Fibroin-based materials support cultivation of limbal stromal cells

Sinfield, Laura J., George, Karina A., Zainuddin, Zainuddin, Chirila, Traian V., Hutmacher, Dietmar, Schwab, Ivan R., & Harkin, Damien G. (2010) Fibroin-based materials support cultivation of limbal stromal cells. Investigative Ophthalmology and Visual Science, 51, p. 6211.

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Purpose: The silk protein fibroin provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial cells (Tissue Eng A. 14(2008)1203-11). We presently extend this body of work to studies of human limbal stromal cell (HLS) growth on fibroin in the presence and absence of serum.

Methods: Primary cultures of HLS cells were established in DMEM/F12 medium supplemented with either 10% fetal bovine serum (FBS) or 2% B27 supplement. Defined keratinocyte serum-free medium (DK-SFM, Invitrogen) was also tested. The resulting cultures were analysed by flow cytometry for expression of CD34, CD90, CD45, and CD141. Cultures grown under each condition were subsequently passaged either onto transparent fibroin membranes prepared from purified fibroin or within 3D scaffolds prepared from partially-solubilised fibroin.

Results: HLS cultures were successfully established under each condition, but grew more slowly and passaged poorly in the absence of serum. Cultures grown in 10% FBS were <0.5% CD34+ (keratocytes) and >97% CD90+ (fibroblasts). Cultures established in 2% B27 formed floating spheres and contained >8% CD34+ cells and reduced CD90 expression. Cultures established in DK-SFM displayed traces of epithelial cell growth (CD141), but mostly consisted of CD90+ cells with <1% CD34+ cells. Cells of bone marrow lineage (CD45) were rarely observed under any conditions. Cultures grown in 10% FBS were able to adhere to and proliferate on silk fibroin 3-D scaffolds and transparent films while those grown serum-free could not. Adhesion of HLS cells to fibroin was initially poorer than that displayed on tissue culture plastic.

Conclusions: HLS cultures containing cells of predominantly fibroblast lineage can be grown on fibroin-based materials, but this process is dependent upon additional ECM factors such as those provided by serum.

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ID Code: 61850
Item Type: Journal Article
Refereed: Yes
Additional Information: [ARVO Meeting Abstracts]
Keywords: Corneal limbus, Stromal cells, Silk fibroin, Corneal transplantation
ISSN: 1552-5783
Subjects: Australian and New Zealand Standard Research Classification > ENGINEERING (090000) > BIOMEDICAL ENGINEERING (090300) > Biomaterials (090301)
Australian and New Zealand Standard Research Classification > TECHNOLOGY (100000) > MEDICAL BIOTECHNOLOGY (100400) > Regenerative Medicine (incl. Stem Cells and Tissue Engineering) (100404)
Australian and New Zealand Standard Research Classification > MEDICAL AND HEALTH SCIENCES (110000) > OPTOMETRY AND OPHTHALMOLOGY (111300) > Ophthalmology (111301)
Divisions: Current > Schools > School of Biomedical Sciences
Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Deposited On: 15 Aug 2013 23:34
Last Modified: 02 Feb 2015 04:05

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