CC Microplate Application Note

CC Microplate Application Note Spheroid Culture and Assay Platform for 3D-Culture Studies Drug discovery has relied on in vitro and in vivo methods to evaluate compound safety, with recent incentives to focus on high-throughput, cost-effective measures like cell-based screening assays. The evolution of 3D cell culture, where cells aggregate into 3D biomimetic structures, has the potential to bridge the gap between traditional in vitro and in vivo compound screens. 1 Unlike monolayer culture, 3D cultures create a microenvironment which allows for cell-cell interactions, matrix production, and metabolic gradient formation. Additionally, 3D spheroids have shown drug responses more similar to those of an in vivo model when compared with traditional 2D cell culture, in cancer therapeutic efficacy, 2 4 hepatotoxicity, 5 9 and other applications. The Likarda CC Microplate is an easy-to-use, high throughput, 3D spheroid generating well plate. The plate is composed of a high-quality glass bottom with spherical microwells etched into the glass surface. Each well contains approximately 150 microwells. Each microwell is 200 µm wide x 100 µm deep, ideal for creating small spheroids which do not undergo hypoxic core necrosis. The well plate is currently available in a 384-well format (Figure 1). Figure 1. The CC Microplate is available in an a sterile, standard 384-well plate format (left). The surface of each well has 150 microwells etched into the glass surface (right). CC Microplate Application Note Rev.A 12/17 1

Quick Start Guide to using the CC Microplate 1 Pre-wet Pre-wet the plate with cell culture medium to ensure cells disperse into the microwells uniformly. Add 15 µl of your desired cell culture medium to each well. Centrifuge the plate at 1200 x g (RCF) for 5-10 minutes.* Check the plate under a microscope to ensure that no air bubbles are present in the microwells as this will interfere with cell seeding (Figure 2). If air bubbles are present, centrifuge again until they are removed. Figure 2. Images of the well surface of the CC Microplate. Air bubbles are present in the microwells before centrifuging (left). After centrifuging the plate, the air bubbles were removed (right). 2 Seeding Using manual or automated pipetting, add the desired number of cells per well. The desired seeding density will need to be determined with each new cell type. The example seeding study used HEK293 cells and the seeding volume was 50 µl (Table 1-2). Table 1. Microplate Specifications. Likarda 384-well CC Microplate Well Diameter (Bottom-mm) 3.2 x 3.2 (square wells) Microwells per Well Single Well Only Total Well Volume (µl) Working Volume (µl) 150 130 10-100 Table 2. Cell Loading Numbers. Cell Number per ml Spheroid Size after 2 days in Culture (µm) Cell Number per Microwell Cell Number per Well 1.79 x 10 6 600 89,400 167 ± 10 8.94 x 10 5 300 44,700 143 ± 9 4.47 x 10 5 150 22,350 120 ± 7 2.24 x 10 5 75 11,175 98 ± 4 1.13 x 10 5 38 5,662 71 ± 8 CC Microplate Application Note Rev.A 12/17 2

3 Incubation Carefully place the plate into an incubator at 37 C and 5% CO 2. 4 Medium Change Gently replenish half of the cell culture medium every 2-3 days using manual or automated pipetting. Aspiration and dispensing speeds should be slow to avoid disrupting the spheroids which can detach and float. 5 Monitor Spheroid Formation Check the plates under the microscope to determine the desired cell culture time to achieve spheroid formation (Figure 3-4). Typically, spheroids are formed after 1-3 days depending on cell type. If cells are left in the well plate too long, they will form large, multiple spheroid cell clusters. 6 Use Spheroids for Intended Application For pharmacologic testing, the spheroids can be tested in the CC Microplate or transferred to a new plate. *The glass bottom of the CC Microplate cannot undergo excessive torque during centrifugation otherwise the surface will crack. Torque can result from uneven contact points on the centrifuge microplate bucket surface. Be sure to check that the centrifuge microplate bucket has a flat surface for the CC Microplate to rest on. CC Microplate Application Note Rev.A 12/17 3

