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To eliminate the influence of temperature, measures were performed within 5 min after taking the culture plates out of the incubator.
Within this time, samples did not show any reading drift. Two hundred microliters of culture medium was added in the upper compartment of the cell culture system. The ohmic resistance of a blank culture insert without cells was measured in parallel. To obtain the sample resistance, the blank value was subtracted from the total resistance of the sample. Correlation coefficient was assessed by Pearson test, while linear regression analyses were used to determine the slope between TEER values and cellular heights.
Cells exhibited disparate architectures when subjected to different media. Overall, both charcoal-stripped media lead to poor morphological quality: Cells were dedifferentiated, flat or dwarf, lost cilia and secretory protrusions Fig. Cells were highly polarized, confluent and retained a mix of ciliated and secretory cells Fig.
Cells are grown in media M1 a , M2 b , M3 c and M4 d , respectively. Shadow area in b highlights a well-differentiated sample with moderate TEER value; shadow area in c highlights a poorly differentiated sample with low TEER value.
Histological investigation further revealed that partway differentiated epithelial layers tend to yield over high TEER output Fig. Contrarily, completely undifferentiated cultures no visible polarization had extremely low TEER values Fig.
All of them demonstrated fully differentiated morphology Fig. As all cultures tested in M4 were well differentiated, we selected medium M4 for further analysis. Negative correlation between TEER and cellular height. We also analyzed the correlation between these two parameters in the hormonal stimulation experiment. The recent advance of filter-supported oviductal epithelial cultures, which proved closer tissue-like morphology and biology than conventional cultures, necessitates a suitable monitoring technique to assess their culture qualities.
After systematic analysis of TEER measurement in conjunction with morphological features of POEC achieved in varied types of media, we clearly revealed how the electrical impedance associates with differentiation status of epithelial cultures. When considering the epithelial layer as a simple electrical structure, it could be described as two parallel units: a transcellular unit and a paracellular unit which is influenced mainly by tight junctions, as well as intercellular spaces and cell-substrate contacts Lo et al.
According to the permeability of epithelial tissues, they could be classified into two categories: leaky and tight epithelia. Oviduct epithelium is described to be leaky Leese ; therefore, in our POEC cultures, the TEER measurement largely reflected the changes in paracellular unit, mainly the elaboration of strands of tight junctions Cereijido et al.
It is widely assumed that low TEER indicates low quality of cultured epithelial cells; however, here we clearly demonstrate that excessive transepithelial resistance in cells originating from a leaky epithelium is associated with a sub-differentiated culture status.
A study comparing both methods on the same cell type and culture system found around threefold lower TEER values when using the Ussing chamber Rajagopal et al.
Apart from the inherent characteristics of epithelial cells, other factors also affect the transepithelial impedance. It has been shown that material and physical properties of the membrane support could affect cell morphology and cell-substrate contacts, therefore leading to divergent TEER values Lo et al. The results we generated in this study are all based on PET 0. Besides, previous studies indicated that temperature has strong influence on the TEER measurement and may skew results Matter and Balda Finally, taking advantage of our standardized POEC model, we were able to quantify the cell polarization along the apical—basal axis and link it to electrical impedance assessment using TEER.
To our knowledge, we could demonstrate for the first time a fine-tuned inversed correlation between cellular height, a parameter reflecting the degree of apical—basal polarity and TEER which mainly corresponds to the strength of cellular junctions. It has been reported that in epithelial tissues, many tight junction complexes e.
However, the underlying molecular mechanisms which co-regulate these two events need to be clarified in further studies. The mathematical equation we provided may facilitate the prediction of porcine oviductal epithelial cell height based on a known TEER value. To conclude, TEER measurement is a non-invasive tool for the quality assessment of filter-based oviduct epithelial cultures.
We hereby presented a proper TEER range which could be used to predict the apical—basal polarization in these cultures. Furthermore, we firstly unveiled the inverse relationship between epithelial height and impedance, which might promote our understanding of the interconnection between apical and basal polarity and tight junction assembly. National Center for Biotechnology Information , U. Histochemistry and Cell Biology. Histochem Cell Biol. Published online Jul Author information Article notes Copyright and License information Disclaimer.
Corresponding author. Accepted Jul 4. This article has been cited by other articles in PMC. Abstract Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphology and functionality.
Electronic supplementary material The online version of this article doi Introduction Culturing epithelial cells on porous filter supports mimics in vivo-like conditions by allowing nutrient supply from the basolateral compartment and therefore promotes cell differentiation and closer morphological as well as functional similarity to the native tissue Zegers et al.
Open in a separate window. TEER negatively correlates with cellular height As all cultures tested in M4 were well differentiated, we selected medium M4 for further analysis. Discussion The recent advance of filter-supported oviductal epithelial cultures, which proved closer tissue-like morphology and biology than conventional cultures, necessitates a suitable monitoring technique to assess their culture qualities.
Compliance with ethical standards Conflict of interest The authors declare that no conflict of interest exists. References Anderson JM. Molecular structure of tight junctions and their role in epithelial transport. News Physiol Sci. Tight junction and polarity interaction in the transporting epithelial phenotype.
Biochim Biophys Acta. In vitro mimicking of estrous cycle stages in porcine oviduct epithelium cells: estradiol and progesterone regulate differentiation, gene expression, and cellular function. Biol Reprod. Long-term culture of primary porcine oviduct epithelial cells: validation of a comprehensive in vitro model for reproductive science.
Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. J Cell Biol. Roles of the oviduct in mammalian fertilization. Glucose transport and metabolism in rabbit oviduct epithelial cells. J Reprod Fertil. Cell polarity: Par6, aPKC and cytoskeletal crosstalk. Curr Opin Cell Biol.
Ian Freshney.
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