The Promise of Using Cell Cultures To Fight SARS-CoV-2

The COVID-19 pandemic has significantly disrupted scientific activities. Labs have been forced to shut down. Researchers have been placed on furloughs and advised to work from home. Animal facilities have also been scaled down and shipping of research supplies has been delayed.

However, despite these challenges, many researchers remain undeterred and are applying their scientific expertise in the fight against SARS-CoV-2, the coronavirus responsible for COVID-19. Portable diagnostic kits with short turnaround time have been invented to facilitate timely and on-field viral detection. Masks made of different materials have been tested for their ability to prevent aerosol transmission and re-usable masks that can be disinfected with electrical fields/heat have been commercialized. Furthermore, the pandemic has significantly accelerated vaccine development and in an unprecedented case, motivated companies with competing interests to work together in efforts to address this global health crisis.

In order to fully win the fight against SARS-CoV-2, it is paramount to understand the biological mechanisms of COVID-19 viral infection. A variety of models have been used, but none as frequently as cell culture. Cell cultures, including immortalized cell lines, stem cell lines and primary cells from patients, are being used as in vitro models to understand viral entry into human cells. They are useful for drug screening purposes and as cellular factories to generate viral particles for testing. This article highlights the promising research being conducted using cell cultures and illustrates how these models are contributing to the fight against COVID-19.

Cell cultures

A variety of in vitro cell cultures exist and depending on the purpose of the experiments, they each have their pros and cons.

Immortalized cell lines

The most common cell cultures are immortalized cell lines, such as HeLa, CHO and Vero cells. As the name suggests, these are cells that either by natural mutation or genetic engineering, have become immortal, enabling them to be grown in culture indefinitely. These cells divide at a relatively fast rate, are cheaper to obtain and maintain, and are generally more homogenous in their biological properties. Cell lines are most suitable for use when there is a demand for high cell numbers such as in an initial phase of high-throughput COVID-19 drug screening to identify a target.

Stem cell lines

Stem cell lines, such as mesenchymal and induced pluripotent stem cells (iPSCs), on the other hand, have the inherent ability to proliferate indefinitely, provided that they receive the appropriate growth signals.

Stem cells can either be obtained directly from their sources or have been treated with a de-differentiation chemical cocktail to revert them to a stem-like state. As they bear more similar biological characteristics to cells in vivo, they are better models for biological testing. However, primary stem cells can be difficult and expensive to maintain. For instance, some primary stem cells require feeder cells to maintain their stemness and most can only grow on surfaces coated with specific biomaterials. In cases where patients’ cells are not easily obtainable, iPSCs can also be differentiated to diverse lineages including neurons, cardiomyocytes, and immune cells for applications like COVID-19 drug testing to investigate potential adverse effects of drugs. iPSCs have also been used to construct 3D cell cultures known as organoids to facilitate biological investigations in a 3D environment in efforts to more closely recapitulate the in vivo setting.

Primary cells

The final type of cell cultures are primary cells from patients. It remains extremely challenging to grow these cells because commercially available products like media are not optimized for them, and thus these cells do not proliferate well ex vivo. Thus far, the exception may be primary leukocytes from patients which can be cultured efficiently ex vivo. This is also partly driven by developments in Chimeric Antigen Receptor (CAR) T-cell therapy to manufacture engineered T cells for cancer immunotherapy. Ex vivo culture of primary immune cells has been particularly useful to study the immune repertoire involved in antigen recognition and antibody production against SARS-CoV-2.

Cell culture for understanding disease

One of the most important questions regarding COVID-19 is the mechanism of viral entry into host cells. Cell culture has been an invaluable tool for discovering the role of the human angiotensin-converting enzyme 2 (hACE2) cell surface receptor in facilitating viral entry into human cells. Shang and colleagues at the University of Minnesota made use of a variety of immortalized cell lines (HEK293T, HeLa, Calu-3 and MRC-5) and found that a viral surface spike protein binds to hACE2 through its receptor-binding domain (RBD) and is proteolytically activated by human host proteases. The coronavirus membrane fusion S2 protein was also discovered as an essential protein for virus–cell membrane fusion. This study suggests that antibody-based therapeutics with high affinity to hACE2 or RBD could inhibit SARS-CoV-2 from associating with the host cell, therefore preventing its entry. Finally, compounds that can inhibit host cell membrane and lysosomal proteases responsible for activating viral entry, may also be useful as therapeutics.

These findings were also supported by a subsequent study by Dr Markus Hoffmann, scientist at the German Primate Center in Göttingen, and colleagues which showed that an inhibitor for the serine protease TMPRSS2 (a cell surface protein) was able to block SARS-CoV-2 infection in lung cells. “We identified the cellular protein TMPRSS2 as a crucial factor for SARS-CoV-2 infection and showed that an existing drug that is approved in Japan for treatment of pancreatitis, camostat mesylate, can inhibit TMPRSS2 activity and thus block SARS-CoV-2 infection in cell culture experiments,” said Hoffmann.

