ABSTRACT
For the process of transient transfection (TTF), DNA is often transported into the cells using polyplexes. The polyplex uptake and the subsequent transient expression of the gene of interest are of great importance for a successful transfection. In this study, we investigated a 3D-printed microfluidic system designed to facilitate direct TTF for suspension of CHO-K1 cells. The results demonstrate that this system achieves significantly better results than the manual approach. Furthermore, the effect of both post-transfection incubation time (t) and temperature (T) on polyplex uptake was explored in light of the membrane phase transitions. Attention was paid to obtaining the highest possible transfection efficiency (TFE), viability (V), and viable cell concentration (VCC). Our results show that transfection output measured as product of VCC and TFE is optimal for t = 1 h at T = 22 °C. Moreover, post-transfection incubation at T = 22 °C with short periods of increased T at T = 40 °C were observed to further increase the output. Finally, we found that around T = 19 °C, the TFE increases strongly. This is the membrane phase transition T of CHO-K1 cells, and those results therefore suggest a correlation between membrane order and permeability (and in turn, TFE).
ABSTRACT
Monoclonal antibodies are increasingly dominating the market for human therapeutic and diagnostic agents. For this reason, continuous methods-such as perfusion processes-are being explored and optimized in an ongoing effort to increase product yields. Unfortunately, many established cell retention devices-such as tangential flow filtration-rely on membranes that are prone to clogging, fouling, and undesirable product retention at high cell densities. To circumvent these problems, in this work, we have developed a 3D-printed microfluidic spiral separator for cell retention, which can readily be adapted and replaced according to process conditions (i.e., a plug-and-play system) due to the fast and flexible 3D printing technique. In addition, this system was also expanded to include automatic flushing, web-based control, and notification via a cellphone application. This set-up constitutes a proof of concept that was successful at inducing a stable process operation at a viable cell concentration of 10-17 × 106 cells/mL in a hybrid mode (with alternating cell retention and cell bleed phases) while significantly reducing both shear stress and channel blockage. In addition to increasing efficiency to nearly 100%, this microfluidic device also improved production conditions by successfully separating dead cells and cell debris and increasing cell viability within the bioreactor.
ABSTRACT
Additive manufacturing (3D printing) enables the fabrication of highly customized and complex devices and is therefore increasingly used in the field of life sciences and biotechnology. However, the application of 3D-printed parts in these fields requires not only their biocompatibility but also their sterility. The most common method for sterilizing 3D-printed parts is heat steam sterilization-but most commercially available 3D printing materials cannot withstand high temperatures. In this study, a novel heat-resistant polyacrylate material for high-resolution 3D Multijet printing was evaluated for the first time for its resistance to heat steam sterilization and in vitro biocompatibility with mouse fibroblasts (L929), human embryonic kidney cells (HEK 293E), and yeast (Saccharomyces cerevisiae (S. cerevisiae)). Analysis of the growth and viability of L929 cells and the growth of S. cerevisiae confirmed that the extraction media obtained from 3D-printed parts had no negative effect on the aforementioned cell types, while, in contrast, viability and growth of HEK 293E cells were affected. No different effects of the material on the cells were found when comparing heat steam sterilization and disinfection with ethanol (70%, v/v). In principle, the investigated material shows great potential for high-resolution 3D printing of novel cell culture systems that are highly complex in design, customized and easily sterilizable-however, the biocompatibility of the material for other cell types needs to be re-evaluated.
