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).

1. DMD Setup
<ol>
	<li>The DMD board, UV light guide (with collimator) and 4x objective lens are all mounted vertically on a vibration isolation table.</li>
		<li>The UV light guide should be adjusted so that the light hits the mirror array at an angle of 45&deg; relative to the plane of the mirrors, and 24&deg; below the plane of the mirrors (Figure 1).</li>
		<li>The objective lens is mounted so that the distance from the DMD to the objective lens corresponds to the focal length associated with the lens.</li>
		<li>An inverted microscope is placed below the objective lens, such that the focused light reflected from the DMD can be visualized through the microscope. The distance from the objective lens to the polymerization surface should approximately correspond to the working distance of the lens. The stage on the microscope can then be used to adjust this distance to finely focus the image. This distance may vary depending on the chosen polymerization surface.</li>
</ol>
2. Dual Hydrogel Constructs for Tissue Explant Cultures
A. DRG explant adhesion
<ol>
<li>Coat the walls of 6-well collagen coated cell culture inserts (Corning) with Rain-X, taking care to avoid the membrane itself. (Alternatively, a hydrophobic barrier pen may be used.)</li>
<li>Hydrate inserts overnight with 1.5 mL of adhesion medium in an incubator at 37 &deg;C and 5% CO2. Adhesion medium is neurobasal medium with 10% fetal bovine serum, 1% Penicillin/streptomycin, 0.5mM L-glutamine, and 20 &mu;g/mL NGF.</li>
<li>Harvest embryonic dorsal root ganglia (DRG) from E-15 rat pups. DRG should be plated onto collagen coated 6-well inserts, as many as four per insert.</li>
<li>Incubate inserts for 2 hours to allow for DRG adherence to the membrane.</li>
</ol>
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.
<ol>
<li>Remove all excess liquid from insert, and add 500 &mu;L of polymerization medium inside the insert. Polymerization medium contains 10% PEG (MW 1000) and 0.5% Irgacure 2959 dissolved in neurobasal medium supplemented with 20 &mu;g/mL NGF and 1% Pen/Strep.</li>
<li>Place the insert underneath the DMD device on a Rain-X treated glass slide.</li>
<li>Load the appropriate black and white image to be used as a "photomask" on the DMD, through the use of the included ALP-3 basic GUI program. For our purposes, a bifurcating shape was chosen to allow for implementation of neurite guidance systems.</li>
<li>Using an inverted microscope for visualization, align the tissue explants with the appropriate location on the photomask using a visible light source reflecting off the DMD.</li>
<li>Switch the visible light source for the UV light source, and illuminate the PEG solution until crosslinking is sufficient. (For the conditions given and 5.0 Watts/cm2 incident on the DMD, crosslinking can be completed in as little as 55 seconds.) Repeat for all four DRG on the insert.</li>
<li>Wash each insert three times with sterile DPBS and 1% Pen-Strep.</li>
<li>Add 1.5mL growth medium underneath the cell culture inserts, and allow to equilibrate in an incubator for 30 minutes. Growth medium is neurobasal medium containing 2% B-27, 1% Pen/Strep, 0.5mM L-glutamine, and 20 &mu;g/mL NGF.</li>
</ol>
C. Secondary hydrogel
Puramatrix
<ol>
<li>For neuronal applications, 1% Puramatrix is diluted according to manufacturer's instructions to 0.15% in sterile H2O and supplemented with 1 &mu;g/mL soluble laminin.</li>
<li>All excess media must be removed from the PEG voids, which contain the DRG explants, using a sterile cotton tip applicator, Kimwipe, or micropipette.</li>
<li>Puramatrix is added to the PEG voids with a micropipette in order to fill the empty space without overflowing. Depending on void volume, typically ~ 1 &mu;L is used per construct.</li>
<li>Puramatrix begins the process of self-assembly immediately upon contact with a physiological salt solution, i.e. the swollen PEG, but 1.5 mL of growth medium is added underneath the insert and placed in the incubator for one hour to ensure total gelation.</li>
<li>Initially, Puramatrix is slightly acidic, so change the media after one hour to allow the pH to equilibrate.</li>
<li>Media changes are required approximately every 48 hours. Be careful that all media is added underneath the insert, to protect the integrity of mechanically weak Puramatrix.</li>
</ol>
Agarose
<ol>
<li>For neuronal applications, agarose is diluted to a 1% solution in growth medium and placed in a 60 &deg;C water bath until the agarose fully dissolves (~1 hr). The solution is then supplemented with 1 &mu;g/mL soluble laminin.</li>
<li>All excess media must be removed from the PEG voids.</li>
<li>Agarose is added to the PEG voids in order to fill the empty space without overflowing. Depending on volume of void, typically ~ 1 &mu;L is used per construct.</li>
<li>1.5 mL of growth medium is pre-chilled (8 &deg;C) in a 6-well plate, and the agarose containing inserts are transferred to the chilled media and maintained in a refrigerator at 8 &deg;C for at least three minutes to allow agarose to gel.</li>
