Molecular Design of Polyampholytes for Vitrification-Induced Preservation of Three-Dimensional Cell Constructs without Using Liquid Nitrogen

Matsumura, Kazuaki; Hatakeyama, Sho; Naka, Toshiaki; Ueda, Hiroshi; Rajan, Robin; Tanaka, Daisuke; Hyon, Suong‐Hyu

doi:10.1021/acs.biomac.0c00293

Cited by 24 publications

(34 citation statements)

References 47 publications

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Section: Discussionsupporting

confidence: 93%

“…Polyampholyte medias have been used to cryopreserve many cell lines and primary cells with high viability (8). Recent work has demonstrated that they can also be used to cryopreserve 3D cell structures (9). We therefore tested the hypothesis that a cryopreserved bank of human lung tissue allows for the in vitro study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

In vitroinfection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response

Schaller

Sharma

Dupee

et al. 2021

Preprint

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Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the in vitro study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 16 donors, most of whom had risk factors for severe illness from COVID-19. Cryopreserved tissues preserved 90% of cell viability and contained heterogeneous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectible with HCoV-OC43 and SARS-CoV-2 coronavirus strains, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL-6, CXCL8 and IFNβ in response to SARS-CoV-2 infection. Treatment of tissues with dexamethasone and the experimental drug, N-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted antiviral activity, suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the in vitro study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.ImportanceThe current biological systems for the study of COVID-19 are in vitro systems that differ from the human lung in many respects, and animal hosts to which the virus is not adapted. We developed another alternative for studying pathogenesis and drug susceptibility of SARS-CoV-2 in a cryopreserved bank of human lung tissues. We consider the importance of this work to relate to the practical use of this culture system as a repeatable and scalable approach that allows for the study of an important infection in relevant tissues.The tissue bank highlights the heterogeneous response to SARS-CoV-2 infection and treatment, which allows researchers to investigate why treatments work in some donors but not others.

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

In vitroinfection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response

Schaller

Sharma

Dupee

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…After cryopreservation, thawed microtissues displayed excellent viability, were metabolically active, and contained the major cell populations of the human lung. Our data agree with previous studies demonstrating the advantages of using polyampholyte-based media as a cryopreservative ( 8 , 9 ). These lung tissues also contain extracellular matrix components that may be lacking in other models, which can be visualized in Figures 1 and 2 .…”

Section: Discussionsupporting

confidence: 93%

“…Polyampholyte media have been used to cryopreserve many cell lines and primary cells with high viability ( 8 ). Recent work with polyampholytes has demonstrated that they can also be used to cryopreserve 3D cell structures ( 9 ). We therefore tested the hypothesis that a cryopreserved bank of human lung tissue allows for the ex vivo study of the interindividual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models.…”

Section: Introductionmentioning

confidence: 99%

Ex vivo SARS-CoV-2 infection of human lung reveals heterogeneous host defense and therapeutic responses

Schaller¹,

Sharma²,

Dupee³

et al. 2021

JCI Insight

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Cell lines are the mainstay in understanding the biology of COVID-19 infection but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue would allow for the ex vivo study of the interindividual heterogeneity of host response to SARS-CoV-2, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 21 donors, many of whom had clinical risk factors for severe COVID-19. Cryopreserved tissues preserved 90% cell viability and contained heterogenous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infected with HCoV-OC43 and SARS-CoV-2 and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL6 , CXCL8 , and IFNB1 in response to SARS-CoV-2. Treatment of tissues with dexamethasone and the experimental drug N-hydroxycytidine suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted but unproven antiviral activity, each suppressed viral replication in tissues from a subset of donors. In summary, we developed a system for the ex vivo study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.

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“…Retention of the highly viscous solution status at higher temperatures due to matrix formation was also observed, which prevents recrystallization during thawing in addition to sufficient dehydration. These results agreed well with previous research, which reported that the glassy state could be stabilized by the polyampholytes [58][59][60] .…”

Section: Resultssupporting

confidence: 93%

Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR

et al. 2021

Self Cite

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Polyampholytes are emerging macromolecular membrane non-penetrating cryoprotectants; however, the mechanism behind their cryopreservation remains unclear. Here, we investigated the mechanism using solid-state NMR spectroscopy. The polymer-chain dynamics and the water and ion mobilities in the presence of various membrane penetrating and non-penetrating cryoprotectants were monitored at low temperatures to mimic cryopreservation conditions. NMR experiments revealed that the water, Sodium-ion, and polymer-chain signals in a carboxylated poly-ʟ-lysine (COOH-PLL) solution broadened upon cooling, indicating increasingly restricted mobility and increased solution viscosity. Moreover, strong intermolecular interactions facilitated the COOH-PLL glass transition, trapping water and salt in the gaps of the reversible matrix, preventing intracellular ice formation and osmotic shock during freezing; this reduced cell stress is responsible for cryoprotection. This simple NMR technique enabled the correlation of the cryoprotective properties of polymers that operate through mechanisms different from those of current cryoprotectants, and will facilitate the future molecular design of cryoprotectants.

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Molecular Design of Polyampholytes for Vitrification-Induced Preservation of Three-Dimensional Cell Constructs without Using Liquid Nitrogen

Cited by 24 publications

References 47 publications

In vitroinfection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response

In vitroinfection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response

Ex vivo SARS-CoV-2 infection of human lung reveals heterogeneous host defense and therapeutic responses

Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR

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