Researchers from the University of Osaka have developed a novel 3D chimeric protein scaffold called Chimera-511, which significantly improves the proliferation of human induced pluripotent stem cells (hiPSCs). This functionalized fibrin gel mimics the physiological interactions of pluripotent stem cells with their surrounding extracellular matrix, making it a more effective tool for biotherapeutic manufacturing and regenerative medicine.
Traditionally, fibrin hydrogels have been utilized as two-dimensional scaffolds for in vitro cell culture. The innovation of Chimera-511 allows for the growth of hiPSCs in three dimensions, thus facilitating the engineering of complex tissue structures that better replicate human tissues, including organoids.
The newly designed scaffold combines the beneficial properties of fibrin gels, such as low immune rejection risk and the ability to be autologously prepared, with the high integrin-binding capacity of laminin. These features are crucial for the efficient expansion of undifferentiated hiPSCs. Senior researcher Kiyotoshi Sekiguchi, PhD, emphasizes that Chimera-511 effectively bridges the gap between in vivo environments and conventional two-dimensional cultures, enabling more complex tissue structures and functions.
According to Sekiguchi, “Chimera-511 could serve as a promising substitute for the current gold standard for 3D stem-cell culture.” He notes that this fibrin gel is the first of its kind to accurately replicate the physiological interactions of pluripotent stem cells with the extracellular matrix. Its potential for clinical application is significant, as Chimera-511 can be produced using cGMP-banked CHO cells, and fibrin gels have a long history of use in surgical applications.
The creation of Chimera-511 involved a direct linkage between the N-terminal self-polymerization domain of fibrinogen and the C-terminal integrin-binding domain of LM-511 through heterotrimeric coiled-coil domains. The researchers ensured that Chimera-511 could anchor to fibrin fibrils via knob and hole interactions, which were essential for hiPSC proliferation. Without these interactions, the proliferation of hiPSCs was minimal.
In their experiments, the team observed that hiPSCs seeded into Chimera-511 experienced more than a 25-fold increase in cell numbers, plateauing around a concentration of 50 nM. In contrast, cells cultured with LM-511 did not exhibit any proliferation. This method of proliferation under three-dimensional conditions allows for serial passaging and scaling up, which is advantageous for biomanufacturers.
After completing five 3D-to-3D serial passages, the number of cells surged from a baseline of 1 million to a staggering 1014. Notably, the cells retained their differentiation capabilities after each passage without initiating differentiation prematurely.
The researchers assert that the fibrin gel functionalized with Chimera-511 is the first 3D cell culture scaffold to display the complete integrin-binding activity of laminin, distinguishing it from mouse tumor-derived basement membrane extracts, which are currently considered the gold standard. This advancement eliminates concerns related to batch variability and xenographic issues associated with mouse-derived extracts.