<li>Finally, the inserts are transferred to 1.5 mL of pre-warmed growth medium (37 &deg;C) and maintained in the incubator at 37 &deg;C, with media changes required every 48 hours.</li>
</ol>
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.
<ol>
<li>Load an appropriate mask on the DMD. For our application, cell survival, we simply chose a cylindrical presentation of Puramatrix to aid in the imaging of cells. Researchers studying cell signaling might be interested in a compartmental geometry to allow for the diffusion of chemotactic molecules. Additionally, a rough approximation of an artery was shown to represent possible application to blood vessel research.</li>
<li>Treat the walls of a polyester cell culture insert with Rain-X, and place under DMD on Rain-X coated slide.</li>
<li>Add 500 &mu;L of polymerization medium to the insert. Induce PEG crosslinking by exposure to UV light for 55 seconds.</li>
<li>Wash three times with sterile DPBS and 1% Pen-Strep.</li>
<li>Remove all excess media from the PEG voids.</li>
<li>Spin down cells at the desired density into a pellet. Care must be taken to remove all medium from the cell pellet prior to mixing, as Puramatrix begins self-assembly immediately upon contact with a salt solution. Suspend the cells inside 0.15% Puramatrix diluted in sterile H2O supplemented with 10% sucrose.</li>
<li>Inject the Puramatrix/cell/sucrose mixture inside voids in PEG.</li>
<li>Add 1.5 mL of growth medium underneath the insert, and allow to gel inside incubator for one hour.</li>
<li>Change media after one hour, and ~every 48 hours thereafter.</li>
</ol>
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.
<ol>
<li>Load an appropriate mask on the DMD. For cell survival studies, we again chose a basic circular mask to represent a cylinder. Masks similar to those shown in method 4 could again be applied for the appropriate research field.</li>
<li>Treat a polyester cell culture insert with Rain-X, and place under DMD on Rain-X coated slide.</li>
<li>Cells at any desired concentration can be added directly to the 10% PEG solution, mixing well to ensure homogenous distribution.</li>
<li>Add 500 &mu;L of polymerization medium to the insert. Induce PEG crosslinking by exposure to UV light for 55 seconds.</li>
<li>Wash three times with sterile DPBS and 1% Pen-Strep.</li>
<li>Fill with growth medium both below and on top of the insert, changing every ~2 days.</li>
</ol>
5. Thin Film Hydrogel Polymerization
<ol>
<li>Load an appropriate mask on the DMD.</li>
<li>Treat a collagen-coated polyester cell culture insert with Rain-X, and place on Rain-X coated slide.</li>
<li>Add sufficient polymerization medium to just cover the bottom of the insert (~250-300 &mu;L for 6-well plate inserts). Allow the media to permeate the insert membrane for 30-45 minutes at room temperature.</li>
<li>Remove excess polymerization medium from the insert with a micropipette or Kimwipe. Add a sufficient amount of UV-transparent oil to completely cover the bottom of the insert. Allow the insert to sit for 15-30 minutes at room temperature, long enough for the oil to form a distinct layer above the polymerization medium saturating the insert membrane.</li>
<li>Place the insert and slide under the DMD. Induce PEG crosslinking by exposure to UV light. Because of the thinness of the PEG layer, crosslinking can be completed in as little as 15 seconds at 5.0 Watts/cm2 incident on the DMD.</li>
<li>Wash the insert three times with sterile DPBS and 1% Pen-Strep. (If the persistence of an oily residue is a concern, a mild detergent such as Tween 20 (1%) may be added to the wash buffer.)</li>
<li>Store the insert in a 6-well tissue culture plate. Add growth medium to suspended cells, in the desired concentration, and pipette a sufficient volume of the cell suspension inside the cell culture insert to obtain the desired cell density. Then, add enough growth medium below the insert to completely maintain cell viability (~1.5 mL).</li>
<li>After 48 hours have elapsed, wash the insert three times with sterile DPBS and 1% Pen-Strep to dislodge any unadhered cells. Change media approximately once every 48 hours.</li>
</ol>
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.
<img src="/files/ftp_upload/2636/2636fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematic illustration of the light path used for photolithography. Inset: The UV light illuminates the DMD at an angle of 45&deg;, and 24&deg; below the plane of the mirror array.
<img src="/files/ftp_upload/2636/2636fig2.jpg" alt="Figure 2" /> 
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).
<img src="/files/ftp_upload/2636/2636fig3.jpg" alt="Figure 3" /> 
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).
<img src="/files/ftp_upload/2636/2636fig4.jpg" alt="Figure 4" /> 
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).
<img src="/files/ftp_upload/2636/2636fig5.jpg" alt="Figure 5" /> 
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.
<img src="/files/ftp_upload/2636/2636fig6.jpg" alt="Figure 6" /> 
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.

