This work was funded by the National Science Foundation (CBET 0845754, AHH).

1. Preparation for Formation of Alginate Discs
<ol>
	<li>Prepare a 4% (w/v) alginate solution in 1x Dulbecco&#39;s Phosphate Buffered Saline without added calcium chloride or magnesium chloride and place in a 37 &#176;C water bath. Concentrations of the alginate solution can vary, but 1 - 4% (w/v) alginate solutions are recommended.</li>
	<li>Mix the alginate solution at a ratio of 1:1 with warm cell culture media base (e.g., Dulbecco&#39;s Modified Eagle Medium) for the desired cell type. The alginate concentration is now half of the original concentration, i.e., 2% (w/v). Sterilize alginate/media solution using sterile 0.2 &#181;m nylon syringe filters.</li>
	<li>Prepare a bath of sterile 102 mM calcium chloride dihydrate in sterile water with enough solution to submerge molds (~ 200 - 300 ml).</li>
	<li>Collect the sterile &#34;gel formation mold&#34; (6 mm diameter x 3 mm tall cylindrical wells in a 3 in x 3 in aluminum plate, see Figure 1) and endplates (Bottom: one 3 in x 3 in aluminum plate, Top: one 1.5 in x 3 in aluminum plate) and prepare molds as follows:
	<ol>
		<li>Cut thick filter paper (blotter filter paper) and the cell microsieve membrane (10 &#181;m pore size) to the sizes of the top and bottom endplates and place them in the calcium chloride bath until saturated, approximately 1 min. If desired, sterilize the thick filter paper and the microsieve membrane via autoclave or ultraviolet light for 30 min prior to use.</li>
		<li>Set-up one half (the bottom half) of the mold construct.
		<ol>
			<li>Place the following items atop one another in the following order and smoothen using a sterile spatula: large endplate (3 in x 3 in), thick filter paper, and then the microsieve membrane.</li>
			<li>Invert and press the mold onto a stack of paper towels to ensure loss of excess calcium chloride solution.</li>
			<li>Place the &#34;gel formation mold&#34; with the cylindrical wells on top of the cell microsieve membrane and gently fasten the mold together on two sides using binder clips on the left and right sides. Make sure to leave enough room for the top endplate (1.5 in x 3 in) to cover the wells completely later.</li>
		</ol>
		</li>
		<li>Set-up the second half (the top half) of the mold construct.
		<ol>
			<li>Place the following items atop one another in the following order and smoothen: small endplate, thick filter paper, microsieve membrane.</li>
			<li>Invert and press this half of the mold onto a stack of paper towels to ensure loss of excess calcium chloride solution. Do not fasten this half of the mold using binder clips at this time.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Culture cells as recommended per manufacturer&#39;s instructions. Harvest the desired cells to embed in the layered alginate hydrogels. The recommended cell density is 1 - 2 x 106 cells/ml, but this can be varied depending on desired experiments. 
	Note: When using this method for making layers with different cell types, make sure to culture two cell types in parallel for layering. Mesenchymal stem cells (MSCs) seeded at a cell density of 1 x 106 cells/ml&#160;will be used as an example for the following protocol. Cell number and disc number can be scaled for experiments as needed.
	<ol>
		<li>One to two weeks prior to embedding, seed mesenchymal stem cells at a cell density of 3,000 - 5,000 cells/cm2 in 10 ml of basal growth media (Dulbecco&#39;s Modified Eagle Medium, 10% Fetal Bovine Serum, 2% L-Glutamine, 1% Non-essential Amino Acids, 1% Penicillin/Streptomycin) in T-75 flasks.</li>
		<li>Remove spent media every 2 - 3 days and replace with 10 ml of basal growth media until the cells are 80 - 90% confluent.</li>
