Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids

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Abstract

An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.
Original languageEnglish
Pages (from-to)165-171
JournalStem Cell Research
Volume28
DOIs
Publication statusPublished - 1 Apr 2018

Fingerprint

Organoids
Swine
Stem Cells
Paneth Cells
Tight Junctions
Poisons
Electric Impedance
Small Intestine
Suspensions
Epithelium
Cell Count
Immunohistochemistry
Food

Keywords

  • Epithelium
  • Intestinal
  • Monolayer
  • Organoids
  • Porcine

Cite this

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title = "Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids",
abstract = "An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.",
keywords = "Epithelium, Intestinal, Monolayer, Organoids, Porcine",
author = "{van der Hee}, B. and L.M.P. Loonen and N. Taverne and J.J. Taverne-Thiele and H. Smidt and J.M. Wells",
year = "2018",
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T1 - Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids

AU - van der Hee, B.

AU - Loonen, L.M.P.

AU - Taverne, N.

AU - Taverne-Thiele, J.J.

AU - Smidt, H.

AU - Wells, J.M.

PY - 2018/4/1

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N2 - An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.

AB - An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.

KW - Epithelium

KW - Intestinal

KW - Monolayer

KW - Organoids

KW - Porcine

U2 - 10.1016/j.scr.2018.02.013

DO - 10.1016/j.scr.2018.02.013

M3 - Article

VL - 28

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EP - 171

JO - Stem Cell Research

JF - Stem Cell Research

SN - 1873-5061

ER -