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Paper Based 3D Scaffold for Multiplexed Single Cell Secretomic Analysis Ruihan Bai, Linmei Li, Meimei Liu, Shiqiang Yan, Chunyue Miao, Ruijun Li, Yong Luo, Tingjiao Liu, Bingcheng Lin, Yibing Ji, and Yao Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00362 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018
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Analytical Chemistry
Ruihan Bai1,2, Linmei Li2, Meimei Liu2, Shiqiang Yan2, Chunyue Miao1,2, Ruijun Li1, Yong Luo3, Tingjiao Liu4, Bingcheng Lin2, Yibing Ji*1, Yao Lu*2 1Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China 2Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China 3State Key Laboratory of Fine Chemicals, Department of Chemical Engineering & School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian, 116024, China 4College of Stomatology, Dalian Medical University, Dalian, 116044, China 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 PVDF membranes and PVDF membrane replicated analog polydimethylsiloxane, into a 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 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.
Cellular analysis has shifted greatly from 2D (two dimensional) to 3D (three dimensional), since cellular microenvironments can greatly influence cell functions like proliferation, migration and activation, etc., and many important cellular functions are significantly altered in 2D.1-4 3D cell culture has been demonstrated to better mimic in vivo conditions to provide more physiologically relevant information. Researchers are establishing “organ-on-chip” to make it possible to study human physiology with cells organized in organ-specific setting, which could potentially be used to replace animal testing during drug development.5-6 However, little attention has been paid to engineer biophysical microenvironment for single cell culture/analysis.7 Among the different types of single cell analysis technologies involving relatively long cell incubation times, single cell protein secretomic analysis provides direct measurement of functional phenotypes, rather than surface makers, to investigate the mechanism of cellular responses as well as potential correlation with therapeutic perturbations.8-9 Currently, the most commonly used tools
for multiplexed single cell secretion analysis includes conventionally used ELISPOT(Enzyme-linked Immunospot Assay),10 ICS(intracellular cytokine staining) (for example, multicolor flow cytometry and mass cytometry),11-12 and recently developed nanowell-based micro-engraving assay and single cell barcode microchip technology (SCBC).13-18 But it remains a unmet need to engineer 3D cell culture physical microenvironments in single live cell secretion analysis platform to investigate the effect of cell culture conditions on cellular functional outcomes in a multiplexed manner. 19 Paper, due to its flexible, multiscale(micro and nano) porous structures provide natural support to mimic cellular microenvironment in terms of physical and mechanical properties, is attracting more attention in recent years for 3D cell culture,20-22 stem cells differentiation,23 and in-vitro disease model construction24. Compared with other types of 3D cell culture scaffolds, such as hydrogels or micro-engineered (micro-fabricated or 3D printed) porous matrix,
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paper has inherent properties such as simplicity, low cost, wide availability, and excellent biocompatibility.2,21-23 Its physical properties such as thickness, pore size, rigidness, etc., chemical composition and surface properties can be flexibly tailored.25 Although paper has demonstrated great potential as a 3D cell culture scaffold for various population based cell applications, its feasibility to be integrated with different types of single cell analysis platform is yet to be explored. Herein, we introduced a simple method to integrate three dimensional cell culture substrates with a single cell analysis microwell array, including PVDF (polyvinylidene fluoride) membrane and PVDF membrane replicated analog PDMS (polydimethylsiloxane, short for pr-PDMS). Applying this platform to cancer cell line U87 to investigate single cell protein secretion on different scaffolds, we revealed protein secretions respond differently with varied physical microenvironments. The technology presented is not only the first report to establish 3D