key: cord-0736727-nhaueml6
authors: Kumar, Subodh; Sarma, Phulen; Kaur, Hardeep; Prajapat, Manisha; Bhattacharyya, Anusuya; Avti, Pramod; Sehkhar, Nishant; Kaur, Harpinder; Bansal, Seema; Mahendiratta, Saniya; Mahalmani, Vidya M.; Singh, Harvinder; Prakash, Ajay; Kuhad, Anurag; Medhi, Bikash
title: CLINICALLY RELEVANT CELL CULTURE MODELS AND THEIR SIGNIFICANCE IN ISOLATION, PATHOGENESIS, VACCINE DEVELOPMENT, REPURPOSING AND SCREENING OF NEW DRUGS FOR SARS-COV-2: A SYSTEMATIC REVIEW
date: 2021-01-26
journal: Tissue Cell
DOI: 10.1016/j.tice.2021.101497
sha: 29f6a837e56a29ae5dc449bde59518f8b57ef711
doc_id: 736727
cord_uid: nhaueml6
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.
. Though CoVs were known to cause milder symptoms, the outbreak of these three strains were deadly with high mortality and shown very adaptive potential as per environmental conditions and classified as "emerging viruses." COVID-19 was initially named "2019-nCoV." but later on, it was changed to "SARS-CoV-2" due to high similarity with severe (4) , and their genetic material is highly susceptible to frequent recombination process, resulting in new strains with alteration in their virulence (5) . The most important structural proteins of SARS-COV-2 are spike (S) protein, which is a trimeric, membrane (M) protein, envelop (E) protein, and the nucleocapsid (N) protein, and all of them are potential drug development targets. Some viruses, such as beta-CoVs, also have hemagglutinin esterase (HE) glycoprotein (4) . The RNA genome of CoV has seven conserved genes (ORF1a, ORF1b, S, OEF3, E, M, and N) arranged in 5' to 3':ORF1a/b alone covers two-third part of its RNA genome. It is responsible for the production of two viral replicase polyproteins which are PP1a and PP1ab. Further processing gives rise to sixteen mature nonstructural proteins (NSPs) and play a crucial role in various viral functions, including the formation of the replicase transcriptase complex (6). The rest of the virus's genomic part encodes the mRNA, which produces its other essential structural proteins, including spike, envelope, membrane, and nucleocapsid (6). HE is another essential envelopassociated protein that is expressed by specific CoV strains (7) . The entire RNA genome of corona virus is packed with nucleocapsid protein under covered with envelope (8).
When SARS-COV-2 enters into the body, the primary target cells are the enterocytes and pneumocytes (8) (9) (10) (11) (12) (13) (14) . In contrast, other target cells include kidney tubular epithelial cells, cerebral J o u r n a l P r e -p r o o f neuronal cells, and immune cells (8, 9) . SARS-CoV-2 entry and infection into the host cells take place through host cell factors, angiotensin-converting enzyme-2 (ACE2), and subsequent proteolytic cleavage on the Spike protein by transmembrane protease serine 2 (TMPRSS2),which is responsible for membrane fusion (10) . In the case of ACE-2, after recognizing the receptor, the virus genome and its nucleocapsid are released in the cytoplasm of the target cells. Two viral polyproteins (pp1a and pp1b) encoded by ORF1a and ORF1b genes are further processed by proteases into 16 NSPs and play a crucial role in the formation of the replication transcription complex (15) . This takes command over host translational machinery for the production of their own proteins (16) . All NSPs have their specific functions required starting from their entry into the target cell to suppression of target gene expression to replicate and translate their genome and necessary proteins and specific functions of NSPs and their cellular production (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) .
Similarly, a recent study suggested that entry and viral spread among other organs such as lungs of the infected host also depend on serine protease TMPRSS-2 activity in S protein priming of SARS-CoV-2 (10). It is suggested that SARS-CoV infection further leads to a surge of proinflammatory cytokines and chemokines, and causes severe pulmonary tissue damage (25), deteriorating lung function and cause lung failure (26) . Both host-directed therapies and virusdirected therapies are under investigation with variable success. The most important among the virus directed therapies, are Lopinavir/Ritonavir combination, hydroxychloroquine, chloroquine, remdesivir as we discussed in our previous studies (24, (27) (28) (29) . Owing to the ease of the
After searching databases and removing duplicates, two authors (HK and SK) independently screened the titles/abstracts using predefined inclusion/exclusion criteria. For relevant articles, full-texts were obtained for further evaluation. In case of any discrepancy, BM was consulted, and the issue was resolved.
