Biomedical Engineering, Tulane University
Curley, J. L., Jennings, S. R., Moore, M. J. Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography. J. Vis. Exp. (48), e2636, doi:10.3791/2636 (2011).
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 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have 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 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. 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 cell-restrictive 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.
1. DMD Setup
2. Dual Hydrogel Constructs for Tissue Explant Cultures
A. DRG explant adhesion
B. Dynamic mask photopolymerization
A digital micromirror device (DMD) is a 1024 x 768 array of individual mirrors, similar to that in projection televisions, which selectively reflects light based on mirror position. For our purposes, the DMD is used to pattern ultraviolet (UV) light onto photocrosslinkable hydrogels, creating specifiable hydrogel geometries in a simple and rapid manner. Figure 1 depicts the setup of the DMD and UV light path. Though our DMD is a standalone unit, the device can also be integrated for use with many existing microscopes.
C. Secondary hydrogel
3. Dual Hydrogel 3D Cell Encapsulation
Dual hydrogel encapsulation is appropriate when using any self-assembling gel. The photocrosslinkable gel, in this case PEG, serves as a structural support for the geometric presentation of the self-assembling gel, for example Puramatrix or agarose. Some of the methods, specifically the type of gel and choice of photomask, will depend on the particular desired application.
4. Single Hydrogel 3D Cell Encapsulation
A single hydrogel encapsulation would be appropriate for any situation where the cells can be examined inside of a photocrosslinkable hydrogel.
5. Thin Film Hydrogel Polymerization
6. Representative Results
Examples of dual hydrogel constructs containing DRG explants are shown in Figure 2. Notice that cellular migration and neurite extension is limited to the cell permissive region of the dual hydrogel construct. Figure 3 depicts dissociated cells encapsulated similarly inside the dual hydrogel constructs. Due to the dynamic nature of the DMD photomask, the geometry available for encapsulation is limited only by the dimensions and resolution of the optics. Cell encapsulation was also possible inside a single photopolymerizable hydrogel, PEG, and a live/dead viability test was performed as evidenced in Figure 4. Encapsulation in PEG is meant only as an example, as PEG does not represent an ideal environment for neural cells. Therefore, the cell viability realized in our PEG constructs is understandably low. Finally, examples of utilizing thin PEG films as a patterned restrictive layer for cell adhesion on cell culture inserts are shown in Figure 5. Additionally, examples of possible "bad" results are offered in Figure 6.
The results represent only a small fraction of the possible uses of the methods developed in our lab. They are meant to demonstrate the ease, versatility and viability of our approach, and could be treated as "proof of principle" for researchers to develop their own possible adaptations.
Figure 1. Schematic illustration of the light path used for photolithography. Inset: The UV light illuminates the DMD at an angle of 45°, and 24° below the plane of the mirror array.
Figure 2. Labeled growth and proliferation in DRG containing dual hydrogel constructs. A-D) Images portraying polymerized PEG constructs (gray) with neurites labeled with Beta III tubulin (green), DAPI stained cell nuclei (blue). The DRG explants are contained in Puramatrix and located in the circular regions of the pattern, with neurites growing towards the bifurcation(s).
Figure 3. Dual hydrogel constructs containing cells labeled with calcein AM, a live cell marker, after 48 hours in growth medium. A-D) Various PEG shapes, filled with Puramatrix containing dissociated DRG neurons (~5x103 cells/mL).
Figure 4. Single hydrogel construct containing live cells labeled with Calcein AM (green) and dead cells labeled with ethidium homodimer-1 (red) after 24 hours in growth medium (5x103 cells/mL).
Figure 5. Cell restrictive PEG polymerized as a thin film using a "test pattern" to selectively adhere dissociated cells to the collagen coated membrane of permeable supports. A, B) Live cells are labeled after 48 hours with Calcein AM (green), while dead cells are labeled with ethidium homodimer-1 (red). Minimal cell adhesion occurs in the area containing the thin PEG film.
