Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage

Abstract

The efficiency of substance exchange may be decreased when the thickness and volume of such a tissue-engineered cartilage that is composed of cultured cells and porous scaffold increase. Moreover, during the transport of this construct with complicated shapes, excessive and focal mechanical loading may cause deformation. The establishment of incubation and transport methods is necessary for the three-dimensional tissue-engineered cartilage. Therefore, we investigated the preparation of an agarose mold with a concavity similar to the shape of 3-dimensional tissue-engineered cartilage to prevent excessive and focal concentration of stress, while avoiding interference with substance exchange as much as possible. Firstly, we investigated the preparation at 1% -4% agarose concentrations. Since the mechanical strength was insufficient at 1%, 2% was regarded as appropriate. Using 2% agarose, we prepared a mold with a 5 × 5 × 5 mm concavity to accommodate tissue-engineered cartilage (5 × 5 × 5 mm mixture of 1.5 × 10 7 cells and collagen gel), and stored the regenerative cartilage in it for 2 and 24 hours. On comparison with storage in a plastic mold with the same shape in which substance exchanged from side and bottom was impossible, although no significant differences were noted in the number or viability of cells after 2 hours, these were markedly reduced in the plastic mold after 24 hours. It was confirmed that favorable cell numbers and viability were maintained by immediately retaining the regenerative cartilage in the culture medium in the agarose mold and keeping the temperature at 37˚C. Since this agarose mold also buffers against mechanical forces loaded on the three-dimensional regenerative tissue, it may be useful as a container for storage and transport of large-sized three-dimensional regenerative tissue.

Focal adhesion signaling affects regeneration by human nucleus pulposus cells in collagen- but not carbohydrate-based hydrogels

“…However, it is difficult to compare hydrogel stiffness between studies because of confounders such as biomechanical loading and confinement, cross-linking methods and sample shape. Generally, hydrogel stiffness is concentration-dependent and power law-dependendent for agarose and gelMA [27,[36][37][38][39][40][41][42][43]. Of course at some point, polymer concentration will likely also inhibit matrix production through nutrient and oxygen deprivation, or changes in fixed charge density [44], although high concentrations of PEG (20% (w/w)) still enabled matrix production by bovine chondrocytes [45,46].…”

Regenerative Cartilage made by Fusion of Cartilage Elements derived from Chondrocyte Sheets prepared in Temperature-Responsive Culture Dishes

“…In the previous reports, the sizes in pellets of mesenchymal stem cells and their analog, cultured chodrocytes, were usually confined within 1 mm, because larger pellets caused necrosis. The chondrocyte sheets of 3 mm square (10-100 μm of thickness) became a pellet of approximately 1 mm agarose mold was employed 15) . Agarose excels at substance permeability and the diffusion of water, nutrients, growth factors, and wastes.…”

“…Agarose molds were prepared according to the previous report 15) . Agarose (Takara Bio, Otsu, Japan) was mixed with MEM at 2% and dissolved at 121°C.…”

Scaffold-free approach produces neocartilage tissue of similar quality as the use of HyStem™ and Hydromatrix™ scaffolds

“…We assumed that we can improve the efficiency of nutrition transfer using the agarose gel-supported well system. Agarose system is also easy to handle, has a low cost and a minimal biological effect on the cells and regenerative tissues [ 34 , 35 ]. The other advantage of using the agarose gels for the cell culture is that the nutrition or oxygen can freely diffuse through the agarose matrix.…”

“…Further, the agarose support could stably hold the assembly of the tissue-engineered constructs. Previously, it has been reported that 3-dimensional tissue-engineered cartilage generated in agarose mold has been used for clinical application [ 35 ].…”