Human Intestinal Enteroids for the Study
of Bacterial Adherence, Invasion, and
Translocation
Nina M. Poole,1,2 Anubama Rajan,1,2 and Anthony W. Maresso1
1
Department of Molecular Virology and Microbiology, Baylor College of Medicine,
Houston, Texas
2
These authors contributed equally to this work.
Adherence, invasion, and translocation to and through the intestinal epithelium are important drivers of disease for many enteric bacteria. However, most
work has been limited to transformed intestinal cell lines or murine models
that often do not faithfully recapitulate key elements associated with human
disease. The recent technological advances in organotypic tissue and cell culture are providing unparalleled access to systems with human physiology and
complexity. Human intestinal enteroids (HIEs), derived from patient biopsy
or surgical specimens of intestinal tissues, are organotypic cultures now being
adapted to the study of enteric infections. HIEs are comprised of the dominant
cell types of the human gastrointestinal epithelium, can be grown in two- or
three-dimensional structures, form a crypt–villus axis with defined apical and
basolateral compartments, and undergo physiologic responses to many different stimuli. Here, we describe a series of protocols that encompass the use of
human enteroids for the measurement of the adherence, invasion, and translocation of E. coli to and through the intestinal epithelium. We also outline the
steps needed to grow and prepare enteroids for this purpose and highlight some
C 2018 by John Wiley & Sons, Inc.
common problems to troubleshoot. Keywords: adhesion r E. coli r bacteria r invasion r human intestinal enteroids
r translocation
How to cite this article:
Poole, N. M., Rajan, A., & Maresso, A. W. (2018). Human intestinal
enteroids for the study of bacterial adherence, invasion, and
translocation. Current Protocols in Microbiology, e55. doi:
10.1002/cpmc.55
INTRODUCTION
The gastrointestinal (GI) tract functions in food digestion, nutrient absorption, and waste
excretion and is a barrier against bacteria that chronically or acutely inhabit this tissue.
The recent explosion of studies associating the resident microbial flora with human health
is further testament to the importance of studying the interaction between bacteria and
the human intestinal epithelium. In addition, the GI tract represents a portal of entry
for pathogenic bacteria, especially those that specifically induce the release of host fluid
as a mechanism to spread progeny into the environment. Pathogen-induced diarrhea,
perhaps the most common GI pathophysiology in humans, is responsible for 2 to
4 billion episodes annually, especially in children (Hodges & Gill, 2010). Diarrhea is
the second leading cause of death in children under the age of 5 years in developing
countries (Wardlaw, Salama, Brocklehurst, Chopra, & Mason, 2010). Chronic diarrhea is
associated with nutritional deficiency, stunted growth, and cognitive impairment (Steiner,
Lima, Nataro, & Guerrant, 1998). Some GI bacteria do not induce diarrhea; instead, they
Poole et al.
Current Protocols in Microbiology e55
Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpmc.55
C 2018 John Wiley & Sons, Inc.
Copyright 1 of 12
are highly invasive of the intestinal epithelium and enter the underlying lymphoid tissue
or even translocate into the blood. This may lead to bacteremia and dissemination to
critical organs often resulting in life-threatening sepsis.
The molecular processes that lead to these pathophysiological effects are largely unidentified. Many rodent models do not recapitulate the hallmark features of human diarrhea.
Larger animal species such as pigs more closely resemble the human condition, but their
use is technically challenging and expensive. In vitro organ cultures of biopsies are short
lived, and transformed culture cells often have aberrant physiology. Recent advances in
organotypic culturing methods of untransformed human tissue may be a viable alternative
to animal models for some of the critical steps in the pathophysiology of these enteric
infections. Of special significance is the progress made in the cultivation and growth of
human intestinal enteroids (HIEs). These “mini guts” are derived from LGR5+ stem cells
of the small intestine and colon (McCracken, Howell, Wells, & Spence, 2011; Sato &
Clevers, 2013; Sato, Stange et al., 2011; Sato, van Es, et al., 2011; Sato et al., 2009) and
are generated by modulating the presence and levels of key stem cell responsive growth
factors, including Wnt3a, R-Spondin, and noggin (Sato, Stange, et al., 2011; Sato, Stange,
et al., 2011; Zachos et al., 2016). Upon removal of Wnt, stem cells differentiate into all
major cell types of the intestinal epithelium, including entero-endocrine, Paneth, and
Goblet cells, and obtain three-dimensional (3D) architecture with an enclosed lumen
and a crypt–villus axis (In et al., 2016). HIEs have now been generated from stomach,
duodenum, jejunum, ileum, and colon and from many different patient donors (Mahe,
Sundaram, Watson, Shroyer, & Helmrath, 2015). Ongoing studies from various groups
are examining the incorporation of immune cells, vasculature, stretch, and flow into
enteroid cultures, as well as “gut-on-a-chip” approaches to model drug metabolism and
multi-organ connections (Dutta & Clevers, 2017; Lee, Choi, & Kim, 2016; Noel et al.,
2017). Here, we describe how to isolate, grow, maintain, store, and infect HIEs to model
various interactions with enteric bacteria.
