Cell culture models for SARS-CoV-2 infectivity and systemic complications

COVID-19 was declared by WHO as a pandemic since March 2020. The vaccination program has been implemented worldwide. Specific antiviral drugs such as remdesivir, molnupiravir and ritonavir-based nirmatrelvir were effective against SARS-CoV-2 infection. However, the new SARS-CoV-2 variants have been elevated due to viral mutation causing vaccine resistance and rapid spreading. Long-term COVID-19 complications have been life-threatening in some recovery cases. To overcome viral adaptation, cell culture model is essential to comprehend SARS-CoV-2 infection, pathophysiology, complications, and drug target alterations. The classical 2D culture cell was frequency used for viral propagation and high-throughput screening. Modern 3D culture has recapitulated key cellular and molecular events of tissue physiology. Here, we reviewed the cell lines, 3D culture, organoid and relevant models for the aforementioned applications

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

<p>Abstract</p> <p>Background</p> <p>The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.</p> <p>Results</p> <p>The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.</p> <p>Conclusion</p> <p>The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.</p

Proteomic Profiles of Mesenchymal Stem Cells Induced by a Liver Differentiation Protocol

The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs

Cytokine-induced killer cells: A novel treatment for allergic airway inflammation.

The effectiveness of cytokine-induced killer (CIK) cells for treatment of cancers has long been appreciated. Here, we report for the first time that CIK cells can be applied to treat allergic airway inflammation. Adopting from an established protocol with some modifications, we generated CIK cells ex vivo from mouse T cells, and examined their effectiveness in treatment of allergic airway inflammation using the ovalbumin-induced model of allergic airway inflammation. Based upon evaluation of bronchoalveolar lavage cellularity, T helper type2 cytokine levels and lung histology, all of which are important parameters for determining the severity of allergic airway inflammation, diseased mice treated with CIK cells showed significant reductions in all the parameters without any obvious adverse effects. Interestingly, the observed effects were comparable to those treated with dexamethasone. Thus, our study provides a novel application of CIK cells in treatment of allergic airway inflammation

Th2 cytokine levels in serum and BAL.

<p>The amount of Th2 cytokines (IL-5, IL-13) in serum (A) and BAL (B) were measured in normal mice (PBS/PBS), OVA-sensitized mice treated with PBS (OVA/PBS), thymocytes (OVA/Thy), dexamethasone (OVA/Dex) or CIK cells (OVA/CIK). Data shown are means ± SEM (n = 4–8) of 4 independent experiments. (ND, not detected; *, P<0.05 and **, P<0.005).</p

A diagram of the protocol for the mouse model of allergic airway inflammation and CIK cells treatment.

<p>C57BL/6 mice were sensitized with an intraperitoneal (i.p.) injection of 50 μg ovalbumin and 25 μg Alum on days 0 and 12. On days 22, 26 and 30, mice were challenged with 2% OVA aerosol (w/v) in saline (Neb) for 30 min. For treatments, 1x10⁷ of CIK cells, PBS or thymocytes were transferred via tail vein on day 21, and 2mg/kg/day dexamethasone was injected i.p. from day 21 to 30. All mice were evaluated 24 h after last challenge.</p

Cellularity of white blood cells in BAL.

<p>Total cell counts (A), numbers of monocytes, lymphocytes and eosinophils (B) were determined in BAL. Mice were sensitized and challenged with OVA and treated with PBS (OVA/PBS), thymocytes (OVA/Thy), dexamethasone (OVA/Dex) or CIK cells (OVA/CIK). Data are means ± SEM (n = 8–12) of 4 independent experiments. (ND, not detected; *, P<0.05; **, P<0.005 and ***, P<0.001).</p