This work was supported by the US National Institutes of Health grant NIH/NICHD #HD051998 (to C.A.M.).

1. Collagen Bead Preparation
<ol>
	<li>Prior to loading EVTs for 3-D cell culture, one needs to prepare the Cytodex-3 microcarrier beads:
	<ol>
		<li>Weigh out the appropriate amount of Cytodex-3 beads required for the experiment. This protocol is adapted for the 10ml RCCS vessel, in which 0.05g of beads are needed. For a 50ml RCCS vessel, scale accordingly. In a 50mL autoclavable conical tube, mix 250 mg Cytodex-3 beads with 12mL Dulbecco phosphate buffered solution (DPBS). This amount is sufficient for 5 RCCS vessels.</li>
	</ol>
	</li>
	<li>Ensure adequate volume is present in the conical tube, as the autoclave process will result in evaporation. Loosely cap the tube and autoclave for 10 min at 110&deg;C.</li>
	<li>Remove the conical tube at the completion of the autoclave cycle and allow the Cytodex-3 bead solution to cool.</li>
	<li>After the conical tube has cooled to room temperature, use sterile technique to bring the total volume to 12.5mL using 1X DPBS.</li>
	<li>Cap and store the prepared Cytodex-3 beads at room temperature. Allow the prepared beads to swell and cool to room temperature prior to use. The above preparation of Cytodex-3 beads will provide for five 10mL RCCS vessels. We have observed that extended storage of Cytodex-3 beads at room temperature will result in diminished performance, as the collagen will destabilize after approximately one month. Cytodex-3 beads range in size from 133-215 &mu;m.</li>
</ol>
2. Media Preparation
<ol>
	<li> Prepare 1L of RCCS optimized GTSF-2 media (adapted from 22), which consists of 40% MEM alpha, plus supplements and 60% L-15 Leibovitz's media (adapted from 22). First, prepare 400mL in total volume of MEM alpha supplemented with: 
	21.2mM sodium bicarbonate 
	0.06% Peptone 
	0.7mM Fructose 
	1.4mM Galactose 
	5.6mM Glucose 
	1% HEPES 
	1% L-glutamine 
	0.5% ITS 
	10% FBS</li>
	<li>To prepare a stock solution of ITS, dissolve in 5mL sterile acidified H2O prepared by addition of glacial acetic acid (approx. 0.05mL). Swirl to dissolve, follow with 45mL sterile water.</li>
	<li>Weigh out enough L-15 Leibovitz's powder for 600ml of medium by dissolving in tissue-culture grade H2O.</li>
	<li>Bring the total volume of cell culture medium to 1L with L-15 Leibovitz's medium. Filter-sterilize and store at 4&deg;C in the dark. Add 1% Penicillin-Streptomycin individually to each aliquot of medium used.</li>
	<li>Many media formulations other than GTSF-2 media have been successfully tested in the RCCS. Individual laboratories must decide for themselves which medium is optimal for the experiment being performed.</li>
</ol>
3. Cells and Bead Incubation
<ol>
	<li>Propagate the EVTs to ~80% confluency, trypsinize and count using accepted cell culture practices.</li>
	<li>Suspend 1x106 EVTs in 4mL of warmed media.</li>
	<li>Gently mix prepared Cytodex-3 beads. Using sterile techniques, remove 2.5mL of prepared beads using a wide tip, 10mL serological pipette and transfer to an unused 15mL conical tube. Note that there may be a small loss of beads as they attach to the pipette.</li>
	<li>Allow the Cytodex-3 beads to settle on the bottom of the tube. After sedimentation, use a pipettor to remove the top layer of DPBS without disturbing the Cytodex-3 beads.</li>
	<li>Mix the 1x106 prepared EVTs in media with the prepared Cytodex-3 beads.</li>
	<li>Incubate the cell-bead mixture at room temperature for 30 min. Periodically mix gently. Incubate for a further 30 min at 37&deg;C and 5% CO2. Periodically mix gently.</li>
</ol>
4. Loading the RWV
<ol>
	<li>In a laminar flow cabinet, remove a 10mL RCCS from the packaging and place it in a sterile, 6-well culture plate for stability. Refer to Figure 1 for a labeled diagram of the RCCS.</li>
	<li>Remove the large stopper from the port.</li>
	<li>Bring the total volume of the incubating cell-bead mixture to 10mL with warmed media.</li>
	<li>Load the cell/bead-mixture into the RCCS through the large port. The RCCS should be tilted at a 45&deg; angle (large port up) when loading to aid in removal of potential air bubbles. Prevent build-up of positive pressure by filling the vessel slowly and steadily.</li>
	<li>Replace the stopper in the large port.</li>
	<li>Remove the pistons from the 3-mL syringes and place the empty syringes onto the small ports. Add 1-3mL of media to each syringe. Slowly open the two valves. Replace the syringe pistons on the syringes gently. Add media from one syringe until all bubbles are removed from the inside chamber.</li>
