Femtosecond Laser Cell Reprogramming Without Viral Vectors
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Solution Overview
Problem
Current methods for reprogramming cells, such as viral reprogramming, are limited by the use of viruses, low efficiency, lengthy production times, inability to produce reprogrammed cells in tissue assemblies, and impossibility of viral reprogramming in patient tissues, hindering clinical applications.
Innovation Solution
A method using a cocktail of transcription factors and microRNA introduced via a femtosecond laser to create transient membrane openings in cells, allowing virus-free optical reprogramming, enabling efficient and rapid conversion of cells into induced pluripotent stem cells or other cell types, including direct reprogramming in tissue structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral reprogramming methods are used to introduce transcription factors into cells, then reprogramming can be achieved, but the use of viruses limits clinical application and increases safety risks
Solution Approach 1:
The patent extracts the harmful viral carrier from the reprogramming process, introducing transcription factors directly into cells without using viruses. This eliminates the safety risks associated with viral integration while maintaining the reprogramming function, thereby improving both reliability and clinical applicability.
Solution Approach 2:
The patent uses an intermediary delivery mechanism (non-viral vector or direct introduction method) to transport transcription factors into cells. This intermediary approach avoids the harmful effects of viruses while still achieving efficient delivery of reprogramming factors, resolving the contradiction between safety and effectiveness.
2Productivity
If traditional reprogramming methods are used, then cells can be reprogrammed, but the production process is too time-consuming and takes at least one week
Solution Approach 1:
The patent employs preliminary optimization of transcription factor delivery and cell culture conditions to accelerate the reprogramming process. By pre-preparing optimized reprogramming cocktails and establishing efficient delivery protocols before actual reprogramming, the method reduces the overall production time from at least one week to a more rapid timeframe.
Solution Approach 2:
The patent changes key parameters of the reprogramming process, including transcription factor concentrations, delivery timing, and cell culture conditions, to optimize reprogramming efficiency and reduce the time required. These parameter optimizations enable faster reprogramming while maintaining high-quality iPS cell generation.
3Productivity
If viral reprogramming is used, then transcription factors can be introduced into cells, but the conversion efficiency is low with too few viable cells
Solution Approach 1:
The patent replaces the mechanical/viral injection system with a non-viral introduction method that is less disruptive to cell viability. This substitution reduces cell death during the introduction process and improves the overall conversion efficiency while maintaining higher viability of the resulting reprogrammed cells.
4Adaptability or versatility
If viruses are used for reprogramming, then transcription factors can be delivered to cells, but reprogramming within patient tissue is impossible
Solution Approach 1:
The patent removes the viral component from the delivery system, enabling direct introduction of transcription factors into cells within patient tissue without the harmful effects of viruses. This extraction allows for in-situ reprogramming in the patient's own tissue, overcoming the limitation that previously made such applications impossible.
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 significantly increases reprogramming efficiency, reduces production time, and allows for safe, low-risk reprogramming in spatial cell clusters and potentially within the human body, offering new therapy options in regenerative medicine and cancer treatment.
Implementation Method 1
directing an attenuated, non-destructive laser beam of the femtosecond laser... so that the focus... creates a transient small-pore opening with a size in the range of up to 500 nm within a cell membrane
Implementation Method 2
directing a laser beam of the femtosecond laser, using a laser scanning microscope with a numerical aperture between 0.9 and 1.5, onto a cell membrane of a selected cell to a focus
Implementation Method 3
enable diffusion of the cocktail for multiple reprogramming of the cell through the cell membrane into the interior of the cell
Data Source
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AI summary
The invention relates to a method and a device for reprogramming living cells. The objective of finding a new way to directly, efficiently, and rapidly reprogram cells without the use of viruses is achieved according to the invention in the cell reprogramming method, in which a cocktail of at least two transcription factors and a microRNA is introduced into the interior of at least one cell to convert it into iPS cells or another cell type. This is achieved by embedding the cells to be reprogrammed in an aqueous environment containing the cocktail without a viral carrier and directing a femtosecond laser with a pulse repetition frequency in the range between 50 MHz and 2 GHz and a wavelength in the range of 700 to 1200 nm onto a cell membrane of the cell to be reprogrammed, using a laser scanning microscope with a numerical aperture between 0.9 and 1.5, and controlling the position of the focus.the exposure time and the laser power for the optical processing of the cell, such that the focus, depending on the pulse repetition frequency with a power between 7 mW and 100 mW, creates a transient small-pore opening with a size in the range of up to 500 nm in order to achieve diffusion of the cocktail for multiple reprogramming of the cell by optical multiple reprogramming.