In the late 19th century, scientists first tried their luck growing tissue outside the body. The earliest attempt was growing chicken embryos in warm saline. Incrementally, scientists grew more savvy. The repertoire of media recipes that simulated conditions within the body ballooned. Immortalized cells grew indefinitely outside the confines of the organ or tumor. Growth factors were titrated with such precision that even the most difficult primary cells could be kept alive, even if the techniques were painstakingly time-consuming and sensitive.
Today scientists not only manipulate the microenvironment, but also the innards of cells, themselves. Gene-editing techniques intersect with powerful three-dimensional and co-culture cell models to yield some of the most physiological relevant models the world has ever seen. Between gloved fingers, scientists hold cells inside of their own carefully designed universe—a universe within a plate.
The ever-evolving science of mimicry
While growing cells in flat sheets has its advantages (fairly inexpensive and easy to maintain), there are shortcomings. “Two-dimensional cultures can be useful for studies about single-cell biochemistry and intracellular signaling pathways, but they tell us little about the signaling and interactions between cells,” says Jennifer Tung, research scientist at ScienCell Research Laboratories.
Gene-editing techniques intersect with powerful 3D and co-culture cell models to yield some of the most physiological relevant models the world has ever seen.
“Three-dimensional cell culture is one step closer to mimicking physiological growing conditions for cells,” points out Tung. “It also enables scientists to look at cells in a system that approximates physiological conditions without using animal models.” Advancements in three-dimensional and co-culturing systems more accurately represent conditions within the body by allowing critical cross-talk between cells. Such signaling enables cells to behave more closely to the way they might in vivo. Cancer cells grown in spheres, for example, model aspects of tumors that ravage the body, oftentimes invading into the surrounding matrix, as might occur during metastasis.
Astrocytes and oligodendrocytes from NSCs
Image: ATCC
Companies are funneling efforts into optimizing cell lines through both improving cellular environments and genetic modifications. Robert E. Newman Jr., director of R&D at ATCC Cell Systems, says that these refinements create cells better suited for research uses, drug and toxicology screening, and bioproduction of therapeutic compounds.
As an example, Newman cites an in vitro kidney-cell model. Through genetic modifications, scientists enabled cells to stably overexpress a vital kidney membrane transporter protein called OAT1 that is often lost in cell culture. “In nature, it is involved in transporting organic ions and toxic substances across the cell membrane allowing [renal] cells to clear unwanted compounds from the blood stream.” He says that they now have an advanced cell model that correctly recapitulates kidney physiology and can be used for toxicology screenings.
Not only are cell models growing more physiologically relevant, they are also growing more accessible. For some, long gone are the days of the time-consuming process of harvesting and growing primary cells or figuring out the perfect constellation of conditions for co-cultures or three-dimensional cultures. Whether generating stable cells, achieving efficient knockdowns, or overexpressing stubborn proteins, much of the guesswork and energy has been replaced by ready-to-use kits.
“We currently offer 3D cell culturing kits that mimic the network formation step of blood vessel formation, tubulogenesis, and pericyte investment,” says Tung. Each kit includes primary cells and everything needed to grow and maintain lines, including protocols. “They are designed to be simple for both the experienced and the novice user, to yield quantifiable and reproducible data.”
ATCC offers similar ready-to-go products such as advanced cancer models to study genetically driven drug resistance or sensitivity, an angiogenesis co-culture model for screening anticancer compounds or regenerative medicine compounds, and ready-to-use neural progenitor cells (NPCs) derived from induced pluripotent stem cells from both normal and disease-state patients. “ATCC’s NPCs can save researchers a minimum of six months’ of development work and provides them with fully authenticated and quality control-tested cells,” adds Newman.
CRISPR—The game changer
CRISPR seems to be at the forefront of most new product development. The advanced cancer model from ATCC, for example, consists of both a parental cell line and one where a point mutation has been precisely introduced into the genome via CRISPR/Cas9 gene editing to confer drug resistance or drug sensitivity.
