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

Published: August 7th, 2016



1Fischell Department of Bioengineering, 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.

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.

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 ....

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1. Preparation for Formation of Alginate Discs

  1. Prepare a 4% (w/v) alginate solution in 1x Dulbecco's Phosphate Buffered Saline without added calcium chloride or magnesium chloride and place in a 37 °C water bath. Concentrations of the alginate solution can vary, but 1 - 4% (w/v) alginate solutions are recommended.
  2. Mix the alginate solution at a ratio of 1:1 with warm cell culture media base (e.g., Dulbecco's Modified Eagle Medium) for the desired cell type. The alginate concent.......

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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.......

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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 .......

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


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Name Company Catalog Number Comments
Alginic Acid sodium salt Sigma Aldrich A1112 Solution made in wt% using DPBS (-/-)
1x Dulbecco's Phosphate Buffered Saline (-/-) Gibco Life Technologies  14190
1x Dulbecco's Phosphate Buffered Saline (+/+) Gibco Life Technologies  14190
Dulbecco's Modified Eagle Medium Gibco Life Technologies  11965 Example. Use desired medium type
Syringe Filters (0.02um Nylon) FisherBrand 0979C
Calcium Chloride Dihydrate Fisher Bioreagents BP510 Prepare solution in sterile water
Criterion Blotter Filter Paper Biorad 1704085 Cut to size of endplates for mold formation
Cell Microsieve Membrane (10um pore size) Biodesign Inc of New York N10R Cut to size of endplates for mold formation
0.25% Trypsin-EDTA (1x), phenol red Gibco Life Technologies  25200 Thaw in 37ᵒC water bath
Sodium citrate dihydrate FisherScience S93364
Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA) FisherBioreagent BP121
Fetal Bovine Serum  Gibco Life Technologies  26140 Used in example mesenchymal stem cell basal growth media
Penicilin/Streptomycin (10000 U/ml) Gibco Life Technologies  15140 Used in example mesenchymal stem cell basal growth media
L-Glutamine (200mM) Gibco Life Technologies  25030081 Used in example mesenchymal stem cell basal growth media
Non-essential Amino Acids (100x) Gibco Life Technologies  11140050 Used in example mesenchymal stem cell basal growth media

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