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1.
Viruses ; 12(6)2020 06 08.
Article in English | MEDLINE | ID: covidwho-1726020

ABSTRACT

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/virology , Pneumonia, Viral/virology , Virus Inactivation/drug effects , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , COVID-19 , Cell Culture Techniques/methods , Chlorocebus aethiops , Containment of Biohazards/methods , Containment of Biohazards/standards , Humans , Nasopharynx/virology , Pandemics , RNA, Viral/isolation & purification , SARS-CoV-2 , Specimen Handling/methods , Vero Cells , Viral Load/methods
2.
Int J Mol Sci ; 23(3)2022 Feb 07.
Article in English | MEDLINE | ID: covidwho-1686815

ABSTRACT

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.


Subject(s)
Cell Culture Techniques/methods , Cryopreservation/methods , Leukocytes, Mononuclear/cytology , Cell Survival , Cells, Cultured , Female , Flow Cytometry , Humans , Leukocyte Count , Leukocytes, Mononuclear/metabolism , Male
3.
J Am Soc Nephrol ; 32(1): 86-97, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496654

ABSTRACT

BACKGROUND: Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line. METHODS: Fourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. RESULTS: Transcripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function. An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins. CONCLUSIONS: No cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.


Subject(s)
Cell Culture Techniques/methods , Kidney Tubules, Proximal/metabolism , Transcriptome , Animals , Biological Transport , Cell Line , Chromatography, Liquid , Gene Expression Profiling , Humans , Internet , Mice , Opossums , Proteomics , RNA-Seq , Rats , Sequence Analysis, RNA , Species Specificity , Swine , Tandem Mass Spectrometry
4.
Biochem Biophys Res Commun ; 577: 146-151, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1401239

ABSTRACT

The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.


Subject(s)
Alveolar Epithelial Cells/virology , COVID-19/virology , Cell Culture Techniques/methods , SARS-CoV-2/physiology , A549 Cells , Alveolar Epithelial Cells/pathology , Cells, Cultured , Disease Susceptibility , Gene Expression Regulation, Neoplastic , Humans , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Up-Regulation/genetics
5.
Methods Mol Biol ; 2305: 129-140, 2021.
Article in English | MEDLINE | ID: covidwho-1355903

ABSTRACT

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.


Subject(s)
Biotechnology/methods , Cloning, Molecular , Insecta/metabolism , Spike Glycoprotein, Coronavirus/biosynthesis , Transfection/methods , Animals , Bioreactors , Cell Culture Techniques/methods , Cell Line , Gene Expression , Glycosylation , Humans , Insecta/cytology , Mammals/genetics , Mammals/metabolism , Plasmids , Proteomics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Reproducibility of Results , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
6.
Biotechnol Bioeng ; 118(7): 2649-2659, 2021 07.
Article in English | MEDLINE | ID: covidwho-1173780

ABSTRACT

The Vero cell line is the most used continuous cell line in viral vaccine manufacturing. This adherent cell culture platform requires the use of surfaces to support cell growth, typically roller bottles, or microcarriers. We have recently compared the production of rVSV-ZEBOV on Vero cells between microcarrier and fixed-bed bioreactors. However, suspension cultures are considered superior with regard to process scalability. Therefore, we further explore the Vero suspension system for recombinant vesicular stomatitis virus (rVSV)-vectored vaccine production. Previously, this suspension cell line was only able to be cultivated in a proprietary medium. Here, we expand the adaptation and bioreactor cultivation to a serum-free commercial medium. Following small-scale optimization and screening studies, we demonstrate bioreactor productions of highly relevant vaccines and vaccine candidates against Ebola virus disease, HIV, and coronavirus disease 2019 in the Vero suspension system. rVSV-ZEBOV, rVSV-HIV, and rVSVInd -msp-SF -Gtc can replicate to high titers in the bioreactor, reaching 3.87 × 107 TCID50 /ml, 2.12 × 107 TCID50 /ml, and 3.59 × 109 TCID50 /ml, respectively. Furthermore, we compare cell-specific productivities, and the quality of the produced viruses by determining the ratio of total viral particles to infectious viral particles.


Subject(s)
Bioreactors/virology , Cell Culture Techniques/methods , Ebola Vaccines , Vesiculovirus/genetics , Animals , COVID-19 Vaccines , Chlorocebus aethiops , Culture Media, Serum-Free , Vero Cells , Viral Vaccines
7.
PLoS Pathog ; 17(3): e1009439, 2021 03.
Article in English | MEDLINE | ID: covidwho-1133695

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.


