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A 3-D cell culture system to study epithelia functions using microcarriers

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Abstract

In vitro cell culture models used to study epithelia and epithelial diseases would benefit from the recognition that organs and tissues function in a three-dimensional (3D) environment. This context is necessary for the development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3D models of epithelium. We engineered 3D epithelia cultures using a kidney epithelia cell line (MDCK) and spherical polymer scaffolds. These kidney epithelia were characterized by live microscopy, immunohistochemistry and transmission electron microscopy. Strikingly, the epithelial cells displayed increased physiological relevance; they were extensively polarized and developed a more differentiated phenotype. Using such a growth system allows for direct transmission and fluorescence imaging with few restrictions using wide-field, confocal and Light Sheet Fluorescence Microscopy. We also assessed the wider relevance of this 3D culturing technique with several epithelial cell lines. Finally, we established that these 3D micro-tissues can be used for infection as well as biochemical assays and to study important cellular processes such as epithelial mesenchymal transmission. This new biomimetic model could provide a broadly applicable 3D culture system to study epithelia and epithelia related disorders.

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Acknowledgments

We wish to thank Uros Kržič and Patrick Theer for technical assistance. This work was funded by BMBF Grant “QuantPro”, Number 0313831D. EGR acknowledges support from the SFI under the Stokes Fellowship Programme.

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    Authors and Affiliations

    1. European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

      Petra H. Jakob, Catharina Wiegel & Uta Haselmann

    2. Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK

      Jessica Kehrer & Markus Meissner

    3. Goethe University Frankfurt, Frankfurt am Main, Germany

      Ernst H. K. Stelzer

    4. School of Biology and Environmental Science, UCD Science Centre West, University College Dublin, Room 141, Science Center West, Belfield, Dublin 4, Ireland

      Peter Flood & Emmanuel G. Reynaud

    Authors
    1. Petra H. Jakob
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    2. Jessica Kehrer
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    3. Peter Flood
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    4. Catharina Wiegel
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    5. Uta Haselmann
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    6. Markus Meissner
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    7. Ernst H. K. Stelzer
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    8. Emmanuel G. Reynaud
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    Corresponding author

    Correspondence to Emmanuel G. Reynaud.

    Ethics declarations

    This study did not involve human participants and/or animals.

    Conflict of interest

    The authors declare no conflict of interest.

    Additional information

    Petra H. Jakob, Jessica Kehrer and Peter Flood have contributed equally to this work.

    Electronic supplementary material

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    Supplementary material 1 (PDF 136 kb)

    Supplementary material 2 (PNG 1022 kb)

    Supplementary material 3 (PDF 80 kb)

    10616_2015_9935_MOESM4_ESM.avi

    Movie S1: A single microcarrier seeded with MDCK cells according to the protocol described in Material and Methods was imaged in bright-field mode. Digital images were acquired with a DeltaVision system (Applied Precision Inc., Issaquah, WA) using time lapse image acquisitions over a 48 hour period at 30 minute intervals (96 frames) (AVI 7358 kb)

    10616_2015_9935_MOESM5_ESM.avi

    Movie S2: A single microcarrier fully seeded with MDCK cells according to the protocol described in Material and Methods was imaged after 2 weeks of growth in bright-field mode by LSFM. A complete stack was acquired to image the entire depth of the Cytodex using a 1 μm spacing (AVI 21987 kb)

    10616_2015_9935_MOESM6_ESM.avi

    Movie S3: A single microcarrier fully seeded with MDCK cells according to the protocol described in Material and Methods was imaged after 1 week of growth in fluorescence. Digital images were acquired with a DeltaVision system (Applied Precision Inc., Issaquah, WA) using time lapse image acquisitions over a 2 hour period at 5 minute intervals. Cells were stably transfected with a nuclear marker (H2B-EGFP, green channel), the transmission channel was displayed in the red channel for convenience. A single plane was focused on close to the coverslip in order to image the Cytodex based monolayer (AVI 5630 kb)

    10616_2015_9935_MOESM7_ESM.avi

    Movie S4: A single microcarrier fully seeded with MDCK cells according to the protocol described in Material and Methods was imaged after a 1 week growth using bright-field mode. Digital images were acquired with a DeltaVision system (Applied Precision Inc., Issaquah, WA) using time lapse image acquisitions over a 72 hour period at 30 minute interval. A single plane was focused on, close to the coverslip to image the Cytodex based monolayer undergoing EMT and moving from the microcarrier to the glass surface of the coverslip. A protocol to centre the microcarrier during the image acquisition process was used to optimize the EMT follow-up over long periods of time. Each experiment was set-up to follow-up 12 Cytodex in a 12 well plate set-up. This movie represents a typical example of the EMT behaviour of a MDCK epithelial Cytodex (AVI 19717 kb)

    10616_2015_9935_MOESM8_ESM.avi

    Movie S5: A single microcarrier fully seeded with MDCK cells was grown for 4 weeks on an agarose pad and let to proceed with EMT for a 96 hour period on a glass bottom dish and was then fixed for imaging. Digital images were acquired in fluorescence mode using a confocal microscope and a stack covering the coverslip monolayer and half of the MDCK epithelial Cytodex was imaged (1 μm interval, 52 μm total). Nuclei (Dapi staining, blue channel), Golgi (Red staining) and Actin (Phalloidin, Green channel) are represented in a volume rendering made using ImageJ 3D viewer (AVI 2033 kb)

    10616_2015_9935_MOESM9_ESM.avi

    Movie S6: A single microcarrier fully seeded with MDCK cells was grown for 4 week on an agarose pad and infected using a thermo-sensitive adenovirus GFP tagged VSVG (Presley et al. 1997) that can be tracked along the secretory pathway and has been reported to traffic through the Golgi apparatus to the basolateral and lateral membranes of MDCK cells (Farr et al. 2009). Digital images were acquired in fluorescence mode by LSFM and a single plane of the MDCK epithelial Cytodex was imaged (2 μm light sheet thickness) over time (1 minute interval, total 16 minutes) (AVI 299 kb)

    10616_2015_9935_MOESM10_ESM.avi

    Movie S7: Enlargement of two cells displayed in MovieS6. A single microcarrier fully seeded with MDCK cells was grown for 4 week on an agarose pad and infected using a thermo-sensitive adenovirus GFP tagged VSVG (Presley et al. 1997) that can be tracked along the secretory pathway and has been reported to traffic through the Golgi apparatus to the basolateral and lateral membranes of MDCK cells (Farr et al. 2009). Digital images were acquired in fluorescence mode by LSFM and a single plane of the MDCK epithelial Cytodex was imaged (2 μm light sheet thickness) over time (1 minute interval, total 16 minutes) (AVI 140 kb)

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    Jakob, P.H., Kehrer, J., Flood, P. et al. A 3-D cell culture system to study epithelia functions using microcarriers. Cytotechnology 68, 1813–1825 (2016). https://doi.org/10.1007/s10616-015-9935-0

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