Established Methods of Cell Culture

Cell culture is a set of biological techniques used to grow cells outside their original in vivo environment, for the purpose of scientific experimentation. It is widely used for in vitro testing in biomedical science to study animal models. However, a great challenge in cell culture techniques is the mimicking of physiologically relevant conditions. Current cell collection techniques are limited to, either the selection of primary cultures taken directly from an organ (or biopsy), or the use of immortalized cell lines (such as HeLa cells) (Skloot, 2010).

Once the sample is collected, there are two main methods of performing cell culture, suspended in flasks containing culture medium, or in the form of 2D cultures (seen above in well plates).

Regardless of the type of sample collected, the cell culture methods present key drawbacks:

• They require a large quantity of reagents and precious samples, as they are conducted in flasks and plates.
• They display limited surface of cell growth and cell surface interactions.
• Stagnation of the nutritive medium occurs, therefore the medium volume must be in excess and changed regularly (every 2-3 days).
• There is no development of extracellular interactions, as witnessed in physiological conditions, i.e. no extracellular matrix development.
• These are not dynamic or physiologically relevant models, which incorporate parameters such as flow of fluids or metabolite interactions.

Therefore, there is an urgent need for physiologically relevant cell culture models, which incorporate 3-dimensional structural organizations that more closely mimic what happens in nature.  For example,  the surface of pulmonary alveoli enables a gas exchange the size of a badminton court (approximately 75m²), thanks to a well-managed 3D organization with precise design ramification (Stanton et al., 2008). These are very sophisticated architectures, not achievable with classic cell culture methods.

The Future of Cell Culture

New technologies are being developed to alleviate the challenges associated with classic cell culture methods, such as multi-compartment devices (i.e transwell), culture on beads, co-culture of different cell types, irrigation systems to renew the medium continuously, and finally microfluidics.

Microfluidics has become particularly interesting to researchers, thanks to its clever use of micron scale spaces (i.e. channels and chambers) that provide large surface-to-volume ratios. These structures – typically fabricated out of polymer materials – have the potential to closely mimic physiological micro-scale environments and can be particularly useful for cell culture.

Miniaturize Your Experiments

Microfluidic was first developed back in the 1960s, and involves the control of fluids in the micron scale, inside of structures such as channels. This field of research, originally limited to the physicists and engineers, has since developed into a vast array of applications in life sciences and healthcare, including medical devices, sequencing, diagnostics, and drug development. It now allows complex analyzes to be performed in a single drop, permitting the reduction of experimental reagents and precious samples used during experiments. It also allows precise liquid control (i.e. single cell sequencing), shorter experimental times, automation of processes, and integration of complex operations into a single device.

Harnessing Microfluidic Cell Culture

In the wake of the miniaturization of electronics, microfluidics has benefited from the understanding of physical phenomena at small scales. It can help advance life science research by providing better in vitro models of cell culture, through the design of miniaturized devices to better study cellular behavior and interactions.  

For example, organ-on-chip devices are now being developed to simulate the properties of certain organs and help reveal new information for drug discovery and process development  in pharmacokinetics and toxicology. These devices may offer a good model for preclinical development of pharmaceutical compounds to reduce and complement the use of animal models.

The Fabrication of Cell Culture Devices

To utilize microfluidics for cell culture, first we need to fabricate a device, and the materials used must be chosen carefully to ensure they fulfill the application requirements. In the case of cell culture device, it is important the material is certified biocompatible and not toxic to the cells. It is important the material is transparent, for fluorescence microscopy. And it must also be gas permeable.

However, engineering aspects must also be considered. The material should provide a user-friendly fabrication protocol and be easy to handle. These features have historically been neglected in microfluidics. But as the number of non-microfluidics specialists developing chip technologies increases, fabrication processes must also evolve and become more accessible.

The polymer polydimethylsiloxane (PDMS) has become a favorite in academia and generally amongst those developing life science applications. It is an elastomer possessing biocompatibility, transparency and a relatively straight forward fabrication protocol, known as soft-lithography. However, the material itself remains expensive, and the fabrication protocol is lengthy, requiring specific microfabrication equipment. A final important limitation of PDMS lies in it not being scalable. Unless you have an army of minions assembling devices, there is little chance of mass production. This PDMS remains limited to research.

Consequently, many industrial players turn to hard thermoplastics for their commercial needs. These materials include polystyrene (PS), cyclic olefin copolymer (COC), polycarbonate (PC), and poly(methyl methacrylate) (PMMA). Thermoplastics are scalable, but generally mean increased prototyping costs and the use of more specialized equipment, facilities and know how. This consequently limits the accessibility of these materials to biologists attempting to develop a commercial technology.

