A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Overview

This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preparation of Organ Culture Plates

NOTE: The following steps should be performed using sterile technique in a tissue culture hood. It is ideal for the dissecting microscope to be located in a sterile field. However, this is not absolutely necessary as long as micro-dissection is performed expeditiously, and instruments are dipped frequently in 70% ethanol.

1. Thaw extracellular matrix (ECM) vial(s) on ice. Dilute the ECM 1:1 with ice-cold Collection media, and mix by pipetting. Pipette slowly to avoid introducing bubbles. Each well of a 6-well tissue culture plate requires 100 µl of this suspension and will accommodate 3-4 organ cultures.
2. Place transwell inserts (pore size 0.4 µM) into each well of the 6-well tissue culture plate.
3. Coat each transwell insert with a thin layer (100 µl) of the ECM/media suspension.
4. Place the plate on ice and immediately proceed to the establishment of organ cultures. Alternatively, prepare the plate prior to tissue collection, in which case wrap it in para-film and store it at 4 °C for later use. Storage longer than 6 hr is not recommended as the ECM will dry out.

2. Establishment of Organ Cultures

NOTE: This step describes how to place decidual organ cultures and villous organ cultures in separate transwells. The tissues are not in contact. Researchers interested in a co-culture technique where decidua and villi are in direct contact with each other can refer to prior literature.

1. Rinse specimens twice in Collection media, and centrifuge at 1,000 x g between each rinse. Transfer tissue to a sterile petri dish and place it on the stage of a dissecting microscope. Keep a 6-well plate on ice adjacent to the microscope.
2. Micro-dissect tissue with sterile springer dissecting scissors and forceps, to establish 2-3 mm villous organ cultures (Figure 1A).
   NOTE: Villous organ cultures are small, well-vascularized "trees" with 2 or 3 branches, and with prominent extravillous trophoblast (EVT) cell columns. It is important to utilize only villous tissue from <9 weeks first trimester specimens, as invasion of extravillous trophoblasts into ECM is most pronounced at these young ages.
3. Select well-vascularized villi with prominent EVT cell columns. EVT cell columns are visualized as bulbous, "fuzzy", "fluffy" ends (refer to arrowheads in Figure 1A).
4. Use sterile forceps to transfer 3 or 4 villous trees into each transwell. Adjust branches so the tree is positioned flat atop ECM and separate clumped branches.
5. Micro-dissect decidual tissue with sterile springer dissecting scissors and forceps, to establish 3 mm³ decidual organ cultures (Figure 1A).
   NOTE: Specimens contain two distinct types of decidual tissues, decidua parietalis and decidua capsularis (left and right tissue fragments, respectively, Figure 1A).
6. Trim with scissors to create 3 mm³ pieces of decidua parietalis. Decidua capsularis is not used for these cultures. Use forceps to tease away any clotted maternal blood.
7. Use sterile forceps to transfer 3-4 pieces of decidua into each transwell.
8. Add 1 ml of Collection media to the bottom of the well. Incubate plate overnight at 37 °C in 5% CO₂.
9. For experiments where it is important to mimic normal first-trimester placental oxygen tension, maintain villous organ cultures in relative hypoxia (3% O₂).
   NOTE: Laboratory options include small hypoxia incubator chambers that fit inside existing incubators or a tri-gas incubator that introduces external N₂ gas to lower oxygen concentration.
10. Add 1 ml of Villous or Decidual medium (Table 1) to the top of the transwell after 12-16 hr of culture.
    NOTE: The absence of media during the first overnight in culture allows the extravillous trophoblast cell columns to invade (villous culture), and for the tissue (both villous and decidual) to become securely embedded in ECM. In our experience, decidual organ cultures remain viable for 72 hr and villous organ cultures for 96 hr. We recommend experimental endpoints that fall within this timeframe.  See Table 1 for the composition of Villous and Decidual medium.  The villous medium has a high percentage of FBS, while the Decidual medium is supplemented with pregnancy hormones (progesterone, 17β-estradiol) that maintain the decidualized state (Table 1).

3. Preparation of L. monocytogenes Cultures

NOTE: For a detailed protocol on the long-term storage of L. monocytogenes, and the culture and growth of this organism in brain heart infusion (BHI) broth and agar, please refer to Wang et al.

