01

Technology

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 a 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 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.1 In 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 gentle nature of mag­netic lev­i­ta­tion allows cul­tures to acquire macroscale mor­phol­ogy that mostly resem­bles its tissue if origin. Bottom-up the 3D cell cul­ture can be ana­lyze 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.

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 uti­lize mild mag­netic forces to induce cell aggre­ga­tion and print one spher­oid at the bottom of each well 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 MagPenTM. 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.

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 NanoShuttleTM-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 iPodTM 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

n3Dock – iPod-based imaging.

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 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.
TitleApplicationYear
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02

Applications & Cell lines

What 3D platform is best for your application?

Stem Cell LinesCancer Cell LinesPrimary Cell LinesOther Cell Lines
Neural stem cellsLN229 – Glioblastoma cellsPrimary Glioblastoma cellsHuman Astrocyte
Mesenchymal stem cellHepG2 — human liver car­ci­noma human lung pri­mary cells:
— epithe­lial
— endothe­lial
— fibrob­lasts
— smooth muscle
3T3 fibrob­lasts
Neural crest-derived mes­enchy­mal stem cellA549 — Lung epithe­lial ade­carcenomaValvular inter­sti­tial cells (VICs)Bend (brain endothe­lial)
Dental pulp stem cellPC3 — Human prostate cancerValvular endothe­lial cells (VECs)Adipocyte
H-4-II-E — Rat hepatoma, liverAortic valve co-cul­tures (AVCCs)Huvec — human umbe­l­i­cal endothe­lial cells
MDA-231 — human breast cancerPrimary mouse heart cellsHPF — human pul­monary fibrob­last
LNCaP — Prostate cancer cell lineHuman Primary vas­cu­lar smooth muscleSMC — tra­cheal smooth muscle cell
Ovarian cancer cellsPrimary Miomytrial Smooth muscleHEK293 — human embry­onic kidney
Panc-1 — pan­cre­atic cancer cellPrimary human hepa­to­cytesMCF-10A — breast epithe­lial cell line
Cancer asso­ci­ated fibrob­lastsPrimary pan­cre­atic cancer cellsFibroblast
Triple neg­a­tive inflam­a­tory breast cancerPrimary tissue from PDXChondrocytes
Caki-1 – human renal cancer cell linePrimary fibrob­lastsT-cells
OsteosarcomaKeratinocytesA10 — Rat vas­cu­lar smooth muscle
HCT116 — colon cancer cell line