Data extraction was done separately by two authors (HK and SK). In articles published in a language other than english, google translate was used to identify relevant data.
After searching a total of 17 literature databases, we identified 1173 relevant articles, which were screened for title and abstract. Following which 272 articles were selected for full-text screening and finally a total of 71 articles were selected for the systematic review. For the review process, various cell lines have been used for SARS-CoV-2 isolation, pathogenesis and therapeutic purpose. The details of the selection process and PRISMA chart are included (figure 1). For studying the pathogenesis and evaluation of vaccines and therapeutics against SARS-COV-2, suitable, rapid, and safe experimental models are a current necessity that could combat the present clinical disease (30, 31) . Various animal models are utilized for SARS-CoV studies, such as golden Syrian hamsters , rabbits, guinea pigs, mouse, ferrets, and non-human primates like rhesus macaques, marmosets, and cats (32-38). The virus specificity to the ACE-2 receptor and TMPRSS-2 (both SARS-CoV-2 and SARS-CoV) was found to be a significant hindrance in developing the animal models for SARS-CoV-2. In this regard, based on the literature available where the viral load of SARS-CoV was either not detected or identified very low, all cells are not susceptible to these viruses (39) . In the present study, we compiled various human and other primates based cell lines used since the outbreak of SARS-CoVs and the compiled list of these cell lines is given in table 1.
Cell culture models can replicate the different properties and functions of the various organs (table 1) . (48) . However, this is a new and unexplored area, and more studies are required in this direction to target the TMPRSS-2 based viral spread and might be a potential therapeutic target for drug development against SARS-Cov-2.
Other important kidney-specific cell lines that have been used for SRS-CoV2 studies include the HEK293 cell line for vaccine production (61), viral pathogenesis, and transmission (10) . (90), and 293F used for monoclonal antibodies production (90, 91) ; whereas, 293T-ACE-2 cell line for vaccine production and antibodies response (92) and viral pathogenesis (87).
Other kidney-specific cell lines used for SARS-CoV-2 study include LLC-MK2 for virus
In the lungs, the majorities of the ACE-2-expressing cells ( ~83%) are alveolar type II cells and may serve as in vitro Model system for Covid 19 studies (96, 97) . Similarly, oral and nasal epithelial cells also showed higher expression of ACE-2 receptors of SARS-CoV-2 (98, 99).
Calu-3 is a lung adenocarcinoma epithelial cell line used for various SARS-CoV-2 studies (48, 100) . Among Calu-3 and its clonally derived Calu-3 2B4, ACE-2 expression was checked, and comparatively, 2B4 have higher ACE2 expression (101), suggesting them a better model system for SARS-CoV2. It has been used to check the viral pathogenesis and transmission (10, 48, 102) and for drug screening of EIDD-2801 (100).
Other (10, 77) , and susceptible to SARS-CoV2; however, in another study, A549 cells did not show TMPRSS2 expression, required for S protein processing and viral spread (48).
J o u r n a l P r e -p r o o f
Huh7 cell line is derived from liver carcinoma of a 57-year-old Japanese male in 1982 and is a potential In Vitro model system for drug screening against many viruses. A recent study shows that SARS-CoV-2 efficiently enters into the Huh-7 cells through ACE-2 receptor binding as a potential In vitro study model (106) . In a recent study published in Cell
Other liver-based cell lines that have been used for SARS-CoV2 susceptibility are HepG2, Hep1-6, HL7702, RHT6.0, X9.0, X9.5, and C3A. HepG2 is used to check the SARS-CoV2
Virus susceptibility (56), infection-induced target cells gene expression (109) while in another study for vaccine production (61) . Similarly, other cell lines, such as Hep1-6, HL7702, RHT6.0, X9.0, X9.5, and C3A are used for vaccine production (61).
Intestinal ACE-2 receptor expression (110) and are very susceptible to 2019-nCoV infection.
Caco-2 cell line has shown very high CoVs viral titer (93) . It has been used for viral pathogenesis and transmission (10), vaccine production (61), and drug repurposing and screening studies of Darunavir (111) against Covid-19 and has proven to be a good in-vitro model system.
There are several other cell lines that have been used for SARS-CoV-2 studies such as T cell
Together In a nut-shell, cells expressing the ACE2 receptor and TMPRSS-2 could be potential
Yes, the Manuscript is submitted with the consent of all authors.
None of the authors declared any conflict of interest.
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Support of Experimental Pharmacology Laboratory (EPL) team, Dept., of Pharmacology, PGIMER Chandigarh is highly acknowledged.