High-density adherent culture of CHO cells using rolled scaffold bioreactor.

Rapid expansion of biopharmaceutical market calls for more efficient and reliable platforms to culture mammalian cells on a large scale. Stirred-tank bioreactors have been widely used for large-scale cell culture. However, it requires months of trials and errors to optimize culture conditions for each cell line. In this article, we extend our earlier studies on rolled scaffold (RS) bioreactors for high-density adherent cell culture and report two new implementations of RSs with greatly enhanced mass-manufacturability, termed as Mesh-RS and Fiber-RS. CHO-K1 cells were successfully expanded in Mesh-RS and Fiber-RS bioreactors with an average growth rate of 1.09 ± 0.04 1/day and 0.95 ± 0.07 1/day, which were higher than those reported in similar studies. Fiber-RS bioreactor exhibited a very high cell density of 72.8 × 106 cells/ml. Besides, a dialyzer was integrated into the RS bioreactor to remove cellular waste and to replenish nutrients without disturbing the cells. By collecting the dialyzed media separately, the dialysis efficiency was significantly improved. In conclusion, the developed RS bioreactor has a strong potential to provide a highly reliable and easily scalable platform for large-scale cell culture in the biopharmaceutical industry.

Development of rolled scaffold for high-density adherent cell culture.

Bioreactors for large-scale culture of mammalian cells are playing vital roles in biotechnology and bioengineering. Various bioreactors have been developed, but their capacity and efficiency are often limited by insufficient mass transfer rate and high shear stress. A rolled scaffold (RS) is a fully defined scaffold for high-density adherent culture of mammalian cells. The RS is a polymer film with spacers, that is rolled into a cylinder with a pre-determined gap between each turn. Cells are cultured on its inner surfaces, while media flows through the gap. The RS exhibits high surface-area-to-volume ratio over 100 cm2/mL and can transport nutrients and gases with significantly reduced shear stress via convection in a unidirectional laminar flow, rather than diffusion and random turbulent flow as in stirred-tank bioreactors. In this paper, we expanded Chinese Hamster Ovary cells with RS bioreactors and demonstrated cell culture density over 60 million cells/mL with a growth rate higher than conventional suspension culture. Besides, murine embryonic stem cells were successfully expanded without losing their pluripotency. The RS will provide an affordable, scalable, and reliable platform for large-scale culture of recombinant cells in biopharmaceutical industries and shear-sensitive stem cells for tissue engineering.

Hollow microcarriers for large-scale expansion of anchorage-dependent cells in a stirred bioreactor.

With recent advances in biotechnology, mammalian cells are used in biopharmaceutical industries to produce valuable protein therapeutics and investigated as effective therapeutic agents to permanently degenerative diseases in cell based therapy. In these exciting and actively expanding fields, a reliable, efficient, and affordable platform to culture mammalian cells on a large scale is one of the most vital necessities. To produce and maintain a very large population of anchorage-dependent cells, a microcarrier-based stirred tank bioreactor is commonly used. In this approach, the cells are exposed to harmful hydrodynamic shear stress in the bioreactor and the mass transfer rates of nutrients and gases in the bioreactor are often kept below an optimal level to prevent cellular damages from the shear stress. In this paper, a hollow microcarrier (HMC) is presented as a novel solution to protect cells from shear stress in stirred bioreactors, while ensuring sufficient and uniform mass transfer rate of gases and nutrients. HMC is a hollow microsphere and cells are cultured on its inner surface to be protected, while openings on the HMC provide sufficient exchange of media inside the HMC. As a proof of concept, we demonstrated the expansion of fibroblasts, NIH/3T3 and the expansion and cardiac differentiation of human induced pluripotent stem cells, along with detailed numerical analysis. We believe that the developed HMC can be a practical solution to enable large-scale expansion of shear-sensitive anchorage-dependent cells in an industrial scale with stirred bioreactors.

Measurement of cell traction force with a thin film PDMS cantilever.

Adherent cells produce cellular traction force (CTF) on a substrate to maintain their physical morphologies, sense external environment, and perform essential cellular functions. Precise characterization of the CTF can expand our knowledge of various cellular processes as well as lead to the development of novel mechanical biomarkers. However, current methods that measure CTF require special substrates and fluorescent microscopy, rendering them less suitable in a clinical setting. Here, we demonstrate a rapid and direct approach to measure the combined CTF of a large cell population using thin polydimethylsiloxane (PDMS) cantilevers. Cells attached to the top surface of the PDMS cantilever produce CTF, which causes the cantilever to bend. The side view of the cantilever was imaged with a low-cost camera to extract the CTF. We characterized the CTF of fibroblasts and breast cancer cells. In addition, we were able to directly measure the contractile force of a suspended cell sheet, which is similar to the CTF of the confluent cell layer before detachment. The demonstrated technique can provide rapid and real-time measurement of the CTF of a large cell population and can directly characterize its temporal dynamics. The developed thin film PDMS cantilever can be fabricated affordably and the CTF extraction technique does not require expensive equipment. Thus, we believe that the developed method can provide an easy-to-use and affordable platform for CTF characterization in clinical settings and laboratories.

Fabrication and characterization of self-folding thermoplastic sheets using unbalanced thermal shrinkage.

Self-folding or micro-origami technologies are actively investigated as a novel manufacturing process to fabricate three-dimensional macro/micro-structures. In this paper, we present a simple process to produce a self-folding structure with a biaxially oriented polystyrene sheet (BOPS) or Shrinky Dinks. A BOPS sheet is known to shrink to one-third of its original size in plane, when it is heated above 160 °C. A grid pattern is engraved on one side of the BOPS film with a laser engraver to decrease the thermal shrinkage of the engraved side. The thermal shrinkage of the non-engraved side remains the same and this unbalanced thermal shrinkage causes folding of the structure as the structure shrinks at high temperature. We investigated the self-folding mechanism and characterized how the grid geometry, the grid size, and the power of the laser engraver affect the bending curvature. The developed fabrication process to locally modulate thermomechanical properties of the material by engraving the grid pattern and the demonstrated design methodology to harness the unbalanced thermal shrinkage can be applied to develop complicated self-folding macro/micro structures.