One of the main methods to investigate cellular function, including stem cell differentiation, pathological state, and drug responsiveness, is by analysing deposition of extracellular matrix (ECM). Current methods such as Western blot, mass spectroscopy, or immunostaining, either require the removal of cells and processing of the ECM prior to analysis, or only allow for the analysis of the ECM of a few hundred cells or less. In the first case, the ECM is destructively harvested and cellular heterogeneity is masked, while in the latter case too few cells are analysed to be able to draw conclusions regarding subpopulations within the cell population. Moreover, these techniques can only be employed to a limited extent to analyse ECM deposition in 3D cell culture. Hence, we propose Extracellular Protein Identification Cytometry (EPIC) as a novel method that allows for the quantification of specific pericellular matrix proteins of individual cells within a 3D microenvironment in an ultra-high-throughput manner.
Human mesenchymal stem cells or chondrocytes were encapsulated in single cell microgels using droplet microfluidics. Standard photolithography and replica moulding was used to create patterned polydimethylsiloxane chips, which were bonded to microscope glass slides. Two different chip designs were used for droplet generation and delayed gelation respectively1. For single cell encapsulation1, detached cells (107 cells/ml) were resuspended in 5% Dex-TA dissolved in PBS mixed with 44 U/ml HRP and 8% OptiPrep. Flow rates were 6 µl/min, 2 µl/min, and 30 µl/min for the 2% Pico-Surf oil phase, aqueous phase with cells, and H2O2 solution respectively. Encapsulated cells were washed and subsequently transferred to chondrogenic medium and cultured for three weeks. For immunostaining, microgels were fixed using 4% PFA, permeated using TritonX-100, and subsequently blocked using 1% BSA for 1 h. Microgels were incubated with primary antibodies at 4ºC overnight, washed with PBS, and incubated with secondary antibodies. DAPI was used as counterstaining. Imaging was performed with a confocal microscope. Quantitative fluorescence cytometry was performed with a FACS Aria II.
Single cells were centred inside micrometre-thin microgels that could be cultured for three weeks with minimal cell escape1 allowing for matrix deposition. As a proof of concept, immunostaining for chondrogenic differentiation markers (e.g. COL II, ACAN, and COL I) was performed. Confocal analysis allowed for visualisation of targeted ECM protein(s) using indirect immunofluorescence. The fluorescent signal could also be detected with a FACS instrument, and flow cytometry analysis of a single antibody staining was performed.
Antibody staining of ECM proteins inside the hydrogel is possible and can be visualised. Also, it was shown that it is possible to detect and quantitate encapsulated cells using FACS, based on the immunostaining of a pericellular matrix protein of interest. As a next step, quantitative multiplexed FACS analysis of several deposited pericellular matrix proteins is going to be investigated.
1.Kamperman, T. et al., Small. 13 (2017)