	<li>Pick up the RCCS and gently tap the side while rotating it in front of you to check for bubbles. If there are any bubbles, you must get them out of the chamber, as air bubbles will interfere with formation of cell aggregates and introduce shear forces. Remove bubbles by rotating the RCCS until the bubbles are under the small port. Then gently push down on the syringe on the opposite side to force the bubbles into the port and out of the chamber.</li>
	<li>Close one side port. Gently push down on the other syringe piston to introduce a small amount of positive pressure into the vessel, which prevents bubbles from forming. Close second valve.</li>
	<li>Load the RCCS onto the rotor. Start the rotation at 19rpm in a 37&deg;C CO2 incubator.</li>
</ol>
5. Changing the Media
<ol>
	<li>Change the media every other day for the first three days, then every day thereafter.</li>
	<li>Turn off the rotor and remove the RCCS. Pull the pistons up to create some suction, remove the syringes from each small port and place the RCCS on an angle so that the beads settle opposite of the large port.</li>
	<li>After the beads have all settled, open one of the small valves and allow the media to flow out of the RCCS and into a waste container. Remove 2/3 of the media in this manner. Be sure to not disturb the beads and to not discard any aggregates.</li>
	<li>Close the small valve and open the large port. Add media back into the RCCS through the large port. Replace the stopper in the large port.</li>
	<li>Repeat steps 4.6 - 4.9.</li>
	<li>As the aggregates increase in size, you need to increase the rotation speed to keep them in suspension at all times, ideally in a small circulatory pattern at optimal speed. Generally, increase the speed between 0.3-0.7 rpm after each feeding once the aggregates start growing visibly.</li>
</ol>
6. Collecting Propagated Cells
<ol>
	<li>Remove the RCCS from the rotor. Remove the syringes from each small port and place the RCCS on an angle so that the beads may settle opposite of the large port.</li>
	<li>After the beads have all settled, open one of the small valves and allow the media to flow out of the RCCS and into a waste container. Remove 1/3 of the media in this manner.</li>
	<li>Close the small valve and open the large port. Gently swirl the vessel to disperse the aggregates back into solution. Empty liquid mixture into a sterile 50ml conical tube. Allow the collected aggregates to settle in the conical tube before using supernatant to thoroughly wash the culture vessel to maximize recovery of aggregates. The aggregates may be used for downstream assays immediately, such as flow cytometry, invasion assays, immunofluorescence and others.</li>
</ol>
7. Representative Results
An example of EVT-like cells (SGHPL-4 trophoblast cell line) grown in the RCCS on Cytodex-3 beads is shown in Figure 2. The EVT-like cell line displays projections extending away from the main cluster and attaching to neighboring clusters. Many of the beads are completely covered with propagating cells. Once removed from the RCCS and plated on an extracellular matrix, the EVT-like 3-D grown cells aggressively invade and/or migrate (Figure 3). RT-PCR data confirms the increased expression of MMPs seen in 3-D aggregates as opposed to traditional cell culture monolayers (Figure 4). Interestingly, genes not associated with invasion are also upregulated in the RCCS (Table 1) and may aid in delineating such areas as immune interactions with invading trophoblasts cells.
<img alt="Figure 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> 
Figure 1. Cartoon depiction of the Rotating Cell Culture System (RCCS).
<img alt="Figure 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> 
Figure 2. Phase contrast micrograph of a representative aggregate after 5 days growth in a RCCS. Arrows indicate invading cells and * denotes the Cytodex-3 microcarrier beads.
<img alt="Figure 3" src="/files/ftp_upload/3367/3367fig3.jpg" /> 
Figure 3. RCCS Grown aggregates were embedded in fibrin gels. Invasion through the fibrin gel was observed as early as (A) 24 hours post-fibrin embedding and (B) continued through 48 hours. Arrows indicate invading cells and * denotes the Cytodex-3 microcarrier beads (Adapted, with permission, from Ref. 7).
<img alt="Figure 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> 
Figure 4. Comparative gelatin zymogram of monolayer and RCCS propagated SGHPL-4 EVT-like cells. Pro- and active forms of MMP-2 and MMP-9 were secreted by the RCCS grown trophoblast cells (Adapted, with permission, from Ref. 7).
<img alt="Table 1" src="/files/ftp_upload/3367/3367table1.jpg" /> 
Table 1. Summary of microarray results from SGHPL-4 cells grown in a RCCS (Adapted, with permission, from Ref. 7).
<ol class="lalpha">
	<li>Fold induction in the 3-D aggregates was calculated as follows: normalized 3-D gene value/normalized monlayer gene value.</li>
	<li>Indicates genes that were below the limit of detection in monolayers.</li>
</ol>

