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Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo.

ABSTRACT: Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic.

SUBMITTER: Chinnadurai R

PROVIDER: S-EPMC4110775 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo.

Chinnadurai Raghavan R Garcia Marco A MA Sakurai Yumiko Y Lam Wilbur A WA Kirk Allan D AD Galipeau Jacques J Copland Ian B IB

Stem cell reports 20140606 1

Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and hum ...[more]

PMID: 25068122

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Project description:BackgroundMesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are a promising cell-free therapy for acute lung injury (ALI). To date, no studies have investigated their biodistribution in ALI or discerned the timing of administration for maximal lung targeting, which are crucial considerations for clinical translation. Our study aimed to characterize a mouse model of ALI and establish the distribution kinetics and optimal timing of MSC-EV delivery during lung injury.MethodsMSC-EVs were isolated by ultracentrifugation alone (U/C) or tangential flow filtration with ultracentrifugation (TFF-U/C) and characterized by nanoparticle tracking analysis and western blot. A lipopolysaccharide (LPS)-induced mouse model of ALI was established to study the inflammatory response over 72 h. ALI was assessed by histological lung injury score, bronchoalveolar lavage fluid cell count and inflammatory cytokines. For biodistribution studies, ALI mice were intravenously administered fluorescently labeled MSC-EVs to determine the optimal timing of administration and organ-specific biodistribution. Live in vivo and ex vivo fluorescence imaging was conducted at various timepoints post-EV injection.ResultsEVs isolated by either ultracentrifugation alone or TFF-U/C displayed comparable size distribution (~ 50-350 nm) and EV marker expression (CD63/81). TFF-U/C generated a 5.4-fold higher particle concentration and 3.9-fold higher total protein when compared to ultracentrifugation alone. From the inflammatory time-course study, cell count and IL-1β peaked in bronchoalveolar lavage fluid at 24 h after ALI induction. MSC-EVs delivered at 24 h (as opposed to 0.5 h, 5 h or 10 h) after disease induction resulted in a 2.7-4.4-fold higher lung uptake of EVs. Biodistribution studies comparing organ-specific MSC-EV uptake showed progressive lung accumulation up to 48 h post-delivery (threefold higher than the spleen/liver), with a decline at 72 h. Importantly, lung EV fluorescence at 48 h in ALI mice was significantly elevated as compared to control mice. The lung tropism of MSC-EVs was further validated as therapeutically inert EVs derived from HEK293T cells accumulated mainly to the spleen and liver with a 5.5-fold lower distribution to the lungs as compared to MSC-EVs.ConclusionMSC-EVs exhibit maximal lung accumulation when administered during heightened inflammation at 24 h after ALI induction. This lung tropism suggests that MSC-EVs may serve as a practical rescue treatment for acute inflammatory respiratory conditions.

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Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo.

Publications

Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo.

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