The core technology of Greiner Bio-One’s Magnetic 3D Cell Culture is the magnetization of cells with biocompatible NanoShuttle™-PL. The reproducible formation of one spheroid per well in an F-bottom plate with Cell-Repellent surface is forced by magnets either by levitation or bioprinting, to form structurally and biologically representative 3D models in-vitro
Advantages of the magnetic 3D cell culture (m3D) technology are:
In contrast to magnetic levitation, with magnetic 3D bioprinting, the magnetized cells in a plate are printed into spheroids by placing atop a drive of magnets. One magnet below each well utilizes mild magnetic forces to induce cell aggregation and print one spheroid at the bottom of each well anywhere within 15 minutes to a few hours. Afterwards the spheroids can be cultured long-term without the use of magnetic force. This system overcomes the limitations of other platforms by enabling rapid formation of spheroids, reproducible and scalable in size for high-throughput formats (96, 384 and 1536 well) and without limitation to cell types. The 3D printing method together with commercially available standardized biochemical assay methods to facilitate continuous assessment of cell viability and other functions, provides an ideal combination for high-throughput compound screening.
By magnetized spheroids, adding and removing solutions is made easy by the use of magnetic forces to hold down spheroids during aspiration, limiting spheroid loss. Spheroids can also be picked up and transferred between vessels using magnetic tools such as the MagPen™. Magnetic forces can also be used to create co-cultures with fine spatial organization.
Magnetic levitation is an easy tool to create native tissue environments in vitro. In magnetic levitation the magnetized cells are levitated off the bottom by a magnet above the plate. By levitating cells off the plate bottom the magnetic forces work as an invisible scaffold that rapidly and gently aggregates cells and induces cell-cell interactions and ECM synthesis. The 3D culture is formed without any artificial substrate or specialized media or equipment and can be cultured long-term. The 3D culture is formed without any artificial substrate or specialized media or equipment and can be cultured long-term.The gentle nature of magnetic levitation allows cultures to acquire macroscale morphology that mostly resembles its tissue of origin. 3D cell cultures can be analyzed using common biological research techniques, such as immunohistochemical analysis and western blotting.
The magnetic levitation has been successfully used to make 3D cultures with different cell types, including different cell lines, including stem cells and primary cells.
GFP labeled Glioblastoma cell line (LN-229).
Immunofluorescence staining of primary cardiomyocytes.
Magnetic levitation in a cell culture dish
The current standards for compound screening are animal models; while representing human tissues of interest, these models are expensive, scarce, and carry ethical challenges. On the other end, 2D in vitro assays poorly mimic native cellular environments and thus human in vivo response, but offer high-throughput testing with ease. There is a demand for in vitro assays that are both predictive of human in vivo response and high-throughput.
As a result, we developed a viability assay, the BiO Assay. Based on magnetic 3D bioprinting, cells magnetized with NanoShuttle™-PL (NS) are printed into spheroids and rings. Immediately after printing, these structures will shrink/close, as a function of cell migration, viability, cell-cell interaction, and/or proliferation, and varies with dosage. Ring closure can be captured using a compact imaging kit (n3Dock) with an iPod™ programmed by a freely available app (Experiment Assistant) to image whole plates at specific intervals, forgoing the need to image well-by-well under a microscope. Culture contraction is generally complete within 24 hours, and images are batch processed to rapidly yield toxicity data Moreover, as the assay is label-free, the remaining rings or spheroids are available for further experimentation (IHC, Western blot, genomics, etc.).
The BiO Assay can be used to track the culture contraction of both rings and spheroids representing different situations. For rings, closure of the ring can represent wound-healing, wherein cells are working to close the void in the middle of the ring. Additionally, rings can represent similarly shaped tissues, like blood vessels, where dilation and contraction can be assayed. For Spheroids, contraction is related to spheroid assembly, with the assay macroscopically measuring how well the cells are interacting and migrating to build a competent structure.
The BiO Assay combines 3D cell culture environments with high-throughput and high-content testing to effectively predict in vivo response in vitro.
