Akademik Veri Yönetim Sistemi
Annual Meeting of the Clinical-Immunology-Society (CIS) / Immune Deficiency and Dysregulation North American Conference, Colorado, Amerika Birleşik Devletleri, 2 - 05 Nisan 2020, cilt.40
Liquid versus Semi-Solid Culture Medium for Differentiation of
Human Mast Cells from Hematopoietic Stem Cells in Bone Marrow
A. Yasemin Göksu-Erol, MD1, Ersin Akıncı, PhD2, Hilmi Uysal, MD3,
Devrim Demir Dora, PhD4, Ozan Salim, MD5
1Assoc. Prof. Dr., MD/Akdeniz University, Faculty of Medicine,
Histology and Embryology Department
2Assist. Prof./Akdeniz University, Faculty of Agriculture, Department of
Agricultural Biotechnology. Head of Enzyme and Microbial
Biotechnology Division.
3Prof./Akdeniz University, Faculty of Medicine, Department of
Neurology
4Assist. Prof./Akdeniz University, Faculty of Medicine, Department of
Pharmacology
5Akdeniz University, Faculty of Medicine, Department of Internal
Medicine, Division of Hematology
Abstract/Case Report Text
Introduction:Mast cells (MCs) are hematopoietic-derived immune cells,
whose precursors migrate within tissues reaching maturation and differentiation.
Masitinib, a selective tyrosine kinase inhibitor, is efficient in
controlling the survival, differentiation, and degranulation of MCs.
Aim: To optimize mast cell-differentiation from human bone marrow (BM)
hematopoietic stem cells, and to find best cell culture conditions for proliferation,
differentiation, and maintenance of MCs, which is important when
studying particularly MCs’ response to cytotoxic compounds.
Material-Methods: To produce MCs in vitro, the first method (M1) we
used was a modified semi-solid culture method (1). Briefly; human BM
mononuclear cells (MNCs) were obtained with Ficoll gradient from BM
sample of a patient with idiopathic thrombocytopenic purpura. Colonyforming
unit (CFU)-mast was developed from MNCs in methylcellulose
medium supplemented with SCF (200ng/ml) + IL-6 (50ng/ml), and IL-3
(1ng/ml; only first week). 5-6 weeks later mast cell colonies were transferred
into suspension cultures, in which MCs matured and multiplied up
to 7-8 weeks and were used in experiments till 10th week of culture. On
the other hand, in our second method (M2); MNCs were separated by
Ficoll, seeded in 6 well-plates with IMDM containing FBS 2%, Pen/
Strep, and a little amount of methylcellulose, and incubated at 37oC,
5%CO2. Cultures were then supplemented with IMDM (FBS 1%) +
SCF (100ng/ml) + IL-6 (50ng/ml) on day 4; and IMDM (FBS 2%) +
SCF (100ng/ml) + IL-6 (50ng/ml) + IL-3 (1ng/ml) on day 9. Beginning
on day 18 till the end, IMDM (FBS 2%) + SCF (100ng/ml) + IL-6
(50ng/ml) were added to cultures.
For both methods, morphological assessment of colonies/cells were evaluated
under an inverted microscope (Figure 1 and 2). Verification ofMCs
was performed by immunoflorescence staining for anti-tryptase and -
chymase antibodies, and by toluidine blue staining. Macrophages were
verified by anti-CD-68 immunoflorescence staining. MCs were exposed
to masitinib or DMSO for the evaluation of dose-related effects of
masitinib, and cytotoxicity was evaluated by MTT assay.
Results: In M2, culture conditions were easier to handle compared toM1.
In M2, high amounts of MCs in immature and pre-mature forms were
appeared as early as 15-18 days, and peak levels of proliferation rate was
around 2-4 weeks of culture, which was about 3 weeks earlier than M1.
Culture could be maintained till 10 weeks in both methods. Although
MCs are non-adherent cells, in liquid method adherent BM cells such
as fibroblasts, endothelial cells and mesenchymal stem cells have adhered
to the plate and grown up, providing an attachment site for MCs and
serving as a naturalBMnest,mimicking in-vivo environment, forMCs to
grow and proliferate (Figure 2). Attachment of MCs has provided medium
exchange available without changing culture dishes.WhenMCs were
exposed to masitinib (0.5, 1, and 2 μM/μl), approximate survival rates
were 75%, 72%, 69%, respectively.
Discussion: In our liquid medium method, the adherent BM cells not only
provided a natural nest supporting MC development and differentiation,
they also served as an attachment site for MCs. As the cells slightly adhered,
when trypsinized shortly, they easily detached and used for experiments.
And we also report for the first time that adding a little amount of methylcellulose
to the liquid medium provides ease of aggregation of CFUs, and
easy development of MCs. We suggest that our liquid culture may be
superior to semi-solid method, that it is faster and easier to handle.
Ref. 1. Ozdemir O. Evaluation of human mast cell-mediated cytotoxicity
by DIOC18 target cell labeling in flow cytometry. Journal of
Immunological Methods. 319, 98–103; 2007.