Hepatocyte Pooling Method Reducing Cryopreservation Stress
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Solution Overview
Problem
Existing methods for pooling and cryopreserving hepatocytes from multiple donors subject cells to physical and chemical stress, leading to cell loss and reduced viability due to density gradient centrifugation and the use of toxic cryopreservative solutions like DMSO.
Innovation Solution
A method involving thawing hepatocytes from multiple sources, pooling them in a preservation solution to dilute DMSO, centrifuging without a density gradient to pellet viable and non-viable cells, and then cryopreserving them in a controlled manner to minimize stress, followed by density gradient fractionation before use to separate viable cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If density gradient centrifugation is used to separate viable cells during pooling, then cell separation precision is improved, but cell loss increases and viability decreases due to mechanical stress
Solution Approach 1:
The patent performs density gradient centrifugation after the final thaw step, which is the preliminary action taken immediately before experimental use. This timing allows cells to be separated just in time, minimizing the duration of stress exposure while still achieving the necessary separation precision for obtaining viable cells.
Solution Approach 2:
The patent eliminates the density gradient centrifugation step from the pooling process itself, rushing through the pooling without this stress-inducing separation. Instead, only simple centrifugation is performed during pooling, and the density gradient step is skipped entirely during this phase, reserving it only for the final preparation before use.
2Measurement precision
If hepatocytes are pooled from multiple donors to represent population diversity, then measurement accuracy of liver function is improved, but process complexity increases
Solution Approach 1:
The patent merges multiple donor hepatocyte samples into a single pooled sample using a simplified protocol. By combining cells from multiple donors in one standardized process without intermediate density gradient separations, the method achieves population-representative measurements while reducing overall process complexity compared to handling each donor separately.
Solution Approach 2:
The patent changes the centrifugation parameters during pooling to optimize for cell recovery rather than separation. By using lower centrifugal forces and simpler protocols tailored to the specific properties of cryopreserved hepatocytes, the method simplifies the pooling process while maintaining the ability to pool diverse donor samples effectively.
3Reliability
If cryopreservative solutions containing DMSO are used throughout the pooling process, then cell preservation is improved, but cell toxicity increases leading to cell death
Solution Approach 1:
The patent extracts or removes the density gradient centrifugation step from the pooling process, eliminating the source of mechanical stress that compounds the chemical stress from DMSO. This removal reduces the cumulative harmful effects on cells while maintaining cryopreservation effectiveness.
Solution Approach 2:
The patent applies density gradient centrifugation only partially - specifically, only after the final thaw and immediately before experimental use, rather than during the pooling process itself. This partial application provides just enough separation precision when needed while avoiding excessive stress exposure that would compound DMSO toxicity.
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 increases the recovery of viable hepatocytes, reducing exposure to mechanical and chemical stress, and allows for a more accurate representation of liver function by maintaining a higher number of viable cells for experimentation.
Implementation Method 1
centrifuging the pooled hepatocytes to cause pelleting of both viable and non-viable hepatocytes
Implementation Method 2
density gradient fractionation to separate viable and non-viable cells immediately prior to performing experiments
Data Source
Figure 1A
Figure 1B
Figure 2A~2F
AI summary
The present invention relates to a novel method for the preparation of a pooled or mixed population of cryopreserved cells (e.g. hepatocytes). In particular, the invention entails the rapid thaw of cells (e.g. hepatocytes), donated from a single individual, which are mixed to create a heterogeneous population and then cryopreserved. The invention also concerns preparations of multi-cryopreserved cells to increase viability prior to immediate use. The process entails reducing exposure to chemical and physical stresses to increase the resultant number of viable cells.