Your Power for Health. 3D Cell Culture. 3D in a fast and easy 2D workflow with products from Greiner Bio-One.

Transcription

1 Your Power for Health 3D Cell Culture 3D in a fast and easy 2D workflow with products from Greiner Bio-One

2 3D Cell Culture in a 2D workflow Cell culture is an essential tool in drug discovery, tissue engineering, toxicology testing, stem cell research, as well as in basic research. In preclinical drug discovery validation processes, monolayer cell cultures are still predominant. Nevertheless, 2D cultures can only mimic the conditions of physiological tissue to a limited extent, whereas cells in vivo are able to interact in a three-dimensional network. Therefore, results generated with 2D cultures may often be of limited relevance for predicting clinical effectiveness and toxicity, contributing to high attrition rates in the drug development process. 1 Cell culturing in 3D enables the expression of extracellular matrix (ECM) components as well as the formation of cell-cell and cell-matrix interactions. These characteristics are important for replicating in-vivo cell differentiation, proliferation, and function in vitro. The employment of spheroid cultures is regarded as an improved approach to developing predictive in-vitro screening assays with high physiological relevance for preclinical drug development, especially in cancer research and toxicology. In spheroid cultures, cells grow in a three-dimensional system with zones of cellular heterogeneity and nutrient and oxygen gradients, to more closely reflect the in-vivo tumor microenvironment. Comparisons of spheroid cultures and 2D monolayer cultures for example of tumor cell lines showed functional differences including alterations in protein expression, phosphorylation patterns and responsiveness to inhibitor molecules. 2,3 Greiner Bio-One developed CELLSTAR cell culture vessels with cell-repellent surface for culturing in 3D. The cell-repellent surface effectively prevents cell adherence and therefore can promote the spontaneous formation of three-dimensional spheroids: a single spheroid per well in round bottom microplates or multiple spheroids in flat bottom plates. For applications such as microscopic analysis of single spheroids under high magnification, where ideal optical properties for high-throughput screening are desired, Greiner Bio-One offers the additional technology of magnetic 3D cell culture. Both system work with the principle and convincement 3D in a fast and easy 2D workflow! Figure 1: Spheroid formed by 3T3-L1 preadipocytes and GFP-expressing mouse bend.3 endothelial cells. Post-adipogenesis spheroids were subjected to immunofluorescence with perilipin antibodies (red) and GFP antibodies (green). Figure 2: Human mesenchymal stem cell spheroid differentiated to chondrocyte lineage. 1) Friedrich J. et al. ( 2007). Experimental anti-tumour therapy in 3-D: Spheroids - old hat or new challenge? Int J Rad Biol. 83(11-12): ) Ekert J E et al. (2014) Three-dimensional lung tumor microenvironment modulates therapeutic compound responsiveness in vitro implications for drug development. PLOS ONE 9(3): e92248.doi /journal.pone ) Vinci, M et al. (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biology 10:29. 2

3 CELLSTAR cell culture vessels with cell-repellent surface Applications: Spheroid culture of tumour cells Aggregation of stem cells Suspension culture of semi-adherent and adherent cell lines 3D culture in hydrogels Also for the successful application of magnetic 3D cell culturing, attachment of the cells to the surface of the culture vessel used must be avoided. The Greiner Bio-One CELLSTAR cell culture vessels with cell-repellent surface provides the perfect match for the magnetic 3D cell culturing approach. Figure 3: Tumor cell spheroids grown in a 96 well U-bottom CELLSTAR cell culture microplate with cell-repellent surface. In contrast to standard tissue culture surfaces which are optimised to enhance conditions for cell attachment, the cell-repellent surface has been developed to effectively prevent cell attachment. CELLSTAR cell culture vessels with a cellrepellent surface reliably prevent cell attachment in suspension cultures of semi- adherent and adherent cell lines where standard hydrophobic surfaces generally used for suspension culture are insufficient. For the formation of spheroids, self-assembled spherical clusters used as 3D cell culture models, the cell-cell interaction must dominate over the interaction between the cells and the culture surface of containment. The same principle is true for the formation of stem cell aggregates, a key step within many protocols for cultivation and differentiation of stem cells. Cell culture vessels equipped with the Greiner Bio-One cell-repellent surface present an ideal platform for cultivating both spheroids and stem cell aggregates. Long-term incubations of hydrogel cultures are frequently performed as an approach to mimic a 3D environment. When standard tissue culture vessels are used in this approach, some cells tend to migrate out of the hydrogel, forming a 2D subculture on the vessel surface. Analysis of such a cell population will therefore result in mixed data from both 2D and 3D cell cultures. CELLSTAR cell culture vessels with a cell-repellent surface can be used for hydrogel cultures due to the fact, that the surface effectively suppress the formation of 2D subcultures. The cell-repellent properties are achieved through an innovative chemical modification of the vessel surface. All cell culture vessels with cell-repellent surface are sterilised by irradiation (SAL of 10-3 ) and controlled for absence of detectable endotoxins, DNase/RNase and human DNA. Vessels with a cell-repellent surface additionally demonstrate no cytotoxic effects. Evaluation of cytotoxicity is done in accordance to EN ISO with mammalian cell lines. As with all Greiner Bio-One microplates, cellrepellent surface microplates are manufactured with a footprint that conforms to the recommendations of the American National Standards Institute (ANSI ) to guarantee compatibility with all widely used lab equipment. In summery Cell culture vessels equipped with the Greiner Bio-One cell-repellent surface present an ideal platform for cultivating suspension cultures of semi-adherent and adherent cell lines as well as the formation of stem cell aggregates and spheroids either with or without magnetic approach. Further information on CELLSTAR cell-repellent surface Forum No. 17: CELLSTAR Cell Culture Vessels with Cell-Repellent Surface (F073777) Application Report Advantage of CELLSTAR Cell Culture Vessels with Cell-Repellent Surface for 3D Cell Culture in Hydrogels (F073792) 3

