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Ex vivo perfusion of mid-to-late-gestation mouse placenta for maternal-fetal interaction studies during pregnancy

Abstract

Ex vivo perfusion systems offer a reliable, reproducible method for studying acute physiological responses of an organ to various environmental manipulations. Unlike in vitro culture systems, the cellular organization, compartmentalization and three-dimensional structure of ex vivo–perfused organs are maintained. These particular parameters are crucial for the normal physiological function of the placenta, which supports fetal growth through transplacental exchange, nutritional synthesis and metabolism, growth factor promotion and regulation of both maternally and fetally derived molecules. The perfusion system described here, which can be completed in 4–5 h, allows for integrated, physiological studies of de novo synthesis and metabolism and transport of materials across the live mouse placenta, not only throughout a normal gestation period but also following a variety of individual or combined genetic and environmental perturbations compromising placental function.

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Figure 1: Overview of the ex vivo perfusion system.
Figure 2: Effect of perfusion rate on placental morphology.
Figure 3: Extraction and isolation of an E18 mouse placenta.
Figure 4: Preparation and perfusion of mouse placental tissue.

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Acknowledgements

This work was supported by the National Institute of Child Health and Human Development (NICHD) (grant 5R21HD065287 to A.B.) and a NARSAD (National Alliance for Research on Schizophrenia and Depression; now the Brain and Behavior Research Foundation) Young Investigator award (to A.B.). We thank P. Levitt for his support during the initial development of this protocol. We acknowledge J. Burford and J. Peti-Peterdi for their valuable contributions in two-photon live imaging.

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Authors and Affiliations

Authors

Contributions

N.G. conducted the experiments. N.G. and A.B. conceived the protocol, interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Alexandre Bonnin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Real-time imaging of flow through the placental vasculature. E18 mouse placenta perfused through the umbilical artery with 70,000 mw Texas Rhodamine Red labeled dextran at 5 μl min-1. The perfusion was imaged in real-time with a Leica SP5 MP 2-photon microscope, demonstrating flow within the placental vasculature during perfusion. All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. (MOV 2316 kb)

Supplementary Video 2

Demonstration of embolism caused by insufficient degassing of perfusate solutions. E18 mouse placenta perfused through the umbilical artery at 5 μl min-1 with PBS + 0.01% FCF that has not been subjected to degassing or pre-warming. All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. (MOV 7559 kb)

Supplementary Video 3

Splitting of the umbilical cord. A demonstration of the technique used when separating the umbilical artery and vein of a mouse placenta of any age. All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. (MOV 11752 kb)

Supplementary Video 4

Cannulation of the uterine artery. A demonstration of the technique used to cannulate the uterine artery of an E14 mouse placenta. All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. (MOV 9708 kb)

Supplementary Video 5

Cannulation of the umbilical artery. A demonstration of the technique used to cannulate the umbilical artery of an E14 mouse placenta. All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. (MOV 7179 kb)

Supplementary Figure 1

Visualization of maternal and fetal blood spaces in ex vivo perfused placenta. E18 mouse placenta perfused through the uterine artery with 500,000 mw FITC labeled dextran (diluted 1:100 with PBS) at 18 μl min-1, and through the umbilical artery with 70,000 mw Texas Rhodamine Red labeled dextran (diluted 1:50 with PBS) at 5 μl min-1. The perfusion was imaged in real-time with a Leica SP5 MP 2-photon microscope, demonstrating visualization of fetal villi (FV - red) surrounding and intermingling with maternal blood space (MBS - green). Scale bar = 150 μm. All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. (PDF 506 kb)

Supplementary Figure 2

Application of surgical thread to prevent leaking from the umbilical artery. The surgical thread is isolated and wrapped around the artery (a, b). The suture is wrapped over itself (c), and then tucked back inside of the loop (d) to create a simple knot. The knot is tightened (e) downstream of the vascular leak, restoring flow to the organ (f). All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. (PDF 419 kb)

Supplementary Figure 3

Placental tissue viability during perfusion. (a) Measurements of fetal volume loss of samples collected from the umbilical vein of E16 mouse placentas (n=3) at 10 min intervals, using an input of 6 μl min-1 as the reference flow rate. A complete volume loss of 6 μl min-1 indicates fully collapsed vasculature. (b) Quantification of LDH activity in the fetal eluate of PBS perfused E16 mouse placentas (n=3), indicating that low and stable LDH activity is present throughout the typical 90 min perfusion. (c-k) Activated caspase-3 staining in the decidua of E14 mouse placentas stained with activated Caspase-3 (green) and DAPI (blue). Fresh, unperfused tissue (c-e) shows little or no activated caspase-3 staining, indicating a lack of cell death. A placenta perfused for 120 mins with fresh PBS (f-h) similarly shows little or no activated caspase-3 positive cells, where as an unperfused placenta (i-k) shows significant activated caspase-3 staining (), indicating the onset of cellular apoptosis. Scale bar = 50 μm. (l) Quantification of co-localized caspase-3 and DAPI positive cells in a 0.6 mm2 area of interest for PBS perfused and unperfused E14 mouse placentas (n=3 each) at several time points. All animal experiments have been conducted in accordance with the National Institutes of Health Animal Use Guidelines and approved by the Institutional Animal Care and Use Committee at The University of Southern California. Error bars indicate standard deviation. (PDF 472 kb)

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Goeden, N., Bonnin, A. Ex vivo perfusion of mid-to-late-gestation mouse placenta for maternal-fetal interaction studies during pregnancy. Nat Protoc 8, 66–74 (2013). https://doi.org/10.1038/nprot.2012.144

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