A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture
Published: October 31, 2023
Abstract
Source: Rizzuto, G. A., et al. Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface. J. Vis. Exp. (2016)
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. Howe…
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…
Declarações
The authors have nothing to disclose.
Materials
| 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, 1×2 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 |
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Citar este artigo
A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture. J. Vis. Exp. (Pending Publication), e21716, doi: (2023).