Pluripotent Stem Cell Sheet Culture via Anti-CD30 Removal
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Solution Overview
Problem
Current methods for producing sheet-shaped cell cultures from pluripotent stem cell-derived cardiomyocytes face challenges in achieving high quality and viability, particularly when removing undifferentiated cells and cryopreserving the cell population, which affects the efficiency and quality of the graft for clinical applications.
Innovation Solution
A method involving anti-CD30 antibody treatment to remove undifferentiated cells, followed by seeding the cell population at confluence and using a Rho-kinase inhibitor in the sheet-forming incubation medium, along with filtering treatment to enhance cell viability and reduce aggregates, thereby improving the quality and efficiency of the sheet-shaped cell culture production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If undifferentiated cells are removed from the cell population, then the safety and quality of the graft is improved, but the cell population density and viability deteriorate
Solution Approach 1:
The patent applies antibody-based removal to extract undifferentiated cells from the cell population. By using antibodies that specifically bind to markers on undifferentiated cells, the method selectively removes these cells while preserving differentiated cardiomyocytes, thus improving graft safety without excessive loss of viable cells.
Solution Approach 2:
The patent optimizes multiple parameters including antibody concentration, incubation time, temperature, and cell density to achieve effective removal of undifferentiated cells while minimizing damage to the overall cell population. By carefully controlling these parameters, the method maintains high cell viability and population density.
2Manufacturing precision
If cell aggregates are removed by strainer, then the purity of cell population is improved, but the cell loss and reduced viability worsen
Solution Approach 1:
The patent changes the physical parameters of the filtering process by using filters with optimized pore sizes and adjusting the pressure gradient during filtration. These parameter changes enable effective separation of cell aggregates while minimizing mechanical stress on individual cells, thus preserving cell viability.
Solution Approach 2:
The patent introduces enzymatic treatment as an intermediary step before filtration. By using enzymes to gently dissociate cell aggregates, the method facilitates easier filtering without requiring harsh mechanical forces that would damage cell viability.
3Adaptability or versatility
If cryopreservation is performed on the cell population, then the storage and transport capability is improved, but the cell viability and functional properties deteriorate
Solution Approach 1:
The patent applies preliminary protective measures before cryopreservation, including optimization of cryopreservation medium composition and pre-conditioning of cells. These preliminary actions prepare the cells for the freezing stress, reducing membrane damage and maintaining viability after thawing.
Solution Approach 2:
The patent optimizes critical parameters including cooling rate, storage temperature, and thawing rate to minimize ice crystal formation and thermal shock. By controlling these parameters, the method preserves cell viability and functional properties after cryopreservation.
4Manufacturing precision
If multiple processing steps are applied to remove undifferentiated cells and aggregates, then the quality of sheet-shaped cell culture is improved, but the process complexity and time consumption worsen
Solution Approach 1:
The patent combines multiple processing steps into an integrated workflow where antibody treatment, filtration, and cryopreservation are sequentially optimized and combined. This merging reduces the number of separate operations and simplifies the overall process while maintaining high quality outcomes.
Solution Approach 2:
The patent optimizes parameters across all processing steps to reduce total processing time, including minimizing incubation times, optimizing flow rates during filtration, and reducing thawing times. These parameter changes maintain quality while reducing process complexity.
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 results in a high-quality sheet-shaped cell culture with minimized undifferentiated cells, suitable for clinical transplantation, maintaining viability even after cryopreservation, and reducing the risk of perforations and breakages.
Implementation Method 1
performing an anti-CD30 antibody treatment
Implementation Method 2
using a Rho-kinase inhibitor in the sheet-forming incubation medium
Data Source
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AI summary
An object is to provide a method for producing a high-quality graft from pluripotent stem cell-derived differentiation-induced cells, a graft produced by using the method, a method for treating a disease using the graft, and the like. By a method for producing a graft including (a) a step of performing an operation for removing undifferentiated cells in a cell population containing pluripotent stem cell-derived differentiation-induced cells, and optionally a step of freezing the cell population and thereafter thawing the cell population; and (b) a step of seeding the cell population obtained in (a) on a culture substrate and performing graft-forming culture, the above problem has been solved.