Microfabricated Cell Culture Device for Stem Cell Bioprocessing with Quantative Online Monitoring

Microfabricated bioreactors have been successfully applied to the fields of microbial fermentation and mammalian suspension cell cultures. Key advantages that make microfabricated bioreactors a cost-effective proposition for early bioprocess development include: significant reduction in reagent use, real-time monitoring and control of process variables, ease of sterilisation via disposable polymer technology, reduced labour due to automation, and the capability to rapidly test different processing conditions. Clearly, a microfabricated, adherent culture device that could translate these advantages to stem cell culture would be of great value to the fields of regenerative medicine and cell therapy where more insight into cell culture processes is direly needed. However, a link must be maintained with conventional culture methods and production systems for the purposes of validation and scale up studies. Further, the device must be highly versatile to facilitate evaluation and optimisation of the wide range of process variables that affect stem cell fate. In particular, this versatility must extend beyond the control of the physico-chemical properties of the culture medium to include auto/paracrine factors, growth surfaces, shear forces, and cell seeding densities. Finally, rapid online assessment of the product (cells) is crucial.

We have developed a microfabricated cell culture device (‘micro bioreactor’) where cell culture growth is monitored online. Using human and mouse embryonic stem cell expansion protocols as a model system, we investigated microfluidic design concepts to enable controlled and low-shear perfusion culture of stem cells that maintain the expression of pluripotency markers. Characterization of our microfabricated cell culture device included fluid flow modeling, device reliability testing, verification of autoclavation as a means of sterilization for our device, and also the careful establishment of protocols to prime these devices in a bubble-free fashion. We showed that with our device, we adhere closely to protocols typically employed for stem cell maintenance in laboratory scale vessels, which will facilitate comparison of results, and creates a link with traditional small-scale culture devices for validation and scale-up studies. To understand both the device and the culture data, an image-processing algorithm rapidly detects cell culture confluency (as an indirect method of cell culture growth). The algorithm is also capable to detect changes in cellular morphology, mean fluorescence expression per confluency (for example to establish time course data of differentiation marker expression), and an estimate of cell density. Our image-processing algorithm furthermore provides heat-map like visualization of temporal and spatial cell growth patterns during microfluidic culture. Bulk and peri-cellular oxygen concentrations are monitored in real time, time course data for 6-day cultures of mouse embryonic stem cells (mESCs) is presented and oxygen profiles analysed, and we have also started with efforts to characterize a multiplexed culture device platform.

References:

Jaccard, N., Griffin, L. D., Keser, A., Macown, R. J., Super, A., Veraitch, F. S., Szita, N. (2013). Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images. Biotechnol Bioeng doi:10.1002/bit.25115.

Kirk, T. V., Szita, N. (2013). Oxygen transfer characteristics of miniaturized bioreactor systems. Biotechnol Bioeng 110(4), 1005-1019 doi:10.1002/bit.24824.

Reichen, M., Veraitch, F. S., Szita, N. (2013). Development of a Multiplexed Microfluidic Platform for the Automated Cultivation of Embryonic Stem Cells. J Lab Autom 18(6), 519-529, doi:10.1177/2211068213499917.  

Reichen, M., Macown, R. J., Jaccard, N., Super, A., Ruban, L., Griffin, L., Veraitch, F. S., Szita, N. (2012). Microfabricated Modular Scale-Down Device for Regenerative Medicine Process Development. 7(12), e52246 doi:10.1371/journal.pone.0052246.