4 Magnetic 3D Cell Culture The core technology of Greiner Bio-One s Magnetic 3D Cell Culture is the magnetisation of cells with NanoShuttle -PL. The cells can be aggregated with magnetic forces, either by levitation or printing, to form structurally and biologically representative 3D models in-vitro. NanoShuttle -PL consists of gold, iron oxide, and poly-l-lysine. These nanoparticles (Ø ~ 50 nm) magnetise cells by electrostatically attaching to cell membranes during an overnight static incubation. Magnetised cells will appear peppered with dark nanoparticles after incubation. NanoShuttle -PL is biocompatible, having no effect on metabolism, proliferation and inflammatory stress. Additionally, it does not interfere with experimental techniques, such as fluorescence or Western blotting. With magnetised spheroids, solution addition and removal is made easy by using magnetic force to hold them in a stationary position during aspiration, thereby limiting spheroid loss. Spheroids can also be picked up and transferred between vessels using magnetic tools such as the MagPen. Advantages of magnetic cell culture: Mimicking native tissue environment Rapid 3D model formation within hours No specialised media Easy to handle / no sample loss Allows co-culture Figure 4: Magnetic levitation, bioprinting and ring formation 4

5 Magnetic 3D Cell Culture Magnetic Levitation Magnetic levitation is an easy tool to create native tissue environments in vitro. Cells are magnetised with NanoShuttle -PL through overnight incubation and dispensed into a cell-repellent dish or multiwell plate, where they are levitated off the bottom by a magnet above the cell-repellent vessel. When levitating, cells come off the stiff well bottom and the magnetic forces work as an invisible scaffold that rapidly aggregates cells to encourage cellcell interactions and induce ECM synthesis. The magnetic levitation method has been successfully used to make 3D cultures with different cell types, including cell lines, stem cells and primary cells. The basic application of this technology is to culture 3D cell cultures under different environmental conditions and then analyse them using common biological research techniques, such as immunohistochemical analysis and western blotting. Figure 5: Magnetic levitation in a cell culture dish Magnetic Bioprinting In contrast to magnetic levitation, with magnetic 3D bioprinting, cells incubated with NanoShuttle -PL overnight are printed into spheroids by placing a microplate containing magnetised cells atop a drive of magnets. The magnets below each well utilise mild magnetic forces to induce cell aggregation and print a spheroid at the bottom of each well. After 15 minutes to a few hours, the plate containing the spheroids can be removed from the magnetic drive and cultured long-term without use of magnetic force. This system overcomes the limitations of other platforms by enabling rapid formation of spheroids unattached to any stiff substrate, reproducible in size without limitation to cell type and scalable in size for high-throughput formats (96 and 384 well). With magnetic 3D bioprinting, viable spheroids can be printed that compact and subsequently grow over time, facilitating continuous assessment of cell viability and other functions using commercially available assays. The 3D printing method, together with commercially available standardised assay methods, provide an ideal combination for highthroughput compound screening. Figure 6: Basic steps of magnetic Bioprinting with magnetization of the cells, transfer to cell-repellent plate and bioprinting with a magnet. 5