Subject(s)
COVID-19/virology , Cell Culture Techniques/methods , SARS-CoV-2/physiology , Antiviral Agents/pharmacology , Containment of Biohazards , Genome, Viral/drug effects , High-Throughput Screening Assays , Humans , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , Virus Replication/drug effects
8.
Methods Mol Biol ; 2273: 131-138, 2021.
Article in English | MEDLINE | ID: covidwho-1092090

ABSTRACT

The current coronavirus disease-19 (COVID-19) pandemic, caused by "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), underscores the threat posed by newly emerging viruses. The understanding of the mechanisms driving early infection events, that are crucial for the exponential spread of the disease, is mandatory and can be significantly implemented generating 3D in vitro models as experimental platforms to investigate the infection substrates and how the virus invades and ravages the tissues.We here describe a protocol for the creation of a synthetic hydrogel-based 3D culture system that mimics in vitro the complex architectures and mechanical cues distinctive of the upper airway epithelia. We then expose the in vitro generated 3D nasal and tracheal epithelia to gold nanoparticles (AuNPs) that display the typical shape and size distinctive of SARS-CoV-2 and of the majority of Coronaviridae presently known.The infection platform here described provides an efficient and highly physiological in vitro model that reproduces the host-pathogen early interactions, using virus-mimicking nanoparticles, and offers a flexible tool to study virus entry into the cell. At the same time, it reduces the risk of accidental infection/spillovers for researchers, which represents a crucial aspect when dealing with a virus that is highly contagious, virulent, and even deadly.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Cell Culture Techniques/methods , Epithelial Cells/cytology , Nanoparticles/metabolism , Respiratory Mucosa/cytology , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/virology , Gold , Humans , Metal Nanoparticles/chemistry , Molecular Mimicry/immunology , Nose/virology , Respiratory Mucosa/virology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Trachea/virology , Vero Cells , Virus Internalization
9.
Stem Cells Transl Med ; 10(6): 883-894, 2021 06.
Article in English | MEDLINE | ID: covidwho-1060671

ABSTRACT

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow-fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore-size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected "free" MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time.


Subject(s)
Bioreactors , COVID-19/therapy , Cell Culture Techniques/methods , Mesenchymal Stem Cell Transplantation/methods , Thrombosis/prevention & control , Animals , Anticoagulants/pharmacology , Blood Coagulation Tests , Bone Marrow Cells/cytology , Cells, Cultured , Dogs , Heparin/pharmacology , Humans , Male , Membranes, Artificial , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Myocardial Infarction/pathology , Myocardial Infarction/prevention & control , SARS-CoV-2 , Swine
10.
Tissue Cell ; 70: 101497, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1049889

ABSTRACT

BACKGROUND: In-Vitro/Cellular evidence is the backbone and vital proof of concept during the development of novel therapeutics as well as drugs repurposing against COVID-19. Choosing an ideal in-vitro model is vital as the virus entry is through ACE2, CD147, and TMPRSS2 dependant and very specific. In this regard, this is the first systematic review addressing the importance of specific cell lines used as potential in-vitro models in the isolation, pathogenesis, and therapeutics for SARS-COV-2. METHODS: We searched 17 literature databases with appropriate keywords, and identified 1173 non-duplicate studies. In the present study, 71 articles are included after a careful, thorough screening of the titles and their abstracts for possible inclusion using predefined inclusion/exclusion criteria (PRISMA Guidelines). RESULTS: In the current study, we compiled cell culture-based studies for SARS-CoV-2 and found the best compatible In-Vitro models for SARS-CoV-2 (Vero, VeroE6, HEK293 as well as its variants, Huh-7, Calu-3 2B4, and Caco2). Among other essential cell lines used include LLC-MK2, MDCKII, BHK-21, HepG2, A549,T cell leukemia (MT-2), stems cells based cell line DYR0100for differentiation assays, and embryo-specific NIH3T3 cell line for vaccine production. CONCLUSION: The Present study provides a detailed summary of all the drugs/compounds screened for drug repurposing and discovery purpose using the in-vitro models for SARS-CoV-2 along with isolation, pathogenesis and vaccine production. This study also suggests that after careful evaluation of all the cell line based studies, Kidney cells (VeroE6, HEK293 along with their clones), liver Huh-7cells, respiratory Calu-3 cells, and intestinal Caco-2 are the most widely used in-vitro models for SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 Vaccines/pharmacology , COVID-19/drug therapy , Cell Culture Techniques/methods , SARS-CoV-2 , Animals , Cells, Cultured , Drug Discovery , Drug Repositioning , Humans , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology
11.
Blood ; 136(25): 2905-2917, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-890058

ABSTRACT

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , Cell Culture Techniques/methods , Immunotherapy, Adoptive/methods , SARS-CoV-2/immunology , Adult , Aged , COVID-19/drug therapy , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunodominant Epitopes/immunology , Male , Membrane Proteins/immunology , Middle Aged , Viral Proteins/immunology , Young Adult
12.
Balkan Med J ; 37(1): 208-214, 2020 06 01.
Article in English | MEDLINE | ID: covidwho-826668