Figure 6. Representative images of undesirable results. A) Partial polymerization of PEG, leading to an unusable PEG construct. Improper polymerization can occur due to the presence of a meniscus in the pre-polymer medium, insufficient amounts of polymerization medium, insufficient UV exposure or improper focus of the optics. B) Image portraying polymerized PEG constructs (gray) with neurites labeled with Beta III tubulin (green), DAPI stained cell nuclei (blue). The neurites were able to grow outside of the patterned PEG channels. This often occurs in the condition that Puramatrix overflows on top of the PEG portion during injection.
The method described herein could be used by any investigator seeking simple and reproducible cell culture systems. Theoretically, due to the wide variety of photopolymerizable hydrogels available, the environment could be tailored to allow for use with any cell type, including whole tissue explants. Additionally, the dual hydrogel system allows for improved spatial control in the presentation of self-polymerizing hydrogels, which tend to form amorphous shapes on their own. The resulting "2.5D" micropatterned hydrogel constructs provide a 3D matrix for neural growth presented in a 2D configuration that enables convenient microscopic evaluation. The substrate on which the gels are polymerized can also be varied, allowing for greater control in experimental design. Our methods are optimized for use with cell culture permeable supports, as we have seen improved viability (Figure 4) as compared to polymerization on glass slides (data not shown). However, other polymerization surfaces may be more applicable for different applications: fabrication on glass slides used in microfluidics experiments or cell aggregate formations, for example.
Our experience with these culture systems has led to the identification of potential areas of difficulty. First, careful techniques are required to maintain the sterility of the constructs. Due to the bulky nature of the DMD setup, it is difficult to operate polymerization steps under sterile conditions. To combat this issue, the rinse step described in the methods is helpful, and antibiotics should be used in all media. Additionally, the final thickness and shape of the polymerized construct is highly dependent on the fluid behavior of the pre-polymer mixture, and the presence of a meniscus can result in gel constructs that are too thin or incompletely polymerized (Figure 6). Two steps can be taken to minimize the formation of a meniscus inside cell culture inserts. For thick hydrogels (>200 μm), a simple coating of Rain-X around the inside wall of the insert is sufficient. However, as briefly described above, for thin constructs (<200 μm), an oil layer is required to both minimize the meniscus and negate oxygen quenching of the free radical polymerization. Resolution was found to be dependent on thickness, with a decrease in feature size realized with increasingly thicker gels. The resolution also varied depending on whether the feature represented a positive or negative relief in the hydrogel. However, we achieved a sufficient resolution for constructs with minimum feature sizes on the scale of ~100 μm using only microscope objectives as focusing optics.
Our experiments have shown that the dual hydrogel constructs described here represent an excellent basis for the formation of basic in-vitro models of neurite growth and guidance. The micropatterning technique employed is an adaptation of existing methods18,19, but our set-up emphasized a simple to implement design and was optimized for production of dual hydrogel constructs on cell culture inserts; cell culture inserts were vital for improving cell viability as well as crosslinking around previously adherent tissue explants. The scope of the results shown is limited by the interests of our lab; however, we believe that the methods described in this publication will prove useful to researchers searching for a relatively cheap, quick and easy to use method for the fabrication of 3D cell culture models.
No conflicts of interest declared.
The authors would like to thank the laboratory of Prof. Anthony Windebank for sharing their expertise on DRG dissection and culture, as well as Prof. Shaochen Chen for helpful discussions regarding the DMD setup. This research was funded in part by Tulane University and grants from the Louisiana Board of Regents (LEQSF[2009-10]-RD-A-18) and the NIH (NS065374).
|Digital Micromirror Device||Texas Instruments||DLPD4X00KIT|
|Collagen Coated Transwell Permeable Support||Corning||3491||Also referred to as Cell Culture Insert in manuscript|
|Polyester Transwell Permable Support||Corning||3412||Also referred to as Cell Culture Insert in manuscript|
|Fetal Bovine Serum||Invitrogen||16000-036|
|Nerve Growth Factor||Invitrogen||13257-019|
|PEG 1000||Polysciences, Inc.||15178|
|Irgacure 2959||Ciba Specialty Chemicals||0298913AB|
|Oil||Have used both canola oil and silicon oil||Needs to be UV transparent, and minimize +/- meniscus formation|
|OmniCure Series 1000||EXFO|