		<li>To harvest cells, remove spent media, and pipette 3 ml of 1x Dulbecco&#39;s Phosphate Buffered Saline without added calcium chloride or magnesium chloride to rinse cells. Remove this solution, pipette 3 ml of 0.25% Trypsin-EDTA onto cells growing in T-75 flasks, and incubate for 5 min at 37 &#176;C. After cells have lifted from the adherent surface, add 6 - 9 ml of basal growth media.</li>
		<li>Centrifuge MSCs at 600 x g for 5 min at room temperature, aspirate the supernatant, and re-suspend in 1 - 5 ml of basal growth media. Count the cells using a hemocytometer using device instructions.</li>
		<li>Remove 1 x 106 cells from the total cell resuspension, centrifuge using the same conditions, and aspirate the supernatant.</li>
	</ol>
	</li>
</ol>
2. Formation of Cell Seeded Alginate Discs
<ol>
	<li>Re-suspend the cell pellet with 1 ml of the sterile alginate/media solution to achieve a cell density of 1 x 106 cells/ml. The cell-alginate mixture will be homogenously cloudy in appearance when cells are mixed in appropriately.</li>
	<li>Pipette 130 &#181;l of the cell-alginate mixture into six 6 mm diameter x 3 mm tall wells in the bottom half of the mold construct dropwise, so as not to create any bubbles. A slight convex meniscus should be visible above the edge of each well.</li>
	<li>Carefully smoothen the top half of the mold construct using a sterile spatula and turn it over, so that the cell microsieve membrane is on top of the wells. Place the mold construct on top of the wells, making sure to cover the wells containing the cell and alginate mixture completely.</li>
	<li>Lift the loaded mold construct and, while pressing down firmly on the center, binder clip the remaining two sides (top and bottom) to fasten the top and bottom halves of the mold construct. The cell-alginate solutions should be securely nestled in the wells at this time.</li>
	<li>Immerse the fastened mold construct in the 102 mM calcium chloride bath, making sure that the entire construct is submerged. Incubate in the cell culture hood at room temperature for 90 min.</li>
	<li>At the end of the 90 min, remove the mold construct from the 102 mM calcium chloride bath and place on a stack of paper towels in the cell culture hood. Remove all four binder clips and separate the two halves of the mold construct. Hydrogels formed in the wells should not have any bubbles and should fill the wells completely.</li>
	<li>Using a spatula, carefully trace the edge of the wells containing the hydrogels to carefully loosen and wedge out the hydrogels. After removing the hydrogels from the mold construct, drop the hydrogels directly into a bath of 1x Dulbecco&#39;s Phosphate Buffered Saline with calcium chloride and magnesium chloride. Cover the gels completely to wash off the excess calcium chloride solution for 1 - 5 min.</li>
	<li>Transfer the hydrogels into basal growth media solution in desired dish (e.g., 6 well plates) that completely covers the hydrogels. Incubate for at least 1 hr in the cell culture incubator at 37 &#176;C and 5% CO2 before continuing to the layering step. 
	Note: Hydrogels containing cells can be kept in the cell culture incubator indefinitely at this time until layering of gels with another cell population is desired, provided that media changes are completed every 2 - 3 days.</li>
</ol>
3. Layering of Alginate Discs
<ol>
	<li>During the last half hour of the 1 hr incubation, collect and prepare sterile molds and solution for annealing the gels.
	<ol>
		<li>Prepare the &#34;cutting mold&#34; by fastening a mold that has wells half of the height of the &#34;gel formation mold&#34; (6 mm diameter x 1.5 mm tall wells in a 3 in x 3 in aluminum plate) to an endplate (3 in x 3 in aluminum plate) using binder clips on the left and right sides.</li>
		<li>Prepare the &#34;annealing mold&#34; by fastening a mold that is 3 mm larger in diameter than the &#34;gel formation mold&#34; (9 mm diameter x 3 mm tall wells in a 3 in x 3 in aluminum plate) to an endplate (3 in x 3 in aluminum plate) using binder clips on the left and right sides.</li>
