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>

<ol>
	<li>Sophia Fox, A. J., Bedi, A., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Basic+Science+of+Articular+Cartilage.">The Basic Science of Articular Cartilage.</a> Sports Health. 1 (6), 461-468 (2009).</li><li>Neidlinger-Wilke, C., 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=Mechanical+loading+of+the+intervertebral+disc:+from+the+macroscopic+to+the+cellular+level.">Mechanical loading of the intervertebral disc: from the macroscopic to the cellular level.</a> Eur. Spine J. 23 (3), 333-343 (2013).</li><li>Klein, T. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zonal+chondrocytes+seeded+in+a+layered+agarose+hydrogel+create+engineered+cartilage+with+depth-dependent+cellular+and+mechanical+inhomogeneity.">Zonal chondrocytes seeded in a layered agarose hydrogel create engineered cartilage with depth-dependent cellular and mechanical inhomogeneity.</a> Tissue Eng. A. 15 (9), 2315-2324 (2009).</li><li>Ng, K. W., 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=A+layered+agarose+approach+to+fabricate+depth-dependent+inhomogeneity+in+chondrocyte-seeded+constructs.">A layered agarose approach to fabricate depth-dependent inhomogeneity in chondrocyte-seeded constructs.</a> J. Orthop. Res. 23 (1), 134-141 (2005).</li><li>Nguyen, L. H., Kudva, A. K., Saxena, N. S., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+articular+cartilage+with+spatially-varying+matrix+composition+and+mechanical+properties+from+a+single+stem+cell+population+using+a+multi-layered+hydrogel.">Engineering articular cartilage with spatially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel.</a> Biomaterials. 32 (29), 6946-6952 (2011).</li><li>Leone, G., 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=Continuous+multilayered+composite+hydrogel+as+osteochondral+substitute.">Continuous multilayered composite hydrogel as osteochondral substitute.</a> J. Biomed. Mater. Res. A. 103 (8), 2521-2530 (2015).</li><li>Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+catalyze+cartilage+formation+by+neonatal+articular+chondrocytes+in+3D+biomimetic+hydrogels.">Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels.</a> Sci. Rep. 3, (2013).</li><li>Vigf&#250;sd&#242;ttir, &. #. 1. 9. 3. ;. T., Pasrija, C., Thakore, P. I., Schmidt, R. B., Hsieh, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Pericellular+Matrix+in+Mesenchymal+Stem+Cell+Deformation+during+Chondrogenic+Differentiation.">Role of Pericellular Matrix in Mesenchymal Stem Cell Deformation during Chondrogenic Differentiation.</a> Cell. Mol. Bioeng. 3 (4), 387-397 (2010).</li><li>Lee, C. S. D., Gleghorn, J. P., Won Choi, N., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+layered+chondrocyte-seeded+alginate+hydrogel+scaffolds.">Integration of layered chondrocyte-seeded alginate hydrogel scaffolds.</a> Biomaterials. 28 (19), 2987-2993 (2007).</li><li>Gleghorn, J. P., Lee, C. S. D., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+laminated+alginate+gels+for+tissue+engineering+of+layered+structures.">Adhesive properties of laminated alginate gels for tissue engineering of layered structures.</a> J. Biomed. Mater. Res. A. 85 (3), 611-618 (2008).</li><li>Gharravi, A. M., Orazizadeh, M., Ansari-Asl, K., Banoni, S., Izadi, S., Hashemitabar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Fabrication+of+Anatomical+Bioreactor+Systems+Containing+Alginate+Scaffolds+for+Cartilage+Tissue+Engineering.">Design and Fabrication of Anatomical Bioreactor Systems Containing Alginate Scaffolds for Cartilage Tissue Engineering.</a> Avicenna J. Med. Biotechnol. 4 (2), 65-74 (2012).</li><li>Xu, 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=Chondrogenic+Differentiation+of+Human+Mesenchymal+Stem+Cells+in+Three-Dimensional+Alginate+Gels.">Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Three-Dimensional Alginate Gels.</a> Tissue Eng. A. 14 (5), 667-680 (2008).</li><li>Yeatts, A. B., Gordon, C. N., Fisher, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+an+aggregated+alginate+construct+in+a+tubular+perfusion+system.">Formation of an aggregated alginate construct in a tubular perfusion system.</a> Tissue Eng. C, Methods. 17 (12), 1171-1178 (2011).</li></ol>

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><tbody><tr><td>Alginic Acid sodium salt</td><td>Sigma Aldrich</td><td>A1112</td><td>Solution made in wt% using DPBS (-/-)</td></tr><tr><td>1x Dulbecco&#039;s Phosphate Buffered Saline (-/-)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>14190</td><td></td></tr><tr><td>1x Dulbecco&#039;s Phosphate Buffered Saline (+/+)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>14190</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco Life Technologies&amp;nbsp;</td><td>11965</td><td>Example. Use desired medium type</td></tr><tr><td>Syringe Filters (0.02 &amp;#956;m Nylon)</td><td>FisherBrand</td><td>0979C</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Fisher Bioreagents</td><td>BP510</td><td>Prepare solution in sterile water</td></tr><tr><td>Criterion Blotter Filter Paper</td><td>Biorad</td><td>1704085</td><td>Cut to size of endplates for mold formation</td></tr><tr><td>Cell Microsieve Membrane (10 &amp;#956;m pore size)</td><td>Biodesign Inc of New York</td><td>N10R</td><td>Cut to size of endplates for mold formation</td></tr><tr><td>Sodium citrate dihydrate</td><td>FisherScience</td><td>S93364</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA)</td><td>FisherBioreagent</td><td>BP121</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco Life Technologies&amp;nbsp;</td><td>26140</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>Penicilin/Streptomycin (10,000 U/ml)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>15140</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>25030081</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>Non-essential Amino Acids (100x)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>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 ...

layered-alginate-constructs-platform-for-co-culture-heterogeneous

Universidad de Cartagena UDEC

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

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 ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Developmental Biology

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Medicine

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, ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...