scaffold microenvironment for microchip based single cell multiplexed proteomic analysis, but also the first demonstration to integrate paper substrates with microchips for single cell analysis. This new biomimetic platform may open up new avenue to investigate the effect of microenvironment conditions on single cell functional proteomic heterogeneity in vitro. EXPERIMENTAL SECTION PDMS microarray stencil fabrication. The positive SU-8 mold to fabricate PDMS stencil was created on silicon wafer with photolithography. After the mold was treated with trimethylchlorosilane (TMCS) (Aldrich) vapor overnight to facilitate PDMS release, well mixed PDMS prepolymer (10: 1, Dow corning SYLGARD 184) was spin coated onto the silicon master at 500 rpm for 18 s followed with 1500 rpm for 60 s. After the PDMS layer was cured at 80oC for 30 minutes, it was carefully detached from the SU-8 mold immersed in ethanol with a tweezer. The PDMS stencil layer was cleaned with ultra-sonication in ethanol for 10 min and dried. It was checked with microarray laser scanner (Molecular devices, USA) to ensure over 95% microwells was completely perforated before use. PVDF membrane replicated analog PDMS (pr-PDMS) fabrication. The PVDF membrane (0.45 um pore size, GE) was attached evenly onto a flat substrate, like silicon wafer or glass slide. PDMS prepolymer was poured onto the membrane and cured at 80oC for 1.5 hrs, in which the micro/nanostructures embedded in the membrane was replicated precisely into PDMS. After that, PDMS was detached from the membrane in ethanol and sonicated with ethanol for 10 min, dried in oven at 80oC for 20 min and be ready for use. Preparation of antibody slide and antibody barcode slide. Antibody slide: Capture antibodies (10 μg/mL, 100 μL for each slide) (full antibody list as shown in Table S1) were coated onto poly-L-lysine coated (Thermo Fisher) glass slide at 4oC overnight. Antibody barcode slide: A poly-L-lysine glass slide was bound with PDMS microchip
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designed for flow patterning and baked at 80oC for 2 hrs. 3 μL of each antibody (250 μg/mL) was then flowed through individual microchannels until complete dry with custom built flow station under 1 psi N2. After the antibodies were immobilized onto glass surface, the glass slide was blocked with 1% BSA for 1 hr. After washing with DPBS, 50/50 DPBS/DI water and DI water sequentially, the glass slide was spun dry in slide centrifuge and stored at 4oC for use. Assembly of PDMS microwell stencil with single cell culture substrates. There were three different substrates for single cell analysis: PVDF membrane, pr-PDMS and plain PDMS (as reference). For pr-PDMS or plain PDMS (5 mm thickness), they were cut into the size of 75x25 mm and aligned with PDMS stencil in ethanol. After the ethanol was aspirated, the device was dried at 80oC for 20 min to evaporate residual liquid to ensure the pr-PDMS or plain PDMS bonded with PDMS microarray stencil tightly. While for PVDF membrane(53x21 mm), one side of PVDF membrane is firstly coated with PDMS prepolymer by attaching onto spin coated PDMS layer (500 rpm for 18 s followed with 3000 rpm for 60 s, the resulting PDMS layer thickness is around 20 μm) to facilitate handling. After curing, PDMS backed PVDF membrane was attached evenly onto a plain PDMS (5 mm thickness) with 53x22 mm size. PDMS microarray stencil was then attached onto PVDF membrane to form 3D single cell culture/analysis sandwich. Cell culture. Human glioblastoma multiforme cell line U87 (American Type Culture Collection) was cultured in DMEM medium with 10% fetal bovine serum (FBS, Gibco, Invitrogen) and 1% antibiotics (100 U/ml of penicillin G sodium, 100 U/ml of streptomycin). After reaching confluent, the cells were trypsinized with 0.25% trypsin-0.02% EDTA for 3 min, centrifuged at 1000 rpm for 5 min and resuspended in fresh medium. MicroELISA procedures. The microELISA was performed on microchannel guided flow patterned antibody glass slide. The PDMS microchannel for antibody patterning was reversibly bonded onto poly-L-lysine glass slide and baked at 80oC for 30 min to enhance bonding. The capture antibodies were then filled into the channels and incubated at room temperature overnight. After washing and blocking for 10 min with 1% BSA, the PDMS layer was peeled off and antibody coated glass slide was dipped into DPBS, 50/50 DPBS/DI water, DI water sequentially and blown dry. Then another