Learn about current and future applications in 3D cell culture. At the 2019 SLAS trade show in Washington, Glauco Souza describes our 3D cell culture products and highlights their advantages and fields of application. Watch the video and get a deeper insight into 3D Cell Culture.
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
Spheroid formation by magnetic levitation, bioprinting or printings of rings.
Advantages of the magnetic 3D cell culture (m3D) technology are:
- 3D in a 2D workflow
- Reproducible spheroid formation
- Scalable – 6 Well to 1536 Well
- Performed on a flat surface optimal for high-resolution microscopy and HTS
- Rapid 3D culture formation within 24 hours for most cell types
- No specialized equipment or media
- Easy media changes and co-culture of different cell types
- Compatible with fluorescence microscopy, Western blotting, qRT-PCR, Flow Cytometry, viability assays, chemiluminescence, etc.
- Ready for automation
- Biocompatible nanoparticles to magnetize cells
- Magnetic 3D Bioprinting
- Magnetic Levitation
- BIO Assay - Magnetic Printing of Rings
- CELLSTAR® Cell-Repellent
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.
Bioprinted 3D co-cultures of lung adenocarcinoma (Calu-3; red) and fibroblasts (green) after 16 hours. Cancer cells reproducibly localized inside, while fibroblasts are mostly at the outside of the co-culture.
Basic steps of magnetic Bioprinting with magnetization of the cells, transfer to cell-repellent plate and bioprinting with a magnet.
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.
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.
Tumor cell spheroids grown in a 96 well U-bottom CELLSTAR® cell culture microplate with cell-repellent surface.
a) LNCaP cells form single spheroids in 96 well U-bottom microplates with cell-repellent surface. 3,000 cells were seeded per well and incubated at 37° C and 5% CO2 over a 7 day period.
b) Aggregate formation of human induced pluripotent stem cells (iPSCs) cultured in a 96 well U-bottom microplate with cell-repellent surface.
Applications & Cell lines
What 3D platform is best for your application?
|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: |
- smooth muscle
|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|
|Magnetic levitation 6 Well / 24 Well||657840||6 Well Bio-Assembler™ Kit|
|Magnetic levitation 6 Well / 24 Well||662840||24 Well Bio-Assembler™ Kit|
|Magnetic levitation 6 Well / 24 Well||662824||24 Well Custom Lid|
|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|
|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|
|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||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||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|
|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|
News / Events
Glauco Souza, Ph.D.
Phone +1 (0)281 939-3407
Greiner Bio-One GmbH
Phone: +49 7022 948-0
E-Mail: [email protected]
Greiner Bio-One worldwide!