Abstract
We describe a small-animal blood exchange approach developed for aging research as an alternative to heterochronic parabiosis or plasma injections. In parabiosis, animals are surgically coupled, which has several disadvantages, including difficulty controlling experimental procedure, the effects of shared organs, environmental enrichment from jointly exploring the housing enclosure, involuntary exercise and an imprecise onset of blood sharing. Likewise, in plasma injections, the added volumes need to be small, and there is little flexibility in changing the relative contributions of ectopic to endogenous blood components. These factors complicate the conclusions and interpretations, including the identification of key mechanisms and molecular or cellular determinants. Our approach, where blood is exchanged between animals without them being surgically coupled, is less invasive than parabiosis. The percentage of exchanged blood or other exchanged fluids is known and precise. The age of plasma and cells can be mixed and matched at all desired relative contributions to the endogenous systemic milieu, and the onset of the effects can be accurately delineated. In this protocol, we describe the preparatory and animal surgery steps required for small-animal blood exchange in mice and compare this process with parabiosis and plasma injections. We also provide the design, hardware and software for the blood exchange device and compare automated and manual exchange methods. Lastly, we report mathematical modeling of the dilution of blood factors. The fluid exchange takes ~30 min when performed by a well-trained biomedical scientist; the entire process takes ~2 h.
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Data availability
The main data discussed in this protocol were generated as part of the studies published in the supporting primary research papers20,21. Blueprints for the fabrication of the dual- and single-pump devices can be found at https://doi.org/10.6084/m9.figshare.19401263.v1. Source data for Fig. 6 can be found at https://doi.org/10.6084/m9.figshare.19401275.v1, and for Fig. 7 at https://doi.org/10.6084/m9.figshare.19401278.v1.
Code availability
The code used in this protocol can be found in Supplementary Information.
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Acknowledgements
We thank E. Watanabe and A. Benoni for help with exchanges and C3 ELISAs. This work was supported by NIH NHLB R01 139605 to I.C. and K.A., 1R01AG071787 to I.C., Open Philanthropy to I.C., PCARI to I.C., Georges Harik Fund to I.C. and NSF predoctoral fellowship to M.M.
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M.M. provided Figs. 3 and 5, participated in the experiments of Fig. 7 and cowrote the manuscript. P.A., J.D. and K.A. designed and fabricated the device, provided Figs. 1,2 and 4 and cowrote the manuscript. K.A. also integrated the study. C.L. performed the experiments for Fig. 7, provided Fig. 7, edited Fig. 4 and cowrote the manuscript. C.S. and C.K. provided the mathematical equations, and C.K. applied these to Figs. 6 and 7. M.J.C. and I.C. planned, directed and integrated the study and wrote the manuscript.
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Key references using this protocol
Rebo, J. et al. Nat. Commun. 7, 13363 (2016): https://doi.org/10.1038/ncomms13363
Mehdipour, M. et al. Aging 12, 8790–8819 (2020): https://doi.org/10.18632/aging.103418
Mehdipour, M. et al. GeroScience 43, 1–18 (2021): https://doi.org/10.1007/s11357-020-00297-8
Extended data
Extended Data Fig. 1 Overview and detailed parts and schematics of single-pump small-animal blood exchange device.
a, Overview of single-pump small-animal blood exchange device and detailed description of the parts involved in constructing the external hardware of the pump including appropriate tubing. b, CAD image for 3D-printed hardware housing of the pump with dimensions. c, Exploded view of the single pump illustrating the intersection of the incorporated components. d, Single-pump electrical wiring diagram.
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Mehdipour, M., Amiri, P., Liu, C. et al. Small-animal blood exchange is an emerging approach for systemic aging research. Nat Protoc 17, 2469–2493 (2022). https://doi.org/10.1038/s41596-022-00731-5
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DOI: https://doi.org/10.1038/s41596-022-00731-5
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