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 cell-repellent surface reliably prevent cell attachment in suspension cultures of semiadherent and adherent cell lines where standard hydrophobic surfaces generally used for suspension culture are insufficient.
For formation of spheroids, stem cell aggregates and 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. Therefore CELLSTAR® cell culture vessels with cell-repellent surface effectively prevent cell adherence and promote the spontaneous formation of three-dimensional spheroids by gravitation: a single spheroid per well in round bottom microplates or multiple spheroids in flat bottom plates, dishes and flasks.
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 to effectively suppress the formation of 2D subcultures.
| Stem Cell Lines | Cancer Cell Lines | Primary Cell Lines | Other Cell Lines |
|---|---|---|---|
| Neural stem cells | LN229 – Glioblastoma cells | Primary Glioblastoma cells | Human Astrocyte |
| Mesenchymal stem cell | HepG2 — Human liver carcinoma | Human lung primary cells: - epithelial - endothelial - fibroblasts - smooth muscle | 3T3 fibroblasts |
| Neural crest-derived mesenchymal stem cell | A549 — Lung epithelial adecarcenoma | Valvular interstitial cells (VICs) | Bend (brain endothelial) |
| Dental pulp stem cell | PC3 — Human prostate cancer | Valvular endothelial cells (VECs) | Adipocyte |
| H-4-II-E — Rat hepatoma, liver | Aortic valve co-cultures (AVCCs) | Huvec - Human umbilical endothelial cells | |
| MDA-231 - Human breast cancer | Primary mouse heart cells | HPF - Human pulmonary fibroblast | |
| LNCaP - Prostate cancer cell line | Human Primary vascular smooth muscle | SMC - Tracheal smooth muscle cell | |
| Ovarian cancer cells | Primary Miomytrial Smooth muscle | HEK293 - Human embryonic kidney | |
| Panc-1 - Pancreatic cancer cell | Primary human hepatocytes | MCF-10A - Breast epithelial cell line | |
| Cancer associated fibroblasts | Primary pancreatic cancer cells | Fibroblast | |
| Triple negative inflammatory breast cancer | Primary tissue from PDX | Chondrocytes | |
| Caki-1 – Human renal cancer cell line | Primary fibroblasts | T-cells | |
| Osteosarcoma | Keratinocytes | A10 - Rat vascular smooth muscle | |
| HCT116 - colon cancer cell line | |||
| Pre-adipocyte stem cell | KPC pancreatic ductal adenocarcinoma | Pancreatic ß-cells (EndoC-ßH3) | |
| DU145 | Murine embryonic fibroblasts (iMEF) | ||
| LOVO cells | DT66066 cells | ||
| Spinal cord cells | |||
| Endothelial cell | |||
| Peripheral blood mononuclear cells (PBMCs) | |||
| Monocytes |
| Type | Order no. | Description |
|---|---|---|
| Spheroid Bioprinting 96 Well / 384 Well | 655840 | 96 Well Bioprinting Kit |
| Spheroid Bioprinting 96 Well / 384 Well | 655841 | 96 Well Bioprinting Kit µClear® |
| Spheroid Bioprinting 96 Well / 384 Well | 655850 | 96 Well Ring Drive |
| Magnetic levitation 6 Well | 657825 | 6 well Intermediate Lid |
| Magnetic levitation 6 Well / 24 Well | 657840 | 6 Well Bio-Assembler™ Kit |
| Magnetic levitation 24 Well | 662825 | 24 well Intermediate Lid |
| Magnetic levitation 6 Well / 24 Well | 662840 | 24 Well Bio-Assembler™ Kit |
| Spheroid Bioprinting 96 Well / 384 Well | 781840 | 384 Well Bioprinting Kit |
| Spheroid Bioprinting 96 Well / 384 Well | 781841 | 384 Well Bioprinting Kit µClear® |
| Spheroid Bioprinting 96 Well / 384 Well | 781850 | 384 Well Ring Drive |
| Screening & Imaging 96 Well / 384 Well | 655846 | 96 Well BiO Assay™ Kit |
| Screening & Imaging 96 Well / 384 Well | 655849 | 96 Well BiO Assay™ Kit & Imaging System |
| Screening & Imaging 96 Well / 384 Well | 781846 | 384 Well BiO Assay™ Kit |
| Screening & Imaging 96 Well / 384 Well | 781849 | 384 Well BiO Assay™ Kit & Imaging System |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657841 | NanoShuttle™-PL Refill |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657843 | NanoShuttle™-PL Refill 3 Pack |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657846 | NanoShuttle™-PL Refill 6 Pack |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657852 | NanoShuttle™-PL Refill 12 Pack |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657850 | MagPen™ 3 Pack |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657847 | NanoShuttle™-PL Refill 6 Pack with free iPod |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657860 | Imaging Kit |
| Consumables / Accessories for Magnetic 3D Cell Culture | 657810 | Battery Power for Imaging Kit |
Product offering may vary depending on geographic location.
