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Optimization of High-Throughput Multiplexed Phenotyping of Extracellular Vesicles Performed in 96-Well Microtiter Plates

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Optimization of High-Throughput Multiplexed Phenotyping of Extracellular Vesicles Performed in 96-Well Microtiter Plates polymers Article Optimization of High-Throughput Multiplexed Phenotyping of Extracellular Vesicles Performed in 96-Well Microtiter Plates Malene Møller Jørgensen 1,2,* , Jenni Kathrine Sloth 1 and Rikke Bæk 1 1 Department of Clinical Immunology, Aalborg University Hospital, 9000 Aalborg, Denmark; [email protected] (J.K.S.); [email protected] (R.B.) 2 Department of Clinical Medicine, Aalborg University, 9000 Aalborg, Denmark * Correspondence: [email protected] Abstract: Extracellular vesicles (EVs) are promising biomarkers for several diseases, however, no simple and robust methods exist to characterize EVs in a clinical setting. The EV Array analysis is based on a protein microarray platform, where antibodies are printed onto a solid surface that enables the capture of small EVs (sEVs) by their surface or surface-associated proteins. The EV Array analysis was transferred to an easily handled microtiter plate (MTP) format and a range of optimization experiments were performed within this study. The optimization was performed in a comprehensive analytical setup where the focus was on the selection of additives added to spotting-, blocking-, and incubation buffers as well as the storage of printed antibody arrays under different temperatures from one day to 12 weeks. After ending the analysis, the stability of the fluorescent signal was investigated at different storage conditions for up to eight weeks. The various parameters and conditions tested within this study were shown to have a high influence on each other. The reactivity of the spots was found to be preserved for up to 12 weeks when stored at room temperature and using blocking procedure IV in combination with trehalose in the spotting buffer. Similar preservation could be Citation: Jørgensen, M.M.; Sloth, J.K.; ◦ Bæk, R. Optimization of High- obtained using glycerol or sciSPOT D1 in the spotting buffers, but only if stored at 4 C after blocking Throughput Multiplexed procedure I. Conclusively, it was found that immediate scanning of the MTPs after analysis was not Phenotyping of Extracellular Vesicles critical if stored dried, in the dark, and at room temperature. The findings in this study highlight the Performed in 96-Well Microtiter necessity of performing optimization experiments when transferring an established analysis to a new Plates. Polymers 2021, 13, 2368. technological platform. https://doi.org/10.3390/ polym13142368 Keywords: extracellular vesicles; microarray; EV array; phenotyping; storage conditions; incubation buffers; spotting buffers Academic Editor: Laura Sola Received: 31 May 2021 Accepted: 8 July 2021 1. Introduction Published: 19 July 2021 Novel extracellular vesicle (EV) based diagnostic techniques are promising non- Publisher’s Note: MDPI stays neutral invasive procedures for early stage disease detection that are gaining increasing importance with regard to jurisdictional claims in in the medical field. EVs are cell derived particles found in body liquids, particularly in published maps and institutional affil- high numbers in blood, from which they are isolated for further analysis [1]. EVs are iations. involved in cell-to-cell communication, they carry biological material from the cell of origin, and they are transported throughout the human body by all body fluids [2,3]. Many stud- ies have suggested that EVs are promising biomarkers for diseases such as metastasis of cancer, diabetes, coronary diseases, inflammatory diseases, infectious diseases, neurologic diseases, and others as reviewed by Schou et al. [4]. Furthermore, EVs can be collected Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. rather non-invasively and give information about the cells from which they originate. This article is an open access article Although the EV phenotype is particularly important in the determination of cellular distributed under the terms and and subcellular origin, it can, in combination with a protein cargo analysis, additionally conditions of the Creative Commons provide clues about the functionality of the EVs. Further refinements of existing methods Attribution (CC BY) license (https:// will not only contribute to broadening our understanding of the biological role of the creativecommons.org/licenses/by/ EVs, but are also likely to accelerate the implementation of EVs as biomarkers in clinical 4.0/). diagnostics [5,6]. Several methods exist to characterize the protein composition of EVs Polymers 2021, 13, 2368. https://doi.org/10.3390/polym13142368 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 2368 2 of 14 related to either a surface marker phenotype or the proteins present in the EV cargo, as reviewed by Revenfeld et al. [7] and Panagopoulou et al. [8]. Detection and molecular profiling of EVs is technically challenging and often requires extensive sample purification and labelling [9–11]. Previously, we developed and described a high-throughput approach for phenotyping EVs [12,13]. This approach, termed the “EV Array”, is