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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 concentration is now half of the original concentration, i.e., 2% (w/v). Sterilize alginate/media solution using sterile 0.2 µm nylon syringe f....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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 (0.02 μm 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 (10 μm pore size)	Biodesign Inc of New York	N10R	Cut to size of endplates for mold formation
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 (10,000 U/ml)	Gibco Life Technologies	15140	Used in example mesenchymal stem cell basal growth media
L-Glutamine (200 mM)	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