Figure 3. Images of HEK cells seeded into CC Microplate. Varying cell concentrations were used to load the plates, ranging from 38 to 600 cells per microwell. Cells were cultured for 2 days with spheroids formed after 1 day in culture. The scale bar represents 400 µm. Figure 4. Magnified images of HEK cells seeded into CC Microplate. The scale bar represents 100 µm. CC Microplate Application Note Rev.A 12/17 4

Many Cell Types Produce Spheroids Easily and Quickly in CC Microplates Most attachment cells produce spheroids using a scaffold-free 3D cell culture method like the CC Microplate. Table 3 provides a list of cell types used in the CC Microplate. Table 3. Cell Types Cultured in the CC Microplate. Cell Name Cell Description Spheroid Produced (Y/N) 22Rv1 Prostate carcinoma (h) Y A-1847 Ovarian carcinoma (h) Y A-375 Skin malignant melanoma (h) N A549 Lung carcinoma (h) Y Caov-3 Ovarian adenocarcinoma (h) Y FaDu Pharynx squamous cell carcinoma (h) Y HEK-293 Embryonic kidney (h) Y HepaRG Hepatocellular carcinoma (h) Y HN-5 Tongue squamous cell carcinoma (h) Y HOS Bone osteosarcoma (h) Y INS-1 832/13 Pancreas insulinoma (r) Y Islet + MSC Co-culture Pancreatic cell; bone marrow stem cell Y Islet cells Primary pancreatic islet cells (r,c,h) Y NCI-H1975 Lung adenocarcinoma; non-small cell lung cancer (h) Y NCI-H358 Lung bronchioalveolar carcinoma; non-small cell lung cancer (h) Y NIH:OVCAR-3 Ovarian adenocarcinoma (h) Y OSC-19 Tongue squamous cell carcinoma (h) Y PANC-1 Pancreas/duct epithelioid carcinoma (h) N h=human; r=rat; c=canine; MSC=Mesenchymal stem cell CC Microplate Application Note Rev.A 12/17 5

Early Drug Discovery Made Possible with the CC Microplate A physiologically-relevant 3D microenvironment is critical for predictive drug screening. Additionally, having a high throughput drug screen allows researchers to test more drugs using less resources and time. The CC Microplate achieves both goals: 1) creating uniform cellular spheroids and 2) in high throughput about 57,000 spheroids are produced in a single 384-well plate. Compounds efficacy and toxicity is easily tested using the CC Microplate. 10-14 Dose-response studies can be conducted in the plate using an increasing dose of the test article. Various endpoint assays are compatible with the CC Microplate. For example, many different viability and metabolism assays can be conducted in the plate to assess how the cellular spheroids respond to treatment. An example of a common viability assay, CellTiter-Glo 3D (Promega), is shown in Figure 5. Cells (22rv1) were loaded into the CC Microplate and allowed to form spheroids for 3 days. Following addition of the CellTiter-Glo 3D reagent to each well, the contents were mixed, incubated, and then the luminescence was recorded (BioTek Cytation 5). A lysed control (Triton X-100) was included in the experiment. The ATP content (RLUs) was easily detected at the various cell loading concentration ranging from 0 to 600 cells per microwell. The assay detection limit will need to be determined for each new application and cell type. Figure 5. An example of a viability assay (CellTiter-Glo 3D) conducted using the CC Microplate. High content imaging is easily conducted with the CC Microplate. Automated imaging systems can be used to capture single spheroid or whole well images. Figure 6 shows an example of spheroids labeled with propidium iodide, a fluorescent marker of necrotic and apoptotic cells. Dead cells (red) are easily distinguished from living cells and percent viability can be measured using manual or automated quantification software. CC Microplate Application Note Rev.A 12/17 6

Figure 6. An example of a high content imaging (propidium iodide) conducted using the CC Microplate. Red fluorescence indicates dead cells. CC Microplate Application Note Rev.A 12/17 7

