July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Stimulation of cell proliferation and collagen synthesis of human corneal stromal cells in vitro for regeneration of ocular surface
Author Affiliations & Notes
  • Meike Hasenzahl
    Pharmazeutische Technologie, TU Braunschweig, Braunschweig, Germany
  • Stephan Reichl
    Pharmazeutische Technologie, TU Braunschweig, Braunschweig, Germany
  • Footnotes
    Commercial Relationships   Meike Hasenzahl, None; Stephan Reichl, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 530. doi:
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      Meike Hasenzahl, Stephan Reichl; Stimulation of cell proliferation and collagen synthesis of human corneal stromal cells in vitro for regeneration of ocular surface. Invest. Ophthalmol. Vis. Sci. 2018;59(9):530.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Due to the lack of donor corneas, alternatives for regenerative surgery are being researched. Vitamin C (vC) is known to stimulate corneal stromal cells to form extracellular matrix in vitro. In this study, we investigated the influence of some growth factors (IGF-2, TGF-β1, PDGF-BB) in combination with fetal calf serum (FCS) and vC on cell proliferation and collagen synthesis of keratocytes as well as the transparency of cell sheets (CS).

Methods : Primary human keratocytes were cultivated for several weeks in basal medium DMEM containing 10 % FCS (BM) supplemented with either 1 ng/mL TGF-β1, 20 ng/mL PDGF-BB or 10 ng/mL IGF-2, with and without 50 µg/mL vC. Proliferation was examined by cell counting and collagen amount (CA) was quantified using a sirius red based assay. For transparency measurements, CS were placed between a two-ring arrangement. Optical density was determined photometrically. Light transmission (LT) was calculated in the range of 400-800 nm.

Results : After 4 weeks of cultivation, TGF-β1 increased not significantly the cell count, but significantly (p<0.0001) CA (71.62±0.81 µg/well; n=3) compared to BM+vC (33.98±1.21 µg/well; n=3). TGF-β1+vC led to higher CA (92.88±1.10 µg/well; n=3; p<0.0001). However, contraction of CS caused by TGF-β1 prevented further determination of CA. After 6 weeks of cultivation, PDGF-BB+vC supplementation resulted in the highest cell count, but had no effect on CA (118.98±16.59 µg/well; n=4) compared to BM+vC (114.74±11.80 µg/well; n=4). With IGF-2 supplementation, neither the cell count, nor CA or LT differed from the respective values of cells cultured with BM. CS cultured with vC, PDGF-BB+vC or IGF-2+vC could be detached from substrate after 7-10 weeks, with TGF-β1+vC after 3 weeks. Adding of TGF-β1 and PDGF-BB had a negative impact on the transparency of the CS. LT of CS cultured with vC was 83 % at 400 and 96 % at 800 nm (n=5), with TGF-β1+vC 62 % and 86 % (n=4) and with PDGF-BB+vC 77 % and 87 %, respectively (n=5).

Conclusions : Due to TGF-β1 supplementation, there was a distinct increase in CA during the first 4 weeks of cultivation. Increased CA allowed earlier detachment of CS. However, adding of TGF-β1 resulted in more intransparent CS. PDGF-BB+vC facilitated cell proliferation and reduced transparency in comparison to BM+vC, whereas IGF-2 had no influence.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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