BASIC
PROTOCOL 1
MAINTENANCE OF ENTEROID CULTURE SYSTEM
The cell culture system described in Basic Protocol 1 is often referred to as an enteroid
or organoid culture system. The technology is novel owing to the use of non-transformed
cells and is an art by itself since the culturing methodology requires technical nuances and
expertise that are vastly different from the traditional cell culture system (i.e., cancer cell
lines). In brief, intestinal crypts containing stem cells are obtained from human biopsy
or surgery samples and are propagated as 3D enteroids embedded inside Matrigel. The
enteroids are allowed to grow for a period of 7 to 10 days before making monolayers or
further passaged. The preparation and growth of the enteroid culture system is described
in detail below and summarized in Figure 1.
Materials
Poole et al.
Complete medium without growth factor (CMGF– ; see recipe)
Human 3D enteroids (see McCracken et al., 2009)
Matrigel, growth factor reduced, phenol-free (e.g., Corning, cat. no. 356231)
Complete medium with growth factor (CMGF+ ; see recipe)
1 mg/ml collagen IV (e.g., Sigma-Aldrich, cat. no. C5533) in 100 mM acetic acid
(cell culture grade)
Phosphate-buffered saline (PBS; see Current Protocols, 2005)
EDTA
0.05% trypsin-EDTA (e.g., Thermo Fisher Scientific, cat. no. 25300-062)
Fetal bovine serum (FBS)
Y-27632
Differentiation medium (see recipe)
2 of 12
Current Protocols in Microbiology
Figure 1 (A) Schematic of enteroid monolayer preparation. Culturing of enteroids is broken
down into three stages. In Stages 1 and 2, 3D enteroids are seeded on Matrigel support in 24-well
plates. The 3D enteroids are maintained in undifferentiated growth conditions (CMGF+ medium)
for 7 to 9 days before either passaging or preparing monolayers. In Stage 3, the monolayers
are made from 3D enteroids and can be seeded on either collagen- or Matrigel-coated 96-well
plates with transwell inserts or cell culture–grade chambered slides and coverslips. After reaching
80% to 90% confluency (usually within 24 to 48 hr), the undifferentiated enteroid monolayers
are differentiated by the addition of differentiation medium to form a monolayer with the different
intestinal epithelial cell types. The differentiation medium is refreshed every other day, and the
infections are carried out after 4 days of differentiation. See Basic Protocol 1 for specifics. (B)
Undifferentiated, healthy 3D enteroid obtained from the jejunum. (C) Differentiated 3D enteroid.
The presence of dead cells (dark zone) in the center of enteroid can be observed. Dead cells
in the center of 3D enteroid culture after 8 days are common. This is due to lack of penetration
of growth medium into the Matrigel. However, if the 3D enteroid appears darker in most parts, it
would indicate the enteroids are dying and not suitable for further proliferation or experiments. (D)
Differentiated enteroid monolayer on 96-well plate.
15-ml and 50-ml conical tubes
Refrigerated centrifuge with swing rotor
24-well plate, Nunclon Delta surface treated (e.g., Thermo Fisher Scientific, cat.
no. 142475)
37°C incubator
1-ml syringe
25-G, 5/8 in. needle
40-µm cell strainer (e.g., Fisher Scientific, cat. no. 352340)
96-well plate or chambered slide or transwell system
NOTE: Perform all steps in a sterile cell culture cabinet.
Stage 1: Growth and maintenance of 3D enteroids
1. Add 10 ml CMGF– to a 15-ml conical tube, and keep on ice.
2. Transfer contents of frozen vial containing enteroids (after thawing) to 15-ml tube
containing 10 ml ice-cold CMGF– .
Poole et al.
3 of 12
Current Protocols in Microbiology
Human 3D enteroids are isolated from intestinal crypts; the protocol for isolation is
based upon a previously established protocol (McCracken et al., 2009). About 100 3D
enteroids in solid Matrigel or at desired confluency should be used after 7 days of growth.
3. Centrifuge tube 5 min at 80 × g, 4°C.
4. Remove supernatant and resuspend pellet in 120 µl Matrigel (enough to seed 4 wells
using 30 µl/well) using cold P200 pipet tips.
Store tip boxes at –20°C to keep them cold.
5. Plate enteroids as droplets without air bubbles in 4 wells of a 24-well plate, and
transfer plate to a 37°C incubator.
6. Allow Matrigel to solidify for 5 to 10 min. Add 500 µl room temperature CMGF+
to each well, and culture in a 37°C incubator.
7. Refresh culture with CMGF+ every other day.
Typically, enteroids are ready to be passaged or made into monolayers after 7 to 10 days
of culturing.
Stage 2: Passage of enteroids
The passage ratio is 1:5 if the growth density is about 100 3D enteroids per well. This
protocol is calibrated for one well only; repeat with other wells. After 7 to 10 days of
culturing, enteroids are ready to be passaged.
8. Remove medium from wells (around the solid Matrigel).
9. Add 500 µl ice-cold CMGF– to well, and mechanically break up Matrigel by pipetting up and down with a P1000 pipettor a couple of times.
10. Aspirate the solution using a 1-ml syringe with 25-G, 5/8 in. needle. Draw the
solution up and down 2 to 3 times per well.