6 Magnetic Printing of Rings and Imaging System Additionally, it was demonstrated that magnetic 3D bioprinting can be used to develop novel migration assays (ring formation) and that automated kinetic imaging can facilitate high-throughput screening. These assays form a foundation for robust screening of compound effects on cell migration. Based on magnetic 3D bioprinting, cells magnetised with NanoShuttle -PL are printed into 3D rings. Immediately after printing, these structures will compact and eventually close, as a function of cell migration and viability. Ring closure can be captured using a compact imaging kit with an ipod* ) programmed by a freely available app (Experiment Assistant) to image whole plates at specific intervals, foregoing the need to image well-by-well under a microscope (Fig. 8). Culture contraction is generally complete within 24 hours, and images are batch processed to rapidly yield toxicity data. Moreover, as closure is label-free, the remaining rings are available for further experimentation (IHC, Western blot, genomics, etc.). Figure 8: ipod* ) -based imaging Figure 7: Cell migration assay with bioprinted 3D rings. Immediately after printing, these structures will compact and eventually close, as a function of cell migration and viability. *) ipod is a trademark of Apple Inc., registered in the U.S. and other countries. 6

7 Yes, NanoShuttle -PL is biocompatible! NanoShuttle -PL is a nanoparticle assembly (~50 nm) consisting of gold, iron oxide, and poly-l-lysine (PLL). The cells are magnetised by electrostatically attaching to cell membranes via PLL at a concentration of around 50 pg/cell. In this mechanism small amounts of NanoShuttle - PL is non-specifically attached to the membrane and sparsely covers the cell, instead of the whole membrane, giving a pepped appearance. 1 The amount is enough to attract cells with mild magnetic forces of 30 pn/cell in magnetic levitation or magnetic bioprinting with fields of around G. Growth, Proliferation and Viablility Neither NanoShuttle -PL nor magnetic forces have any effect on the proliferation of valvular interstitial cells (VIC, left) 7 and 3T3 fibroblasts 6 (right). NanoShuttle -PL: Consists of biocompatible components: Iron oxide and PLL are recognised as safe by the FDA 2,3. Gold nanoparticles are in clinical trials for therapeutic use, with no indications for system toxicity 4. Will not affect proliferation 6,7, viability 6, metabolism 7,8, inflammatory 7 or oxidative stress 5, phenotype 5,7,8, and other macro cell functions. Does not bind any specific receptors, works with all cell types. Will release off the cell over 7-8 days into the surrounding extracellular matrix, as shown by transmission electron microscopy (TEM). Requires magnetic forces (30 pn) only strong enough to aggregate but not harm cells. Does not cause any chromosomal abnormalities in cells, as shown by comparative genomic hybridisation (CGH) and demonstrates that NanoShuttle -PL does not lead to genomic instability. NanoShuttle -PL has no effect on viability, as demonstrated by live/dead staining (live = green, red = dead) on magnetically 3D bioprinted spheroids of 10,000 HepG2 hepatocellular carcinoma cells in a 384-well plate. Scale bar = 500 µm (left). The comparison of LN-299s growth in magnetic levitation (3D) shows a much better result over time than in a monolay (2D) (right). 1 Transmission Electron Microscopy After 24 h (left), NanoShuttle -PL is localised with the cells, but by 8 d (right) move out of the cell and into the extracellular space 1. In flammatory stress Overall, NanoShuttle -PL is biocompatible and facilitates rapid 3D culture formation. NanoShuttle -PL has also been shown not to induce any inflammatory stress in cells. When lung cells (BEpiC, SMC, PF, PEC) were exposed to NanoShuttle -PL for 6h, no significant difference in interleukin (IL)-6 and IL-8 production were found aside from IL-6 production in SMCs. 7 1) Souza, G. R., J. R. Molina, R. M. Raphael, M. G. Ozawa, D. J. Stark, C. S. Levin, L. F. Bronk, J. S. Ananta, J. Mandelin, M.-M. Georgescu, J. A. Bankson, J. G. Gelovani, T. C. Killian, W. Arap, and R. Pasqualini. Three-dimensional tissue culture based on magnetic cell levitation. Nat. Nanotechnol. 5:291 6, ) GRN No. 135, FDA. (2004) 3) CFR, 21.I.A (1994) 4) ClinicalTrials.gov #NCT (2014) 5) Haisler, W. L., D. M. Timm, J. A. Gage, H. Tseng, T. C. Killian, and G. R. Souza. Three-dimensional cell culturing by magnetic levitation. Nat. Protoc. 8:1940 9, ) Daquinag, A. C., G. R. Souza, and M. G. Kolonin. Adipose tissue engineering in three-dimensional levitation tissue culture system based on magnetic nanoparticles. Tissue Eng. Part C. Methods 19:336 44, ) Tseng, H., J. A. Gage, R. M. Raphael, R. H. Moore, T. C. Killian, K. J. Grande-Allen, and G. R. Souza. Assembly of a three-dimensional multitype bronchiole coculture model using magnetic levitation. Tissue Eng. Part C. Methods 19:665 75, ) Tseng, H., L. R. Balaoing, B. Grigoryan, R. M. Raphael, T. C. Killian, G. R. Souza, and K. J. Grande-Allen. A three-dimensional co-culture model of the aortic valve using magnetic levitation. Acta Biomater. 10:173 82,