PDMS layer with microchannels was bonded onto antibody coated glass slide perpendicular to antibody lines. The PDMS microchannel was treated with O2 plasma to be hydrophilic before bonding to facilitate liquid handling. After blocking with 1% BSA for 1 hr to reduce nonspecific absorption, samples (cell culture supernatants) were added into different microchannel for each sample and incubated overnight. After that, standard ELISA procedures were performed to obtain detectable sandwich complex. The fluorescence results were read and analyzed with Genepix scanner and software. Intracellular protein staining. U87 cells were cultured in custom made PDMS microwells (6 mm diameter) on
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Analytical Chemistry
plain PDMS and pr-PDMS respectively. Brefeldin A (Topscience, USA) was added to cells after 2 hrs, when the cells were fully adhered onto substrates. After incubation, the cells were washed with PBS and fixed with 4% Paraformaldehyde. They were incubated with primary antibodies (Invitrogen) and stained with Alexa Fluor 649 conjugated goat anti-mouse antibody (Invitrogen). Then the nucleus of cells were stained with DAPI (4',6-diamidino-2-phenylindole) following manufacturer’s instructions. After intensive washing, stained cells on different substrates were imaged with Nikon Eclipse TiE microscope (Andor Zyla 4.2 PLUS sCMOS Camera). Single cell assay and Imaging. The PDMS microarray stencil for single cell assay was sterilized under ultraviolet light for 30 min and treated with O2 plasma (Harrick Plasma PDC-32G) for 2 min. Then it was blocked with cell culture medium immediately to maintain surface hydrophilic to facilitate cell loading. Cells were stained with cell viability dye Calcein-AM green at 37oC for 30 min and resuspended into fresh medium contained 20% FBS. The stained cells were loaded onto chips at 2x105 cells/mL cell density, 200 μL/chip. The single cell device was clamped at finger tight to form enclosed microchamber for trapped single cells. The single cell assembly was imaged on Nikon Eclipse TiE microscope equipped with an automatic stage to scan images (both bright field and 470 fluorescence) of the microwell array, which provides information on cell number/position within each microwell. After cells were incubated for the time period specified, the clamp was untightened to remove glass slide to finish the sandwich immunoassay procedures. The glass slide was incubated with a mixture of detection antibodies for 1 hour and stained with streptavidin-APC or streptavidin-PE (eBioscience, 1: 100 dilution) for 30 min. After the slide was washed with DPBS, 50/50 DPBS/DI water and DI water sequentially, the glass slide was spun dry in slide centrifuge and scanned with a GenePix 4300A microarray scanner (Molecular Devices). Data analysis. Optical and fluorescence images obtained with microscope can be processed in Nikon software (NIS-Elements Ar Microscope Imaging Software) by defining threshold on each image to realize automated cell counting. The fluorescence detection image was analyzed with GenePix Pro software (Molecular Devices) by aligning the microwells array template followed by extraction of fluorescence results. The cell counts can be matched with the extracted fluorescent data to their respective microwells. Excel (Microsoft) and Graphpad Prism or Origin was used to compile extracted data. RESULTS AND DISCUSSION Integration of paper based 3D scaffold with single cell secretomic analysis chip. The configuration of the single cell secretomic analysis platform with paper-based 3D scaffold is comprised of three functional components arranged in sandwich format as shown in Figure. 1A: top layer: antibody glass slide for surface immunoassay; middle layer: high throughput PDMS microwell array stencil for