Hoffmann added that “we are currently exploring the antiviral effects of camostat mesylate and related drugs in culture systems that represent the human respiratory tract, and we are collecting data on the required dosage and longevity of the drugs. Finally, we are planning to do efficacy testing of the most promising drugs in non-human primates that have been experimentally infected with SARS-CoV-2.”

Studies using cell cultures have also provided insights into symptoms of COVID-19 infection. To understand why COVID-19-positive patients suffer from a persistent cough and other respiratory effects, Jia and colleagues analyzed ACE2 receptor expression in cell cultures of primary human airway epithelia, and found high receptor expression in these airway cells, as well as a correlation between ACE2 expression and susceptibility to SARS-CoV-2 infection. Researchers have also identified high ACE2 expression on human neurons, particularly the olfactory neuronal cells, possibly explaining the loss of smell experienced in some patients. Recently, a greater ratio of ACE2 positive cells were found to be present in the digestive tract compared to the lungs, and the receptor expression was higher in gastric cancer cells, potentially explaining the symptom of diarrhea.

This myriad of COVID-19 related symptoms has motivated the search for cellular tropism by SARS-CoV-2. For instance, Chu and colleagues systematically investigated the replication rate and cellular damage due to SARS-CoV-2 in cells from different species (humans, non-human primates, cats, rabbits, and pigs) and organs. Their study revealed the range of cells that SARS-CoV-2 is able to infect efficiently for generating physiologically relevant animal models for studying the disease.

To better generate policies for managing the spread of SARS-CoV-2, scientists also made use of cell cultures to understand the possible routes of virus entry into the body. For instance, Xu and co-workers made use of a bulk RNA sequencing technique and found that ACE2 was highly expressed on the mucosa of the oral cavity, especially in epithelial cells on tongue tissue derived from patients. This finding suggests that the oral cavity is a susceptible route for SARS-CoV-2 entry, in addition to the lungs.

Cell culture for drug screening

Cell cultures are invaluable tools for high-throughput drug screening to identify therapeutics capable of inhibiting entry and replication of SARS-CoV-2. Touret and colleagues screened 1,520 US Food and Drug Administration (FDA)-approved drugs in vitro for their anti-viral properties using VeroE6 and Caco-2 immortalized cell lines. From this study, they identified 90 compounds spanning different drug categories such as antibiotics and proton pump inhibitors, that may be therapeutically relevant. Similarly, Ianevski and co-workers made use of VeroE6 cell lines and found that a combination of orally-available virus-directed nelfinavir and host-directed amaodiaquine exhibited the best therapeutic effects against SARS-CoV-2 across 136 broad spectrum antiviral products.

Recently, Daniloski and colleagues also performed a knock-out screening of genes in the human genome to identify which are needed for SARS-CoV-2 infection of human alveolar epithelial cell lines. They discovered that the most important genes included those encoding the vacuolar ATPase proton pump and Arp2/3 complex which they also validated using RNA interference knock-out and small molecule inhibitors.

Going a step further, by using both in vitro cell culture and computational analysis approaches, Prof. Tudor Oprea and his team screened almost 4,000 approved drugs and identified those with structural similarity to hydroxychloroquine. In their study, they discovered that zuclopenthixol and nebivolol blocked COVID-19 infection at a low concentration with minimal side effects, and proposed that these drugs may be further tested for their therapeutic value.

“Critical to our work was not just the computer-guided effort, but also the dual (independent) experimental confirmation of these drugs in vitro. Experiments conducted first at University of New Mexico Health Sciences Center (Steven Bradfute lab) were later confirmed at the University of Tennessee Health Sciences Center (Colleen Jonsson lab),” said Oprea.

“Given the documented cases of reinfection, it is possible that vaccines may not work against SARS-CoV-2, so we need to keep pursuing effective therapeutic approaches. Combining drugs with synergistic effects may be the best way to go forward. The thought behind this is to give a lower dose of each drug which can be safer and accessible because some drugs are in shortages. At the same time, this approach delivers a two-pronged attack against virus which is prone to develop drug resistance when subjected to monotherapy,” Oprea added.

Cell culture for producing viral particles

To study SARS-CoV-2 transmission and infection, such as testing how different mask materials block transmission of SARS-CoV-2, it is necessary to obtain samples of the virus. Attributing to their lower costs of maintenance and fast cell division, immortalized cell lines have been used as factories to generate viral particles. This facilitates high-throughput production of viruses for testing and greatly enhances scientific study when it is challenging to access patients’ samples for extracting viruses. Kaye and co-workers showed that viral replication of severe acute respiratory syndrome–associated coronavirus (SARS-CoV) occurs efficiently in different cell lines. The viruses could then be isolated at high titers in the absence of specific cytopathic effects. Similar technology can likely be applied to SARS-CoV-2 as it belongs to the same virus family.

Conclusion

Cell cultures have facilitated studies investigating the biology of SARS-CoV-2 infection and have been harnessed for drug screening, they are also useful as a means for producing viral particles and therapeutics. Cell cultures have been an invaluable tool for biological studies and will be a key contributor in the ongoing fight against COVID-19.