| Type | Order no. | Description |
|---|---|---|
| Dishes with Cell-Repellent Surface | 627979 | Cell Culture Dish, Ø 35x10 mm, PS, cell-repellent surface, clear, sterile |
| Dishes with Cell-Repellent Surface | 628979 | Cell Culture Dish, Ø 60x15 mm, PS, cell-repellent surface, clear, sterile |
| Dishes with Cell-Repellent Surface | 664970 | Cell Culture Dish, Ø 100x20 mm, PS, cell-repellent surface, clear, sterile |
| Flasks with Cell-Repellent Surface | 690980 | Cell Culture Flask, 50 ml, PS, cell-repellent surface, sterile, white screw cap |
| Flasks with Cell-Repellent Surface | 690985 | Cell Culture Flask, 50 ml, PS, cell-repellent surface, sterile, white filter screw cap |
| Flasks with Cell-Repellent Surface | 658980 | Cell Culture Flask, 250 ml, PS, cell-repellent surface, sterile, white screw cap |
| Flasks with Cell-Repellent Surface | 658985 | Cell Culture Flask, 250 ml, PS, cell-repellent surface, sterile, white filter screw cap |
| Flasks with Cell-Repellent Surface | 660980 | Cell Culture Flask, 550 ml, PS, flat flask design, cell-repellent surface, sterile, white screw cap |
| Flasks with Cell-Repellent Surface | 660985 | Cell Culture Flask, 550 ml, PS, flat flask design, cell-repellent surface, sterile, white filter screw cap |
| Flasks with Cell-Repellent Surface | 661980 | Cell Culture Flask, 650 ml, PS, high flask design, cell-repellent surface, sterile, white screw cap |
| Flasks with Cell-Repellent Surface | 661985 | Cell Culture Flask, 650 ml, PS, high flask design, cell-repellent surface, sterile, white filter screw cap |
| Multiwell Plates with Cell-Repellent Surface | 657970 | 6 well Multiwell Plate, PS, cell-repellent surface, clear, with lid, sterile |
| Multiwell Plates with Cell-Repellent Surface | 665970 | 12 Well Multiwell Plate, PS, cell-repellent surface, clear, sterile, with lid |
| Multiwell Plates with Cell-Repellent Surface | 662970 | 24 well Multiwell Plate, PS, cell-repellent surface, clear, with lid, sterile |
| Multiwell Plates with Cell-Repellent Surface | 677970 | 48 well Multiwell Plate, PS, cell-repellent surface, clear, with lid, sterile |
| Microplates with Cell-Repellent Surface | 655970 | 96 well Microplate, PS, F-bottom/chimney well, cell-repellent surface, clear, with lid, sterile |
| Microplates with Cell-Repellent Surface | 655976 | 96 well Microplate, PS, F-bottom/chimney well, cell-repellent surface, black, μClear®, with lid, sterile |
| Microplates with Cell-Repellent Surface | 655976-SIN | 96 well Microplate, PS, F-bottom/chimney well, cell-repellent surface, black, μClear®, with lid, sterile |
| Microplates with Cell-Repellent Surface | 650970 | 96 well Microplate, PS, U-bottom, cell-repellent surface, clear, with lid, sterile |
| Microplates with Cell-Repellent Surface | 651970 | 96 well Microplate, PS, V-bottom, cell-repellent surface, clear, with lid, sterile |
| Microplates with Cell-Repellent Surface | 650979 | 96 well Microplate, PS, U-bottom, cell-repellent surface, clear, with lid, sterile |
| Microplates with Cell-Repellent Surface | 781970 | 384 well Microplate, PS, cell-repellent surface, clear, with lid, sterile |
| Microplates with Cell-Repellent Surface | 781974 | 384 Well Microplate, PS, cell-repellent surface, white, μClear®, sterile, with lid |
| Microplates with Cell-Repellent Surface | 781976 | 384 well Microplate, PS, cell-repellent surface, black, μClear®, with lid, sterile |
| Microplates with Cell-Repellent Surface | 781976-SIN | 384 well Microplate, PS, cell-repellent surface, black, μClear®, with lid, sterile |
