Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model
The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in v...
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Published in | ACS biomaterials science & engineering Vol. 7; no. 7; pp. 3030 - 3042 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
American Chemical Society
12.07.2021
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Abstract | The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel–tumor cell coculture assay. |
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AbstractList | The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel–tumor cell coculture assay. |
Author | Laoukili, Jamila García, Silvia Bonilla van den Bent, Lotte Kranenburg, Onno Queiroz, Karla Poghosyan, Susanna Frenkel, Nicola Alarcón, Carmen Rubio Vulto, Paul Rinkes, Inne Borel Hagendoorn, Jeroen |
AuthorAffiliation | Mimetas BV UMC Utrecht Cancer Center |
AuthorAffiliation_xml | – name: UMC Utrecht Cancer Center – name: Mimetas BV |
Author_xml | – sequence: 1 givenname: Nicola surname: Frenkel fullname: Frenkel, Nicola organization: UMC Utrecht Cancer Center – sequence: 2 givenname: Susanna surname: Poghosyan fullname: Poghosyan, Susanna organization: UMC Utrecht Cancer Center – sequence: 3 givenname: Carmen Rubio surname: Alarcón fullname: Alarcón, Carmen Rubio organization: UMC Utrecht Cancer Center – sequence: 4 givenname: Silvia Bonilla surname: García fullname: García, Silvia Bonilla organization: Mimetas BV – sequence: 5 givenname: Karla surname: Queiroz fullname: Queiroz, Karla organization: Mimetas BV – sequence: 6 givenname: Lotte surname: van den Bent fullname: van den Bent, Lotte organization: UMC Utrecht Cancer Center – sequence: 7 givenname: Jamila surname: Laoukili fullname: Laoukili, Jamila organization: UMC Utrecht Cancer Center – sequence: 8 givenname: Inne Borel surname: Rinkes fullname: Rinkes, Inne Borel organization: UMC Utrecht Cancer Center – sequence: 9 givenname: Paul orcidid: 0000-0002-1525-1545 surname: Vulto fullname: Vulto, Paul organization: Mimetas BV – sequence: 10 givenname: Onno surname: Kranenburg fullname: Kranenburg, Onno organization: UMC Utrecht Cancer Center – sequence: 11 givenname: Jeroen surname: Hagendoorn fullname: Hagendoorn, Jeroen email: j.hagendoorn-3@umcutrecht.nl organization: UMC Utrecht Cancer Center |
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