11. Transfer the whole contents into a 15-ml tube.
12. Add equal volume of ice-cold CMGF– .
13. Centrifuge 5 min at 80 × g, 4°C.
14. Remove supernatant or medium, and keep the tube on ice.
15. Resuspend enteroid pellet in Matrigel using cold P200 pipet tips.
Calculate the amount of Matrigel that will be needed based on 30 µl/well.
16. Add 30 µl/well enteroid–Matrigel mixture as a droplet into a 24-well plate using
cold P200 pipet tips. Repeat steps 6 and 7 for revival of enteroids.
Stage 3: HIE monolayers (HIEM)
17. Prepare either collagen or Matrigel to coat wells. For collagen, prepare a 1:30
dilution of 1 mg/ml collagen IV solution in water. For Matrigel, prepare a 1:40
dilution of Matrigel in ice-cold PBS.
For coating wells, both collagen and Matrigel can be used. See Critical Parameters and
Troubleshooting for information regarding selecting the appropriate coating solution.
18. To coat with collagen, add 100 µl diluted collagen solution, and incubate at 37°C
for 2 hr. To coat with Matrigel, add 100 µl diluted Matrigel solution, and incubate
at 37°C for 20 min.
Poole et al.
4 of 12
Current Protocols in Microbiology
19. After 7 to 9 days of culturing 3D enteroids in CMGF+ , collect desired number of
3D enteroids from the wells, and wash with 500 µl ice-cold PBS containing 0.5 mM
EDTA.
20. Centrifuge 5 min at 80 × g (1000 rpm), 4°C.
21. Remove the wash medium, and dissociate the enteroids by adding 1 ml of 0.05%
trypsin-EDTA. Incubate at 37°C for 4 min.
22. Inactivate trypsin by adding 1 ml CMGF– containing 10% (v/v) FBS.
23. Dissociate enteroids by vigorously pipetting up and down 50 times using a P1000
pipettor.
24. Pass the dissociated enteroids into 50-ml conical tube containing a 40-µm cell
strainer. Pre-wet the strainer with CMGF– containing 10% (v/v) FBS, and rinse 2
times after passing the enteroids.
25. Centrifuge 5 min at 120 × g (1500 rpm), 4°C.
26. Resuspend the pellet in the required volume of CMGF+ containing 10 µM Y-27632.
27. Add 100 µl resuspended pellet to 1 well of a 96-well plate or chambered slide or to
the upper compartment of a transwell insert. If using a transwell system, add 500 ml
CMGF+ containing 10 µM Y-27632 (no pellet) to the lower compartment.
28. On the following day or the day after, refresh the medium with differentiation
medium to allow proliferation of different types of intestinal epithelial cells.
FREEZING AND STORING OF ENTEROIDS
While the enteroid culture system can be continuously grown for several passages, it is
always good practice to freeze and store some early passage lines of enteroids. This is
useful in case of contamination or failure to grow well at later passages for unknown
reasons, as well as just as a good laboratory practice.
SUPPORT
PROTOCOL
Additional Materials (also see Basic Protocol 1)
Human 3D enteroids grown for 7 to 10 days (see Basic Protocol 1)
Recovery Cell Culture Freezing Media (e.g., Thermo Fisher Scientific, cat. no.
12648-010)
Refrigerated centrifuge with swing rotor
2-ml cryovial (e.g., Corning, cat. no. 430659)
Freezing container
Liquid nitrogen storage system
1. Grow entreroids as in Basic Protocol 1 steps 1 through 7.
After 7 days of enteroid growth, enteroids are ready to be frozen.
2. Repeat steps 8 and 9 of Basic Protocol 1, Stage 2: Passage of enteroids.
3. Centrifuge 5 min at 80 × g, 4°C.
4. Remove the supernatant or medium, and keep the tube on ice.
5. Resuspend enteroids by combining 1 ml freezing medium with the contents of 2 wells
enteroids into 1 cryovial.
6. Place cryovial in freezing container, and store at –80°C overnight. The following day
transfer cryovials into liquid nitrogen.
Poole et al.
5 of 12
Current Protocols in Microbiology
BASIC
PROTOCOL 2
ADHERENCE TO HUMAN INTESTINAL ENTEROID MONOLAYERS
It is now well known and appreciated that most commensal and pathogenic bacteria
interact with host cells by expressing adhesive molecules on their surfaces that engage
host-cell receptors or soluble macromolecules. This host–microbe interaction, referred
to as bacterial adherence, is often the first step of pathogenesis, thereby making adhesins,
proteins that promote adhesion, a target for therapeutic development. E. coli is a very
large and diverse group of bacteria with different pathotypes producing multiple adhesins
that associate with the host cell. The following protocol uses HIEM to evaluate E. coli
adherence to intestinal epithelial cells. For adherence, dilute and wash E. coli grown normally in suitable bacteriologic medium. Resuspend washed bacteria in enteroid CMGF–
medium, and add desired multiplicity of infection (MOI) to the differentiated HIEM.
Then, incubate for 1 to 24 hr at 37°C in the presence of 5% CO2 in a humidified incubator. Next, wash monolayers, lyse or scrape cells, and enumerate bacteria by plating serial
dilutions on Luria-Bertani (LB) agar plates with or without antibiotic supplementation.
