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    Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells
    (2022)
    Markus Uhrig
    ;
    EZQUER, EDUARDO FERNANDO  
    ;
    EZQUER, EDUARDO MARCELO  
    <jats:p>Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4–7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2–3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.</jats:p>
      6Scopus© Citations 34
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    Integrin Alpha E (CD103) Limits Virus-Induced IFN-I Production in Conventional Dendritic Cells
    (2021)
    Vikas Duhan
    ;
    Vishal Khairnar
    ;
    Simo Kitanovski
    ;
    Thamer A. Hamdan
    ;
    KLEIN POSTERNACK, ANDRES DAVID  
    <jats:p>Early and strong production of IFN-I by dendritic cells is important to control vesicular stomatitis virus (VSV), however mechanisms which explain this cell-type specific innate immune activation remain to be defined. Here, using a genome wide association study (GWAS), we identified Integrin alpha-E (<jats:italic>Itgae</jats:italic>, CD103) as a new regulator of antiviral IFN-I production in a mouse model of vesicular stomatitis virus (VSV) infection. CD103 was specifically expressed by splenic conventional dendritic cells (cDCs) and limited IFN-I production in these cells during VSV infection. Mechanistically, CD103 suppressed AKT phosphorylation and mTOR activation in DCs. Deficiency in CD103 accelerated early IFN-I in cDCs and prevented death in VSV infected animals. In conclusion, CD103 participates in regulation of cDC specific IFN-I induction and thereby influences immune activation after VSV infection.</jats:p>
      1Scopus© Citations 4
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    Concurrent Versus Sequential Intrapleural Instillation of Tissue Plasminogen Activator and Deoxyribonuclease for Pleural Infection
    (2018)
    Fayez Kheir
    ;
    George Cheng
    ;
    Estefania Rivera
    ;
    Alejandro Folch
    ;
    Erik Folch
      5Scopus© Citations 25