Research progress on optimization of in vitro isolation, cultivation and preservation methods of dental pulp stem cells for clinical application

Abstract

Due to high proliferative capacity, multipotent differentiation, immunomodulatory abilities, and lack of ethical concerns, dental pulp stem cells (DPSCs) are promising candidates for clinical application. Currently, clinical research on DPSCs is in its early stages. The reason for the failure to obtain clinically effective results may be problems with the production process of DPSCs. Due to the different preparation methods and reagent formulations of DPSCs, cell characteristics may be affected and lead to inconsistent experimental results. Preparation of clinical-grade DPSCs is far from ready. To achieve clinical application, it is essential to transit the manufacturing of stem cells from laboratory grade to clinical grade. This review compares and analyzes experimental data on optimizing the preparation methods of DPSCs from extraction to resuscitation, including research articles, invention patents and clinical trials. The advantages and disadvantages of various methods and potential clinical applications are discussed, and factors that could improve the quality of DPSCs for clinical application are proposed. The aim is to summarize the current manufacture of DPSCs in the establishment of a standardized, reliable, safe, and economic method for future preparation of clinical-grade cell products.

Keywords: 3D cell culture; clinical application; cryopreservation; dental pulp stem cells; serum-free culture; stem cell property.

FIGURE 1

Schematic representation of optimization studies for the extraction, culture, cryopreservation, and resuscitation of DPSCs. To prepare DPSCs that conform to standards and maintain stem cell characteristics for clinical applications, optimization of many critical steps is essential. The extraction of DPSCs can be achieved using enzymatic digestion, tissue block culture, or a combination of enzymatic digestion and tissue block culture. Culture optimization of DPSCs includes not using fetal bovine serum (FBS), cultivation under low oxygen conditions, transitioning from two-dimensional (2D) culture to three-dimensional (3D) culture that is more suitable for cellular physiological environments, pathway regulation, and gene introduction for immortalization of cells. Cryopreservation optimization of DPSCs includes not using dimethyl sulfoxide (DMSO) or reducing its concentration, not using FBS, using a simpler uncontrolled-rate freezing method, freezing with a 3D scaffold, and directly freezing teeth or dental pulp tissue. Resuscitation optimization of DPSCs includes the addition of growth factors or other additives. Figure 1 was created with BioRender.com.

FIGURE 2

3D Culture of DPSCs. The 3D culture of DPSCs includes two methods: scaffold culture and scaffold-free culture. Scaffold culture commonly employs materials such as hydrogels, collagen fibers, alginate, and decellularized matrix. Special techniques used for scaffold-free culture include hanging drop method, magnetic levitation method, spontaneous spheroid formation, and rotational culture method. 3D cell culture can simulate the interactions between cells and the extracellular matrix, as well as cell-to-cell interactions, more closely resembling the physiological environment of cells. This is beneficial for gene expression and signal transduction within the cells and can be used for tissue engineering, organoid construction, drug screening, and other applications. Figure 2 was created with BioRender.com.

FIGURE 3

Optimization of DPSCs Cryopreservation. The traditional method for DPSCs cryopreservation involves programmed freezing in the presence of a cryoprotectant, usually, dimethyl sulfoxide (DMSO), combined with fetal bovine serum (FBS). Optimization methods include reducing the concentration of DMSO to 3% or replacing it with other low-toxicity cryoprotectants, avoiding the use of hazardous FBS, and adopting a convenient and rapid direct freezing method, followed by storage at −80°C or in liquid nitrogen at −196°C. In addition, 3D scaffold cryopreservation can eliminate the long waiting period for patients with acute diseases, while direct cryopreservation of teeth or dental pulp tissue can reduce costs and the risk of tissue contamination. Figure 3 was created with BioRender.com.