single cell capture and the bottom layer: 3D scaffold for cell culture. Antibodies (100 μL volume at 10 μg/mL for each antibody) were uniformly immobilized onto a high quality poly-L-lysine coated glass (C.V.=5.2%, characterized by fluorescence labelled BSA coating within each glass slide, Supporting Figure. S1). A high-throughput PDMS microwell array stencil (15x7 blocks, 10,500 microwells in total, each square microwell is 100 μm x 100 μm) was fabricated by spin coating PDMS onto SU-8 hard mold with resulting thickness to be 120 μm (Supporting Figure. S2), which determined the volume of each microwell to be 1.2 nL corresponding to the volume of each cell occupied in population experiments. Scanning Electron Microscope (SEM) was used to check the PDMS microwell array stencil for complete perforation; however SEM is difficult to scale for large area validation. Here we proposed using microarray laser scanner for large scale PDMS stencil validation, which confirmed that over 98% of microwells are completely perforated (Figure. 1B and Supporting Figure. S3). PVDF membrane (0.45 μm pore size, 170 μm thickness), in which aligned cellulose fibers formed porous micro/nano structures, was chosen in this study as a low cost paper substrate for 3D single cell culture/analysis due to its established use in conventional ELISPOT assays26 which proved its excellent biocompatibility. Its natively hydrophobic surface helped to minimize the protein diffusion and crosstalk between neighboring microwells (the contact angle is around 120oC, Supporting Figure. S4). However, as PVDF membrane is opaque, making it difficult for imaging during cell analysis, we also proposed to fabricate a replicated analog with PDMS as a low cost alternative 3D cell culture substrate to overcome this issue. The PVDF membrane and its replicated analog PDMS were characterized with SEM (Figure. 1C) and surface profiler (Supporting Figure. S5 & S6), from which we can see both substrates embedded randomly oriented microstructures in the range of 10 μm to submicron with nice consistency and stability which would provide reliable three dimensional cell culture physical support at multiscale. The PDMS microwell array stencil was then attached onto the paper or PDMS substrate to form a high throughput 3D microwell array (Figure. 1D). The configuration proposed to assemble the 3D single cell culture scaffold in microwells is simple and easily adapted to some other substrates like glass, polystyrene, or microspot array, etc. (Supporting Figure. S7). We used cancer cell line U87 cells cultured on different substrates to characterize whether 3D cell culture microenvironment was established successfully. The imaging results indicated that the U87 cells exhibited excellent viability on these substrates and formed tumor cell spheroids/aggregates on PVDF membrane and pr-PDMS, which is the characteristics of 3D cell culture system and is very similar with cells cultured on a commercial 3D substrate (Figure. 1E). While U87 cells cultured on conventional tissue culture petri dish only exhibited monolayer cell morphology which is typical in 2D cell culture. To further validate the established 3D scaffolds, we detected protein secretions from population U87 cells cultured on PVDF membrane, pr-PDMS, 3D culture plate and conventional
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Figure 1. Paper-based 3D scaffold for multiplexed single cell secretomic analysis. (A) Schematic illustration of integration of 3D scaffold with single cell microdevice and workflow of single cell secretomic assay; (B) Enlarged view of PDMS microarray stencil (one block, 100 microwells) with different methods(optical, fluorescence scanning and SEM); (C) SEM images showing the porous or protruding multiscale structures in 3D scaffold substrates: PVDF membrane (left) and pr-PDMS (middle) and their comparison with plain PDMS (right) (2000x and 6000x respectively); (D) Optical images showing the assembly of PDMS microarray stencil with 3D scaffold; (E) Characterization of U87 cells’ morphology on PVDF membrane and pr-PDMS and their comparisons with TCPD(tissue culture petri dish) and commercial 3D culture plate (SCIVAX's NanoCulture® Plate); (F) Comparison of U87 cells’ proteins secretion profile on PVDF membrane and pr-PDMS and comparison with commercial 3D culture plate and conventional TCPD (96 well plate).
TCPD (96 well plate) (Figure. 1F), from which no signifi-
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Analytical Chemistry
cant differences were observed for highly secreted cytokines such as IL-8 (Interleukin 8), MCP-1 (Monocyte chemotactic protein 1 (CCL2)), or low secreted cytokines such as TNF-a (Tumor necrosis factor alpha), Rantes (Chemokine (C-C motif) ligand 5), MIP-1a (Macrophage inflammatory protein 1-alpha) and MIP-1b (Macrophage inflammatory protein 1-beta) (p>0.05, n=3, biological triplicates). The antibodies used in this paper (summarized in Table S1) were validated with their corresponding recombinant proteins to ensure high detection sensitivity (Supporting Fig. S8) and minimum cross reactivity (