8 Best matching 3D platform for your application What 3D platform is best for you? Magnetic Levitation Spheroid Bioprinting Well Number 35 mm dish Application Cancer Cardiotoxicity Hepatotoxicity Wound Healing Viability Assays Organoids Genomics Western Blotting 8

9 Ordering - CELLSTAR Cell-Repellent Surface CELLSTAR Cell Culture Vessels With Cell-Repellent Surface REF Description Pieces / Bag Pieces / Case Cell Culture Dishes Cell culture dish, 35 x 10 mm, PS, cell-repellent surface, clear, sterile Cell culture dish, 60 x 15 mm, PS, cell-repellent surface, clear, sterile Cell culture dish, 100 x 20 mm, PS, cell-repellent surface, clear, sterile 1 5 Cell Culture Multiwell Plates well Multiwell Plate, PS, cell-repellent surface, clear, with lid, sterile well Multiwell Plate, PS, cell-repellent surface, clear, with lid, sterile well Multiwell Plate, PS, cell-repellent surface, clear, with lid, sterile 1 5 Cell Culture Microplates well Microplate, PS, F-bottom/chimney well, cell-repellent surface, clear, with lid, sterile well Microplate, PS, F-bottom/chimney well, cell-repellent surface, black, µclear, with 8 32 lid, sterile SIN 96 well Microplate, PS, F-bottom/chimney well, cell-repellent surface, black, µclear, with 1 32 lid, sterile well Microplate, PS, U-bottom, cell-repellent surface, clear, with lid, sterile well Microplate, PS, V-bottom, cell-repellent surface, clear, with lid, sterile well Microplate, PS, cell-repellent surface, clear, with lid, sterile well Microplate, PS, cell-repellent surface, black, µclear, with lid, sterile SIN 384 well Microplate, PS, cell-repellent surface, black, µclear, with lid, sterile 1 32 Cell Culture Flasks Cell Culture Flask, 50 ml, PS, cell-repellent surface, sterile, white screw cap Filter Cap Cell Culture Flask, 50 ml, PS, cell-repellent surface, sterile, white filter screw cap Cell Culture Flask, 250 ml, PS, cell-repellent surface, sterile, white screw cap Filter Cap Cell Culture Flask, 250 ml, PS, cell-repellent surface, sterile, white filter screw cap Cell Culture Flask, 550 ml, PS, flat flask design, cell-repellent surface, sterile, white screw cap Filter Cap Cell Culture Flask, 550 ml, PS, flat flask design, cell-repellent surface, sterile, white filter screw cap Cell Culture Flask, 650 ml, PS, high flask design, cell-repellent surface, sterile, white screw cap Filter Cap Cell Culture Flask, 650 ml, PS, high flask design, cell-repellent surface, sterile, white filter screw cap

