Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis.

Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients' health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium-supplemental hormone epithelial medium (SHEM)-supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.

Human Amniotic Membrane: A review on tissue engineering, application, and storage.

Human amniotic membrane (hAM) has been employed as scaffolding material in a wide range of tissue engineering applications, especially as a skin dressing and as a graft for corneal treatment, due to the structure of the extracellular matrix and excellent biological properties that enhance both wound healing and tissue regeneration. This review highlights recent work and current knowledge on the application of native hAM, and/or production of hAM-based tissue-engineered products to create scaffolds mimicking the structure of the native membrane to enhance the hAM performance. Moreover, an overview is presented on the available (cryo) preservation techniques for storage of native hAM and tissue-engineered products that are necessary to maintain biological functions such as angiogenesis, anti-inflammation, antifibrotic and antibacterial activity.

Study on microbial safety of HAM in preparation and preservation / 重庆医科大学学报

Objective: To evaluate the microbial safety of human amniotic membrane(HAM) in preparation and preservation,and to establish effective methods of microbe control for HAM bank.Methods: Human amnions were collected from elective caesarean sections in 20 healthy women,and divided into groups based on different sterilization procedures and preservation methods.Bacteria,fungi and mycoplasmas were detected at 24h,1m,3m and 12m after preservation.Other amnions collected in eutocia from 5 healthy women were detected bacteria,fungi,lactobacilli and mycoplasmas.The series of serological determination for the parturients were followed up in 6 months.Results: Two HAM samples only soaked in NS were detected to be contaminated with E.coli and/or S.epidermidis,respectively from two caesarean sections.There is no microbe detected in all of the amnions from caesarean sections which were soaked in the antibiotics solution.One HAM sample from eutocia was yet detected to have lactobacilli after soak.The series of serological determination were all negative.Conclusion: During one year storage,HAM only from caesarean sections in healthy women,can successfully avoid contamination if soaked in the antibiotics solution then prepared and preserved with aseptic technique.