References 1. Pampaloni, F.; Reynaud, E. G.; Stelzer, E. H. The Third Dimension Bridges the Gap between Cell Culture and Live Tissue. Nature Reviews Molecular Cell Biology 2007, 8, 839 845. 2. Vinci, M.; Gowan, S.; Boxall, F.; et al. Advances in Establishment and Analysis of Three-Dimensional Tumor Spheroid-Based Functional Assays for Target Validation and Drug Evaluation. Bmc Biol 2012, 10, 1 21. 3. Mehta, G.; Hsiao, A.; Ingram, M.; et al. Opportunities and Challenges for Use of Tumor Spheroids as Models to Test Drug Delivery and Efficacy. J Control Release 2012, 164, 192 204. 4. Lee, J.; Mhawech-Fauceglia, P.; Lee, N.; et al. A Three-Dimensional Microenvironment Alters Protein Expression and Chemosensitivity of Epithelial Ovarian Cancer Cells in Vitro. Lab Invest 2013, 93, 528 542. 5. Lauschke, V.; Hendriks, D.; Bell, C.; et al. Novel 3D Culture Systems for Studies of Human Liver Function and Assessments of the Hepatotoxicity of Drugs and Drug Candidates. Chem Res Toxicol 2016. 6. Dambach, D.; Andrews, B.; Moulin, F. New Technologies and Screening Strategies for Hepatotoxicity: Use of In Vitro Models. Toxicol Pathol 2005, 33, 17 26. 7. Godoy, P.; Hewitt, N.; Albrecht, U.; et al. Recent Advances in 2D and 3D in Vitro Systems Using Primary Hepatocytes, Alternative Hepatocyte Sources and Non-Parenchymal Liver Cells and Their Use in Investigating Mechanisms of Hepatotoxicity, Cell Signaling and ADME. Archives of Toxicology, 2013, 87. 8. McKim, J. M. Building a Tiered Approach to In Vitro Predictive Toxicity Screening: A Focus on Assays with In Vivo Relevance. Combinatorial Chemistry & High Throughput Screening 2010, 13, 188 206. 9. Soldatow, V.; LeCluyse, E.; Griffith, L.; et al. In Vitro Models for Liver Toxicity Testing. Toxicol Res 2012, 2, 23 39. 10. Ott L.M.; Ramachandran K.; Stehno-Bittel L. An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D. SLAS Discov 2017, 22, 614-625. 11. Ramachandran K.; Peng X.; Bokvist K.; Stehno-Bittel L. Assessment of re-aggregated human pancreatic islets for secondary drug screening. Br J Pharmacol 2014, 171, 3010-3022. 12. Amin J.; Ramachandran K.; Williams S.J.; Lee A.; Novikova L.; Stehno-Bittel L. A simple, reliable method for high-throughput screening for diabetes drugs using 3D β-cell spheroids. J Pharmacol Toxicol Methods 2016, 82, 83-89. 13. Rawal S.; Williams S.J.; Ramachandran K.; Stehno-Bittel L. Integration of mesenchymal stem cells into islet cell spheroids improves long-term viability, but not islet function. Islets 2017, 9, 87-89. 14. Cole C.; Burgoyne T.; Lee A.; Stehno-Bittel L.; Zaid G. Arum Palaestinum with isovanillin, linolenic acid and β-sitosterol inhibits prostate cancer spheroids and reduces the growth rate of prostate tumors in mice. BMC Complement Altern Med 2015, 15, 264. CC Microplate Application Note Rev.A 12/17 8

For more details and a complete list of Likarda products and services, contact info@likarda.com, and visit www.likarda.com. Author Lindsey Ott, PhD Director of Product Innovation Likarda, LLC Kansas City, Missouri, USA lindsey.ott@likarda.com For research use only. Not for use in diagnostic procedures. All trademarks and copyrights belong to Likarda, LLC or its affiliates, or to their respective third-party owners. The information contained herein is believed to be correct and corresponds to the latest state of scientific and technical knowledge. However, no warranty is made, either expressed or implied, regarding its accuracy or the results to be obtained from the use of such information and no warranty is expressed or implied concerning the use of these products. The buyer assumes all risks of use and/or handling. Any user must make his own determination and satisfy himself that the product supplied by Likarda, LLC or its affiliates and the information and recommendations given by Likarda, LLC or its affiliates are (i) suitable for intended process, (ii) in compliance with environmental, health and safety regulations, and (iii) will not infringe any third party s intellectual property rights. 2017 Likarda, LLC. All rights reserved. CC Microplate Application Note Rev.A 12/17 9