Proper pretreatment of BMSCs before in vivo transplantation enhances its proliferation, survival, and osteogenic activity and is thus essential for the further clinical application of BMSCs [22, 23]. This study provides an in vitro pretreatment method for BMSCs(combination of interrupted hypoxia pretreatment and three-dimensional culture) for efficient transplantation in vivo.
The cell behaviors change during the in vitro culture due to the significant changes in the growth environment[24, 25]. Moreover, only a few primary MSCs can be directly obtained from the body. However, a sufficient number of cells can be obtained by generating 3D culture microspheres after a certain period of in vitro culture [26, 27]. Herein, although the cells were cultured in a non-physiological monolayer during cell amplification, hypoxic pretreatment was used to simulate the ischemia and hypoxia environments in vivo a. As a result, cells could survive in an anoxic environment, thus ensuring that the cells maintain amplification and proliferate and survive in an anoxic environment. However, the adverse effects of hypoxia on cells were not ignored. The results showed that continuous hypoxia had a certain inhibitory effect on the osteogenic differentiation of BMSCs. Therefore, the effects of different hypoxic pretreatment methods on cells were analyzed, the traditional hypoxic culture method was optimized, and finally the intermittent hypoxic culture method was selected for further analysis. BMSCs could proliferate and survive in an anoxic environment and maintain osteogenic differentiation to the maximum extent under the intermittent hypoxic culture method. Moreover, all aspects of biological properties of cells could be balanced, thus suitable in subsequent application.
The 3D cell culture technology can simulate the physiological environment of cells in organisms and maintain the contact between cells. Therefore, it is more suitable for maintaining biological behavior of isolated cells. Currently, 3D cell culture techniques are grouped into two main categories: those with and without scaffolds . The 3D cell culture techniques with scaffolds, such as hydrogel technology, mainly rely on the support and guidance of natural or artificial scaffolds to enable cell growth on scaffolds and are widely used at present . The 3D cell culture techniques without scaffolds mainly include suspension drop technology, suspension technology, and ultra-low adhesion surface. Stent-free-3D cell culture technology mainly uses a specific culture plate to suspend and aggregate cells into a 3D sphere [30, 31]. Compared with 3D cell culture technology with scaffolds, 3D cell culture without scaffolds can obtain a single cell sphere without dealing with cell scaffolds. Moreover, the volume and number of cell spheres are easier to control when using 3D cell culture without scaffolds. Herein, cell suspension was added above the hole of GravityPLUS™ automatic suspension plate. The geometry of the hole plate guides the cells and culture medium through the hole to form a stable suspension. Each hole forms a droplet, and each droplet contains a sphere whose size can be controlled by the density of the inoculated cells. GravityPLUS™ automatic hanging drop board can be sterilized and reused, thus significantly saving costs. As a result, this stent-free 3D cell suspension culture technique was used in the in vitro cell experiments.
Although the 3D culture represented by microspheres simulates the 3D growth environment of cells in vivo, the influence of large microspheres on cell apoptosis cannot be ignored. The diameter of microspheres affects plasma osmotic pressure, oxygen content, and nutrient supply in cell culture microspheres . In this study, the cells at the center of the microspheres rapidly underwent apoptosis in a short period when the diameter of BMSCs microspheres was too large. Although increasing the inoculation density can successfully generate microspheres with a larger volume, it does not necessarily indicate that the cells can maintain a high survival rate. Therefore, BMSCs microspheres should have a certain range of diameter to improve good cell performance and efficiency of cell application. Moreover, waste resources are minimized, thus obtaining a good practical application and improving the clinical application potential of BMSCs, such as seed cells.