Paper-Based 3D Scaffold for Multiplexed Single Cell Secretomic Analysis

Abstract

Despite rapid progresses in single-cell analysis technologies, efforts to control the three-dimensional microenvironment for single cell measurements have been lacking. Here, we report a simple method to incorporate three-dimensional scaffolds, including polyvinylidene fluoride (PVDF) membranes and PVDF membrane replicated analog polydimethylsiloxane, into multiplexed single cell secretomic analysis platforms (including a microwell array and a single cell barcode microchip) to mimic the extracellular physical matrix and mechanical support for single cells. Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in three-dimensional (3D) microenvironments. This finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other three-dimensional cell culture scaffolds or other single cell assays and may become a broadly applicable three-dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.

Application of microfluidic chip technology in pharmaceutical analysis: A review

“…This review provides a new insight into the research of drug analysis. Table 1 describes the significant use of microfluidic chips in this area and summarizes the device features and related representative literature [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67].…”

Single-Cell Omics Analyses Enabled by Microchip Technologies

“…By combining spectral encoding with different fluorescent colors and spatial encoding with 15 stripes, the platform achieved simultaneous measurement of 42 effector proteins (130), representing the highest multiplexing to date for a single-cell protein secretion assay. The device's compatibility with live cell culture and microscope imaging also enabled the correlative study between protein secretion profiles and cellular migration or interaction (129,(138)(139)(140)(141). To date, this platform has been applied to the study of functional heterogeneity in cell signaling, immune responses, tumorigenesis, and cellular therapies (126,129,130,135,(142)(143)(144).…”

Paper Microfluidics for Cell Analysis

“…To meet this need, Bai et al developed a simple and robust method to incorporate 3D scaffolds, including paper (PVDF membrane) and paper replicated analog polydimethylsiloxane, into the single cell analysis system for multiplexed secretomic analysis ( Figure ). They applied this platform into the investigation of brain tumor U87 single cell protein secretion behavior on different substrates, which revealed single cell proteins secretion was regulated differently by 3D microenvironment, demonstrating the value of 3D physical conditions provided by paper substrate …”

“…Comparison of multiplexed single cell secretions (IL‐6/IL‐8/MCP‐1) with brain tumor cell U87 on 2D substrate (PDMS) and 3D substrates (PVDF and PVDF membrane replicated analog polydimethylsiloxane). Reproduced with permission . Copyright 2018, American Chemical Society.…”