 17 days. This is the first report of proliferation associated with long-term survival of culture derived macrophages in a serum-free, cytokine-free system.",
keywords = "Defined medium, Differentiation, Monocytes, Monokine, Surface antigen",
author = "S. Bennett and Por, {S. B.} and Stanley, {E. R.} and Breit, {S. N.}",
year = "1992",
month = "8",
day = "30",
doi = "10.1016/0022-1759(92)90323-L",
language = "English (US)",
volume = "153",
pages = "201--212",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Monocyte proliferation in a cytokine-freem serum-free system

AU - Bennett, S.

AU - Por, S. B.

AU - Stanley, E. R.

AU - Breit, S. N.

PY - 1992/8/30

Y1 - 1992/8/30

N2 - The aim of this study was to establish a cytokine-free, serum-free system which would enable the long-term survival and proliferation of human peripheral blood monocytes. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by adherence to untreated plastic petri dishes and maintained up to 6 weeks in serum-free medium (SFM) consisting of IMEM, insulin, transferrin, sodium selenite and BSA. Maximal cell proliferation occurred during the first 2 weeks of culture and corresponded to the appearance of large numbers of pure, nonadherent culture-derived macrophages. Monocyte maturation was characterised by the modulation of specific cell surface antigens. The percentage of cells staining for the transferrin receptor increased with time, whereas the percentages of cells expressing CD11b, CD11c and HLA-DR remained greater than 60% for the 15 days studied. The mean fluorescent intensities (MFI) of all these antibodies increased significantly with time. The only differences found between the adherent and nonadherent cells, using the above antibodies, were with the MFI for CD11b and CD11c. In both cases, the intensity of staining was significantly greater in the adherent cells. Estimation of cytokine production by cells maintained for 5 weeks in SFM found that they constitutively produced large amounts of macrophage colony-stimulating factor (M-CSF) in the absence of any exogenous stimuli. These cells were also found to secrete high levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during the 1st week and granulocyte macrophage colony-stimulating factor (GM-CSF) during the 3rd week. However, the addition of exogenous GM-CSF (5 U/ml, S5) was found to significantly inhibit monocyte proliferation up to 17 days. This is the first report of proliferation associated with long-term survival of culture derived macrophages in a serum-free, cytokine-free system.

AB - The aim of this study was to establish a cytokine-free, serum-free system which would enable the long-term survival and proliferation of human peripheral blood monocytes. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by adherence to untreated plastic petri dishes and maintained up to 6 weeks in serum-free medium (SFM) consisting of IMEM, insulin, transferrin, sodium selenite and BSA. Maximal cell proliferation occurred during the first 2 weeks of culture and corresponded to the appearance of large numbers of pure, nonadherent culture-derived macrophages. Monocyte maturation was characterised by the modulation of specific cell surface antigens. The percentage of cells staining for the transferrin receptor increased with time, whereas the percentages of cells expressing CD11b, CD11c and HLA-DR remained greater than 60% for the 15 days studied. The mean fluorescent intensities (MFI) of all these antibodies increased significantly with time. The only differences found between the adherent and nonadherent cells, using the above antibodies, were with the MFI for CD11b and CD11c. In both cases, the intensity of staining was significantly greater in the adherent cells. Estimation of cytokine production by cells maintained for 5 weeks in SFM found that they constitutively produced large amounts of macrophage colony-stimulating factor (M-CSF) in the absence of any exogenous stimuli. These cells were also found to secrete high levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during the 1st week and granulocyte macrophage colony-stimulating factor (GM-CSF) during the 3rd week. However, the addition of exogenous GM-CSF (5 U/ml, S5) was found to significantly inhibit monocyte proliferation up to 17 days. This is the first report of proliferation associated with long-term survival of culture derived macrophages in a serum-free, cytokine-free system.

KW - Defined medium

KW - Differentiation

KW - Monocytes

KW - Monokine

KW - Surface antigen

UR - http://www.scopus.com/inward/record.url?scp=0026657071&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026657071&partnerID=8YFLogxK

U2 - 10.1016/0022-1759(92)90323-L

DO - 10.1016/0022-1759(92)90323-L

M3 - Article

VL - 153

SP - 201

EP - 212

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 1-2

ER -