Short-term cultivation of murine thymic epithelial cells in a serum-free medium |
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Authors: | Carsten Ropke Bo van Deurs Ole W Petersen |
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Institution: | (1) Department of Anatomy A, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark;(2) Department of Medical Anatomy A, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark |
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Abstract: | Summary Thymic epithelial cells were grown in defined medium without unknown serum factors and without concurrent growth of other
cell types. Thymic tissue was obtained from 1- to 4-wk-old mice, disaggregated, and incubated in a mixture of collagenase-dispase-DNAse.
The resulting organoids were seeded on collagen-coated flasks. The culture medium consisted of DME-F12 with low or high concentration
of Ca2+ supplemented with insulin, epidermal growth factor, cholera toxin, hydrocortisone, and transferrin. Under these conditions,
explants attached to the substrate within 2 d, and expanding epithelioid monolayer islets emerged from the organoids during
the following days. 3H]Thymidine incorporation revealed a growth fraction of the cells close to 5%. By omitting either epidermal growth factor,
insulin, or cholera toxin from the medium, pronounced reduction in sizes of islets and in 3H]thymidine incorporation was found. Throughout the culture period, the islets appeared as continuous sheets of polygonal
cells. The epithelial nature of the expanding cell islets was confirmed by demonstration of cytokeratins and of desmosomes.
Ultrastructural evaluation of early cultures revealed clusters of epithelial cells intermixed with lymphocytes, and late cultures
showed a typical pattern of stratified keratinizing epithelium. However, squamous metaplasia was avoided by the use of low
Ca2+ medium, which also proved essential for cell transfer. MHC class II antigen was detected on the majority of the cultured
cells, and culture supernatants contained co-mitogenic activity for thymocytes and GM-colony stimulating activity.
This work supported by The Danish Research Council, grant 12-8148. |
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Keywords: | thymic epithelial cells serum-free culture |
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