hUC-MSC Purity and Population Doubling Time by Flow Cytometry
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Solution Overview
Problem
Existing methods lack a reliable and efficient method for determining the purity and population doubling time of human umbilical cord mesenchymal stem cells (hUC-MSCs), which are crucial for controlling the quality of these cells.
Innovation Solution
A method involving mixing cultured hUC-MSCs with buffer solution to create a cell suspension, incubating with fluorescein-labeled antibodies, centrifuging and cleaning, then immobilizing with anhydrous ethanol, followed by PI staining and analysis using Multicycle for DNA software to determine purity and population doubling time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometry with fluorescein-labeled antibodies is used to determine cell purity, then measurement precision is improved, but device complexity and operation difficulty increase
Solution Approach 1:
The patent uses fluorescein-labeled antibodies as intermediaries that specifically bind to cell surface markers. These labeled antibodies serve as mediators between the flow cytometer detection system and the target cells, enabling precise purity determination through fluorescent signal detection while maintaining a relatively simple operational workflow.
2Measurement precision
If cell cycle analysis with PI staining and Multicycle software is performed, then measurement precision of population doubling time is improved, but operation difficulty and time consumption increase
Solution Approach 1:
The patent performs preliminary cell fixation with anhydrous ethanol and staining with propidium iodide (PI) before flow cytometry analysis. These preliminary actions prepare the cell samples in advance with stable DNA content, enabling accurate population doubling time measurement without requiring complex real-time analysis during the flow cytometry process.
3Loss of information
If multiple cell surface markers are detected simultaneously, then information completeness is improved, but measurement complexity and time consumption increase
Solution Approach 1:
The patent combines multiple fluorescein-labeled antibodies against different cell surface markers (such as CD73, CD90, CD105 for MSC identification) into a single flow cytometry measurement. This merging approach allows simultaneous detection of multiple markers in one experiment, providing complete cell characterization without requiring separate measurements for each marker.
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
Enables accurate analysis of cell surface markers and cell cycles, providing a precise determination of hUC-MSC purity and population doubling time, ensuring quality control.
Implementation Method 1
Mixing and incubating the cell suspension obtained in step 1) with a fluorescein-labeled antibody to obtain an incubated substance
Implementation Method 2
Centrifuging the incubated substance obtained in step 2), cleaning the precipitate obtained
Implementation Method 3
Mixing the cultured hUC-MSCs with the buffer solution and an anhydrous ethanol solution to obtain an immobilized substance after immobilization
Implementation Method 4
Centrifuging the immobilized substance obtained in step 4), mixing, incubating and centrifuging the precipitate obtained with a PI working solution
Data Source
AI summary
The present invention relates to the technical field of stem cell detection, and particularly relates to a determination method for purity and population doubling time of human umbilical cord mesenchymal stem cells (hUC-MSCs). The cultured hUC-MSCs are mixed with the buffer solution to obtain cell suspension; the cell suspension is mixed and incubated with a fluorescein-labeled antibody to obtain an incubated substance; and the incubated substance is centrifuged, and the precipitate obtained is cleaned and then mixed with the buffer solution to obtain a substance to be tested. The present invention analyzes cell surface markers and cell cycles by using a flow cytometer and monoclonal antibodies labeled with different fluoresceins of hUC-MSCs as well as PI staining, and further defines a determination method for purity and population doubling time of hUC-MSCs.