Hydrogel Modulus Affects Proliferation Rate and Pluripotency of Human Mesenchymal Stem Cells Grown in Three-Dimensional Culture

Hydrogels have been used extensively with various cell types in three-dimensional (3D) culture, including with human mesenchymal stem cells (hMSCs). Here, we report on the use of poly(ethylene glycol) (PEG)-conjugated fibrinogen hydrogels to grow bone marrow-derived hMSCs in 3D culture. The initial modulus of the PEG-fibrinogen (PF) hydrogels was varied to study the influence of the stiffness on the proliferation response of the cells growing within. Shear rheology was used to quantify the changes to the initial material properties; the shear storage modulus of the PF was controlled by changing the concentration of synthetic PEG cross-linker, while keeping the fibrinogen concentration constant. Cell culture was performed during a 14-day experiment to quantify the cell proliferation response in the different modulus materials tested. The hMSCs were recovered from the hydrogels by mild enzymatic dissolution, and characterized for proliferation and cell number using cytometry. The results indicate a modulus-dependent response from the cells, and the ability to preferentially define initial hydrogel modulus that favors higher proliferation and multipotency of the hMSCs. Bioactive supplements added to the hydrogels, including exogenous fibronectin (Fn) and von willebrand factor (vWf) were used to further stimulate the proliferation response of the hMSCs in the hydrogel cultures, without altering their multipotency. These insights underscore the importance of mechanical properties in regulating cell proliferation in a 3D culture milieu. The versatility of the hydrogel culture environment and the ability to control mechanical properties for cell-fate determination further highlight the significance of a modular approach when developing materials that ultimately optimize stem cell cultures.