MSC-Derived Exosome Production with Two-Phase Media
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Solution Overview
Problem
The low production yield of exosomes limits their potential therapeutic applications, particularly in clinical settings, necessitating the development of efficient methods for large-scale production compliant with good manufacturing practices (GMP).
Innovation Solution
A method involving culturing mesenchymal stem cells (MSCs) in a functionally closed bioreactor, initially with human platelet lysate to 80-90% confluency, followed by a switch to lysate-free media, collecting conditioned media fractions, and isolating exosomes using ultracentrifugation and electroporation in a serum-free system to load therapeutic agents like cytokines or nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional exosome production methods are used, then the process is simple, but the production yield is low
Solution Approach 1:
The production process is segmented into distinct phases: initial expansion phase with PLT-containing media, followed by exosome production phase with PLT-free media. This segmentation allows optimization of each phase for its specific purpose, achieving high yield while maintaining manageable complexity
Solution Approach 2:
MSCs are pre-cultured in PLT-containing media to achieve high confluency and activate exosome production pathways before switching to PLT-free media. This preliminary action prepares the cells for maximum exosome secretion during the production phase
2Reliability
If multiple washing steps are added to maintain exosome integrity, then exosome quality is improved, but the production process time increases
Solution Approach 1:
The method extracts and removes PLT from the media after the initial expansion phase, allowing exosomes to be secreted into a clean environment without requiring extensive washing steps. This extraction approach maintains integrity while reducing time
Solution Approach 2:
The production process maintains continuous exosome secretion by keeping cells in a controlled environment with consistent conditions, avoiding interruptions from multiple washing steps while preserving exosome integrity through the PLT-free media system
3Productivity
If PLT is continuously present in media, then cell growth is enhanced, but exosome purity is reduced
Solution Approach 1:
The media composition is segmented into two distinct phases: PLT-containing media for cell growth and expansion, followed by PLT-free media for exosome production. This temporal segmentation ensures both cell growth and exosome purity are optimized in their respective phases
Solution Approach 2:
The media is periodically changed from PLT-containing to PLT-free composition at the appropriate growth stage. This periodic action allows the system to benefit from PLT-induced cell proliferation initially, then switch to purity-optimized conditions for exosome harvest
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 method achieves a high yield of exosomes, up to 15×1012, maintaining their integrity for clinical use without additional washing steps, suitable for treating diseases such as cancer and immune disorders.
Implementation Method 1
culturing the MSCs in a functionally closed bioreactor to confluency (e.g., 75-95% or 80-90% confluency, such as about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%) in media comprising human platelet lysate (PLT)
Implementation Method 2
isolating exosomes from the conditioned media fractions
Implementation Method 3
isolating exosomes using ultracentrifugation and electroporation in a serum-free system to load therapeutic agents like cytokines or nucleic acids
Data Source
AI summary
Provided herein are methods of manufacturing clinical grade exosomes derived from mesenchymal stem cells (MSCs). Further provided are methods of loading the exosomes with therapeutic agents, such as siRNA. Also provided herein are methods of treating diseases by administering the clinical grade exosome.