Materials
E. coli cultures with or without antibiotic resistance cassettes
Sterile LB medium
Appropriate antibiotics for E. coli cultures (optional)
PBS (see Current Protocols, 2005)
CMGF– (see recipe)
Differentiated (4 days) HIEM in:
96-well plate (see Basic Protocol 1)
Chambered slides, coverslips, or transwell inserts (see Basic Protocol 1)
Differentiation medium (see recipe)
0.25% trypsin-EDTA (e.g., Fisher Scientific, cat. no. 25-053-CI)
Hanks balanced salt solution (HBSS), calcium- and magnesium-free (e.g., Fisher
Scientific, cat. no. 20-021-CV)
Triton X-100 (e.g., Sigma-Aldrich, cat. no. T-8787)
LB agar plates, with our without appropriate antibiotics
Methanol, paraformaldehyde, or glutaraldehyde
37°C, 5% CO2 , humidified incubator with shaker
Spectrophotometer
Microcentrifuge
1.5-ml microcentrifuge tubes (e.g., Fischer Scientific, cat. no. 05-408-129)
Hemocytometer
Cell scraper
Stage 1: Bacterial culture preparation
1. Grow E. coli strains from a single colony in LB medium with or without antibiotic
supplementation at 37°C with aeration overnight (16 to 18 hr; Son & Taylor, 2012).
If experiment requires bacteria in log phase, subculture the overnight samples and
grow accordingly.
2. Measure the optical density of bacterial cultures using a spectrophotometer, and
estimate the number of colony forming units (CFU) based on bacterial growth
correlation curve.
3. Wash cultures in equal volume PBS one time, and resuspend in equal volume of
CMGF– . Pellet bacteria by microcentrifuging 3 min at 13,793 × g (13,000 rpm),
room temperature.
Poole et al.
6 of 12
Current Protocols in Microbiology
Stage 2: HIEM infection
4. Remove differentiation medium from HIEM cultured in 96-well plates (from Basic
Protocol 1 Stage 3), and replace with 100 µl fresh CMGF– (if infection is only 1 to
2 hr) or differentiation medium (if infection is 3 to 24 hr).
5. Determine the number of cells for MOI calculation by removing 1 HIEM in the
96-well plate. Add 200 µl trypsin to the monolayer, and incubate at 37°C in the
presence of 5% CO2 in a humidified incubator for 5 min. Stop trypsinization by
transferring cells to a 1.5-ml microcentrifuge tube containing 800 µl HBSS, and
mix by pipetting. Next, load 10 µl cell suspension to a hemocytometer, count cells,
and calculate the number of cells/ml according to manufacturer’s instructions. Since
the volume of the seeded cell suspension is 100 µl per well, calculate the number of
cells for the monolayer by dividing the number of cells/ml by 10.
The number of cells for the monolayer ranges from 50,000 to 100,000. Calculate the
desired MOI (MOI 1 to 200 for 1-hr infection, MO1 1 to 10 for 3- to 6-hr infection, MO1
0.01 to 0.1 for overnight infection).
6. Add each strain at the calculated MOI to the assigned wells of the 96-well plate, and
incubate for the desired time at 37°C in the presence of 5% CO2 in a humidified
incubator.
7. Wash monolayers three times with PBS. Lyse with 100 µl cold PBS containing 0.1%
(v/v) Triton X-100, or remove monolayer by scraping using only PBS. Enumerate
the bacteria by plating serial dilutions (10-2 to 10-4 ) on LB agar plates with or without
antibiotics.
Stage 3: Adhesion using staining methods
8. Use HIEM cultured on chambered slides, coverslips, or transwell inserts from Basic
Protocol 1 Stage 3 for studying adherence by staining methods.
9. Repeat steps 1 through 6 to perform bacterial infection to evaluate adherence.
To visualize adhered bacteria, various staining methods such as Giemsa-Wright staining,
immunofluorescence, and electron microscopy can be used.
10. To fix HIEM, incubate at room temperature in methanol for 5 min, paraformaldehyde
for 30 min, or glutaraldehyde for 20 min. Alternatively, incubate at 4°C overnight
for all the three fixatives.
BACTERIAL INVASION OF HUMAN INTESTINAL ENTEROID
MONOLAYERS
BASIC
PROTOCOL 3
Invasive E. coli strains are categorized according to their clinical manifestation as nondiarrheagenic or diarrheagenic. Determining how these strains penetrate the intestinal
epithelium after host colonization is crucial to understanding the initial steps of infection. The methods applied here are also applicable to other invasive species, including
shigella, salmonella, and listeria. The following protocol describes how to use HIEM
to evaluate invasion. This protocol was modified from Pı́zarro-Cerdá, Lecuit, & Cossart
(2002) where transformed Caco-2 cells were used as the model system to test Listeria
monocytogenes invasion. Briefly, as in Poole et al. (2017), dilute and wash E. coli cultures. Resuspend washed bacteria in enteroid CMGF– medium, and add desired MOI to
the differentiated HIEM. Incubate for 1 to 24 hr at 37°C in the presence of 5% CO2 in
a humidified incubator (Poole et al., 2017). Next, wash monolayers, and add gentamicin
for 2 hr to kill extracellular bacteria. Then, lyse cells, and enumerate bacteria by plating
serial dilutions on LB agar plates with or without antibiotic supplementation.
