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 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, and after gel formation a clear separation of the two cell populations within the gel is visible with no initial overlapping region.

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 filters.
3. Prepare a bath of sterile 102 mM calcium chloride dihydrate in sterile water with enough solution to submerge molds (~ 200 - 300 ml).
4. Collect the sterile "gel formation mold" (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:
   1. Cut thick filter paper (blotter filter paper) and the cell microsieve membrane (10 µm 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.
   2. Set-up one half (the bottom half) of the mold construct.
      1. 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.
      2. Invert and press the mold onto a stack of paper towels to ensure loss of excess calcium chloride solution.
      3. Place the "gel formation mold" 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.
   3. Set-up the second half (the top half) of the mold construct.
      1. Place the following items atop one another in the following order and smoothen: small endplate, thick filter paper, microsieve membrane.
      2. 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.
5. Culture cells as recommended per manufacturer'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 will be used as an example for the following protocol. Cell number and disc number can be scaled for experiments as needed.
   1. 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's Modified Eagle Medium, 10% Fetal Bovine Serum, 2% L-Glutamine, 1% Non-essential Amino Acids, 1% Penicillin/Streptomycin) in T-75 flasks.
   2. Remove spent media every 2 - 3 days and replace with 10 ml of basal growth media until the cells are 80 - 90% confluent.
   3. To harvest cells, remove spent media, and pipette 3 ml of 1x Dulbecco'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 °C. After cells have lifted from the adherent surface, add 6 - 9 ml of basal growth media.
   4. 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.
   5. Remove 1 x 10⁶ cells from the total cell resuspension, centrifuge using the same conditions, and aspirate the supernatant.

2. Formation of Cell Seeded Alginate Discs

1. 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.
2. Pipette 130 µl 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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'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.
8. 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 °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.

3. Layering of Alginate Discs

1. During the last half hour of the 1 hr incubation, collect and prepare sterile molds and solution for annealing the gels.
   1. Prepare the "cutting mold" by fastening a mold that has wells half of the height of the "gel formation mold" (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.
   2. Prepare the "annealing mold" by fastening a mold that is 3 mm larger in diameter than the "gel formation mold" (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.
   3. Prepare a solution of 100 mM sodium citrate/30 mM EDTA (sodium citrate dihydrate, ethylenediaminetetraacetic acid tetrasodium salt dihydrate) in sterile water.
2. Remove gels of desired cell populations (in this example, two discs with hMSCs) from the media in the dish, and place into the "cutting mold" wells. Each gel should fit snugly into the wells with half of the gel protruding above the mold.
3. 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.
4. 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.
5. 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 µl is sufficient for four wells.
6. 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.
7. For each annealed gel, transfer one sodium citrate/EDTA-treated half of the hydrogel to the prepared "annealing mold" with the cut surface facing upwards. This will be one half of the annealed construct.
8. 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 "annealing mold", flipping this second half-gel so that the cut surface is in contact with the cut surface of the gel already in the "annealing mold".
9. 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.
10. Gently lift the "annealing mold" and submerge it in the 102 mM calcium chloride bath for 30 min. Do not cover the wells with an endplate.
11. After the 30min incubation, remove the "annealing mold" and place it onto paper towels. Remove the binder clips and separate the mold from the endplate.
12. Using a spatula, collect the annealed gels and place them into a 1x Dulbecco'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.

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