03

Products

TypeOrder no.Description
Magnetic lev­i­ta­tion 6 Well / 24 Well6578406 Well Bio-Assembler™ Kit
Magnetic lev­i­ta­tion 6 Well / 24 Well66284024 Well Bio-Assembler™ Kit
Magnetic lev­i­ta­tion 6 Well / 24 Well66282424 Well Custom Lid
Spheroid Bioprinting 96 Well / 384 Well65584096 Well Bioprinting Kit
Spheroid Bioprinting 96 Well / 384 Well65584196 Well Bioprinting Kit µClear®
Spheroid Bioprinting 96 Well / 384 Well65585096 Well Ring Drive
Spheroid Bioprinting 96 Well / 384 Well781840384 Well Bioprinting Kit
Spheroid Bioprinting 96 Well / 384 Well781841384 Well Bioprinting Kit µClear®
Spheroid Bioprinting 96 Well / 384 Well781850384 Well Ring Drive
Screening & Imaging 96 Well / 384 Well65584696 Well BiO Assay™ Kit
Screening & Imaging 96 Well / 384 Well65584996 Well BiO Assay™ Kit & Imaging System
Screening & Imaging 96 Well / 384 Well781846384 Well BiO Assay™ Kit
Screening & Imaging 96 Well / 384 Well781849384 Well BiO Assay™ Kit & Imaging System
Consumables / Accessories for Magnetic 3D Cell Culture657841NanoShuttle™-PL Refill
Consumables / Accessories for Magnetic 3D Cell Culture657843NanoShuttle™-PL Refill 3 Pack
Consumables / Accessories for Magnetic 3D Cell Culture657846NanoShuttle™-PL Refill 6 Pack
Consumables / Accessories for Magnetic 3D Cell Culture657852NanoShuttle™-PL Refill 12 Pack
Consumables / Accessories for Magnetic 3D Cell Culture657850MagPen™ 3 Pack
Consumables / Accessories for Magnetic 3D Cell Culture657847NanoShuttle™-PL Refill 6 Pack with free iPod
Consumables / Accessories for Magnetic 3D Cell Culture657860Imaging Kit
Consumables / Accessories for Magnetic 3D Cell Culture657810Battery Power for Imaging Kit
TypeOrder no.Description
Dishes with Cell-Repellent Surface 627979Cell Culture Dish, Ø 35×10 mm, PS, cell-repel­lent sur­face, clear, ster­ile
Dishes with Cell-Repellent Surface 628979Cell Culture Dish, Ø 60×15 mm, PS, cell-repel­lent sur­face, clear, ster­ile
Dishes with Cell-Repellent Surface 664970Cell Culture Dish, Ø 100×20 mm, PS, cell-repel­lent sur­face, clear, ster­ile
Flasks with Cell-Repellent Surface690980Cell Culture Flask, 50 ml, PS, cell-repel­lent sur­face, ster­ile, white screw cap
Flasks with Cell-Repellent Surface690985Cell Culture Flask, 50 ml, PS, cell-repel­lent sur­face, ster­ile, white filter screw cap
Flasks with Cell-Repellent Surface658980Cell Culture Flask, 250 ml, PS, cell-repel­lent sur­face, ster­ile, white screw cap
Flasks with Cell-Repellent Surface658985Cell Culture Flask, 250 ml, PS, cell-repel­lent sur­face, ster­ile, white filter screw cap
Flasks with Cell-Repellent Surface660980Cell Culture Flask, 550 ml, PS, flat flask design, cell-repel­lent sur­face, ster­ile, white screw cap
Flasks with Cell-Repellent Surface660985Cell Culture Flask, 550 ml, PS, flat flask design, cell-repel­lent sur­face, ster­ile, white filter screw cap
Flasks with Cell-Repellent Surface661980Cell Culture Flask, 650 ml, PS, high flask design, cell-repel­lent sur­face, ster­ile, white screw cap
Flasks with Cell-Repellent Surface661985Cell Culture Flask, 650 ml, PS, high flask design, cell-repel­lent sur­face, ster­ile, white filter screw cap
Multiwell Plates with Cell-Repellent Surface6579706 well Multiwell Plate, PS, cell-repel­lent sur­face, clear, with lid, ster­ile
Multiwell Plates with Cell-Repellent Surface66297024 well Multiwell Plate, PS, cell-repel­lent sur­face, clear, with lid, ster­ile
Multiwell Plates with Cell-Repellent Surface67797048 well Multiwell Plate, PS, cell-repel­lent sur­face, clear, with lid, ster­ile
Microplates with Cell-Repellent Surface65597096 well Microplate, PS, F-bot­tom/chim­ney well, cell-repel­lent sur­face, clear, with lid, ster­ile
Microplates with Cell-Repellent Surface65597696 well Microplate, PS, F-bot­tom/chim­ney well, cell-repel­lent sur­face, black, μClear®, with lid, ster­ile
Microplates with Cell-Repellent Surface655976-SIN96 well Microplate, PS, F-bot­tom/chim­ney well, cell-repel­lent sur­face, black, μClear®, with lid, ster­ile
Microplates with Cell-Repellent Surface65097096 well Microplate, PS, U-bottom, cell-repel­lent sur­face, clear, with lid, ster­ile
Microplates with Cell-Repellent Surface65197096 well Microplate, PS, V-bottom, cell-repel­lent sur­face, clear, with lid, ster­ile
Microplates with Cell-Repellent Surface65097996 well Microplate, PS, U-bottom, cell-repel­lent sur­face, clear, with lid, ster­ile
Microplates with Cell-Repellent Surface781970384 well Microplate, PS, cell-repel­lent sur­face, clear, with lid, ster­ile
Microplates with Cell-Repellent Surface781976384 well Microplate, PS, cell-repel­lent sur­face, black, μClear®, with lid, ster­ile
Microplates with Cell-Repellent Surface781976-SIN384 well Microplate, PS, cell-repel­lent sur­face, black, μClear®, with lid, ster­ile
Microplates with Cell-Repellent Surface787979384 well Microplate, PS, U-bottom, cell-repel­lent sur­face, black, μClear®, with lid, ster­ile