Poole et al.
7 of 12
Current Protocols in Microbiology
Materials
500× (50 mg/ml) gentamicin sulfate (e.g., Santa Cruz Biotechnology, cat. no.
sc-29066A)
CMGF– (see recipe)
PBS (see Current Protocols, 2005)
Triton X-100 (e.g., Sigma-Aldrich, cat. no. T-8787)
LB agar plates, with our without appropriate antibiotics
Additional reagents and equipment for bacterial culture preparation and HIEM
infection (see Basic Protocol 2)
1. Repeat steps 1 through 6 of Basic Protocol 2 Stages 1 (bacterial culture preparation)
and 2 (HIEM infection).
2. Make a working concentration of 50 µg/ml gentamicin sulfate by adding 1 µl/ml of
a 50 mg/ml stock solution to CMGF– .
The manufacturer recommends working concentrations of gentamicin sulfate in a range
of 0.5 to 50 µg/ml. According to the Clinical and Laboratory Standards Institute (CLSI),
the minimum inhibitory concentration interpretive standard for most Enterobacteriaceae
for gentamicin is 4 µg/ml.
3. After infection (1 to 24 hr), wash HIEM three times with PBS. Then, add 100 µl of
50 µg/ml gentamicin sulfate, and incubate for an additional 2 hr to kill extracellular
bacteria.
4. Wash monolayers one time with PBS to remove antibiotic, and lyse with 100 µl cold
PBS containing 0.1% (v/v) Triton X-100. Enumerate by plating undiluted sample and
serial dilutions (10-1 to 10-2 ) on LB agar plates with or without antibiotics.
BASIC
PROTOCOL 4
BACTERIAL TRANSLOCATION THROUGH HUMAN INTESTINAL
ENTEROID MONOLAYERS
E. coli can cause infections distal to the intestine and is the most frequently isolated
bacterium in Gram-negative-associated bacteremia and sepsis. The mechanism by which
certain E. coli strains breach the intestinal epithelial barrier and disseminate to extraintestinal sites is poorly understood. The translocation assay using HIEM was modified from Kortman, Boleij, Swinkels, & Tjalsma (2012), who evaluated how Salmonella
typhimurium and other enteric pathogens passage across polarized Caco-2 cell monolayers. Briefly, check the polarization of differentiated HIEM seeded on transwell inserts
in a 24-well plate by measuring the trans-epithelial electrical resistance (TEER). Dilute,
wash, and resuspend cultures of E. coli with or without antibiotic resistance cassettes
in enteroid differentiation medium. Apically infect HIEM with an MOI of 1 to 100,
and determine the number of translocated bacteria after 1, 2, 4, and 6 hr by removing
medium from the bottom chamber. Enumerate bacteria by plating serial dilutions on LB
agar plates with or without antibiotic supplementation. Monitor the effect of bacteria on
monolayer integrity by measuring TEER at 0, 3, and 6 hr.
Materials
Day 5 differentiated and polarized HIEM seeded on transwell inserts in a 24-well
plate (see Basic Protocol 1)
Differentiation medium (see recipe)
0.25% trypsin-EDTA (e.g., Fisher Scientific, cat. no. 25-053-CI)
HBSS, calcium- and magnesium-free (e.g., Fisher Scientific, cat. no. 20-021-CV)
LB agar plates, with our without appropriate antibiotics
R
Poole et al.
Millicell ERS-2 Voltohmmeter (e.g., Millipore Sigma, cat. no. MERS00002)
8 of 12
Current Protocols in Microbiology
Razor blade, sterile
1.5-ml microcentrifuge tubes (e.g., Fischer Scientific, cat. no. 05-408-129)
37°C, 5% CO2 , humidified incubator
Hemocytometer
Additional reagents and equipment for bacterial culture preparation (see Basic
Protocol 2)
1. Repeat steps 1 through 3 of Basic Protocol 2 Stage 1 (bacterial culture preparation).
2. On day 5 of differentiation of HIEM on a transwell insert in a 24-well plate (Basic
Protocol 1 Stage 3), replace differentiation medium with fresh medium in the upper
and lower compartment. Increase the volume to 200 µl in the upper compartment to
R
measure TEER. Use the Millicell ERS-2 Voltohmmeter, which measures membrane
potential and resistance of epithelial cells in culture, to determine TEER values of
HIEM seeded on transwell inserts following the manufacturer’s instructions.
Polarized monolayers have a TEER 800 Ω. If the TEER of monolayers does not reach
this value, do not use the monolayers to measure translocation in HIEM.
3. To determine the number of cells for MOI calculation, remove 1 HIEM by using a razor
blade to cut the filter off the transwell insert. Place cut filter in a 1.5-ml microcentrifuge
tube containing 200 µl trypsin, and incubate at 37°C in the presence of 5% CO2
in a humidified incubator for 5 min. Dislodge cells from filter by pipetting. Stop
trypsinization by adding 800 µl HBSS, and mix by pipetting. Use a hemocytometer
to count the cells and calculate the desired MOI as in Basic Protocol 2.
For this system an MOI ranging from 1 to 100 has been used.