		<li>Prepare a solution of 100 mM sodium citrate/30 mM EDTA (sodium citrate dihydrate, ethylenediaminetetraacetic acid tetrasodium salt dihydrate) in sterile water.</li>
	</ol>
	</li>
	<li>Remove gels of desired cell populations (in this example, two discs with hMSCs) from the media in the dish, and place into the &#34;cutting mold&#34; wells. Each gel should fit snugly into the wells with half of the gel protruding above the mold.</li>
	<li>Using a scalpel, slice the gel along the surface of the mold (this will cut the hydrogel in half). Flip the top half of the gel and place it into an open mold well. The half of the gel should also fit snugly into the mold well, but now both half gels should be the height of the mold with the cut inner surface visible. Repeat with second gel. 
	Note: Warning: Only the inner cut surfaces will form layered discs. Using the outer surfaces will result in the halves separating. It is suspected that texture from the microsieve membrane transferred onto the gel surface prevents success of the annealing process.</li>
	<li>Place a piece of dry cell microsieve membrane on top of the wells, making sure that the microsieve membrane is in contact with all of the gel halves to be annealed and covering them entirely. Place thick filter paper on top of the microsieve membrane, making sure to cover the gels completely.</li>
	<li>Pipette a solution of 100 mM sodium citrate/30 mM EDTA (Sodium citrate dihydrate, Ethylenediaminetetraacetic acid tetrasodium salt dihydrate) onto the thick filter paper until it is saturated. Approximately 750 &#181;l is sufficient for four wells.</li>
	<li>Incubate the gels for 1 min at room temperature. Then, remove the cell microsieve membrane and thick filter paper and discard them. Remove the binder clips and open the mold.</li>
	<li>For each annealed gel, transfer one sodium citrate/EDTA-treated half of the hydrogel to the prepared &#34;annealing mold&#34; with the cut surface facing upwards. This will be one half of the annealed construct.</li>
	<li>Using a spatula, lift and place a second sodium citrate/EDTA-treated half-gel (may contain a different cell type) onto the gel already in the &#34;annealing mold&#34;, flipping this second half-gel so that the cut surface is in contact with the cut surface of the gel already in the &#34;annealing mold&#34;.</li>
	<li>Press gently down on the two gels using a spatula to remove any bubbles between to the two gels. Also, reposition the gels as needed to make sure that they are directly on top of one another.</li>
	<li>Gently lift the &#34;annealing mold&#34; and submerge it in the 102 mM calcium chloride bath for 30 min. Do not cover the wells with an endplate.</li>
	<li>After the 30min incubation, remove the &#34;annealing mold&#34; and place it onto paper towels. Remove the binder clips and separate the mold from the endplate.</li>
	<li>Using a spatula, collect the annealed gels and place them into a 1x Dulbecco&#39;s Phosphate Buffered Saline with calcium chloride and magnesium chloride bath to wash the gels. Subsequently, transfer annealed hydrogels to cell culture media in a desired dish (e.g., 6-well plate) for culture.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+stratified+articular+cartilage+from+chondrocyte+subpopulations.">Tissue engineering of stratified articular cartilage from chondrocyte subpopulations.</a> Osteoarth. Cartilage. 11 (8), 595-602 (2003).</li><li>Han, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+Grafts+for+Articular+Cartilage+Defects.">Scaffold-free Grafts for Articular Cartilage Defects.</a> Clin. Orthop. Relat. R. 466 (8), 1912-1920 (2008).</li><li>Ng, K. W., Ateshian, G. A., Hung, C. 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The authors have nothing to disclose.

