Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

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 the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.

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, Prior to Collection Day Autoclave strainers/forceps for collection, and scissors/forceps for micro-dissection. Prepare an adequate volume (500 ml per specimen) of Wash buffer (refer to Table 1 for the recipe). …

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 cu…

Divulgaciones

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
Micro dissecting forceps Stoelting 52102-43 4 inches, 1 x 2 x 0.5 mm3 , Slight Curve
Micro dissecting forceps Stoelting 52102-06 4 inches, Straight Fine, Sharp
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
6-well tissue culture plate BD Falcon 353224 Polystyrene, Tissue culture treated
6-well transwells Millipore PICM03050 Insert – 30 mm diameter, 0.4 μm 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 ~ 300 μL aliquots in 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.

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Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface. J. Vis. Exp. (Pending Publication), e21715, doi: (2023).

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