Organelle Complex Isolation for Intact Mitochondrial Transfer
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Solution Overview
Problem
There is a need for scalable and high surfactant-compatible methods to isolate mitochondria and other organelles from cells while retaining their functional and structural integrity for treating or preventing diseases associated with mitochondrial dysfunction.
Innovation Solution
The method involves isolating organelle complexes, including mitochondria and other organelles, by incubating cells with a surfactant at or above its critical micellar concentration (CMC), followed by tangential flow filtration and centrifugation to remove cytosolic macromolecules, maintaining structural and functional integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional mitochondrial isolation methods are used, then mitochondria can be isolated from cells, but structural and functional integrity is compromised and scalability is limited
Solution Approach 1:
The patent changes the chemical parameters of the isolation medium by formulating a specific buffer composition with controlled pH, ionic strength, and additive concentrations. This optimized buffer system enables scalable isolation while preserving mitochondrial membrane potential and structural integrity, resolving the contradiction between scalability and integrity maintenance.
Solution Approach 2:
The patent introduces a specialized buffer system as an intermediary medium that facilitates gentle separation. This buffer acts as a mediator between the cell lysis step and mitochondrial isolation, providing a controlled environment that maintains mitochondrial functionality while enabling scalable processing.
2Productivity
If surfactants are used to enhance organelle isolation, then isolation efficiency improves, but cytosolic macromolecule contamination increases
Solution Approach 1:
The patent applies local quality by using surfactants selectively and controllably - adding them only when needed during specific isolation steps and using them at optimized concentrations. This localized application enhances isolation efficiency for certain organelle types while minimizing cytosolic contamination through controlled, step-specific surfactant usage.
Solution Approach 2:
The patent carefully controls surfactant concentration parameters and timing in the isolation protocol. By adjusting surfactant addition timing, concentration levels, and exposure duration, the method achieves efficient organelle release while minimizing cytosolic macromolecule contamination, resolving the contradiction between efficiency and purity.
3Quantity of substance
If harsh isolation procedures are applied to increase yield, then mitochondrial quantity increases, but functional capability is lost
Solution Approach 1:
The patent optimizes multiple parameters including buffer composition, temperature control, centrifugation forces, and incubation times to achieve high mitochondrial yield while preserving function. The controlled parameter changes throughout the protocol ensure that mitochondria are recovered in high quantities but maintain their structural and functional integrity.
Solution Approach 2:
The patent incorporates protective measures beforehand by pre-chilling buffers, adding protective agents to the isolation medium, and controlling temperature throughout the protocol. These preemptive protective measures prevent damage during the isolation process, enabling high yield recovery while maintaining functional capability.
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 at least 80% of mitochondria with intact structural integrity and functional capability, enabling superior incorporation into host cells, with a 2-fold to 6-fold greater mitochondrial DNA copy number compared to homogenized mitochondria.
Implementation Method 1
cells contacted with a surfactant at a concentration at or above the critical micellar concentration (CMC) for the surfactant
Implementation Method 2
followed by tangential flow filtration and centrifugation to remove cytosolic macromolecules
Implementation Method 3
followed by tangential flow filtration and centrifugation to remove cytosolic macromolecules
Data Source
AI summary
Disclosed herein include organelle complexes populations. The organelle complexes can comprise mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the organelle complexes are isolated or derived from floating cells and/or frozen cells. In some embodiments, the organelle complexes are isolated or derived from cells contacted with a surfactant at a concentration at or above the critical micellar concentration (CMC) for the surfactant. At least about 80% of the mitochondria of the organelle complexes are capable of maintaining structural integrity in an extracellular environment. Also provided herein are methods for generating first organelle complexes populations.