Cellular Uptake Measurement in CD31+ CD45+ Hepatic Cells
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Solution Overview
Problem
There is a lack of in vitro cell-based assessment systems that accurately mimic in vivo conditions for quantifying FcγRIIB expression and immune complex uptake in human and monkey cells, which are essential for predicting pharmacokinetics and screening drug candidates.
Innovation Solution
A method involving the separation of human and monkey hepatic nonparenchymal cells into CD31+ CD45+ populations and sorting them based on CD31 and CD45 expression levels to measure immune complex and antibody uptake, using flow cytometry to identify specific cell populations for accurate quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional in vitro cell-based assessment systems are used, then ease of operation is improved, but measurement precision deteriorates because they do not accurately mimic in vivo conditions for quantifying FcγRIIB expression and immune complex uptake
Solution Approach 1:
The hepatic nonparenchymal cell population is segmented into specific subsets expressing CD31 and CD45 markers. This segmentation allows isolation of the precise cell population (CD31+CD45+) that mimics in vivo conditions for FcγRIIB expression and immune complex uptake, thereby improving measurement precision while maintaining operational feasibility through flow cytometry-based sorting
Solution Approach 2:
The invention changes the parameter of cell population selection by specifically identifying and isolating cells based on CD31 and CD45 expression levels. This parameter change transforms the assessment system from using general hepatic nonparenchymal cells to using a precisely defined subset (CD31+CD45+), which accurately reflects in vivo conditions for measuring FcγRIIB-mediated immune complex uptake
2Reliability
If animal testing is used to predict pharmacokinetics, then reliability is improved, but loss of time and resources increases due to the complexity and duration of animal studies
Solution Approach 1:
The invention creates an in vitro copy of the in vivo system by using human and monkey hepatic nonparenchymal cells that express FcγRIIB and naturally take up immune complexes. This cell-based assessment system copies the essential pharmacokinetic processes (FcγRIIB expression, immune complex formation, and cellular uptake) that occur in vivo, providing reliable predictions without requiring animal studies
Solution Approach 2:
The invention introduces an intermediary assessment system that mediates between in vitro simplicity and in vivo complexity. The CD31+CD45+ cell population serves as an intermediary model that captures the key pharmacokinetic behaviors of FcγRIIB-mediated uptake, allowing reliable drug candidate screening and pharmacokinetic prediction without direct animal testing
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 precise quantification of immune complex and antibody uptake in vitro, facilitating pharmacokinetic predictions and drug candidate screening that reflect human and monkey biophenomena, reducing the need for animal testing.
Implementation Method 1
sorting them based on CD31 and CD45 expression levels to measure immune complex and antibody uptake, using flow cytometry to identify specific cell populations for accurate quantification
Implementation Method 2
measuring uptake of the immune complex consisting of IgE and an anti-IgE antibody, which demonstrated the uptake of the immune complex into CD146+CD45low LSECs
Data Source
AI summary
The present invention provides a method for measuring a cellular uptake amount of a molecule, comprising (i) adding the molecule to an organ-derived cell population to perform incubation, (ii) sorting the organ-derived cell population based on the expression levels of CD31 and CD45, and (iii) after steps (i) and (ii), measuring the amount of the molecule incorporated into the cell population sorted in the step (ii), wherein the molecule is incorporated into cells via a cell surface receptor.