Cryopreserved Apheresis NK Cell Expansion Without Density Separation
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Solution Overview
Problem
Existing methods for producing natural killer (NK) cells from cryopreserved apheresis samples are inefficient and result in reduced cell viability and functionality, making them less effective as immunotherapeutic agents compared to fresh samples, and the use of density gradient separation further complicates the process, reducing NK cell yield and increasing processing time.
Innovation Solution
A method for producing expanded NK cells from cryopreserved apheresis samples that omits density gradient separation, involving thawing, depleting CD3+ cells, and culturing with membrane-bound IL-21 to expand NK cells, resulting in higher yields and equivalent functionality to fresh samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If density gradient separation is used to process cryopreserved apheresis samples, then PBMC isolation is achieved, but NK cell yield is reduced and processing time is increased
Solution Approach 1:
The patent extracts and eliminates the density gradient separation step from the processing protocol. By removing this complex step, the method directly thaws cryopreserved apheresis samples and proceeds to CD3+ cell depletion and NK cell expansion, thereby simplifying the process while improving NK cell yield and reducing processing time.
Solution Approach 2:
The patent segments the processing workflow into distinct functional steps: thawing, CD3+ cell depletion, and NK cell expansion. This segmentation allows each step to be optimized independently, with the removal of the density gradient step allowing direct progression from thawing to depletion to expansion, improving efficiency.
2Adaptability or versatility
If cryopreserved apheresis samples are used, then storage flexibility is improved, but cell viability and functionality are reduced
Solution Approach 1:
The patent applies preliminary actions by immediately thawing the cryopreserved samples and proceeding directly to CD3+ cell depletion and NK cell expansion without intermediate steps. This rapid sequential processing minimizes the time cells are exposed to suboptimal conditions, thereby maintaining viability and functionality while utilizing the storage flexibility of cryopreserved samples.
Solution Approach 2:
The patent changes the processing parameters by eliminating the density gradient separation step and implementing a direct thaw-and-expand protocol. This parameter change in the processing methodology allows cryopreserved samples to maintain their storage flexibility advantage while achieving comparable or superior cell viability and functionality through optimized downstream processing.
3Productivity
If conventional processing methods are used, then standard protocols are followed, but processing time is increased and cost is increased
Solution Approach 1:
The patent extracts and removes the density gradient separation step from the conventional processing protocol. This elimination directly reduces processing time and associated costs while maintaining or improving NK cell yield and functionality, thereby enhancing manufacturing efficiency.
Solution Approach 2:
The patent implements continuous useful action by transitioning directly from thawing to CD3+ cell depletion to NK cell expansion without interruption or unnecessary intermediate steps. This continuous processing approach maximizes productivity by eliminating idle time and reducing overall processing duration.
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 achieves higher NK cell yields and equivalent functionality compared to fresh samples, reducing processing time and cost, and allows for multiple manufacturing campaigns from a single donor, enhancing manufacturing efficiency and reducing batch variability.
Implementation Method 1
culturing the PBMCs in a medium with membrane-bound IL-21 to expand NK cells contained in the PBMCs
Data Source
AI summary
Provided herein are methods of producing expanded natural killer cells from cryopreserved blood samples, compositions comprising natural killer cells produced by the methods, and uses thereof.