Cells are cultured in laboratories around the world every day. They are typically used in a large number of applications, including screening of new drug-leads, cytotoxicity testing of new biomaterials or chemicals, cancer research, stem cell research, tissue engineering, gene therapy, and for the production of cell culture products such as vaccines, hormones, enzymes, antibodies, and other therapeutics. Can it be done reliably in 3D, on a large scale, with minimal damage to harvested cells?
Culturing cells in a laboratory is conventionally done using a two-dimensional (2D) cell culture tray. It has some pitfalls though. It is highly labour-intensive and does not replicate the complexity of the three-dimensional (3D) environment of natural tissues. “Cells grown on 2D surfaces tend to lose their native characteristics and display vast differences to their in vivo counterparts. The methods used to remove these cells from the surface that they have grown on are also damaging to the cells,” explains Avashnee Chetty, who heads up the CSIR’s team developing an automated cell culturing system.
From 2D to 3D
Chetty says that the need existed to develop a system for culturing cells that would allow them to grow in 3D, as they would do in their natural environment, without too much human intervention. This would reduce the chances of contaminating the cells. The system also needed to spontaneously release the cells from the surface they have grown on without damaging them. This was achieved through the CSIR’s cell culturing bioreactor, a cost-effective bench-top system that allows for non-invasive, high-density, 3D culture of adherent cells.
Chetty describes the bioreactor: “It is a small cylindrical glass vessel housed in a stainless steel casing, which is perfused with oxygenated media with a peristaltic pump allowing sufficient oxygen and nutrient delivery to the cells. It contains stacked scaffolds – highly porous 3D non-woven matrices onto which the cells are seeded and where they grow.”
The scaffolds are made from a3D, highly porous, non-woven material that was manufactured at the CSIR’s facilities in Port Elizabeth. The non-woven material has been treated with a thermos-responsive polymer. The polymer changes from a hydrophilic (water-loving) to a hydrophobic state depending on the temperature. At temperatures above 32°C, cells happily attach to the polymer-covered scaffolds and grow in 3D into all the spaces of the porous scaffold. If the temperature is lowered to around 20°C, the polymer that they have attached to, changes its state and releases the cells in clusters from the scaffolds without damaging it.
“What is important is that the bioreactor delivers large numbers of 3D cell clusters by simply cooling the culture media, and without damaging the cell membrane,” says Chetty. “These cells also have enhanced metabolic activity and gene expression, which we have demonstrated in our labs and would be far more valuable for use in, for example, drug screening,” says CSIR candidate researcher, Claire Rossouw.
A perfused bioreactor system containing a 3D thermos-responsive scaffold allowing for high-density, non-invasive culture of adherent cells does not exist anywhere in the world. The novelty of the CSIR’s system is the combination of three key
components into one device, that is, the thermo-responsive cell release mechanism, the use of a 3D scaffold, and a perfused bioreactor. The CSIR’s cell culturing bioreactor has been tested using mammalian hepatocyte cells (liver cells). It has demonstrated that cells attach and grow on the scaffold, easily yielding high density cultures (approximately 45 million cells were achieved after 14 days of continuous culture with 10 times increase in cell number). Says Chetty: “We have also proven the bioreactor’s ability to release the cells spontaneously without requiring enzymes, and we have shown that even after 21 days of continuous culture, the cells remain healthy and viable.”
The CSIR’s cell culture system received a positive International Preliminary Report on Patentability from the EPO (European Patent Office), and is patented in South Africa (2010/02052), with a patent application pending in the USA (12,678,450). “With respect to bioreactors and automated systems, there are several types commercially available. However, destructive cell release methods are often still used to release confluent cells. The CSIR’s cell culturing bioreactor combines the advantages of the use of a 3D scaffold, the thermos-responsive polymer and a perfused bioreactor to provide a superior system for cell culture which currently is not available,” explains Chetty.
Work to be done
According to Chetty, the CSIR hopes to sell the cell culturing bioreactor in its final form as an automated cell culture system. “We can develop the bioreactor further so that it becomes an automated cell culture system. To get it to this final form, we would need to optimise the system for high-density cell culture and optimal cell release, and automate it. This
will include incorporating a programmable logic control system to automate the manual steps, as well as a user interface.”