| Microplates with Cell-Repellent Surface | 787979 | 384 well Microplate, PS, U-bottom, cell-repellent surface, black, μClear®, with lid, sterile |
| Microplates with Cell-Repellent Surface | 782974 | 1536 Well Microplate, PS, cell-repellent, white, HiBase, μClear®, sterile, with lid |
| Microplates with Cell-Repellent Surface | 782976 | 1536 Well Microplate, PS, cell-repellent, black, HiBase, μClear®, sterile, with lid |
| Microplates with Cell-Repellent Surface | 783976 | 1536 Well Microplate, PS, cell-repellent, black, LoBase, μClear®, sterile, with lid |
Product offering may vary depending on geographic location.
Brief description of the topic/short abstract
with a few learning objectives
The growing push for 3D cell culture models is limited by technical challenges in handling, processing, and scalability to high-throughput applications. To meet these challenges, Greiner Bio-One offers an innovative technology called magnetic 3D bioprinting, in which cells are individually magnetized and assembled with magnetic forces without changing the phenotype or the physiology of these cells. Magnetization of cells not only makes routine cell culture and experiments feasible and scalable, but also enables scientist to have fine spatial control in the formation of spheroids or more complex structures.
This presentation will focus on recent developments using magnetic 3D bioprinting, particularly in cancer biology and immunology. Specifically, it will demonstrate a method for phenotypic profiling of cell types within spheroids using real-time high-throughput imaging.
Learning objectives:
Bio-sketch of the presenter
Dr. Souza is the Director of Global Business Development and Innovation, 3D Culture at Greiner Bio-One and Adjunct Assistant Professor at the University of Texas Health Science Center at Houston. He is one of the creators of magnetic 3D cell culture, including magnetic 3D bioprinting. Results using these groundbreaking technologies have been reported in various high-impact scientific journals, including Nature Nanotechnology, Proceedings of the National Academy of Sciences, Nature Protocols, Biomaterials, and Nature Reviews Cancer. Recently, his work was selected for the Short List of The 2017 Lush Prize for outstanding research producing an effective non-animal safety test. He was also an invited speaker at TEDx Houston. Dr. Souza’s research has been funded by grants from National Science Foundation (NSF), National Institute of Health (NIH), Department of Defense (DOD), Center for Advancement of Science in Space (CASIS), and Texas Emerging Technology Fund (ETF).
| Application | Title | Author |
|---|---|---|
| General Protocol | Three-dimensional cell culturing by magnetic levitation | Haisler et al. |
| General Protocol | Protocol for Invasion Assay in a 384-well plate | Glauco R Souza |
| General Protocol | Protocol for magnetizing cells in suspension with NanoShuttle using centrifugation | Glauco R Souza |
| General Protocol | Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts | Noel et al. |
| General Protocol | Protocol for Handling Small Spheroids <5,000 cells in a 384-well plate | Glauco R Souza |
| Immunohistochemistry | Fixing and Embedding Magnetically Levitated Cultures in Paraffin | Glauco R Souza |
| Immunohistochemistry | Immunohistochemistry of Paraffin Embedded Tissue | Glauco R Souza |
| Immunohistochemistry | Immunohistochemistry of Non-Paraffin Embedded Tissue | Glauco R Souza |
| qRT-PCR | RNA Isolation in Levitated 3D Cultures | Glauco R Souza |
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