Pariduval Mesenchymal Stem Cell Preparation Using Optimized Digestion and Selective Medium
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Solution Overview
Problem
Current methods for preparing and recovering pariduval mesenchymal stem cells (PMSCs) face challenges in promoting cell growth, achieving high concentrations, and maintaining cell viability and biological function, leading to poor quality and variable batch results.
Innovation Solution
A method involving tissue digestion with high-glucose DMEM and collagenase, followed by cultivation in a selective serum-free medium with growth factors, and cryopreservation in a serum-free medium, along with a recovery method using a selective medium to ensure consistent cell viability and growth.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional tissue digestion and cultivation methods are used, then the process is simple, but cells are difficult to climb out of tissue and grow adherently, resulting in low cell concentration and poor quality
Solution Approach 1:
The patent changes the chemical parameters of the digestion solution by specifying precise concentrations of Tryple (40-60%), EDTA (4-8 mM), and collagenase type II (8-12 mg/ml), which significantly improves cell detachment efficiency and growth adherence compared to conventional methods
Solution Approach 2:
The patent applies preliminary action by pre-treating tissues with the optimized digestion solution before cultivation, ensuring cells are properly detached and prepared for adherence, which prevents poor growth during the cultivation phase
2Productivity
If conventional complete medium with FBS is used for cultivation, then the medium is simple to prepare, but it cannot effectively promote PMSC growth, resulting in low cell concentration
Solution Approach 1:
The patent transforms the medium from a simple complete medium with FBS to a complex selective serum-free medium containing specific growth factors (bFGF at 18-25 ng/ml, EGF at 16-22 ng/ml, SCGF at 6-12 ng/ml), which dramatically enhances PMSC proliferation and achieves high cell concentration
Solution Approach 2:
The patent creates a composite medium formulation combining multiple growth factors, amino acids, and supplements in specific concentrations, which works synergistically to promote PMSC growth more effectively than simple complete medium
3Reliability
If conventional cryopreservation method with DMSO is used, then the protocol is standard, but it cannot effectively maintain cell viability and biological function, resulting in variable batch results
Solution Approach 1:
The patent replaces DMSO with Cryosure-DEX-40 at 18-25% concentration in the cryopreservation solution, which significantly improves cell viability and maintains biological function after thawing, eliminating the variability seen with conventional DMSO-based protocols
Solution Approach 2:
The patent uses a disposable cryopreservation solution without DMSO, which avoids the toxicity and variability issues of DMSO while maintaining simplicity in the overall process
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
This approach enhances PMSC growth, purity, and expansion, maintaining cell viability and biological function, resulting in high-quality cells with consistent batch results that effectively inhibit tumor cell proliferation.
Implementation Method 1
subjecting the tissue blocks to digestion with a tissue digestion solution under constant temperature oscillation, where the tissue digestion solution is a high-glucose DMEM that includes a Tryple-ethylenediaminetetraacetic acid (EDTA) enzyme of 40% to 60% in volume concentration and collagenase type II of 8 mg/ml to 12 mg/ml
Implementation Method 2
centrifuging to obtain a first precipitate
Implementation Method 3
adding a cryopreservation solution to a resulting precipitate, and cryopreserving in a liquid nitrogen tank after programmed cooling
Data Source
AI summary
The present disclosure discloses a preparation method and a recovery method of pariduval mesenchymal stem cells (PMSCs). In the preparation method, a high-glucose Dulbecco's Modified Eagle Medium (DMEM) that includes a Tryple-ethylenediaminetetraacetic acid (EDTA) enzyme of 40% to 60% in volume concentration and collagenase type II of 8 mg/ml to 12 mg/ml is used as a tissue digestion solution to digest tissue blocks, which facilitates PMSCs to climb out of the tissue blocks and grow adherently; and a serum-free DMEM is adopted as a selective medium to terminate the digestion and resuspend PMSCs, which helps to improve a purity of PMSCs, accelerate the growth of PMSCs, and achieve the rapid expansion of PMSCs in vitro.