4. Add each E. coli strain at the calculated MOI to the upper compartment of the assigned
transwell insert, and incubate for 1, 2, 4, and 6 hr at 37°C in the presence of 5% CO2
in a humidified incubator.
5. To determine the number of translocated bacteria, remove medium from the lower
compartment at each time point, and enumerate by plating undiluted samples and
serial dilutions (10-1 to 10-4 ) on LB agar plates with or without antibiotic supplementation. During the infection, monitor the effect of bacteria on monolayer integrity by
taking TEER measurements at 0, 3, and 6 hr.
REAGENTS AND SOLUTIONS
CMGF– (complete medium without growth factor)
500 ml Advanced Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (e.g.,
Thermo Fisher Scientific, cat. no. 12634028)
5 ml 100× GlutaMAX (e.g., Thermo Fisher Scientific, cat. no. 35050-061)
5 ml 1 M HEPES (e.g., Thermo Fisher Scientific, cat. no. 35630-080)
5 ml 100× penicillin-streptomycin (e.g., Thermo Fisher Scientific, cat. no.
15140-122)
Store at 4°C for up to 4 weeks
CMGF+ (complete medium with growth factor)
1.5 ml CMGF–
5 ml Wnt3A conditioned medium (ATCC #CRL-2647)
200 µl 50× B27 supplement (e.g., Thermo Fisher Scientific, cat. no. 17504-044)
100 µl 100× N2 supplement (e.g., Thermo Fisher Scientific, cat. no. 17502-048)
20 µl 500 mM n-acetylcysteine (1 mM final; e.g., Sigma-Aldrich, cat. no. A9165)
2 ml Rspo-1 conditioned medium (R-Spondin HEK293T cells)
Poole et al.
9 of 12
Current Protocols in Microbiology
1 ml noggin conditioned medium (noggin expressed in HEK293 cells)
10 µl 50 µg/ml mouse epidermal growth factor (EGF) in PBS (50 ng/ml final; e.g.,
Thermo Fisher Scientific, cat. no. PMG8043)
10 µl 10 µM [Leu15 ]-gastrin I in PBS (10 nM final; e.g., Sigma-Aldrich, cat. no.
G9145)
100 µl 1 M nicotinamide (10 mM final; e.g., Sigma-Aldrich, cat. no. N0636)
10 µl 500 µM A 83-01 in DMSO (500 nM final; e.g., Tocris, cat. no. 2939)
10 µl 10 mM SB202190 in DMSO (10 µM final; e.g., Sigma-Aldrich, cat. no.
S7067)
Store at 4°C for up to 2 weeks
Differentiation medium
Prepare CMGF+ without Wnt3A, nicotinamide, or SB202190, and reduce the RSpondin and noggin conditioned medium to half of the concentration. Store at 4°C
for up to 2 weeks.
COMMENTARY
Background Information
Caco-2 cells and other transformed cancer
cell line models have been used for several
years to determine and evaluate the virulence
factors and steps in pathogenesis of different enteric pathogens such as E. coli, Listeria,
Salmonella, and Shigella. Even though these
models have been instrumental in identifying
key components of the pathogenic process, the
data collected for these experiments do not always translate to what happens in vivo. Therefore, the ex vivo model described in this unit
can serve as a human surrogate and a bridge to
fill the gap between in vitro and in vivo models currently used to evaluate gastrointestinal
infections.
Critical Parameters and
Troubleshooting
Poole et al.
Growth of 3D enteroids
Enteroid cultures are typically grown in
very low or no antibiotic medium, and this
makes them prone to contamination. To prevent contamination, perform all steps in a sterile environment. If absolutely required, amphotericin A and gentamicin can be added to
prevent contamination. However, this can reduce the growth of enteroids and is usually not
recommended. During passage of enteroids,
if you observe more dead cells (which usually appear as dark patches of 3D enteroids),
incorporating an additional wash may help in
removal of dead cells, and survival of enteroids
can be increased by adding more Roc inhibitor.
A dedicated enteroid culture room equipped
with biosafety hoods, separate incubators, and
entry only allowed for those culturing the enteroids significantly cuts down on the level of
culture contamination.
Ratio of 3D enteroids to monolayers
If the density of 3D enteroids is about 100
per well, then use the following recommended
ratio for making monolayers: (1) use 1 3D well
to make 3 wells of monolayers on plastic 96well plate; (2) use 2.5 3D wells to make 1 transwell insert; (3) use 1 3D well to make 2 wells
of monolayers on plastic chambered slides;
and (4) use 1 3D well to make 1 monolayer on
cell culture–grade modified glass chambered
slides or coverslips.
Choice of collagen or Matrigel
For plastic surfaces both collagen and Matrigel provide optimal conditions for seeding
and growth of enteroids. For glass surfaces,
Matrigel works better and helps in formation
of a uniform monolayer. While working with
Matrigel, using previously chilled pipet tips
allows for easy aliquoting of Matrigel without
the formation of air bubbles.
