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Secondcell Bio Hopes to Make Creation of Cell Lines as Easy as Building Legos

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NEW YORK – A new company called Secondcell Bio plans to make a cell engineering method that can create diverse cell lines that more accurately mimic human biology widely available to the research community.

According to the developer, the method, called Chromovert, works faster and more efficiently than current cell line development approaches, which took off in 1951 when the first immortal human cell line was established from a cervical cancer sample taken from Henrietta Lacks.

Chromovert, a combination of the words chromosomes and convert, was invented in 2002 by Secondcell Bio founder Kambiz Shekdar when he was a graduate student at Rockefeller University.

At the time, he was creating new cell lines, using an inducible expression system on pre-engineered, commercially available cell lines to make them express genes he was interested in studying. However, the cells started to malfunction due to a quality control issue, and the company's customer service was unable to resolve the issue for a year. With the dean's office pressuring him to finish his experiments and his Ph.D., Shekdar decided to try something else.

He had heard about other labs using molecular beacons — oligonucleotide hybridization probes that can report the presence of specific nucleic acids — and thought they could be used in combination with fluorescence-activated cell sorting to detect cells that were highly expressing one or more genes of interest. In the simplest terms, that's how Chromovert works, he said.

Following several rounds of improvement and optimization, it now uses molecular beacons or fluorogenic oligonucleotide signaling probes to isolate individual living cells expressing one or more transfected or endogenously expressed genes. Once they hybridize, the probes undergo a fluorogenic conformational change that displaces the fluorophore from the quencher. Positive cells that fluoresce above the background are then detected and isolated using flow cytometry.

Thousands of individual clones can be isolated and then expanded using automated cell culture methods, and functional testing over time in the absence of selective pressure is used to select the final clones.

There's a second part to the platform called Chromo-Tags — 3' untranslated RNA sequence tags subcloned for expression downstream of the stop codon of cDNAs of interest. Unlike epitope tags, Chromo-Tags are not translated into protein, leaving only native protein sequences to be expressed in the cell. Chromo-Tags streamline the cell engineering process by obviating the need to design and develop gene-specific probes.

In March, Shekdar and his team published details of Chromovert for the first time, in a paper in Biotechnology Letters.

The technology can be combined with any other cell culture or engineering technique, such as CRISPR, for the creation of rare or interesting cells, Shekdar said. Because Chromovert can isolate even vanishingly rare cells for the viable and stable expression of what may be challenging genes, it can be used to scan for cells expressing the target genes, which can then be multiplied using cell culture methods. It could even be used to scan laboratory cell cultures, he said, which may exhibit high levels of cell-to-cell genetic variability, even if they don't contain very high levels of the desired cells, and pick out the rare ones for cell culture.

Shekdar has now established Secondcell Bio to make the technology available to the wider research community. The startup, which is based in New York City, is ramping up operations — it will have about 10 employees by the end of the week. Shekdar said he's privately funding the company currently, but is in talks with investors as well as a university-backed research park about setting up a "cell line production farm" to ramp up production of cell lines for various drug targets. The company primarily offers Chromovert to create stable cell lines overexpressing any one or more proteins and will also create libraries of cell lines for drug discovery at scale.

"Cell engineering is challenging. We're making these things available so that people can get it in their own hands and tinker with [the technology]," he said.

The company will likely be coming up against some stiff competition. Firms likes Sigma-Aldrich, Labome, ATCC, Lonza, Abcam, and dozens of others have been selling human and animal cell lines for decades, including commonly used human cancer cell lines such as HeLa and HEK293T cells. Some are now using CRISPR to create and sell single-gene knockout cell lines.

According to Secondcell Bio's website, what the company offers is greater stability and yield, as well as streamlined production of stable cell lines expressing multiple genes without the need for customers to design or optimize their own gene-specific materials or methods.

And while Secondcell Bio may be new, Chromovert has already been used for close to 20 years in drug discovery and other applications. By the time Shekdar graduated from Rockefeller in 2003, he had incorporated a company called Chromocell to get the technology working. That company, where he served as CSO, has raised more than $100 million in funding from large corporate partners who have used its cell lines to discover compounds that affect taste and pain receptors.

