A major drawback of most chemo- and radiotherapeutic cancer treatments is their toxicity to the ovaries, meaning they may affect fertility and/or endocrine function of female patients. The state-of-the-art method nowadays is the cryopreservation of the ovaries. However, with transplantation of ovarian tissue after the end of cancer treatment there is always the risk to also transfer malignant cells back into the patient which could lead to disease recurrence. A safe alternative to restore fertility in female cancer patients could be to develop a bioengineered ovary using different biomaterials, which would be used as a safe, temporary environment for the patient’s follicles. Like the natural ovary, it would contain the patient’s follicles, ensure its survival, and support its growth. In this study, we develop 3D scaffolds consisting of different biomaterials to culture cells for several days. We will evaluate cell viability, morphology, and proliferation in response to the different hydrogel formulations.
Scaffolds for cell culture were fabricated using a combination of the synthetic polymer polyethylene glycol (PEG) and the natural biomaterial collagen type I. Collagen type I was incorporated into the hydrogels to enable cell adhesion within the bioinert PEG hydrogels. Another approach to promote cell adhesion is to incorporate RGD into the hydrogels, which was investigated as well. Different concentrations of collagen were used, and the hydrogels were either supplemented with RGD, or not. In a first approach, hydrogels were seeded with human bone marrow-derived mesenchymal stromal cells (BM-MSCs) at two different concentrations. Cell morphology, proliferation and viability were evaluated over seven days using brightfield imaging, and by quantifying the DNA content of hydrogels directly after fabrication and after one week of culture.
With the help of established protocols to seed PEG hydrogels with BM-MSCs, it was possible to successfully seed cells in PEG-collagen composite hydrogels. Using brightfield imaging it was observed that BM-MSCs start to spread faster in hydrogels containing collagen, compared to those fabricated without. This observation was the same for hydrogels prepared with or without RGD and for both cell seeding densities. Cells proliferated in all hydrogel compositions during seven days of culture, and no difference in proliferation between hydrogels prepared with and without collagen, using the higher cell concentrations, could be detected.
To conclude, a protocol has been established to seed BM-MSCs into PEG-collagen hydrogels with different concentrations of collagen. Future experiments will be carried out incorporating other ECM components into the hydrogels and using fetal membrane cells as more sensitive cells to test for differences in cell proliferation and viability depending on the hydrogel composition. In addition, matrix deposition of cells will be detected using immunofluorescence staining for fibronectin and subsequent imaging. The morphology of the cells within the hydrogel scaffolds will be evaluated by staining actin and cell nuclei. The stability of hydrogel composition will be evaluated by measuring the collagen content after different number of days in culture.