Non-enzymatic Stem Cell Harvesting via Sodium Citrate Chelation
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Solution Overview
Problem
Current methods for harvesting and passaging human pluripotent stem cells, such as mechanical scraping and enzymatic treatments, are labor-intensive, inefficient, and can lead to low viability and karyotypic instability, especially in multilayer cell culture vessels, limiting scalability and commercial production.
Innovation Solution
A non-enzymatic formulation using sodium citrate, optimized for osmolarity and Ca2+ chelator concentration, allows for high-yield harvesting and passaging of pluripotent stem cells as clusters without mechanical scraping, maintaining high viability and karyotypic stability, suitable for multilayer cell culture vessels and scalable manufacturing processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If mechanical scraping is used for harvesting hPSCs, then cells can be detached from the culture surface, but cell viability decreases and the process becomes labor-intensive
Solution Approach 1:
The patent replaces mechanical scraping with a chemical solution-based detachment method. The formulation containing sodium citrate and other components chemically disrupts cell-adherens junctions and cell-matrix interactions, allowing cells to be harvested by gentle rinsing rather than mechanical force, thereby maintaining high cell viability while achieving efficient detachment
Solution Approach 2:
The patent introduces a chemical intermediary formulation that mediates between the cell culture surface and the harvesting process. This formulation acts as a buffer that facilitates cell detachment through chemical interactions rather than direct mechanical contact, serving as an intermediary that protects cells from mechanical damage while enabling efficient harvest
2Productivity
If enzymatic treatment is used for cell detachment, then cells can be harvested, but residual enzymes require additional washing steps and may affect cell quality
Solution Approach 1:
The patent extracts and eliminates the need for enzymatic treatments from the cell detachment process. By using a non-enzymatic chemical formulation, the patent removes the harmful element (enzymes) that would require additional washing steps, simplifying the overall process while maintaining effective cell detachment capability
Solution Approach 2:
The patent employs a disposable chemical formulation that performs its function and can be easily removed or diluted, replacing the need for complex enzymatic systems that require careful control and removal. The formulation acts as a single-use solution that simplifies the process workflow
3Adaptability or versatility
If conventional passaging methods are used in multilayer vessels, then cells can be passaged, but scalability is limited due to inability to access cells for scraping
Solution Approach 1:
The patent replaces mechanical scraping, which is incompatible with multilayer vessels, with a chemical detachment method that can be applied to multilayer configurations. The formulation can penetrate and act on cells in multilayer vessels without requiring physical access for scraping, enabling scalability while maintaining ease of operation
Solution Approach 2:
The patent creates a universal passaging method that works across different culture formats including traditional flasks and scalable multilayer vessels. The chemical formulation provides a multi-functional solution that adapts to various vessel types and scales, eliminating the need for format-specific techniques
4Productivity
If single-cell passaging is performed, then cells can be distributed, but cloning efficiency is low and karyotypic stability is compromised
Solution Approach 1:
The patent applies local quality control by allowing cells to detach and be passaged as small clusters rather than complete single cells. This partial clustering maintains the benefits of distribution while preserving enough cellular context to maintain karyotypic stability and cloning efficiency, optimizing both productivity and genetic integrity
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 sodium citrate formulation enables over 90% harvesting with >90% viability, maintaining pluripotency and karyotypic stability, reducing labor intensity and process time, and supporting scalable production and cryopreservation with high post-thaw recovery and plating efficiency.
Implementation Method 1
The present invention provides a non-enzymatic reagent formulation and a method of harvesting and subsequently passaging pluripotent stem cells as clusters with high yield and high post-detachment cell viability... the formulation includes, for example, sodium citrate... which disrupts the cell-surface bond and cell-cell association by chelating/sequestering Ca2+, the divalent cation required for cell-surface and cell-cell binding
Data Source
AI summary
Formulations and methods are disclosed for the harvesting and subsequent passaging of human pluripotent stem cells without the use of enzymes and/or scraping to dislodge cells from cell culture vessels. The formulations and methods permit the harvesting of cells as large clusters from the surface of various cell culture vessels including multilayer cell culture vessels. Further, the formulations and methods provide high yields of harvested cells for subsequent passaging and high post-harvest cell viability. Pluripotent stem cells passaged with the formulations according to the methods remain undifferentiated and express typical stem cell markers, while, at the same time, they retain the differentiation capability and are able to differentiate into the cells in all three germ layers and generate teratomas, even after numerous rounds of harvesting and passaging. These hPSCs also maintain normal karyotype after passaged with the formulations for extended period of time.