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A three-dimensional culture method to expand limbal stem/progenitor cells.

ABSTRACT: The current standard method to culture human limbal stem/progenitor cells (LSCs) in vitro is to culture limbal epithelial cells directly on a layer of murine 3T3 feeder cells (standard method). The direct contact between human cells and murine feeder cells poses the potential risk of incomplete removal of feeder cells after culture and cross-contamination in clinical applications. We present here a novel three-dimensional (3D) sandwich method in which LSCs and feeder cells were separately cultured on opposite sides of a porous membrane. Limbal epithelial cells in the form of single-cell suspensions, cell clusters, and tissue explants were subjected to standard culture or to a 3D sandwich culture method. The 3D sandwich method consistently yielded LSCs derived from cell clusters and tissue explants. The expanded LSCs exhibited a small, compact, cuboidal stem-cell morphology and stem cell phenotypes comparable to those of LSCs derived from the standard culture method. Limbal epithelial cell clusters cultured with the sandwich method had a significantly higher proliferation rate than did those cultured with the standard method. The 3D sandwich method did not favor the propagation of single LSCs. In summary, the 3D sandwich method permits complete separation between cultured cells and feeder cells, while providing an even and maximal proximity between them. This alternative method permits culturing of LSCs without the risk of feeder cell contamination.

SUBMITTER: Mei H

PROVIDER: S-EPMC4005490 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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A three-dimensional culture method to expand limbal stem/progenitor cells.

Mei Hua H González Sheyla S Nakatsu Martin N MN Baclagon Elfren Ray ER Lopes Vanda S VS Williams David S DS Deng Sophie X SX

Tissue engineering. Part C, Methods 20131107 5

The current standard method to culture human limbal stem/progenitor cells (LSCs) in vitro is to culture limbal epithelial cells directly on a layer of murine 3T3 feeder cells (standard method). The direct contact between human cells and murine feeder cells poses the potential risk of incomplete removal of feeder cells after culture and cross-contamination in clinical applications. We present here a novel three-dimensional (3D) sandwich method in which LSCs and feeder cells were separately cultur ...[more]

PMID: 24047104

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Project description:BackgroundIntestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine.ObjectivesTo describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities.Study designDescriptive study.MethodsIntestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed.ResultsIntestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle.Main limitationsTissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time.ConclusionsThe successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).

Dataset Information

A three-dimensional culture method to expand limbal stem/progenitor cells.

Publications

A three-dimensional culture method to expand limbal stem/progenitor cells.

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