Overcoming Cell Culture Device Challenges with Flexdym

More recently, new materials are being developed to bridge this gap which exists between academia and industry, and between research and commercial product development. Eden Tech’s Flexdym is one such example. Flexdym has been specifically designed for microfluidics life science applications, and consequently has been experiencing international success in cell culture applications. It is a block co-polymer combining the advantages of both PDMS and thermoplastics. It retains the soft elastomeric properties of PDMS, as well as high optical transparency, certified biocompatibility, and gas permeability. But it has also been developed to provide user-friendly device fabrication for life science researchers. And similarly to thermoplastics, it can be thermo-molded via hot embossing and can be scaled up for commercial purposes.

	Flexdym™	TP	PDMS
Transparency	+++	+	+++
Molding	+++	+	+++
Gas permeability	++	+	+++
Cost ($/kg)	0.2–60	0.2–20	50–200
Reversible bonding	+++	0	−
Irreversible bonding	++	−	+
Scalability	++	++	– –

With a continuously growing community of Flexdym users around the world, publications utilizing this material for device fabrication are appearing in peer-reviewed journals. Recently, a research team from the Biomedical Engineering Department at McGill University, has shown the advantages of using Eden Tech’s Flexdym technology to develop a cell culture device in house, that is easy fabricate and low-cost (Salmon et al.,2020). In less than an hour, they were able to prototype a fully interfaced chip, capable of integrating a reservoir or transducer for cell experiments using continuous flow. The Flexdym polymer used is molded at low temperatures,  presents adhesive properties, is recyclable, and has adjustable thickness. The researchers, led by Hugo Salmon, were able to fabricate their prototype devices and replicate in less than 2 minutes. They also tested its capability to be irrigated by a passive perfusion or connected to a fluidic circuit. In both cases they attested to a leak-free device, which is an improvement since the old techniques.

They additionally used Eden Tech’s Sublym hot embossing machine to mold their prototype in minutes. The set up was conducted outside of a clean room, a feature that is not common in biological laboratory settings.

To learn more about his experience with Flexdym, we sat down with Dr. Hugo Salmon, first author of the paper published in Engineering Reports, earlier this year(Salmon et al.,2020). For him the main advantage of Flexdym for cell culture devices, lies in its ability to build reversibly sealable devices. It allows for switching from an open cell culture device format with (good oxygenation and sampling possibilities), to a perfusable one (with medium renewal and more physiologically relevant). These material capabilities are particularly interesting in biomedical sciences, for the development of alternatives to animal models.

“There are no miracle materials, but Flexdym has a lot of aspects that can be exploited to open up possibilities in microfluidics” – Dr. Hugo Salmon, Researcher at University of Paris

Nowadays, Dr. Salmon is an Assistant Professor and researcher at University of Paris, in the Department of Fundamental Science and Biomedical Engineering. He continues to use Eden Tech’s Flexdym technology for studying extracellular vesicles and neuronal myelination.

“As history shows, I will keep using Flexdym for years”

Indeed, the Flexdym material has proven to be very useful for cell culture device fabrication in both academic labs and industrial R&D settings. Flexdym has several unique features that make it ideal for this application:

• It is optically transparent with high transmittance from 295-800 nm.
• It is certified biocompatible ISO 10993 (part 4, 5, 6, 10 and 11) and UPS Class VI, corresponding to systemic toxicity.
• It is a gas permeable material.
• Flexdym bonding can be conduct at room temperature with no harsh treatments.
• At small scales, Flexdym devices can be produced in a matter of minutes via hot embossing.
• At large scales, Flexdym devices can be mass produced through injection molding.

If you are interested in finding out more about Flexdym, other user-friendly fabrication equipment, or anything else, please contact us!

Bibliography

1. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License
2. Rebecca Skloot, La vie immortelle d’Henrietta Lacks, Ed. Calman-Levy, 2010, p. 78-81
3. Stanton Bruce, Koeppen Bruce  Berne & Levy physiology (6th ed.), Elsevier, 2008, ISBN 978-0-323-04582-7
4. Hugo Salmon, M. Reza Rasouli, Nicholas Distasio and Maryam Tabrizian, Facile engineering and interfacing of styrenic block copolymers devices for low‐cost, multipurpose microfluidic applications, Wiley, 2021, DOI 10.1002/eng2.12361