HCEC Isolation Using Affinity Reagent Selection
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Solution Overview
Problem
Current methods for isolating human corneal endothelial cells (HCECs) face challenges due to the lack of specific surface markers, making it difficult to confirm cell identity, separate HCECs from contaminating cells, and achieve high clinical efficacy, particularly in cultures where keratocytes outcompete HCECs.
Innovation Solution
Utilizing positive and negative selection processes with affinity reagents that selectively bind to specific corneal proteins unique to HCECs or contaminants, such as keratocytes, to enrich and isolate HCECs from mixed cell populations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional isolation methods using morphology and functional genes are used, then HCECs can be identified, but the identification is not specific enough to distinguish HCECs from contaminating cells
Solution Approach 1:
The patent introduces surface markers (CD markers, integrins, and other cell surface proteins) as intermediary identification tools. These markers serve as mediators between the cell and the identification method, providing a more specific and reliable way to distinguish HCECs from contaminating cells compared to morphology and functional genes alone.
Solution Approach 2:
The patent changes the identification parameters from general morphology and functional genes to specific surface marker expression profiles. By measuring the expression levels of multiple surface markers simultaneously, the method achieves more precise and reliable cell identification.
2Quantity of substance
If HCECs are expanded ex vivo for increased cell numbers, then tissue availability is overcome, but the lack of specific surface markers makes it difficult to maintain purity and select high-efficacy cells
Solution Approach 1:
Surface markers serve as intermediaries for cell selection during expansion. By using these markers as targets for sorting and selection, the method maintains cell purity while expanding the total cell number, solving the contradiction between quantity and precision.
Solution Approach 2:
The patent changes from monitoring only morphology to monitoring surface marker expression parameters during cell expansion. This allows for real-time identification and selection of high-efficacy HCECs, maintaining manufacturing precision while increasing cell quantity.
3Quantity of substance
If the peel-off method is used to obtain HCECs from intact corneas, then HCECs can be collected, but corneal keratocytes are co-collected as contaminants that outcompete HCECs in culture
Solution Approach 1:
The patent extracts HCECs from the mixed cell population by targeting specific surface markers that are present on HCECs but absent or at low levels on keratocytes. This extraction process separates the desired cells from contaminants, removing the harmful factor while preserving the yield.
Solution Approach 2:
Surface markers act as intermediaries that enable selective identification and separation of HCECs from keratocytes. By using these markers as targets for magnetic sorting or flow cytometry, the method achieves selective extraction of HCECs while leaving keratocytes behind.
4Measurement precision
If current identification criteria based on morphology and functional genes are used, then HCECs can be recognized, but it is difficult to identify the subset of HCECs with highest clinical efficacy
Solution Approach 1:
The patent changes from using single-parameter identification (morphology or one functional gene) to multi-parameter identification using multiple surface markers. This provides more information about cell state and efficacy potential, preventing loss of important efficacy-related information.
Solution Approach 2:
Surface markers serve as intermediaries that provide additional information about cell characteristics and efficacy potential. These markers act as information carriers that reveal subsets of HCECs with different clinical efficacy without losing the fundamental cell identity information.
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
Enriched HCEC populations of 50% or more can be achieved, with up to 95% purity, suitable for clinical therapy and research, overcoming the limitations of existing identification and isolation methods.
Implementation Method 1
contacting the cell population with one or more affinity reagents that selectively bind to cells other than HCECs
Data Source
Figure 1A~2
Figure 3A~3C
Figure 4A~4D
AI summary
The present invention provides methods for the identification, isolation and/or enrichment of human corneal endothelial cells (HCECs). In some embodiments, the method comprises a positive selection process in which a cell population containing human corneal cells is contacted with a positive affinity reagent that selectively binds to HCECs relative to cells other than HCECs (e.g., corneal keratocytes, etc.) in the population and/or a negative selection process in which a cell population containing HCECs is contacted with a negative affinity reagent that selectively binds to cells other than HCECs in the population relative to HCECs. The present invention also provides reagents and kits for the identification, isolation and/or enrichment of HCECs as well as compositions that are enriched in HCECs.