December 1, 2014 (Vol. 34, No. 21)

Gail Dutton

Cytoo Manipulates Cell Deposition to Develop 3D Models that Balance Quality (High Content) and Quantity (High Throughput)

French contract research organization (CRO) Cytoo specializes in developing high-content, three-dimensional cellular assays for muscle and skin. It does so by means of micropatterns, which are initiated photolithographically and are ultimately elaborated at the cellular level. According to Cytoo, micropatterns reproduce microenvironmental cues that control the shape, organization, polarity, spreading and internal organization of attached cells, through a strong impact on genome transcription.

“If you want to exist as a CRO, you must have a niche,” insists Luc Selig, Ph.D., the company’s chief business officer. “Large CROs are providing lots of big assays that are quite common and are expanding their portfolios.” Cytoo, in contrast, is focusing its efforts on developing specialized, physiologically relevant cell models.

Typically, companies test a few millions of compounds in biochemical or irrelevant cell models, looking for compounds that affect the target. “That approach offers very high throughput, but adds little value,” Dr. Selig says.


High Throughput versus High Content

Drug developers, realizing that high-throughput approaches can be limiting if physiological relevance is lacking, are reweighing their options. They are less inclined to embrace high-throughput approaches simply because they are convenient. Instead, more and more drug developers, says Dr. Selig, are using high-content phenotypic screens.

“High-content screens offer a lot of value in the form of data regarding the localization of proteins, the expression levels of proteins, and the involvement of multiple proteins or organelles,” he explains. Such screens involve multiple biological processes simultaneously and thus more closely reproduce conditions in the body. “You get more information about your compound in a cell-based model,” points out Dr. Selig, “and [this information can be] representative of a specific pathology.”

Ideally, high-throughput screening would connect multiple organs on chips, but that’s “not possible now,” Dr. Selig says. Instead, he adds, there is a “trade-off between sophisticated cellular models and high-throughput screening.”

Cytoo positions itself in that middle ground. With the new cellular models it has developed, Cytoo is able to offer, in Dr. Selig’s words, “high-throughput screens that also generate qualitative data.”


Business Approach

“We want to achieve credibility for developing highly innovative cell-based assays that are more relevant to the human body than existing assays,” Dr. Selig says. “We focus on a few key tissues or organs.” The idea is to develop something unique, not just a larger portfolio.

Cytoo’s key areas of interest are muscle, skin, and 3D tumor spheroid models. Specifically, the CRO works with pharmaceutical companies using its fully mature micro-muscles—used to assess sarcopenia, cachexia, and muscle-related disorders—and with cosmetics companies using its skin models.


3D Approach, Still HCS Compatible

Typically, cells grow in vitro as unorganized monolayers. Cytoo’s technology causes them to grow with physical guidance either as quasi-plane microtissues or as three-dimensional spheroids that mimic in vivo organization.

“When companies screen drugs using monolayers of cancer cells, for example, they test only the external, proliferating zone,” Dr. Selig remarks. But tumors are composed of three layers: the central necrotic zone, which cannot access oxygen or nutrients; the quiescent layer, which is trying to survive; and the external layer, which proliferates.

Metastasis is caused by the external, proliferating layer but also can be triggered by cells in the quiescent zone. Therefore, excluding the quiescent cells yields an incomplete screen. 3D cellular models overcome that limitation.

“Skin, muscle, and 3D spheroids are our main areas,” states Dr. Selig. Next, he avers, Cytoo plans to expand its cellular assays to include kidney proximal tubes, followed eventually by hepatocytes and cardiomyocytes.

“In spring 2014, we released the first cellular model of fully mature human myotubes, the basic element in creating skeletal muscle,” Dr. Selig says. Cytoo’s technology causes primary myoblasts to grow by groups, fuse, and self-organize into myotubes. “We have a number of parameters we can adjust to push maturation and differentiation.”

Cytoo calls this developmental process micropatterning. “Basically, we ask the cells to adhere to a position on the microplate, where their depositions are defined by size, shape, coating protein, and other parameters,” Dr. Selig explains.

The constraints at each position cause the cells to grow at specified densities within the specified parameters for that type of cellular model. “It takes about 120 hours (five days) to develop a mature myotube with all the characteristics of a fully mature myocyte,” notes Dr. Selig.

“The key when developing assays is to add more physiology to the model while maintaining the throughput and the robustness of the assay,” he continues. “Micropatterning allows fine-tuning of morphology, size, and growth rate. The cells are located at defined plate positions, which enables multiple parameters to be captured simultaneously and in a timely manner.”

There is little product variability among plates. “It’s more predictive than mouse models and is faster and less expensive,” he tells GEN. “And, it reduces the numbers of research mice needed.”

Micropatterning is not without challenges, though. “Every time you change the cell type, you have to find the right conditions (shape, size, cell density, culture medium, etc.) and essentially reinvent the wheel,” he admits. “Also, the technology doesn’t work for every type of cell.” To minimize those challenges, Cytoo focuses on a few, related cell types that Dr. Selig says have a high chance of success. “We’re capitalizing on our experience.”    


Cytoo has developed a myotube model by fully maturing human primary myoblasts. Myotubes show a high level of striation, high fusion index with aligned nuclei, and low morphological variability enabling high Z’ factor and high-content screening. Applications include atrophy and hypertrophy assays and metabolic studies including glucose uptake.

Cytoo

Location: Minatec – BHT – Bât. 52, 7 parvis Louis Néel, 38040 Grenoble cedex 9, France

Phone: 33 (0)4 38 88 47 05

Website: www.cytoo.com

Principal: Marc Le Bozec, CEO

Number of Employees: 34

Focus: Cytoo is a contract research organization focused on the development of muscle and skin-related cellular assays. The company also uses its systems and tools to create 3D tumor spheroids that are compatible with high-content analysis.



























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