(334h) 3D Culture of Trabecular Meshwork Cells

Glaucoma is a disease in which damage to the optic nerve leads to progressive, irreversible vision loss. The primary risk factor for glaucoma is intraocular pressure (IOP), which is associated with resistance to aqueous humor outflow. The tissue closely associated with IOP is the trabecular meshwork (TM). To understand the relation between the TM and its effect on IOP it has been proposed to create a biomimetic scaffold that exhibits the same behavior as the native TM. To mimic the tissue structure of the trabecular meshwork, tissue scaffolds have been constructed using the same materials found in the native TM. The scaffold architecture is tuned to promote TM cells to exhibit the same morphology as the native TM.

Problem Statement:

Uniaxially aligned collagen scaffolds containing chrondoitin sulfate have been used to reconstruct the extracellular matrix found in native TM. TM cells proliferate on these scaffolds in static culture and become restricted after one week. The pore sizes of the collagen scaffolds restrict the proliferation of TM cells to the surface. The effect of different glycosaminoglycans (GAGs) in the trabecular meshwork ECM is not understood. It has been shown that TM cells perfused with media with at least 1 µL/min show significantly more cell growth for a longer period of culture. We are now investigating dynamic cell culture to simulate perfusion conditions found in the native TM tissue, and investigating the effects on cell morphology, gene expression and proliferation. Pressure change across the perfusion chamber is due to cellular activity resulting in a decrease in porosity.

Approach:

Scaffolds were made from collagen type I and chondroitin sulfate, using freeze casting to produce uniaxially aligned pores. Trabecular meshwork (TM) cells were isolated from porcine eyes.

TM cells were grown on collagen scaffolds inside a perfusion reactor to measure the effect of dynamic culture on cell proliferation. A perfusion reactor was built so that scaffolds could be placed inside and cell media perfused through it in the axial direction. Flow rates varying from 10 µL/min to 100 µL/min were used to measure diffusion and convection controlled growth. A pressure transducer was used to quantify the pressure change across the perfusion reactor. By measuring pressure change, we can measure changes in porosity, and obtain information about cell interactions with the scaffold over time, as well as screen the effects of potential therapeutics for glaucoma.

The cells were also grown on scaffolds with varying pore sizes. This increase in pore size from ~10 µm to ~50 µm allows cells to proliferation deeper into the scaffold.

TM cells were grown on collagen scaffolds containing chondroitin sulfate (CS), and hyaluronic acid (HA) and a mixture of CS and HA to measure the effect that each GAG had on the growth and proliferation.

Results:

TM cells cultured on collagen-GAG scaffolds in the perfusion reactor were found to proliferate more quickly compared to those cultured on collagen-GAG scaffolds in static culture. Additionally, the cells migrated deeper into the scaffolds under perfusion compared to those in static culture. Once the cells were confluent, they exhibited morphology more closely resembling native TM. The pressure change of the perfusion reactor increased as the growth of the TM cells decreased the porosity of the scaffold.

TM cells cultured on collagen scaffolds with large pore sizes had increased growth compared to those of the small pore sizes. The larger pore sizes allowed for proliferation deeper in to the interior of the scaffold.

TM cells cultured on the collagen-CS, collagen-HA, and collagen-CS-HA all had similar rates of proliferation showing that the effect of different GAG components have a small effect on the proliferation of cells. (Here I am not sure what these different GAGs will do but I hypothesize they will have a greater effect on gene expression than on proliferation alone)

Conclusions:

The perfusion reactor is a powerful tool to be used in the study of the Trabecular meshwork. As shown above, TM cells grow much better under perfuse conditions. The use of the perfusion reactor replicates the perfuse conditions of the native TM tissue. This setup can be used to model both healthy and glaucomatous tissue by measuring pressure change in real time over a longer period of time. Potential therapeutics for glaucoma can be investigated in vitro before use in clinical studies. Further studies will show the benefit of tissue culture under perfuse conditions.

The microarchitecture of tissue scaffolds has been shown to have a large effect on cell growth and behavior. As shown above cells have a much higher growth rate on larger pores that on smaller pores.

The composition of tissue scaffolds can have a large impact on how cells interact and differentiate, however in the TM the presence of different GAG molecules has shown to have little effect on the overall proliferation of TM cells.