In drug discovery, companies often want to generate cell lines that stably express ion channels or G protein-coupled receptors they have identified as drug targets for high-throughput screening. "Chromovert lets us scan orders of magnitude greater numbers of cells than previously possible to fish out the ones" where the genes have stably integrated and which express the correct stoichiometry, he said.

For example, in 2016, researchers at Pfizer and elsewhere identified the voltage-gated sodium channel blocker NaV1.7 in the propagation of pain signals from the peripheral nervous system to the brain. Pfizer's identification of a truncating mutant in NaV1.7 that stopped some people from feeling pain made researchers think that chemically blocking the gene could provide a pain treatment that wouldn't be addictive, since the pain signal to the brain came from the periphery and not the central nervous system.

Chromocell created cells that expressed NaV1.7. "Where we came out ahead was in creating a cell line that [expressed] not only the alpha subunit, but the accessory factors of the NaV1.7 channel," Shekdar said. "We did high-throughput screening and found a prospective drug candidate that in animal models works better than morphine."

The US Food and Drug Administration has fast-tracked Chromocell's candidate, and the company has completed clinical Phase I trials, with Phase II trials soon to start.

Chromocell's other area of focus is taste. The company has made taste receptor cell lines in collaboration with Coca-Cola, Kraft, and Nestle in order to do what Shekdar called "turbo taste testing" of natural extracts derived from fruits, vegetables, and other foods. These cells express taste receptor genes, essentially acting like miniature taste buds in a dish, to help the food companies find combinations of the natural extracts that enhance salty or sweet tastes that can help them cut the sodium and sugar in their products.

"Once we made our cell lines for these taste receptors, our cell-based assay results were exactly matching human sensory data," he said. "Chromovert helps select cells that are more physiologically relevant."

In 2014, Shekdar also established the Research Foundation to Cure AIDS, which aims to help develop an inexpensive cure for the disease, and Chromocell licensed its technology to RFTCA for AIDS research in 2019.

The foundation's approach is based on editing the CCR5 gene, which encodes a co-receptor the HIV virus uses to enter a cell, in hematopoietic stem cells. Multiple groups are working on disrupting CCR5 and then infusing the engineered stem cells back into the body, Shekdar said, but they tend to produce too few cells that are optimally modified on both chromosomes.

"We are planning to use Chromovert to see if we can piggyback onto these established methods that have generated positive proof of concept in the lab," he noted, adding that Chromovert may be able to increase the efficiency of these existing strategies in order to cure HIV.

The RFTCA submitted two grant applications to the National Institute of Allergy and Infectious Diseases this September covering in vivo and ex vivo strategies using Chromovert to help develop a cure, he added.

Shekdar hopes the technology's previous success shows that it is a true platform technology. "It means a lot for me to put this out into the research space and make it available for other researchers," he said.

He has stepped down from his role at Chromocell and is mostly engaged with Secondcell Bio and the RFTCA, but he is also working on Chromovert's potential for discovering more about drugs, especially those that affect the central nervous system and brain, and reducing their side effects.

"Part of the problem is, these ion channel drug targets have 19 or 20 types of subunits that come together to form pentameric channels. The number of combinations [of drug targets] possible are more than 10,000, and people are targeting drugs to the most abundant subunit combinations. … [B]ut we have no idea which other combinations even exist. Do our drugs interact with those? It's a total black box."

With Chromovert, he said, it's possible to scale up the development of cell lines through an automated process and to faithfully reproduce these thousands of ion channel combinations. Not all of them actually exist in the brain, he added, but once the panel exists, "you can take drugs that are on the market, you can take drugs that were pulled off the market because they caused suicidal ideation, you can take drugs that failed, and you can start to correlate which subunits, which cell lines, which subunit combinations correlate with desired efficacy, which ones correlate with side effects. And you can use that, then, to guide improved discovery of safer drugs with fewer side effects."

After that, Shekdar said, it's no longer about making one cell line for a complex target, but making thousands of cell lines to test multiple targets.

"I think of Legos," he said. "We have 19 Lego bricks that we've identified, but no one knows how they actually come together. So if we make cell lines that provide all possible combinations of these pieces and start to use those cell lines to see which combinations accurately correlate with in vivo function, this technology has a chance to tease apart some of the complexity of these CNS drug targets."

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