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No conflicts of interest declared.

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 (&gt;200 &mu;m), a simple coating of Rain-X around the inside wall of the insert is sufficient. However, as briefly described above, for thin constructs (&lt;200 &mu;m), 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 &mu;m 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.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Digital Micromirror Device</td>
<td>&nbsp;</td>
<td>Texas Instruments</td>
<td>DLPD4X00KIT</td>
<td>&nbsp;</td>
<tr>
<td>Collagen Coated Transwell Permeable Support </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3491</td>
<td>Also referred to as Cell Culture Insert in manuscript</td>
</tr>
<tr>
<td>Polyester Transwell Permable Support</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3412</td>
<td>Also referred to as Cell Culture Insert in manuscript</td>
</tr>
<tr>
<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-036</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-164</td>
<td>&nbsp;</td>
<tr>
<td>Nerve Growth Factor</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>&nbsp;</td>
<tr>
<td>Puramatrix</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354250</td>
<td>&nbsp;</td>
<tr>
<td>PEG 1000</td>
<td>&nbsp;</td>
<td>Polysciences, Inc.</td>
<td>15178</td>
<td>&nbsp;</td>
<tr>
<td>Irgacure 2959</td>
<td>&nbsp;</td>
<td>Ciba Specialty Chemicals</td>
<td>0298913AB</td>
<td>&nbsp;</td>
<tr>
<td>Oil</td>
<td>&nbsp;</td>
<td>Have used both canola oil and silicon oil</td>
<td>&nbsp;</td>
<td>Needs to be UV transparent, and minimize +/- meniscus formation</td>
</tr>
<tr>
<td>OmniCure Series 1000</td>
<td>&nbsp;</td>
<td>Exfo</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rain-X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of micropatterned hydrogels for neural culture systems using dynamic mask projection photolithography

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 (&lt;200 &mu;m) 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.

Watch this Scientific Journal Video about Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography at JoVE.com

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

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

fabrication-micropatterned-hydrogels-for-neural-culture-systems-using

Research

JoVE Journal

Bioengineering

18.7K Görüntüleme.  Tulane University. Hızlı, düzenli hücre kültürü yüzeylerde dinamik maske projeksiyon litografi için bir dijital micromirror cihaz kullanarak microfabricated sinir kültür sistemleri üretimi için basit teknikleri açıklanmıştır. Bu kültür sistemleri doğal biyolojik mimarisi daha iyi temsil olabilir, ve açıklanan teknikleri çok çeşitli uygulamalar için adapte olabilir.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...