Tissue Engineering
GFP-Tagged Huh7 Hepatoma Cells in Glycosaminoglycan-Based Capsules: Studying the Effects of Intra-Capsular Collagen on Cell Aggregation and Morphology.
Rafael Ramos, B.S Biomedical Engineering; M.S. Bioengineering (he/him/his)
MD-PhD Student
Wayne State University School of Medicine
Detroit, Michigan, United States
Howard W.T. Matthew
Professor, Dept. of Chemical Engineering & Materials Science, College of Engineering
Wayne State University, United States
Wanqing Liu
Professor, Dept. of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Science
Wayne State University, United States
Creation of in-vitro tissue models which mimic hepatic structure and function could offer enormous benefits in generating high throughput screening templates to improve upon the 90% failure rate of drugs in clinical trial. Traditional drug metabolism and toxicity in vitro approaches utilize dish-cultured cells on a two-dimensions soft material substrate. These models are limited by key factors such as rapid degeneration of the template environment, along with the restrictive inability to replicate the three-dimensional structural complexity of the native liver’s microenvironment and the synergism between cells in vivo. While modern biomanufacturing techniques have made significant strides in the creation of improved templates, these models continue to make use of top-down manufacturing approaches which continue to present restrictive limitations in total construct size, as well as the lack of incorporated vascular elements 1,2.
We previously reported a method of encapsulating hepatocytes within glycosaminoglycan (GAG) stabilized chitosan membranes generated by polyanion-polycation electrostatic interactions. These hollow capsules allow for cellular organization within a defined spherical volume whose size and microenvironment can be tailored through the inclusion of extracellular matrix (ECM) components 3,4.
Given our interest in developing a robust testing template for the study of novel small molecule drugs, in this study we seek to explore the role of additional intra-capsular ECM components in the culturing of Huh-7 hepatoma cells, as this model cell line has been extensively used to study pathologic states such as HCV infection and in the testing of GalNAc-conjugated short interfering RNAs (siRNAs).
GFP-transfected Huh-7 Hepatoma cells were cultured to confluence in high glucose DMEM media. Cells were trypsinized and encapsulated within GAG-chitosan membranes in solution formulations of hyaluronic acid (HA), chondroitin sulfate (CS), and collagen type I dissolved in a HEPES-Sorbitol buffer at a concentration of 10 million cells per mL of GAG solution. Concentrations of HA and CS were kept consistent at 1 w/v% and 2 w/v% respectively, with neutralized type 1 collagen (collagen in 1mM HCl mixed with 10X L-DMEM in a 9:1 ratio) added to create one of four final collagen concentrations (0, 0.2, 1, and 2 mg/mL) in the solution. Changes to this variable would allow a change in the amount of collagen that was entrapped inside the capsule lumen along with the cells. Droplets of the cell suspension were generated using air entrainment and captured into stirred chitosan solution as previously described. Care was taken to generate uniform spherical droplets.
Once stabilized and washed, capsules were cultured on six well plates under static conditions. Culture medium was changed daily and assayed for cell function and growth. Capsules were imaged daily using brightfield and fluorescence microscopy to observe the interaction between encapsulated cells and the ECM components entrapped in the capsule lumen and the membrane, as well as changes in cell morphology and aggregation. Capsules were also collected and fixed for histology or scanning electron microscopy imaging (SEM).
The procedures above produced hollow capsules 750 mm in diameter with a membrane thickness of 8 mm. Huh-7 cells maintained viability through the encapsulation protocol and proliferated well inside the capsules. The use of GFP-transfected cells allowed the cell bodies to be observed and tracked through the capsule lumens across the culture period.
In the absence of co-encapsulated collagen, encapsulated cells weakly adhered to the capsule wall within the first 24 hrs. These cells then formed spheroids which increased in size over time. It was noted that spheroid sizes ranged from 50-70 mm on day 2 to up to 300 mm on day 5. Spherical aggregate morphology and low interaction with the membrane was also observed via histology and SEM, with histology also indicating that even the larger aggregates did not exhibit cellular necrosis in the core of the aggregate.
In contrast, the addition of collagen to the GAG formulation at any concentration enhanced the adhesivity of the capsule wall which allowed cells to form flattened/spread aggregates which strongly adhered to the internal capsule wall. This flattened morphology was observed as early as day 2 using fluorescence microscopy and was also seen in SEM images. Across subsequent days of culture, this internal fibrillar collagen network allowed cells to grow in one of two distinct morphologies, as layers of cells which grew on top of each other which appeared attached to the capsule membrane, or grafted onto a network of collagen fibers which transected the capsule lumen. The transecting aggregates were present primarily in capsules containing a final concentration of 1 mg/mL, while capsules containing 0.2 mg/ml collagen appeared to show cells in flattened morphology adhering to the internal capsule membrane.
Encapsulation of Huh7 cells within GAG-chitosan capsules appears to be a viable approach for rapidly generating three dimensional cellular aggregates, when compared to other approaches available in the literature 5. Ongoing studies are exploring the characteristics of this culture system as a model of specific liver disease modalities and therapeutic applications.