10 Ordering - Magnetic 3D Cell Culture Magnetic levitation Single Well / 6 Well / 24 Well REF Description Single Well Bio- 6 Well Bio- 24 Well Bio- 24 Well Custom Lid Assembler Kit Assembler Kit Assembler Kit Content NanoShuttle -PL (2) Levitating Drives (3) Concentrating Drives (3) 35 mm Cell Culture Dishes with Cell-Repellent Surface (10) Holding lid (1) NanoShuttle -PL (2) Levitating Drive (1) Concentrating Drive (1) 6 Well Cell Culture Multiwell Plates with Cell- NanoShuttle -PL (2) Levitating Drive (1) Concentrating Drive (1) 24 Well Cell Culture Multiwell Plates with Cell-Repellent Surface (2) Custom lid (1) 24-well special lid Spheroid Bioprinting 96 Well / 384 Well REF Description 96 Well Bioprinting Kit 96 Well Bioprinting Kit 96 Well Ring Drive Content NanoShuttle -PL (3) 96 Well Cell Culture Microplate (clear) with Cell-Repellent Surface (2) NanoShuttle -PL (3) 96 Well Cell Culture Microplate (black, µclear ) with Cell-Repellent Surface (2) Ring Drive REF Description 384 Well Bioprinting Kit 384 Well Bioprinting Kit 384 Well Ring Drive Content NanoShuttle -PL (2) 384 Well Cell Culture Microplate (clear) with Cell-Repellent Surface (2) NanoShuttle -PL (2) 384 Well Cell Culture Microplate (black, µclear ) with Cell-Repellent Surface (2) Ring Drive 10

11 Ordering - Magnetic 3D Cell Culture Screening & Imaging 96 Well / 384 Well REF Description 96 Well BiO Assay Kit 96 Well BiO Assay Kit & Imaging System 384 Well BiO Assay Kit 384 Well BiO Assay Kit & Imaging System Content NanoShuttle TM -PL (3) NanoShuttle TM -PL (3) NanoShuttle TM -PL (2) NanoShuttle TM -Pl (2) Levitating Drive (1) Levitating Drive (1) Levitating Drive (1) Levitating Drive (1) Ring Drive (1) 96-Well Deep Well Plate (1) 6-Well Cell Culture Multiwell Plates with Cell- 96-Well Cell Culture Microplates with Cell- Ring Drive (1) 96- Well Deep Well Plate (1) 6-Well Cell Culture Multiwell Plates with Cell- 96-Well Cell Culture Microplates with Cell- 96-Well Deep Well Plate (1) 6-Well Cell Culture Multiwell Plates with Cell- 384-Well Cell Culture Microplates with Cell- 96-Well Deep Well Plate (1) 6-Well Cell Culture Multiwell Plates with Cell- 384-Well Cell Culture Microplates with Cell- Imaging System (1) Imaging System (1) Light Pad (1) Light Pad (1) Cooling Fan (1) Cooling Fan (1) AC-to-USB Adapter (1) AC-to-USB Adapter (1) Extension Cord (1) Extension Cord (1) Analytica Software - link Analytical Software - link Consumables / Accessories for Magnetic 3D Cell Culture REF Description NanoShuttle -PL Refill NanoShuttle -PL Refill 3 Pack NanoShuttle -PL Refill 6 Pack NanoShuttle -PL Refill 12 Pack Content 600 µl vials of NanoShuttle -PL (1) 600 µl vials of NanoShuttle -PL (3) 600 µl vials of NanoShuttle -PL (6) 600 µl vials of NanoShuttle -PL (12) REF Description MagPen 3 Pack NanoShuttle -PL Refill 6 Pack with free ipod* ) Imaging Kit Battery Power for Imaging Kit Content Teflon caps (3) NanoShuttle -PL (6) Imaging System (1) Batteries (2) Magnets (3) Free ipod* ) (with purchase of either or ) Light Pad (1) Cooling Fan (1) AD-to-USB Adapter (1) Extension Cord (1) *) ipod is a trademark of Apple Inc., registered in the U.S. and other countries. 11

12 Your Power for Health For further information, please visit our website or contact us: Germany (Main office) Greiner Bio-One GmbH Maybachstraße 2 D Frickenhausen Phone +49 (0) Fax +49 (0) info@de.gbo.com Austria Greiner Bio-One GmbH office@at.gbo.com Belgium Greiner Bio-One BVBA/SPRL info@be.gbo.com Hungary Greiner Bio-One Hungary Kft. office@hu.gbo.com Italy Greiner Bio-One Italia S.r.l office@it.gbo.com Spain Vacuette Espana S.A. info@vacuette.es UK Greiner Bio-One Ltd. info@uk.gbo.com Brazil Greiner Bio-One Brasil office@br.gbo.com China Greiner Bio-One Suns Co., Ltd. office@cn.gbo.com France Greiner Bio-One SAS accueil@fr.gbo.com Japan Greiner Bio-One Co. Ltd. info@jp.gbo.com Netherlands Greiner Bio-One B.V. info@nl.gbo.com Portugal Vacuette Portugal S.A. info@vacuette.pt USA Greiner Bio-One North America Inc. office@us.gbo.com Revision: January 2018 F