Trypsinization of enteroids
For trypsinizing 3D enteroids, collect 8 to
12 wells into a single 15-ml conical tube, and
incubate with 1 ml trypsin. If there are more
3D wells, use a different tube. Do not combine more than 12 wells of 3D enteroids in
a single 15-ml conical tube, as it can result
in poor or improper digestion. The timing of
trypsinization varies for every segment of intestine used. Use the following recommended
timing as a guideline for trypsin digestion:
(1) for jejunum and colon incubate at 37°C
for 4 min; (2) for duodenum incubate at
37°C for 4.5 min; and (3) for ileum incubate
at 37°C for 5 min. Longer incubation duration
can result in death of enteroid cells.
10 of 12
Current Protocols in Microbiology
Preparation of monolayers
Failure to form a uniform monolayer could
be due to many reasons. Careful consideration of parameters like centrifugation speed,
air bubble formation, and trypsin digestion
timing can help in troubleshooting. Do not increase the centrifugation speed, and strictly
follow the suggested timing and temperature.
The enteroid cells are very sensitive to centrifugal forces, and higher speed and inappropriate temperature can reduce viability and
increase the number of dead cells. If under
visual examination using a microscope one
observes dead cells in the 3D format, which
are marked by presence of dark and denser enteroids (whereas undifferentiated 3D enteroids
appear clear and globular), addition of 10 µM
Y-27632 to growth medium may help in rescuing the culture. Avoid formation of air bubbles
by pipetting inside the liquid volume available.
Air bubbles can result in loss of total available
cells. If the number of cells is lower while
seeding, a uniform monolayer is less likely to
be formed.
Differentiation of enteroids
Differentiated enteroid monolayers provide
a great platform to study host–pathogen interactions in a physiologically relevant model
system. Typically, after plating the enteroid
monolayers, they can settle and grow to 80%
to 90% confluency for 1 or 2 days if required.
The day the differentiation medium is changed
or introduced is considered day 0, and day 4
or 5 is considered as an ideal day for infection. However, the cells are ready for infection
between days 3 and 5 of the differentiation
process.
Time Considerations
Basic Protocol 1: Plating of enteroid monolayers will be completed over a period of 2
weeks, depending upon the growth of the cells.
For a researcher or technician who has previous cell culturing experience and is handling
enteroid cultures for the first time, it is recommended to begin with 4 wells of 3D enteroids
until comfortable with all the procedures. After thawing the frozen enteroid vials, seeding
and growing 3D enteroids (Stage 1) will take
about 15 min to seed and feed the enteroids.
For passaging of enteroids (Stage 2), it can take
about 30 to 60 min depending on the number
of wells handled. After coating the wells with
Matrigel (20 min) or collagen (2 hr), preparation of enteroid monolayers (Stage 3) can take
about 30 to 50 min to plate enteroid mono-
layers depending on the number of 3D wells
used.
Basic Protocol 2: Bacterial adherence as
determined by the plating method will be completed over 48 to 72 hr. The time for this
protocol does not include the number of days
for HIEM preparation and differentiation on
96-well tissue culture dishes (see time considerations for Basic Protocol 1). This protocol
was adapted to E. coli but can be optimized for
other bacteria. Step 1 (growth of E. coli strains
overnight) will take 16 to 18 hr. If the experiment requires bacteria in log phase, growth of
sub-cultures the day of the experiment takes
2 to 3 hr. Steps 2 through 4, including washing and resuspending the bacteria, counting
the numbers of cells in the HIEM, and changing the HIEM medium, will take 1 to 2 hr
depending on the number of samples. Step
5 (infecting the HIEM) ranges from 1 to 24
hr depending on MOI used. Step 6 (washing,
dissociating or lysing the HIEM, and plating
serial dilutions) will take 30 to 60 min depending on the number of samples. The plates will
be incubated overnight and counted the next
day.
Bacterial adherence: The bacterial adherence assay has the same time considerations as
Basic Protocol 2; however, histological staining methods may involve an additional 1 to 2 hr
depending on the reagents used. Immunofluorescence may take an additional 24 to 48 hr
depending on the antibody used, and electron
microscopy may involve an additional 72 hr
based on the drying method chosen.
Basic Protocol 3: The measurement of
bacterial invasion has the same time considerations as adherence in Basic Protocol 2; however, invasion requires an additional 2-hr incubation with gentamicin to kill extracellular
bacteria after infection.
Basic Protocol 4: Bacterial translocation
will be completed over 48 hr. The time for
this protocol does not include the number
of days for HIEM preparation and differentiation on transwell inserts (see time considerations for Basic Protocol 1). The growth
of overnight and sub-cultures, determination
of the CFU number, preparation of bacteria,
removing and counting the numbers of cells in
the HIEM on the transwell insert, and changing the HIEM medium will take 1 to 2 hr depending on the number of samples. Measuring
the TEER value for each HIEM will take 15
to 60 min depending on the number of samples and transwells. Apical HIEM infection
and plating serial dilutions of medium from the
bottom chamber of each sample to determine
Poole et al.
11 of 12
Current Protocols in Microbiology
the number of translocated bacteria will be 7
hr. The plates will be incubated overnight and
counted the next day.
Acknowledgements
This work was supported by the U.S.
National Institutes of Health grants U19
AI11497, T32 AI055413-14, and K12
GM084897.
Literature Cited
Current Protocols (2005). Commonly used
reagents and equipment. Current Protocols
in Microbiology, 00, A.2A.1–A.2A.15. doi:
10.1002/9780471729259.mca02as00.