1. Pick a single L. monocytogenes colony from a streaked BHI agar plate and inoculate 3 ml of BHI broth.
2. Incubate the L. monocytogenes culture overnight (16 hr), in a slanting position, at 30 °C.
   NOTE: These culture conditions allow bacterial growth to reach the stationary phase. L. monocytogenes is flagellated at 30 °C, which increases host cell invasion.

4. Infection of Organ Cultures with L. monocytogenes

1. Warm phosphate-buffered saline (PBS) and Villous or Decidual medium (Table 1) to 37 °C.
2. Use sterile tweezers to remove any organ culture pieces that did not invade the ECM, and are therefore floating in the well.
3. Carefully aspirate media from the bottom of the transwell. Rinse upper and lower transwells twice by gently pipetting 1 ml of warm PBS into the well, and carefully aspirating in between. Gently pipette 1 ml of antibiotic-free Villous or Decidual media to the upper and lower transwell. Be careful not to disturb the organ culture.
4. Let the tissue incubate in antibiotic-free media for at least an hour to ensure that antibiotics have been removed.
   NOTE: At the stationary phase, the density of the L. monocytogenes culture is approximately 1 x 10⁹ colony-forming units (CFU) per ml. The number of bacteria used to infect organ cultures should be empirically determined to meet experimental needs.
5. For wild-type L. monocytogenes routine infection experiments show robust invasion with a 1:50 dilution of the overnight (4 x 10⁷  L. monocytogenes in 1 ml of media per well). Resuspend the appropriate number of bacteria in an antibiotic-free Villous or Decidual medium.
6. Aspirate media from the top of transwells. Gently add 1 ml of inoculum. Incubate plates at 37 °C in 5% CO₂ to allow for bacterial invasion.
7. Remove extracellular bacteria by aspirating media from upper and lower transwells. Rinse wells twice with warm PBS. Add 1 ml of Villous or Decidual media supplemented with 50 µg ml^-1 Gentamicin. Incubate plates at 37 °C in 5% CO₂.
   NOTE: L. monocytogenes is a facultative intracellular bacterium that can grow in an extracellular medium, grow inside of cells, and spread from cell to cell without accessing the extracellular space. Gentamicin has a bactericidal effect on extracellular L. monocytogenes, and thus allows measurement of intracellular L. monocytogenes growth and spread through the organ culture.
   NOTE: Reproducible L. monocytogenes infection of villous and decidua organ cultures occurs with 5 hr incubation times. The time of incubation before the addition of gentamicin can be modified based on experimental needs and the ability of the organism to invade the tissue. To better understand the anatomic sites most vulnerable to infection, shorter incubation times can be used.
8. Maintain plates at 37 °C in 5% CO₂ for the duration of the experiment. Change the whole media in each well daily.
   NOTE: In our experience, L. monocytogenes-infected decidual organ cultures remain viable for 48 hr and villous organ cultures for 72 hr.

Table 1: Media recipes. Components and concentrations for preparation of Wash buffer, Collection medium, Villous medium, and Decidual medium

Wash buffer	Collection medium	Villous medium	Decidual medium
PBS	DMEM/F-12 with GlutaMAX	DMEM/F-12 with GlutaMAX	DMEM/F-12 with GlutaMAX
Penicillin 100 IU/ml	Fetal Bovine Serum 2.5%	Fetal Bovine Serum 20%	Fetal Bovine Serum 2.5%
Streptomycin 100 μg/ml	Penicillin 100 IU/ml	Penicillin 100 IU/ml	17β-estradiol 300 pg/ml
Gentamicin 50 μg/ml	Streptomycin 100 μg/ml	Streptomycin 100 μg/ml	Progesterone 20 ng/ml
Amphotericin B 1.25 μg/ml	Gentamicin 50 μg/ml
	Amphotericin B 1.25 μg/ml

Representative Results

Figure 1: Villous organ cultures – Representative gross and microscopic images.

(A) Two terminal villous trees with a gestational age of 6 weeks, as viewed under a dissecting microscope. Note the "fluffy" ends (arrowheads) and prominent fetal vasculature coursing through the branches of the tree on the left that make this piece suitable for organ culture. (B) Immunofluorescence microscopy of organ culture (gestational age 8.3 weeks) 72 hr post-infection with Listeria monocytogenes, highlighting heavy bacterial burden [green] in extravillous trophoblasts. DAPI is shown in blue, and βHCG+ syncytiotrophoblasts in red. Scale bars = 1 mm (A), 250 µm (B).