04

Publications

YearAuthor/Title/Link
2018Eckhardt, B. L. et al. Clinically rel­e­vant inflam­ma­tory breast cancer patient-derived xenograft-derived ex vivo model for eval­u­a­tion of tumor-spe­cific ther­a­pies. PLOS ONE. 13, 5 (2018).
2018Hou, S. et al. Advanced Development of Primary Pancreatic Organic Tumor Models for High-Throughput Phenotypic Drug Screening. SLAS DISCOVERY. 0, 0 (2018).
2017Noel, P. et al. Preparation and Metabolic Assay of 3-dimen­sional Spheroid Co-cul­tures of Pancreatic Cancer Cells and Fibroblasts. J. Vis. Exp. 126, 56081 (2017).
2017 Desai, P. K., Tseng, H. & Souza, G. R. Assembly of hepa­to­cyte spher­oids using mag­netic 3D cell cul­ture for CYP450 inhibition/induction. Int. J. Mol. Sci. 18, 1085 (2017).
2017Souza, G. R. et al. Magnetically bio­printed human myome­trial 3D cell rings as a model for uter­ine con­trac­til­ity. Int. J. Mol. Sci. 18, 683 (2017).
2016Pan, Y. et al. miR-509–3p is clin­i­cally sig­nif­i­cant and strongly atten­u­ates cel­lu­lar migra­tion and multi-cel­lu­lar spher­oids in ovar­ian cancer. Oncotarget. 7.18, 25930–25948 (2016).
2016Tseng, H. et al. A high-through­put in vitro ring assay for vasoac­tiv­ity using mag­netic 3D bio­print­ing. Sci. Rep. 6, 30640 (2016).
2016Hogan, M. et al. Assembly of a func­tional 3D pri­mary car­diac con­struct using mag­netic lev­i­ta­tion. AIMS Bioeng. 3, 277–288 (2016).
2016Lin, H. et al. Nanoparticle improved stem cell ther­apy for erec­tile dys­func­tion in a rat model of cav­ernous nerve injury. J. Urol. 195, 788–95 (2016).
2015Tseng, H. et al. A spher­oid tox­i­c­ity assay using mag­netic 3D bio­print­ing and real-time mobile device-based imag­ing. Sci. Rep. 5, 13987 (2015).
2014Jaganathan, H. et al. Three-dimen­sional in vitro co-cul­ture model of breast tumor using mag­netic lev­i­ta­tion. Sci. Rep. 4, 6468 (2014).
2013Tseng, H. et al. A three-dimen­sional co-cul­ture model of the aortic valve using mag­netic lev­i­ta­tion. Acta Biomater. 10, 173–82 (2013).
2013Timm, D. M. et al. A high-through­put three-dimen­sional cell migra­tion assay for tox­i­c­ity screen­ing with mobile device-based macro­scopic image analy­sis. Sci. Rep. 3, 3000 (2013).
2013Haisler, W. L. et al. Three-dimen­sional cell cul­tur­ing by mag­netic lev­i­ta­tion. Nat. Protoc. 8, 1940–9 (2013).
2013Tseng, H. et al. Assembly of a three-dimen­sional mul­ti­type bron­chi­ole cocul­ture model using mag­netic lev­i­ta­tion. Tissue Eng. Part C. Methods 19, 665–75 (2013).
2013Becker, J. L. & Souza, G. R. Using space-based inves­ti­ga­tions to inform cancer research on Earth. Nat. Rev. Cancer 13, 315–27 (2013).
2012Daquinag, A. C., Souza, G. R. & Kolonin, M. G. Adipose tissue engi­neer­ing in three-dimen­sional lev­i­ta­tion tissue cul­ture system based on mag­netic nanopar­ti­cles. Tissue Eng. Part C. Methods 19, 336–44 (2012).
2010Souza, G. R. et al. Three-dimen­sional tissue cul­ture based on mag­netic cell lev­i­ta­tion. Nat. Nanotechnol. 5, 291–6 (2010).
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Contact us







Contact Persons

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Glauco Souza, Ph.D.

Director of Global Business Development & Innovation 3D Cell Culture Bioscience

Phone +1 (0)281 939‑3407
[email protected]

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Dr. Jennifer Bischoff

Produktmanager / Product Manager

Phone +49 (0)7022 948–317
[email protected]

Greiner Bio-One GmbH
Maybachstr. 2
D-72636 Frickenhausen
Germany
Phone: +49 7022 948–0
E-Mail: [email protected]