Macromolecular crowding in equine bone marrow mesenchymal stromal cell cultures using single and double hyaluronic acid macromolecules.

Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition in eukaryotic cell culture. Single hyaluronic acid (HA) molecules have not induced a notable increase in the amount and rate of deposited ECM. Thus, herein we assessed the physicochemical properties and biological consequences in equine bone marrow mesenchymal stromal cell cultures of single and mixed HA molecules and correlated them to the most widely used MMC agents, the FicollⓇ cocktail (FC) and carrageenan (CR). Dynamic light scattering analysis revealed that all HA cocktails had significantly higher hydrodynamic radius than the FC and CR; the FC and the 0.5 mg/ml 100 kDa and 500 kDa single HA molecules had the highest charge; and, in general, all molecules had high polydispersity index. Biological analyses revealed that none of the MMC agents affected cell morphology and basic cell functions; in general, CR outperformed all other macromolecules in collagen type I and V deposition; FC, the individual HA molecules and the HA cocktails outperformed CR in collagen type III deposition; FC outperformed CR and the individual HA molecules and the HA cocktails outperformed their constituent HA molecules in collagen type IV deposition; FC and certain HA cocktails outperformed CR and constituent HA molecules in collagen type VI deposition; and all individual HA molecules outperformed FC and CR and the HA cocktails outperformed their constituent HA molecules in laminin deposition. With respect to tri-lineage analysis, CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. This work opens new avenues in mixed MMC in eukaryotic cell culture. STATEMENT OF SIGNIFICANCE: Mixed macromolecular crowding (MMC) in eukaryotic cell culture is still under-investigated. Herein, single and double hyaluronic acid (HA) macromolecules, along with the traditional MMC agents FicollⓇ cocktail (FC) and carrageenan (CR), were used as MMC agents in equine mesenchymal stromal cell cultures. Biological analysis showed that none of the MMC agents affected cell morphology and basic cell functions. Protein deposition analysis made apparent that CR outperformed all other macromolecules in collagen type I and collagen type V deposition, whilst FC, the individual HA macromolecules and the HA cocktails outperformed CR in collagen type III deposition. Tri-lineage analysis revealed that CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. These data illustrate that MMC agents are not inert macromolecules.

Hyaluronic Acid as Macromolecular Crowder in Equine Adipose-Derived Stem Cell Cultures.

The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the highest negative charge and hydrodynamic radius, and the 10 mg/mL 100 kDa HA exhibited the lowest polydispersity index and the highest % fraction volume occupancy. Although HA had the potential to act as a macromolecular crowding agent, it did not outperform carrageenan and Ficoll®, the most widely used macromolecular crowding molecules, in enhanced and accelerated collagen I, collagen III and collagen IV deposition.

Preparation and Characterization of Tissue Surrogates Rich in Extracellular Matrix Using the Principles of Macromolecular Crowding.

Tissue engineering by self-assembly allows for the fabrication of living tissue surrogates by taking advantage of the cell's inherent ability to produce and deposit tissue-specific extracellular matrix. However, the long culture periods required to build a tissue substitute in conducive to phenotypic drift in vitro microenvironments result in phenotype and function losses. Although several biophysical microenvironmental modulators (e.g., surface topography, substrate stiffness, mechanical stimulation) have been used to address these issues, slow extracellular matrix deposition remains a limiting factor in clinical translation and commercialization of such therapies. Macromolecular crowding is an alternative in vitro microenvironment modulator that has been shown to accelerate extracellular matrix deposition by several orders of magnitude, thereby decreasing culture periods required for the development of an implantable device, while maintaining cell phenotype and function. Herein, we provide protocols for the production of tissue surrogates rich in extracellular matrix from human dermal fibroblasts, equine tenocytes, and equine adipose-derived stem cells using the principles of macromolecular crowding and the subsequent characterization thereof by means of immunofluorescent staining and complementary fluorescence intensity analysis.