“The advent of efficient genome editing technologies, such as CRISPR Cas9, has been a game changer,” says Gregory Alberts, global subject matter expert at Lonza Pharma Bioscience Solutions. He notes, “CRISPR can be used to insert a specific gene, and even a promoter, stably into the genome of the cell line, and is likely to be more effective than the standard technique of forcing random integration of a gene or construct into the genome.”
Miguel Dominguez, global distribution manager at Mirus Bio, says, “CRISPR offers a much faster approach for generating a targeted event with the genome. Previous stable cell-line development relied on much longer timelines to select for desired modification via homologous recombination.”
This same gene-editing technology is being applied to large-scale production of biotherapeutic peptides, proteins, and monoclonal antibodies that are used in medicinal applications or therapies. According to Alberts, refinements made to the host cell line can improve both the amount and quality of the expressed protein. He says that identifying and altering post-translational modifications of proteins, such as glycosylation, can have a significant impact on the final protein product, with respect to efficacy, activity, and reduced immunogenicity.
“There is tremendous potential to engineer these little cellular protein production ‘factories’ and create customized versions of the cell lines that allow greater total yields [or] humanized proteins with far less immunogenicity,” explains Alberts. “[CRISPR] has helped to create a process for the practical modification of protein production cell lines.” Dominguez says that CRISPR formats must be carefully considered. “While we see that expression of the entire CRISPR system (i.e., Cas9 and gRNA) from plasmid DNA is the most widely used due to its low-cost, it may not be as efficient and accurate as the other delivery formats.”
According to Dominguez, an exclusive RNA approach offers more effective targeting without plasmid integration. Cas9 RNP, while more costly, is the most accurate with minimal chance of off-target cleavage events, since it bypasses transcription and translation. He explains, “Some experiments show improved genome-editing success moving from DNA to RNA to RNP in difficult-to-transfect cell lines.”
Additionally, scientists must think about delivering CRISPR components to the cell. “One common pain point is cytotoxicity,” says Dominguez. Liposomal formulations may be destructive to the cellular membrane and organelles within the cell. On the other hand, polymer-based formulas are less toxic but may have a lower efficiency. “We employ a combination of both strategies to formulate lipid and polymer-based solutions (nonliposomal) to address a broad spectrum of cell types with minimal toxicity.”
Lonza’s Nucleofector Technology is another delivery system option for DNA, RNA, and protein. Described as “improved electroporation technology,” it promises high efficiency and limited cytotoxicity. “If we move away from some of the easily transfected cell lines [HEK, CHO] into other cell types, you may well find that lipids become less effective at transfection, whereas nucleofection will remain effective,” says Alberts.
Preventing unintended consequences
Alberts warns, “There is always the law of unintended consequences.” Scientists must keep in mind that “every cell is made up of a host of interrelated biochemical pathways with elaborate feedback mechanisms.” Manipulating one aspect of the pathway can affect another aspect that may not always be apparent. “When we upset the balance of these delicate mechanisms, we can create an unintended result.”
Newman advises researchers to evaluate the product quality control testing, authentication, characterization data, and application data that supports the model. Without doing this, he cautions that “scientists could waste significant time on models that are not suitable for their research.” Newman encourages scientists to ask a lot of questions before moving forward: “For example, how extensive is the off-target testing to show that the cells do not have aberrant mutations in genetic locations where there is an increased likelihood of introducing an aberrant mutation by using the CRISPR technique? Have suppliers demonstrated the desired functionality of the cells?”
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Researchers must also consider the expense associated with developing advanced cell models and integrating new techniques into the lab as well as the long-term price-rage of maintaining models. “The cost of media … can be quite high because of the expense of components, such as growth factors,” notes Newman.
As gene-editing techniques and advanced cell culture systems rapidly evolve and integrate into mainstream science, so too, do the potential issues that arise from inadequate design and characterization. “It will be important for scientists to thoroughly understand the changes and modifications that they choose to make, and the possible ramifications of these changes,” says Alberts.
Not only can poor quality models or misidentified cell lines consume an enormous amount of time and money, but Newman says they ultimately impair the progress of science.
Images: Mirus Bio and ATCC