3D cell culturing by magnetic levitation

Standard monolayer cell culturing on tissue culture plastic has notably improved our understanding of basic cell biology, but it does not replicate the complex 3D architecture of in vivo tissue, and it can significantly modify cell properties. This often compromises experiments in basic life science, leads to misleading drug-screening results on efficacy and toxicity, and produces cells that may lack the characteristics needed for developing tissue regeneration therapies.[1][2][4][5][6][7][8][9]

The future of cell culturing for fundamental studies and biomedical applications lies in the creation of multicellular structure and organization in three dimensions.[4][5][7][8][10][11][12] Many schemes for 3D culturing are being developed or marketed, such as bio-reactors[13] or protein-based gel environments.[7][14]

A 3D cell culturing system known as the Bio-Assembler uses biocompatible polymer-based[1] reagents to deliver magnetic nanoparticles to individual cells so that an applied magnetic driver can levitate cells off the bottom of the cell culture dish and rapidly bring cells together near the air-liquid interface. This initiates cell-cell interactions in the absence of any artificial surface or matrix. Magnetic fields are designed to rapidly form 3D multicellular structures in as little as a few hours, including expression of extracellular matrix proteins. The morbius, protein expression, and response to exogenous agents of resulting tissue show great similarity to in vivo results.[1]

3D cell culturing by magnetic levitation method (MLM) was developed from collaboration between scientists at Rice University and University of Texas MD Anderson Cancer Center in 2008.[1] Since then, this technology has been licensed and commercialized by Nano3D Biosciences.[15]

Above is a picture showing 3D cell culturing through magnetic levitation with the Bio-Assembler cell culturing system. (A) A magnetic iron oxide nanoparticle assembly known as Nano shuttle is added and dispersed over cells and the mixture is incubated. (B) After incubation with Nanoshuttle, cells are detached and transferred to a petri dish. (C) A magnetic drive is then placed on top of a petri dish top. (D) The magnetic field causes cells to rise to the air–medium interface. (E) Human umbilical vein endothelial cells (HUVEC) levitated for 60 minutes (left images) and 4 hours (right images) (Scale bar, 50 μm). The onset of cell-cell interaction takes place as soon as cells levitate, and 3D structures start to form. At 1 hour, the cells are still relatively dispersed, but they are already showing some signs of stretching. Formation of 3D structures is visible after 4 hours of levitation (arrows).[1][2]

Protein expression in levitated cultures shows striking similarity to in vivo patterns. N-cadherin expression in levitated human glioblastoma cells was identical to the expression seen in human tumour xenografts grown in immunodeficient mice, while standard 2D culture showed much weaker expression that did not match xenograft distribution as shown in the picture below.[1] The transmembrane protein N-cadherin is often used as an indicator of in-vivo-like tissue assembly in 3D culturing.[1]

In the picture above, distribution of N-cadherin (red) and nuclei (blue) in human brain cancer mouse xenograft (left, human brain cancer cells grown in a mouse brain), brain cancer cells cultured by 3D magnetic levitation for 48 h. (middle), and cells cultured on a glass slide cover slip (2D, right). The 2D system shows N-cadherin in the cytoplasm and nucleus and notably absent from the membrane, while in the levitated culture and mouse, N-cadherin is clearly concentrated in the membrane, and also present in cytoplasm and cell junctions.[1]

Listed below are the cell types (primary and cell lines) that have been successfully cultured by the magnetic levitation method. The second table is the same but with images included. More images are available at Nano3D Biosciences, Inc.

Same table as above, but with images.

Cells	Cell line	Organism	Organ tissue	Image
Murine endothelial[16]	Cell line	Mouse	Vessel	[16]
Murine adipocyte[16]	Cell line	Mouse	Adipose	Adipocytes cultured by MLM acquire in vivo morphology in adipospheres [16]
Rattus norvegicus hepatoma	Cell line	Rat	Liver	Hepatoma cultured by MLM for different cell numbers at 24 hours
Pulmonary fibroblasts (HPF)[17]	Primary	Human	Lung	Primary pulmonary fibroblasts cultured by MLM compared to in vivo [17]
Pulmonary endothelial (HPMEC)[17]	Primary	Human	Lung
Small airway epithelial (HSAEpiC)[17]	Primary	Human	Lung	Primary small airway epithelial cells cultured by MLM compared to in vivo [17]
Bronchial epithelial[17]	Primary	Human	Lung
Human alveolar adenocarcinoma[17]	A549	Human	Lung	Human alveolar adenocarcinoma cell line - A549 cultured by MLM at different time points and cell numbers [17]
Type II alveolar [17]	Primary	Human	Lung
Tracheal smooth muscle (HTSMC)[17]	Primary	Human	Lung	SMC cultured by MLM with IHC staining [17]
Mesenchymal stem cells (HMSC)	Primary	Human	Bone marrow	Human mesenchymal stem cells cultured by MLM
Bone marrow endothelial cells (HBMEC)	Primary	Human	Bone marrow
Dental pulp stem cells	Primary	Human		Dental pulp cultured by MLM (3D vs. 2D) - 5 days
Human umbilical vein endothelial cells (HUVEC)	Primary	Human		HUVEC cultured by MLM in 24 Well format
Murine chondrocytes	Primary	Mouse	Bone	Chondrocytes cultured in 3D by MLM at different time points
Murine adipose tissue[16]	Primary	Mouse		Primary WAT cells cultured by MLM assemble into vascularized adipospheres [16]
Heart valve endothelial	Primary	Porcine
Pre-adipocytes fibroblasts[16]	3T3	Mouse
Neural stem cells	C17.2	Mouse	Brain	Levitating neural stem cells cultured by MLM
Human embryonic kidney cells	HEK293	Human	Kidney	HEK 293 cultured by MLM and tested with ibuprofen using bioassay
Melanoma	B16	Mouse	Skin
Astrocytes[1]	NHA	Human	Brain	Astrocyte and glioblastoma invasion assay performed after 3D culturing by MLM [1]
Glioblastomas[1]	LN229	Human	Brain	Glioblastoma cultured with Bio-Assembler vs. Matrigel at 24 hours [1]
T-cells and antigen presenting cells		Human
Mammary epithelial	MCF10A	Human	Breast	Bio-Assembler vs. Matrigel with human mammary epithelial cells
Breast cancer	MDA231	Human	Breast
Osteosarcoma	MG63	Human	Bone