Abstract
The cellular events underlying intestine regrowth in the sea cucumber Holothuria glaberrima have been described by our group. Currently, the molecular and signaling mechanisms involved in this process are being explored. One of the limitations to our investigations has been the absence of suitable cell culture methodologies, required to advance the regeneration studies. An in vitro system, where regenerating intestine explants can be studied in organ culture, was established previously by our group. However, a detailed description of the histological properties of the cultured gut explants was lacking. Here, we used immunocytochemical techniques to study the potential effects of the culture conditions on the histological characteristics of explants, comparing them to the features observed during gut regeneration in our model in vivo. Additionally, the explant outgrowths were morphologically described by phase-contrast microscopy and SEM. Remarkably, intestine explants retain most of their original histoarchitecture for up to 10 days, with few changes as culture time increases. The most evident effects of the culture conditions on explants over culture time were the reduction in the proliferative rate, the loss of the polarity in the localization of proliferating cells, and the appearance of a subpopulation of putative spherulocytes. Finally, cells that migrated from the gut explants could form net-like monolayers, firmly attached to the culture substrate. Overall, regenerating explants in organ culture represent a powerful tool to perform short-term studies of processes associated with gut regeneration in H. glaberrima under controlled conditions.
Highlights
Explants are non-disaggregated tissue or organ fragments removed from an organism
The intestine rudiment area and the expression and localization of markers for muscle dedifferentiation, cell proliferation, mesothelium, nerve fibers and neuron-like cells, and components of the connective tissue were determined in the above-mentioned cultured gut tissues and were compared to explants dissected at 5 dpe but not cultured
The gut rudiment grows as intestine regeneration proceeds in H. glaberrima in vivo [16]
Summary
Explants are non-disaggregated tissue or organ fragments removed from an organism. In mammals, cultured explants are generally used to obtain primary cell lines constituted by the cells that migrate out from the tiny explants as outgrowths, forming monolayers around them. The arm explant model has been the most used in echinoderms for in vitro studies. Arm explants have been utilized in organ culture to study regenerative processes, mainly using histological methods, while some groups have focused on describing their outgrowths. We have established the explant culture, which was recently used to gain new insights about the signaling mechanisms involved in intestine regrowth [8,11] and the dedifferentiation mechanisms during radial nerve cord regeneration [12] in our model. We emphasized the effects of the culture conditions on cellular processes that have been well documented during gut regeneration in vivo in our model, such as muscle dedifferentiation, cell proliferation, changes in the extracellular matrix components, and the distribution of cell populations, such as neurons and mesothelial cells in explants. The explant outgrowth characteristics were described by phase-contrast microscopy and SEM
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