ABSTRACT

Background: Brain metastasis is a major cause of cancer death in patients with lung cancer. Sirtuin 1 and hsa-miR-217 have been identified to mediate the development of non-small cell lung cancer. Aims: To investigate the roles of hsa-miR-217, its target sirtuin 1, and the P53/KAI1 axis in the brain metastasis from non-small cell lung cancer. Study Design: Cell culture study. Methods: Human pulmonary adenocarcinoma brain metastasis cell line PC-14/B were incubated and treated with constructed lentiviral plasmids expressing miR-217 and/or sirtuin 1. BEAS-2B cell line was used as a control. The targeted regulation of miR-217 to sirtuin 1was examined using a dual-luciferase reporter assay. Cell proliferation, migration, invasion, and related protein expression were detected to examine the effect of the miR-217/sirtuin 1 expression on metastasis. Results: PC-14/B cells expressed higher sirtuin 1 and lower P53 and KAI1 compared with BEAS-2B control cells (p<0.05). Sirtuin 1 was a direct target of miR-217. MiR-217 expression suppressed PC-14/B cell invasion (p=0.004), migration (p=0.001), and proliferation (p<0.05), whereas sirtuin 1 overexpression reversed all processes. sirtuin 1 expression inhibited P53, KAI1/CD82, matrix metalloproteinase-9, and ß-catenin but upregulated E-cadherin protein. MiR-217 overexpression induced reverse changes. Conclusion: Hsa-miR-217 and its target sirtuin 1 acted as metastasis suppressor and promoter gene in non-small cell lung cancer, respectively. The hsa-miR-217/sirtuin 1/P53/KAI1 metastasis regulatory pathway showed novel and crucial roles in brain metastasis from non-small cell lung cancer. This axis might be a potential target for the treatment of brain metastasis of lung cancer.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Kangai-1 Protein/drug effects , MicroRNAs/pharmacology , Sirtuin 1/drug effects , Tumor Suppressor Protein p53/drug effects , Brain Neoplasms/etiology , Brain Neoplasms/physiopathology , Brain Neoplasms/prevention & control , Carcinoma, Non-Small-Cell Lung/physiopathology , Cell Culture Techniques/methods , Cell Proliferation/drug effects , Humans , MicroRNAs/therapeutic use , Signal Transduction/drug effects
13.
mSphere ; 5(5)2020 09 02.
Article in English | MEDLINE | ID: covidwho-742194

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/growth & development , Cell Culture Techniques/methods , Coronavirus Infections/virology , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , COVID-19 , Pandemics , RNA, Viral/analysis , RNA, Viral/isolation & purification , SARS-CoV-2 , Virus Replication/drug effects
15.
Virol Sin ; 35(3): 311-320, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-617330

ABSTRACT

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury (AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells (KPTECs) in 2D and 3D culture systems using conditional reprogramming (CR) and organoids techniques. These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters (SLC34A3 and cubilin). They also expressed angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3D organoids culture compared to that in 2D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike (S) protein was able to enter CR cells with luciferase reporter. This integrated 2D CR and 3D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.


Subject(s)
Betacoronavirus/metabolism , Cell Culture Techniques/methods , Coronavirus Infections/virology , Coronavirus/metabolism , Epithelial Cells/virology , Kidney/virology , Pneumonia, Viral/virology , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/pathogenicity , COVID-19 , Cell Line , Coronavirus/pathogenicity , DNA Damage , Disease Models, Animal , Humans , Organoids , Pandemics , Peptidyl-Dipeptidase A/metabolism , Receptors, Cell Surface/metabolism , SARS Virus/metabolism , SARS Virus/pathogenicity , SARS-CoV-2 , Sodium-Phosphate Cotransporter Proteins, Type IIc/metabolism , Spike Glycoprotein, Coronavirus/metabolism
16.
Cytotherapy ; 22(8): 458-472, 2020 08.
Article in English | MEDLINE | ID: covidwho-209852

ABSTRACT

BACKGROUND AIMS: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow-derived MSCs. METHODS: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. RESULTS: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. CONCLUSIONS: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.


Subject(s)
Blood Platelets/cytology , Cell Culture Techniques/methods , Filtration , Mesenchymal Stem Cells/cytology , Nanotechnology , Adipogenesis/drug effects , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Culture Media/pharmacology , Extracellular Vesicles/metabolism , Gene Expression Profiling , Humans , Immunophenotyping , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Mesenchymal Stem Cells/drug effects , Osteogenesis/drug effects , Particle Size , Serum/chemistry
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