Dental Pulp Cryopreservation from Definitive Teeth for Stable DPSC Isolation
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Solution Overview
Problem
Current methods fail to efficiently generate large numbers of pure dental pulp stem cells (DPSCs) for therapeutic applications due to limited availability, age-related regenerative potential, and challenges in isolation and expansion, leading to senescence and DNA aberrations.
Innovation Solution
A method for cryopreservation and isolation of dental pulp from healthy teeth, followed by rapid thawing and expansion in a specific culture medium, ensuring high viability and chromosomal stability of DPSCs, using a simplified protocol that maintains their pluripotency and differentiation capacity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current methods are used to isolate and expand DPSCs, then some cells can be obtained, but large numbers of pure DPSCs cannot be efficiently generated
Solution Approach 1:
The tooth is segmented into multiple sections to access different dental pulp regions. Multiple holes are drilled at different locations (mesial, distal, buccal, lingual surfaces) to extract pulp from various segments, increasing the total yield of DPSCs while maintaining purity through controlled segmentation of the extraction process
Solution Approach 2:
The tooth undergoes preliminary disinfection and drying before cell extraction. The root canals are treated with sodium hypochlorite and ethanol, and the tooth is dried with absorbent paper before drilling, ensuring that the DPSCs are isolated from a pre-prepared, contamination-free environment, thus improving both quantity and purity
2Reliability
If complex isolation protocols are used, then cell purity may be maintained, but process complexity and cost increase
Solution Approach 1:
Different regions of the tooth are treated with different approaches based on their specific characteristics. The crown and root canals receive specific disinfection treatments, while the dental pulp chambers are accessed through strategically placed holes. Each location is processed according to its local requirements, simplifying the overall protocol while maintaining high purity
Solution Approach 2:
The dental pulp is extracted directly from multiple locations in the tooth using a simple drilling and aspiration method. The pulp tissue is taken out as discrete samples from each drilled location, avoiding complex isolation procedures while ensuring purity through the selective extraction of fresh pulp tissue immediately before cell culture
3Productivity
If DPSCs are expanded in culture, then large numbers of cells can be obtained, but chromosomal instability and DNA aberrations occur
Solution Approach 1:
The dental pulp is extracted and processed immediately before cell culture initiation. The pulp tissue is aseptically removed and placed directly into culture medium without prolonged storage or complex processing, reducing stress on the cells and maintaining chromosomal stability during the critical initial expansion phase
Solution Approach 2:
The culture conditions are optimized with specific parameters including defined medium composition, controlled pH (7.2-7.4), regulated CO2 levels (5%), and controlled temperature (37°C). These parameter optimizations enable robust cell expansion while maintaining chromosomal stability through controlled cellular metabolism and reduced stress responses
4Duration of action of stationary object
If teeth are stored for long periods, then availability for therapy increases, but viability of dental tissue decreases
Solution Approach 1:
The teeth undergo preliminary disinfection of root canals with sodium hypochlorite and ethanol, and thorough drying with absorbent paper before storage. This pre-treatment creates a sterile, low-moisture environment that prevents microbial degradation and maintains dental pulp viability during long-term storage at room temperature or in controlled conditions
Solution Approach 2:
The tooth structure itself serves as a protective cushion for the dental pulp during storage. The intact tooth architecture, combined with the disinfection and drying treatments, creates a protective environment that shields the pulp tissue from degradation, allowing long-term storage while maintaining cell viability for future therapeutic use
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method enables the isolation and expansion of high-quality DPSCs with stable chromosomal integrity, capable of differentiating into multiple cell types, suitable for regenerative therapies, including bone, nervous, and vascular regeneration, while reducing complexity and cost.
Implementation Method 1
cryopreservation solution comprising: a culture medium xeno-free, human serum off the clot type AB, without heparin, dimethyl sulfoxide (DMSO)
Implementation Method 2
The teeth must be cryopreserved in liquid nitrogen (gas phase) and subjected to rapid thawing
Implementation Method 3
The teeth must be cryopreserved in liquid nitrogen (gas phase) and subjected to rapid thawing in a water bath at 37° C.
Data Source
AI summary
The present invention relates to a process for preparation and cryopreservation of dental pulp teeth and products thereof resulting in innovative cellular systems useful for therapeutic application based on the mesenchymal stem cells, so called dental pulp stem cells (DPSCs).The objective of this invention is to provide the most adequate cellular isolates from dental pulp tissue from a tooth of a human subject. Fast expanding populations of DPSCs can be obtained, while maintaining their chromosomal stability, and determined to present the phenotypical and functional characteristics desired of such populations.In another aspect, the present invention provides a novel and simplified method increasing thSpece viability of the dental tissue during the storing and banking. Also, the isolation of DPSCs from these teeth is improved and herein disclosed.Therefore, the present invention is in the field of cell-based therapies, regenerative medicine, and optimized processes for obtaining the desired cell-isolates.