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Anim. 33, 344-344 (1997).</li><li>GE Healthcare. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+-+Principles+and+Methods.">Microcarrier Cell Culture - Principles and Methods.</a> Handbooks. , (2005).</li></ol>

We have nothing to disclose.

The culture technique presented here provides investigators with highly invasive EVT-like cells. It has now been recognized that a loss of differentiation occurs in monolayers due to inhibition of cellular responses to chemical and molecular cues in three dimensions (apical, basal, and lateral cell surfaces)10,13. This technique reflects characteristics noted in utero on invading EVT cells. As the procedure mimics conventional monolayer tissue culture time kinetics, but provides cells with differentia...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Cytodex microcarrier beads</td>
 <td>Sigma-Aldrich</td>
 <td>C3275</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rotating Cell Culture System (RCCS)</td>
 <td>Synthecon</td>
 <td>RCCS-D</td>
 <td>Includes rotor base, power supply, 4 disposable RCCS units</td>
 </tr>
 <tr>
 <td>RCCS Disposable Units</td>
 <td>Synthecon</td>
 <td>Contact Synthecon</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3ml Luer-Lock tip syringe</td>
 <td>BD</td>
 <td>309585</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10ml wide-tip serological pipette</td>
 <td>BD</td>
 <td>357504</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MEM Alpha</td>
 <td>Invitrogen</td>
 <td>12561-072</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 medium, powder</td>
 <td>Invitrogen</td>
 <td>41300-039</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>H2O, Endotoxin free</td>
 <td>Fisher</td>
 <td>MT-25-055-CM </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate</td>
 <td>Sigma-Aldrich</td>
 <td>S-7795</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Peptone</td>
 <td>Fisher Scientific</td>
 <td>BP1420-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fructose</td>
 <td>Sigma-Aldrich</td>
 <td>F3510-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Galactose</td>
 <td>Sigma-Aldrich</td>
 <td>G5388-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G7528-250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Invitrogen</td>
 <td>15630-080</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Invitrogen</td>
 <td>25030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Insulin-Transferrin-Sodium Selenite (ITS)</td>
 <td>Sigma-Aldrich</td>
 <td>I1884</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Invitrogen</td>
 <td>10437</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

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

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 ...

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JoVE Journal

Bioengineering

16.4K ビュー.  Tulane University Medical School. 伝統的な、二次元細胞培養技術は、しばしば、分化マーカー、サイトカインおよび増殖因子に関する変更特性になる。 extravillous栄養膜細胞株を以下に示すように回転する細胞培養系における三次元細胞培養（RCCS）は、これらの要因の多くの表現を再確立します。

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

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 ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

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 ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...