based on a protein microarray platform. Antibodies are printed onto epoxy-coated glass slides, which enable the capture of small EVs (sEV) by their surface or surface-associated proteins. Afterwards, profiling of the EVs is performed by detection with selected biotinylated antibodies for sEVs, e.g., anti-CD9, -CD63, and -CD81. The EV Array analysis requires manual handling of the glass slides throughout all steps. Handling slides requires a fast and trained workflow to avoid inappropriate drying of the surface and great precautions not to touch the spotted microarray. To run the analysis in a high-throughput manner, the slides were assembled in a multi-well gasket, where a silicone membrane was divided into 96 wells. A perfect assembling is crucial to avoid leakages between the well. To bring the EV Array technology into a clinical setting in the future will require development of a more robust and easily handled analysis, which is why a replacement for the slides as microarray substrates is needed. Variants and derivatives of immunological analyses in 96-well microtiter plates (MTP) have become assay workhorses of laboratories, and a host of compatible reagents, consum- ables, plate washers, multi-channel pipettes, robotic liquid handlers, and assay formats have been developed and are available from multiple vendors. An alternative format suitable for use in research labs is a MTP-based microarray printed directly onto the bottom of a 96-well plate [14]. This method has been used by different investigators and companies. For example, SearchLight™ arrays (Pierce Biotechnology, Rockford, IL, USA) have up to 16 assays per well in a sandwich format similar to other multiplexed sandwich microELISA arrays [15]. Likewise, our EV Array analysis was transferred to such a MTP format and a range of optimization experiments were performed. To capture EVs onto a surface, the maintenance of the capture bioactivity and perfor- mance of antibodies on microarray substrates for relatively long periods (under storage or shipping) requires carefully designed and optimized conditions. Here, we focused on the selection of additives added to spotting-, blocking-, and incubation buffers and the storage of printed antibody arrays under different temperatures from one day to 12 weeks. After the analysis ended, the stability of the fluorescent signal was investigated at different storage conditions for up to eight weeks. 2. Materials and Methods 2.1. Production of the EV Array For this test, 18 MTPs of the type Microfluor 2 (96-wells, polystyrene, round well, flat bottom, black, Thermo Fisher Scientific, Waltham, MA, USA) were prepared using a sciFLEXARRAYER S12 microarray printer installed with a piezo dispense capillary (PDC) size 60 with coating type 3 (Scienion AG, Berlin, Germany. The printing procedure was performed under strict humidity (55–65%) and temperature control (18–20 ◦C). The design of the array (shown in Figure1) consisted of three anti-human antibodies against CD63 (Bio-rad Laboratories Inc., Hercules, CA, USA), CD9, and CD81 (Ancell Corporation, Stillwater, MN, USA) at a concentration of 100 µg/mL, biotinylated goat anti- mouse IgG (Novus Biologicals, Centennial, CO, USA) in two concentrations (10 µg/mL and 50 µg/mL) as positive controls, and buffer as the negative control. The antibodies and controls were prepared in three different spotting buffers: 5% glycerol in PBS (denoted “gly”), 50 mM trehalose in PBS (denoted “tre”), and 1 × sciSPOT Protein D1 buffer (2 × con- centrate, denoted “D1”) in PBS (Scienion AG, Berlin, Germany), which were all included in the same print design. When the printing was finalized, the MTPs were incubated in the controlled climate of the printer for 10 h to allow adsorption of the antibodies. Polymers 2021, 13, 2368 3 of 14 concentrate, denoted “D1”) in PBS (Scienion AG, Berlin, Germany), which were all in- Polymers 2021, 13,cluded 2368 in the same print design. When the printing was finalized, the MTPs were incu- 3 of 14 bated in the controlled climate of the printer for 10 h to allow adsorption of the antibodies. Figure 1. (A) Sketch of the microarray printed in each MTP well. A sub-array was created for each Figure 1. (A) Sketch of the microarray printed in each MTP well. A sub-array was created for each of the spotting buffers of the spotting buffers (containing glycerol, trehalose, or sciSPOT D1) and each analyte was printed (containing glycerol, trehalose, or sciSPOT D1) and each analyte was printed in triplicate. (B) Example of one MTP well in triplicate. (B) Example of one MTP well fluorescently scanned after the EV Array analysis. The fluorescentlymagnified scanned spot after illustrates the EV Arraythe template analysis. for Thethe calc magnifiedulation spotof the illustrates mean spot the intensity template and for local the back- calculation of the mean spotground intensity subtraction. and local background subtraction. 2.2. Blocking and Storage 2.2. Blocking and Storage All blocking procedures were performed at room temperature (RT). Three of the All blocking procedures were performed at room temperature (RT). Three of the blocking procedures were initiated prior to storage. Each MTP was divided into four blocking procedures were initiated prior to storage.
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