Living in Three Dimensions: 3D Nanostructured Environments for Cell Culture and Regenerative Medicine

Schindler, Melvin; Nur-E-Kamal, Atom; Ahmed, Ijaz; Kamal, Jabeen; Liu, Hsing-Yin; Amor, Nathan; Ponery, A. S.; Crockett, David P.; Grafe, Timothy H.; Chung, Heesung; Weik, Thorn; Jones, Elizabeth; Meiners, Scott J.

doi:10.1385/cbb:45:2:215

Cited by 118 publications

(83 citation statements)

References 118 publications

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AbstractIncreasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities [1][2][3] . Studies in fields such as cancer research , and cell-matrix interaction 7,8 have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.

Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization [9][10][11][12] .

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mentioning

confidence: 99%

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Curley

Jennings

Moore

2011

JoVE

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Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities [1][2][3] . Studies in fields such as cancer research , and cell-matrix interaction 7,8 have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization [9][10][11][12] . Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning . Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cellrestrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (<200 μm) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast. Video LinkThe video component of this article can be found at https://www.jove.com/video/2636/ Protocol 1. DMD Setup 1. The DMD board, UV light guide (with collimator) and 4x objective lens are all mounted vertically on a vibration isolation table. 2. The UV light guide sh...

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Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization [9][10][11][12] .

…”

mentioning

confidence: 99%

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Curley

Jennings

Moore

2011

JoVE

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“…Microscale technologies are emerging powerful tools for tissue engineering that can help in generating physiologically relevant, reproducible and well controlled cell-based systems [44]. Microfabrication techniques allowed the development of a wide range of synthetic nanostructured 3-D substrates, now available for cell culturing, that are promising for ensuring more reliable and specific 3-D microenvironments for cell models [75]. Coupling microfabrication of physically and chemically defined 3-D surfaces/scaffolds with advanced photo patterning, soft lithography techniques and microfluidics has led to a great enhancement in the complexity and biomimetic properties of engineered cell constructs [44].…”

Section: Tissue Engineering and Microtechnologies: The Perspectivesmentioning

confidence: 99%

Modelling tissues in 3D: the next future of pharmaco-toxicology and food research?

2008

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The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.

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“…Gels were formed by placing 50 mL=well of ECM extract in a 96-well plate and incubating the solution for 1-4 h at 378C. Subsequently, 2Â10 4 HUVECs, a cell number commonly used for Matrigel morphogenesis assay, 18,19 were seeded on the ECM gels. The cells were incubated at 378C overnight and imaged 24 h later.…”

Section: Cell Culturementioning

confidence: 99%

“…1,2 Historically, two-dimensional (2D) culture systems have been used to gain insight into cell-extracellular matrix (ECM) interactions; however, cell responses to ECM function differently in 2D versus 3D systems. 1,[3][4][5] Cells cultured in, or on, ECM gels recapitulate many of their in vivo functions as they are exposed to physical and mechanical conditions that more closely approximate the in vivo environment. 4 ECM-based biomaterials have played a prominent role in tissue engineering and regenerative medicine.…”

Section: Introductionmentioning

confidence: 99%

“…1,[3][4][5] Cells cultured in, or on, ECM gels recapitulate many of their in vivo functions as they are exposed to physical and mechanical conditions that more closely approximate the in vivo environment. 4 ECM-based biomaterials have played a prominent role in tissue engineering and regenerative medicine. Clinically successful tissue regeneration has been observed with collagen-based materials, small intestine submucosa, and decellularized tissues.…”

Section: Introductionmentioning

confidence: 99%

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Extraction and Assembly of Tissue-Derived Gels for Cell Culture and Tissue Engineering

Uriel

Labay

Francis-Sedlak

et al. 2009

Tissue Engineering Part C: Methods

108

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Living in Three Dimensions: 3D Nanostructured Environments for Cell Culture and Regenerative Medicine

Cited by 118 publications

References 118 publications

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Modelling tissues in 3D: the next future of pharmaco-toxicology and food research?

Extraction and Assembly of Tissue-Derived Gels for Cell Culture and Tissue Engineering

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