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

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…

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.