Ex Vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement.
Available from: Shiplu ROY Chowdhury
- "However, studies employing human serum have reported conflicting results regarding their ability to maintain the biological characteristics of MSC with some studies demonstrating inhibitory effects on MSC biology (Kuznetsov et al., 2000). Previous study has shown that human serum as a growth supplement is used in ex vivo expanded autologous limbal epithelial cells on amniotic membrane (Shahdadfar et al., 2011). In this study, patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. "
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ABSTRACT: The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. The present study focused on investigating the effects of human serum (HS) on the qualitative and quantitative properties of the human respiratory epithelium compared to the fetal bovine serum (FBS), as a supplement in culture. Respiratory epithelial (RE) cells derived from human nasal turbinate were co-cultured with fibroblasts, subsequently separated at 80–90% confluency by differential trypsinization. RE cells were then sub-cultured into 2 different plates containing 5% allogenic HS and FBS supplemented media respectively up to passage 1 (P1). Cell morphology, growth rate, cell viability and population doubling time were assessed under light microscope, and levels of gene expression were measured via real time reverse transcriptase-polymerase chain reaction (qRT-PCR). RE cells appeared as polygonal shape and expanded when cultured in HS whereas RE cells in FBS were observed to be easily matured thus limit the RE cells expansion. Proliferation rate of RE cells in HS supplemented media (7673.18 ± 1207.15) was 3 times higher compared to RE in FBS supplemented media (2357.68 ± 186.85). Furthermore, RE cells cultured in HS-supplemented media required fewer days (9.15 ± 1.10) to double in numbers compared to cells cultured in FBS-supplemented media (13.66 ± 0.81). Both the differences were significant (p < 0.05). However, there was no significant differences in the viability of RE cells in both groups (p = 0.105). qRT-PCR showed comparable expressions of gene Cytokeratin-14 (CK-14), Cytokeratin-18 (CK-18) and Mucin-5 subtype B (MUC5B) in RE cells cultured in both groups (p > 0.05). In conclusion, HS is a comparatively better choice of media supplement in accelerating growth kinetics of RE cells in-vitro thus producing a better quality of respiratory epithelium for future tracheal reconstruction.
Available from: Aftab Ahmad
- "This may have important clinical implications when bioengineering tissues for clinical use, as it provides a safer and xenobiotic-free culture system. 43 Cultivation of limbal cells was also studied by Shahdadfar et al (2012) on amniotic membrane. Although in his study he did not use CBS but he compared FBS with human serum as the sole supplement and found them equally efficient for ex vivo growth of limbal epithelial cells. "
Available from: Réka Albert
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ABSTRACT: A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2×2×0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.
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