A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Abstract

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.

M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells

“…41,42 Drugs, cell survival and proliferation The M2 agonist Arecaidine was purchased from SigmaAldrich. The muscarinic antagonists used were: gallamine, (final concentration 10 ¡6 M, Sigma, Cat.…”

“…28 Cell growth of T24 cell lines (2000 cells/well) and human dermal fibroblasts 41,42 was quantified using a colorimetric method (CellTiter 96 AQueus One Solution Cell Proliferation Assay, Promega) according to the manufacturer's instructions. 42,43 M2 receptor silencing The human M2 muscarinic receptor (CHRM2) (ID1129) expression was inhibited in T24, 5637 and HT1197 cells using 4 different siRNAs. The Chromo GAPDH siRNA (20 nM/well) were also used as positive control of transfection.…”

Culture of Human Limbal Epithelial Stem Cells on Tenon’s Fibroblast Feeder-Layers: A Translational Approach

“…We suggest to test products from three different companies and to evaluate at least three different lot numbers to identify the best product. In our experience, we have demonstrated that in TFs cultures AS and FBS exhibit comparable proliferative properties (13). In addition, considering that the percentage of AS in the coculture media is extremely low (0.2 %), the replacement with FBS should not compromise the cell-system and it should contain the minimum risk.…”

“…We recently demonstrated that TFs are eligible candidates as GMP-grade replacement in compliance with European directives and standards (13,14). Herein we propose a double scenario where autologous or heterologous TFs can sustain LSC growth in vitro.…”

The adipose tissue of origin influences the biological potential of human adipose stromal cells isolated from mediastinal and subcutaneous fat depots

“…ASC cultures were maintained at a cell density 4000 cells/cm 2 and passaged when reached 80-90% of confluence. Cell growth/viability was assessed by Trypan Blue (Siciliano et al, 2015a;De Falco et al, 2013) and measured by the automatic cell counter Countess® (Life Technology, Monza, Italy). The number of doubling cells was calculated as the following ratio: 3.322 log 10 N/N 0 (N is the final number of cells obtained and N 0 is the number of cells plated) (De Falco et al, 2013).…”