Materials

Name	Company	Catalog Number	Comments
Sterilization pouches	Fisher Scientific	01-812-54	For autoclaving individual dissecting tools
Fine mesh strainer	Cuisinart (Amazon.com)	NA	Wrap completely in aluminum foil and autoclave prior to tissue collection.
Carboy with spigot	Fisher Scientific	03-007-647	For large volume preparation of Wash buffer.
Ice packs	Nortech labs	GB8818	These do not have to be purchased, rather they can be recycled/reused from any routine laboratory shipment that includes them in the packaging.
70% Ethanol	VWR	V1001	70% solution made by adding dH20 to 190 or 200 proof research grade alcohol.
10% Bleach	Waxie Sanitary Supply	CLO 30966	10% solution made by adding dH20.
Light Box	Litebox Lumina (dickblick.com)	55305-2009	Note replacement bulbs also purchased on Blick (55305.0100)
Micro dissecting forceps	Stoelting	52102-43	4in, 1x2 0.5mm, Slight Curve
Micro dissecting forceps	Stoelting	52102-06	4in, Straight Fine, Sharp
Micro dissecting vannas spring scissors	Stoelting	52130-01P	McPherson-Vannas Spring Scissors, 8.5cm, 0.33 Tip, Slight Curve
Dissecting microscope	Leica Microsystems	MZ16 or M60	We have had success with the listed models. External gooseneck flexible light sources are helpful but not necessary.
50 mL conical tubes	Sigma-Aldrich (Corning)	CLS4558
Phosphate Buffered Saline	Gibco (ThermoFisher Scientific)	10010023	We purchase from our university Tissue Culture Core facility, alternate options such as this are available.
10X Phosphate Buffered Saline	Teknova	P0195	For preparation of Wash buffer we use 10X PBS
DMEM/F-12 nutrient mixture (Ham's) with GlutaMAX	Gibco (Life Technologies)	10565-018	We purchase this specific media formulation, containing 2.438 g/L sodium bicarbonate, 55 mg/L sodium pyruvate, and 4.5 g/L glucose
Gentamicin	Thermofisher Scientific	15710072	1000X stock. Recommend to prepare and store aliquots at -20 °C to avoid freeze/thaw.
Penicillin/Streptomycin	Gibco (ThermoFisher Scientific)	15140-122	100X stock. Recommend to prepare and store aliquots at -20 °C to avoid freeze/thaw.
Amphotericin B	Gibco (ThermoFisher Scientific)	15290-018	500X stock. Recommend to prepare and store aliquots at -20 °C to avoid freeze/thaw.
progesterone	Sigma-Aldrich	P8783	A 1 mM stock solution is made by reconstituting 15.7 mg progesterone powder in 15.7 mL Ethanol and adding 34.3 mL PBS. Solution is sterile filtered, aliquoted, and stored at -20 °C.
17β-estradiol	Sigma-Aldrich	E2758	A 10 μM stock solution is made by reconstituting 13.5 mg estradiol powder in 10 mL ethanol and adding 40 mL PBS. Solution is sterile filtered, aliquoted, and stored at -20 °C.
Bottle-top vaccum filter (0.22 μm)	Sigma-Aldrich (Corning)	CLS430769	For sterile filtration of Collection media after preparation
6-well tissue culture plate	BD Falcon	353224	Polystyrene, Tissue culture treated
6-well transwells	Millipore	PICM03050	Insert - 30mm diameter, 0.4μm pore size hydrophilic PTFE membrane
Extracellular Matrix (for example, Matrigel Matrix)	BD Biosciences	354234	We have utilized Matrigel Matrix in our studies. It is a solid at room temperature and at -20 °C. Avoid repeat freeze/thawing. Thaw bottle to viscous solution at 4 °C, and prepare ~300μL aliquotsin the cold room with chilled pipette tips. Store aliquots at -20 °C.
Paraformaldehyde, 16% w/v aqueous solution	Alfa Aesar	30525-89-4	For tissue fixation, a fresh preparation of 4% paraformaldehyde is made by diluting this stock in PBS.
Tissue culture incubator, maintained at 37 °C, 5% CO2, 3% oxygen (optional for villous organ cultures)			For some experiments, hypoxia may be preferred. This can be established multiple ways, including addition of exogenous nitrogen via gas cylinder, Tygon tubing, and a regulator.
Bench top centrifuge