Dutta, D., & Clevers, H. (2017). Organoid culture systems to study host-pathogen interactions.
Current Opinion in Immunology, 48, 15–22. doi:
10.1016/j.coi.2017.07.012.
Hodges, K., & Gill, R. (2010). Infectious diarrhea:
Cellular and molecular mechanisms. Gut Microbes, 1, 4–21. doi: 10.4161/gmic.1.1.11036.
In, J. G., Foulke-Abel, J., Estes, M. K., Zachos,
N. C., Kovbasnjuk, O., & Donowitz, M. (2016).
Human mini-guts: New insights into intestinal
physiology and host-pathogen interactions. Nature Reviews Gastroenterology & Hepatology,
13, 633–642. doi: 10.1038/nrgastro.2016.142.
Kortman, G. A., Boleij, A., Swinkels, D. W.,
& Tjalsma, H. (2012). Iron availability increases the pathogenic potential of Salmonella
typhimurium and other enteric pathogens at
the intestinal epithelial interface. PLoS One, 7,
e29968. doi: 10.1371/journal.pone.0029968.
Lee, J., Choi, J. H., & Kim, H. J. (2016).
Human gut-on-a-chip technology: Will this
revolutionize our understanding of IBD and
future treatments? Expert Review of Gastroenterology & Hepatology, 10, 883–885. doi:
10.1080/17474124.2016.1200466.
Pizarro-Cerdá, J., Lecuit, M., & Cossart, P.
(2002). 8 Measuring and analysing invasion
of mammalian cells by bacterial pathogens:
The Listeria monocytogenes system. Methods in Microbiology, 31, 161–177. doi:
10.1016/S0580-9517(02)31009-2.
Poole, N. M., Green, S. I., Rajan, A., Vela,
L. E., Zeng, X. L., Estes, M. K., &
Maresso, A. W. (2017). Role for FimH
in extraintestinal pathogenic Escherichia coli
invasion and translocation through the intestinal epithelium. Infection and Immunity, 85, e00581–17. doi: 10.1128/IAI.0058117.
Sato, T., & Clevers, H. (2013). Growing selforganizing mini-guts from a single intestinal
stem cell: Mechanism and applications. Science,
340, 1190–1194. doi: 10.1126/science.1234852.
Sato, T., Stange, D. E., Ferrante, M., Vries, R.
G., Van Es, J. H., Van den Brink, S., . . .
Clevers, H. (2011). Long-term expansion of
epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology, 141, 1762–1772. doi:
10.1053/j.gastro.2011.07.050.
Sato, T., van Es, J. H., Snippert, H. J., Stange, D.
E., Vries, R. G., van den Born, M., . . . Clevers,
H. (2011). Paneth cells constitute the niche for
Lgr5 stem cells in intestinal crypts. Nature, 469,
415–418. doi: 10.1038/nature09637.
Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., . . . Clevers,
H. (2009). Single Lgr5 stem cells build cryptvillus structures in vitro without a mesenchymal niche. Nature, 459, 262–265. doi: 10.1038/
nature07935.
Son, M. S., & Taylor, R. K. (2012). Growth
and maintenance of Escherichia coli laboratory strains. Current Protocols in Microbiology, 27, 5A.4.1–5A.4.9. doi: 10.1002/
9780471729259.mc05a04s27.
Mahe, M. M., Sundaram, N., Watson, C. L.,
Shroyer, N. F., & Helmrath, M. A. (2015). Establishment of human epithelial enteroids and
colonoids from whole tissue and biopsy. Journal of Visualized Experiments, 97, e52483. doi:
10.3791/52483.
Steiner, T. S., Lima, A. A., Nataro, J. P., & Guerrant,
R. L. (1998). Enteroaggregative Escherichia coli
produce intestinal inflammation and growth impairment and cause interleukin-8 release from
intestinal epithelial cells. Journal of Infectious
Diseases, 177, 88–96. doi: 10.1086/513809.
McCracken, K. W., Howell, J. C., Wells, J. M.,
& Spence, J. R. (2011). Generating human intestinal tissue from pluripotent stem cells in
vitro. Nature Protocols, 6, 1920–1928. doi:
10.1038/nprot.2011.410.
Wardlaw, T., Salama, P., Brocklehurst, C., Chopra,
M., & Mason, E. (2010). Diarrhoea: Why
children are still dying and what can be
done. Lancet, 375, 870–872. doi: 10.1016/
S0140-6736(09)61798-0.
Noel, G., Baetz, N. W., Staab, J. F., Donowitz,
M., Kovbasnjuk, O., Pasetti, M. F., & Zachos,
N. C. (2017). A primary human macrophageenteroid co-culture model to investigate
mucosal gut physiology and host-pathogen interactions. Scientific Reports, 7, 45270. doi:
10.1038/srep45270.
Zachos, N. C., Kovbasnjuk, O., Foulke-Abel, J., In,
J., Blutt, S. E., de Jonge, H. R., . . . Donowitz,
M. (2016). Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology.
Journal of Biological Chemistry, 291, 3759–
3766. doi: 10.1074/jbc.R114.635995.
Poole et al.
12 of 12
Current Protocols in Microbiology