A microwell array platform to print and measure biomolecules produced by single cells.

Fikri Abali,Leonie Zeune,Richard B M Schasfoort,Arjan Tibbe,Leon W M M Terstappen,Joska Broekmaat

doi:10.1039/c9lc00100j

Fikri Abali, Leonie Zeune + Show 4 more

Open Access

https://doi.org/10.1039/c9lc00100j

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Abstract

Here we describe a combined method to monitor the secretion of molecules produced by single cells, followed by a method to isolate the individual cells that produced these molecules. The method is based on a self-sorting microwell chip that is connected to an activated membrane that collects the produced molecules. The produced molecules are printed by diffusion in small spots onto the membrane. The location of the printed spots can be correlated to the microwell number and the cell that produced these molecules. To demonstrate the method, we used the EpCAM antibody producing hybridoma cell line VU1D9 and a genetically engineered CHO cell-line producing Her2. VU1D9 cells produced 4.6 ± 5.6 pg (mean ± SD) of EpCAM antibody per 24 h and CHO cells 6.5 ± 8.2 pg per 24 h of Herceptin antibody.

Highlights

The ability to measure biomolecules such as cytokines, hormones and antibodies secreted by individual cells over a period of time under various conditions increases our understanding of complex biological processes
The microwell array comprises 6400 individual microwells contained within a surface of 8 × 8 mm
Fluidic forces drag the single cell into the microwells

Summary

Introduction

The ability to measure biomolecules such as cytokines, hormones and antibodies secreted by individual cells over a period of time under various conditions increases our understanding of complex biological processes. Clonality is a requirement for ensuring that the produced antibody is monoclonal.[3] An improvement to this method is the use of fluorescenceactivated cell sorting (FACS). There are lab-on-a-chip based approaches that use microwells to sort single cells and measure the secreted products.[16,17,18] Single cells are sorted into individual wells and their secreted products were captured onto glass slides coated with antibodies against the secreted proteins. The method comprises of three simple steps i.e. 1) distribute single cells in individual wells with high cell capture efficiency,20 2) measure the molecule secretion rate and quality of the secreted product at the single cell level, 3) cells of interest can be isolated by punching and clonally expanded. Interesting cells can be isolated and retrieved and expanded into a new cell line

Results

Discussion

Online Methods