Modeling SARS-CoV-2 infection in vitro with a human intestine-on-chip device

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1. 

   ScreenIT

   Sep 9, 2020

   SciScore for 10.1101/2020.09.01.277780: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	The kit eliminates duplication bias in PCR and sequencing steps, by using unique molecular identifier (UMI) of 8 random bases to label the pre-amplified cDNA molecules.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cord and cultured in Endothelial Cell Medium purchased from ScienCell Research Laboratories, inc	HUVEC suggested: KCB Cat# KCB 200648YJ, RRID:CVCL_2959)
   Then, Caco-2 cells (∼ 1×105 cells) and HT29 cells …

   SciScore for 10.1101/2020.09.01.277780: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	The kit eliminates duplication bias in PCR and sequencing steps, by using unique molecular identifier (UMI) of 8 random bases to label the pre-amplified cDNA molecules.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cord and cultured in Endothelial Cell Medium purchased from ScienCell Research Laboratories, inc	HUVEC suggested: KCB Cat# KCB 200648YJ, RRID:CVCL_2959)
   Then, Caco-2 cells (∼ 1×105 cells) and HT29 cells (∼ 1×104 cells) were mixed and seeded into the upper channel under static cultures.	HT29 suggested: None
   Virus: A clinical isolate SARS-COV-2 strain 107 was obtained from Guangdong Provincial Center for Disease Control and Prevention, China, and propagated in Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   The virus titers were (infectious titers of virus) were determined by a TCID50 assay on Vero cells.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   At day 3 post-infection, the epithelial cells and endothelial cells cultured on chip were fixed for immunofluorescence analysis or lysed for RNA-sequencing analysis, respectively. Immunostaining: Caco-2 cells cultured on well plate were washed with PBS and fixed with 4% PFA at 4°C overnight.	Caco-2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Image processing was done using ImageJ (NIH).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   RNA-sequencing analysis: Raw sequencing data was first filtered by Trimmomatic (version 0.36), low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed.	Trimmomatic suggested: (Trimmomatic, RRID:SCR_011848)
   They were mapped to the reference genome of Homo sapiens from Ensembl database (ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna/) using STAR software (version 2.5.3a) with default parameters.	Ensembl suggested: (Ensembl, RRID:SCR_002344) STAR suggested: (STAR, RRID:SCR_015899)
   Reads mapped to the exon regions of each gene were counted by featureCounts (Subread-1.5.1; Bioconductor) and then RPKMs were calculated.	featureCounts suggested: (featureCounts, RRID:SCR_012919) Bioconductor suggested: (Bioconductor, RRID:SCR_006442)
   Genes differentially expressed between groups were identified using the edgeR package (version 3.12.1).	edgeR suggested: (edgeR, RRID:SCR_012802)
   Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis for differentially expressed genes were both implemented by KOBAS software (version: 2.1.1) with a corrected P-value cutoff of 0.05 to judge statistically significant enrichment.	KEGG suggested: (KEGG, RRID:SCR_012773) KOBAS suggested: (KOBAS, RRID:SCR_006350)
   Statistical analyses: Data were collected in Excel (Microsoft).	Excel suggested: None

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   One potential limitation of this work is the use of immortalized intestinal epithelial cell lines (e.g., Caco-2) originally isolated from human colon tumors. However, Caco-2 cells have been widely used to recapitulate many physiological and pathological functions of human intestine in vivo (38, 39). The intestinal epithelium differentiated from primary intestinal stem cells may be selected for further studies in later time. Overall, in this work, we provide the proof-of-concept to build an intestine-on-chip infection model at organ-level that permits to closely mirror the intestinal pathophysiology and human response to SARS-CoV-2 infection, which is impossible to achieve by existing in vitro culture models. It can not only supply a unique, rapid and low-cost in vitro platform for viral infection, but also provide a complement to animal models to study disease progress, virus transmission and host-virus interactions in a more realistic manner, thereby accelerating the COVID-19 research and development of novel therapeutics.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 17 and 19. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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2. 

   Version published to 10.1101/2020.09.01.277780v1 on bioRxiv

   Sep 2, 2020