Serum-Free Media Composition for Stable CAR-T Cell Expansion
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Solution Overview
Problem
Existing cell culture methods for genetically engineered cells, such as those expressing chimeric antigen receptors, face challenges with serum variability, L-glutamine degradation, and precipitation, which affect cell viability and expansion.
Innovation Solution
A serum-free media formulation using a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the base medium and a separate frozen supplement with free L-glutamine, along with human proteins like albumin, to stabilize and provide efficient glutamine supply, reducing degradation and precipitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If serum is used in cell culture media, then cell viability is maintained, but serum variability causes inconsistency in cell expansion and therapy production
Solution Approach 1:
The invention removes serum from the cell culture media, extracting the problematic variable component while retaining essential nutrients through alternative sources. This eliminates serum variability that causes inconsistency in cell expansion across different patient samples, while maintaining cell viability through defined serum-free formulation containing dipeptide-L-glutamine, proteins, and other essential components.
Solution Approach 2:
The invention changes the chemical composition parameters of the culture media by replacing serum with defined serum-free components. Specifically, it uses dipeptide-L-glutamine (0.5-5 mM) instead of free L-glutamine, and incorporates specific protein concentrations (e.g., albumin 2.5-7.5 mg/mL) to achieve consistent cell expansion across diverse samples without serum variability.
2Productivity
If free L-glutamine is added to media, then cell growth is supported, but L-glutamine degrades and precipitates affecting cell viability
Solution Approach 1:
The invention changes the chemical form of glutamine from free L-glutamine to dipeptide-L-glutamine (e.g., L-alanyl-L-glutamine). This parameter change in molecular structure prevents degradation and precipitation while maintaining stability in the media at physiological pH and temperature, ensuring consistent cell expansion without loss of glutamine functionality.
Solution Approach 2:
The invention uses dipeptide-L-glutamine, which is a composite structure combining two amino acids (alanine and glutamine). This composite form provides enhanced stability compared to free L-glutamine, preventing degradation and precipitation while still serving as an effective nutrient source for cell growth and expansion.
3Productivity
If high concentration of L-glutamine is used to support cell expansion, then cell growth increases, but precipitation occurs reducing media quality
Solution Approach 1:
The invention changes the solubility parameter of glutamine by using dipeptide form instead of free amino acid form. This allows high concentrations (0.5-5 mM) to be maintained in the media without precipitation, ensuring consistent media composition and quality while supporting high cell expansion rates throughout the culture period.
Data Source
AI summary
Provided herein is a serum-free media for culturing, such as cultivating, preparing and/or producing cells, such as immune cells, such as genetically engineered cells. Also provided is a liquid basal media and frozen supplements that can be used to produce serum-free media. The provided embodiments include methods for producing serum-free media and methods for culturing cells, such as activating, transducing, cultivating or expanded cells, in the presence of serum-free media.