Here, we describe a protocol for the formation of layered alginate hydrogel discs for studying co-cultures of multiple cell populations, such as those in physiologically layered tissues, e.g., cartilage. Layered structures, such as the described culture platform, can be used to examine the interplay between two distinct cell populations subjected to the same culture environment or under load.
Alginate is an anionic linear polysaccharide that has been found to be biocompatible and has ...

Compressive load bearing tissues such as articular cartilage or intervertebral discs consist of heterogeneous tissue regions that are critical for both biomechanical function and appropriate mechano-transduction in the tissue. Not only is cellular organization and function distinct in different regions, but the extracellular matrices (ECM) are also varied in composition and organization. For example, articular cartilage consists of three primary zones with varying cell morphology, mechanical function, and ECM. Differences in their ECM lead to differential load bearing responsibilities; the superficial layer is primarily involved in tensile response to load, while the middle and deep zones are mainly accountable for response to compression 1. Similarly, in the intervertebral disc, a gel-like nucleus pulposus is surrounded by a lamellar annulus fibrosis and the cells within these two distinct areas experience different types of biophysical stimuli 2. In these types of tissues, cells and the extracellular matrices within the tissue layers interact with each other as the tissue undergoes and responds to mechanical forces.
Recapitulation of such heterogeneous tissue structures remains a challenge in tissue engineering and regenerative medicine, and our understanding of their biological significance is limited. There is a need for culturing platforms for analyzing stratified tissues as well as co-cultures of different populations of cells within one construct. In articular cartilage tissue engineering, scaffold-less layered constructs have been constructed by harnessing the ability of zonal chondrocytes to deposit varied ECM to mimic the different layers of this tissue 3,4. However, layered hydrogel constructs provide an opportunity for investigating the interaction of diverse types of cell populations that lack the ability to form a robust tissue independently. For example, different populations of mesenchymal stem cells can be co-cultured within layered constructs. Such layered scaffolds have been used with both chondrocytes and differentiating mesenchymal stem cells for improved tissue engineering 5. Not only can different cell populations be co-cultured in similar hydrogel layers, but a single cell type can also be cultured within layers that have been manipulated to have varying stiffness or biochemical content to elicit different responses from cells 6,7.
Many different biomaterial hydrogels have been used to layer cell populations for cartilage tissue engineering such as those using polyethylene glycol or poly vinyl alcohol bases 7-9. However, alginate hydrogels are one of the simplest biomaterials from which to create layered scaffolds for studying heterogeneous cell populations in co-culture. While agarose hydrogels are also easily formed, alginate hydrogels have the added benefit of allowing easy isolation of cells from the 3-D construct for analysis of individual cells as has been described previously 10. In previous studies, bi-layered alginate hydrogels have been formed in thin sheets and from these sheets, sections were sliced (e.g., using a biopsy punch) for particular applications such as for analysis of biochemical content or interfacial shear properties 11,12. Another method for forming thin alginate sheets has been described with the potential for stratification into multiple layers, but still would require alteration to the hydrogel for use in mechanical testing 13.
Here, we present a method for reproducibly creating bi-layered alginate hydrogel discs for use in co-culturing different populations of cells. This alginate disc platform possesses several advantages. Primarily, the reproducible shape and small size is conducive for mechanical stimulation of the embedded cells without requiring a biopsy punch or other physical alteration to the hydrogel for many applications. Additionally, cell viability remains high during the layering process,&#160;and after gel formation a clear separation of the two cell populations within the gel is visible with no initial overlapping region.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Alginic Acid sodium salt</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td style="width:119px;">A1112</td>
			<td style="width:295px;">Solution made in wt% using DPBS (-/-)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1x Dulbecco&#39;s Phosphate Buffered Saline (-/-)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">1x Dulbecco&#39;s Phosphate Buffered Saline (+/+)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">11965</td>
			<td>Example. Use desired medium type</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Syringe Filters (0.02um Nylon)</td>
			<td style="width:180px;">FisherBrand</td>
			<td style="width:119px;">0979C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Calcium Chloride Dihydrate</td>
			<td style="width:180px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP510</td>
			<td>Prepare solution in sterile water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Criterion Blotter Filter Paper</td>
			<td style="width:180px;">Biorad</td>
			<td style="width:119px;">1704085</td>
			<td>Cut to size of endplates for mold formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Cell Microsieve Membrane (10um pore size)</td>
			<td style="width:180px;">Biodesign Inc of New York</td>
			<td style="width:119px;">N10R</td>
			<td>Cut to size of endplates for mold formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">0.25% Trypsin-EDTA (1x), phenol red</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">25200</td>
			<td>Thaw in 37&#7506;C water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium citrate dihydrate</td>
			<td style="width:180px;">FisherScience</td>
			<td style="width:119px;">S93364</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA)</td>
			<td style="width:180px;">FisherBioreagent</td>
			<td style="width:119px;">BP121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fetal Bovine Serum&#160;</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">26140</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Penicilin/Streptomycin (10000 U/ml)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">15140</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">L-Glutamine (200mM)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">25030081</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Non-essential Amino Acids (100x)</td>
			<td style="width:180px;">Gibco Life Technologies&#160;</td>
			<td style="width:119px;">11140050</td>
			<td>Used in example mesenchymal stem cell basal growth media</td>
		</tr>
	</tbody>
</table>

layered alginate constructs: a platform for co-culture of heterogeneous cell populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Figure 1 depicts the formation and layering of the alginate hydrogels. Completed bi-layered gels exhibit a complete initial separation of cell populations as shown in Figure 2. Cell viability of human mesenchymal stem cells) embedded within these hydrogels and layered remains high and comparable to the bulk hydrogels as shown in Figure 3. Viability was assessed after annealing, slicing the gels vertically to access the center and then sta...

Watch this Scientific Journal Video about Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations at JoVE.com

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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Research

JoVE Journal

Bioengineering

7.8K Views.  University of Maryland. Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct. 

Video: Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

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 ...

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, ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.