The overall goal of this procedure is it establish primary human placental and decidual organ cultures for the study of the infection of the maternal-fetal interface. This method can help answer key questions in the fields of placental biology and infectious diseases, such as where do pathogens cross the maternal-fetal interface during pregnancy? The main advantages of this simple and efficient technique are that fresh human specimens are used and the three dimensional tissue structure is maintained in the organ culture.
This method can be used to study infections in the maternal-fetal interface. It can also be applied to other areas of research, such as placental development. Visual demonstration of this method is critical as the selection of appropriate tissue for culturing is difficult, without prior knowledge of the micro-anatomy and demonstrating this method will be Dr.Gabrielle Rizzuto.
Using sterile technique in a tissue culture hood, fill extra-cellular matrix vials on ice, then dilute the warmed extra-cellular matrix at a one to one with ice cold collection medium. Mix the suspension with pipetting, taking care not to induce bubbles. Next, place trans-well inserts with 0.4 micron pores into individual wells of a six well tissue culture plate, and coat each insert with 100 micro-liters of the extra-cellular matrix suspension.
Place the plate on ice, and rinse the specimens two times in collection medium, transferring the tissue to a sterile petri dish under a dissecting microscope after the second wash. The specimens contain two distinct types of decidual tissues, decidua parietalis, and decidua capsularis. Using sterile springer dissecting scissors and forceps, micro-dissect the decidua parietalis into 3 cubic millimeter pieces, using the forcepts to tease away any clotted maternal blood, and use forceps to transfer three or four pieces of decidua into each trans-well.
Identify the well vascularized villi, with prominent extra-villous trophoblast cell columns and use forceps to transfer three or four villous trees into each trans-well. Adjust the branches so that the trees are positioned flat atop the extra-cellular matrix, and separate any clumped branches. Then add one milliliter of collection medium to the bottom of each well and incubate the cultures overnight at 37 degrees Celsius in 5%carbon dioxide.
12 to 16 hours later, add one milliliter of the appropriate culture medium to the top of each trans-well. Before beginning the infection, inoculate a single L.monocytogenes colony from a streaked brain-heart infusion agar plate in three milliliters of brain-heart infusion broth. Incubate the culture overnight in a slanted position at 30 degrees Celsius to allow the bacterial growth to reach a stationary phase.
The next morning, use sterile tweezers to remove any floating organ culture pieces that did not invade into the extra-cellular matrix and carefully aspirate the medium from the bottom of each trans-well. Next, gently pipette one milliliter of warm PBS into the upper and lower trans-wells two times, removing each wash with careful aspiration. After the second wash, gently pipette one milliliter of the appropriate medium to the upper and lower trans-wells, taking care not to disturb the organ cultures and return the cultures to the incubator for one hour.
At the end of the incubation, replace the medium in the top of the trans-wells with one milliliter of inoculum, and place the plates back in the cell culture incubator to facilitate the bacterial invasion, replacing all of the medium in each well daily. For villous organ cultures it is essential to dissect only the placental villous branches that terminate in well vascularized fluffy, or fuzzy, extra-villous trophoblast columns. In contrast, villous trees with not readily visualized vasculature and extra villous trophoblasts are suboptimal for culture.
Analysis of a villous organ culture at 72 hours after infection with listeria monocytogenes demonstrates a heavy bacterial burden in the extra-villous trophoblasts, and a relative lack of bacteria within the tree lining syncytiotrophoblast layer. For decidual organ cultures it is important to know that early gestational specimens consist largely of decidua capsularis, easily distinguished by its thin, membranous nature and decidua parietalis, which is trimmed as demonstrated and used for the cultures. H and E-staining of paraffin embedded decidual organ culture reveals epithelial lined glands and endothelial lined vasculature positioned heterogeneously within the decidual stromal cell compartment.
48 hours after infection CD-14 positive macrophages localize near the vasculature and scatter throughout the decidual stroma, while large aggregates of listeria monocytogenes cluster predominantly within the stroma. Once mastered the organ cultures can be established in less than 60 minutes per specimen if prepared properly. While attempting this procedure, it is important to remember to maintain sterility in the tissue culture hood and while working at the dissecting microscope.
Following their preparation, these organ cultures can be infected with the relevant bacterial, fungal, or viral pathogens of interest, to answer additional questions about which cell types are most vulnerable to infection. After its development, this technique paved the way for microbiologists to explore the pathogenesis of placental infections with clinically relevant pathogens such as listeria monocytogenes. After watching this video, you should have a good understanding of how to establish decidual and villous organ cultures on extra-cellular matrix coated tissue culture trans-well inserts.
