Ex vivo expansion of conjunctival and limbal epithelial cells using cord blood serum-supplemented culture medium.

PURPOSE: Conventional cell culture methods use fetal bovine serum (FBS) as a growth supplement. The purpose of this study was to develop a xenobiotic-free culture system using umbilical cord blood serum (CBS) as an alternative growth supplement for the cultivation of human conjunctival and limbal epithelial cells. METHODS: Human conjunctival and limbal epithelial cells were cultivated in varying concentrations of CBS-supplemented medium and compared with FBS-supplemented medium. Bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and a number of cell generations were analyzed. Cytokeratin expression of cultured cells was evaluated (K3, K4, K12, K13, K14, K15, K19, and PanCK). The authors compared the cytokine and growth factor levels in CBS, FBS, and adult serum using antibody array assays. RESULTS: Conjunctival and limbal cells cultivated in 0.25% CBS- and 0.5% CBS-supplemented culture media demonstrated the highest proliferative capacity in terms of BrdU proliferation assay, CFE, and number of cell generations. These results were comparable to FBS-supplemented medium. Cultured epithelial cells retained their normal cytokeratin expression. Cytokines brain-derived neurotrophic factor, growth-related oncogene, and leptin and growth factors EGF, HGF, FGF-6, IGF-1, PDGF, and IGFBP were present in higher concentrations in CBS than in FBS and adult serum. CONCLUSIONS: CBS-supplemented culture medium supported the proliferation and differentiation of conjunctival and limbal epithelial cells. CBS contained a higher concentration of growth factors and cytokines than FBS and adult serum. CBS may be a viable and safer alternative to FBS as a growth supplement in the culture medium for culturing epithelial cells, which may have important clinical implications when bioengineering tissues for clinical use.

Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency.

PURPOSE: To determine the feasibility of cultivated conjunctiva as a viable epithelial sheet for transplantation and corneal resurfacing in eyes with limbal stem cell deficiency (LSCD). METHODS: Human corneal epithelial (HCE) and human conjunctival epithelial (HCjE) cells were cultivated on human amniotic membrane (AM) to confluence and then air lifted to allow further stratification and differentiation. Denuded AM and cultivated HCE and cultivated HCjE cells were then transplanted into 18 eyes of rabbits with induced LSCD. The cultivated and engrafted epithelia were examined by transmission electron microscopy (TEM) and immunohistochemistry. Two weeks after transplantation, the eyes were examined by slit lamp biomicroscopy and scored on epithelial integrity, corneal haze, and corneal neovascularization. RESULTS: Both cultivated and engrafted HCjE sheets demonstrated confluent epithelial sheets with five to six layers of well-stratified epithelium. TEM examination of engrafted HCjE revealed numerous microvilli, desmosomes, and hemidesmosomes, identical with in vivo corneal epithelium. Immunohistochemical analysis of both HCjE and HCE cells showed the presence of CK3, CK4, and CK12, with absence of Muc5AC. Clinical outcomes for eyes receiving HCjE transplants and HCE transplants were comparable, with most having transparent, smooth corneas, free of epithelial defects. CONCLUSIONS: The study showed that microscopically, HCjE cells have features similar to HCE cells, with clinically equivalent outcomes. The ex vivo cultivation of conjunctiva to form transplantable epithelial sheets for corneal replacement is a promising new treatment modality in patients with LSCD.

Characteristics of dual species Candida biofilms on denture acrylic surfaces.

UNLABELLED: Biofilms contribute to the pathogenesis of oral candidiasis, some 15% of which may be due to dual Candida species. Despite extensive studies on monospecies biofilms (MSB) on denture acrylic surfaces, few have investigated the characteristics of dual species Candida biofilms (DSB). OBJECTIVES: To examine interactions of DSB of Candida albicans and Candida krusei on denture acrylic surfaces. METHODS: Two isolates each of C. albicans (Ca) and C. krusei (Ck), with high (Ca(h), Ck(h)) and low (Ca(l), Ck(l)) biofilm-forming ability were used. The biofilms were developed on acrylic surfaces aerobically at 37 degrees C in yeast nitrogen base (YNB) medium, and growth quantified by colony-forming unit (CFU) assay. We determined: (i) the population profiles of DSB comprising each pair of Candida species, of a total of four combination pairs, after 12 h, (ii) the effect of a constant concentration of Ca(h) (10(7)cells/ml) on varying concentrations of Ck(h) (10(3)-10(7)cells/ml) on DSB development and (iii) the effect of saliva on the growth of DSB. RESULTS: (i) DSB exhibited a lower cell population after 9 or 12 h in comparison to MSB (P<0.05), (ii) C. albicans (10(7)cells/ml) co-cultured with varying initial concentrations of C. krusei was inhibited at high concentrations of the latter (10(6)-10(7)cells/ml) (P<0.05) and (iii) only the MSB development of C. krusei was affected by saliva (P<0.05). CONCLUSION: Our data suggest that the competitive interactions of fungal species are likely to be important in biofilm formation on acrylic surfaces and human saliva may further modulate this process.

In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and anaerobic conditions.

An understanding of biofilm behavior of Candida species under different environmental conditions is key to the development of effective preventive measures for candidal infections. Hence in this study we assessed the impact of the environmental milieu on Candida biofilm formation using polystyrene, flat-bottomed 96-well microtiter plates. A total of 20, comprising 10 clinical isolates each of Candida albicans and, non-albicans species of Candida were compared for their biofilm forming ability both under aerobic and anaerobic conditions, and static and dynamic conditions. XTT reduction assay was used to quantify the sessile growth. Biofilm formation of all 10 C. albicans isolates differed significantly between dynamic and static states under both atmospheric conditions (P<0.05). For non-albicans Candida species, a significant difference in biofilm growth between dynamic and static states was noted only when incubated aerobically (P<0.05), and no significant difference in biofilm formation was noted between aerobic and anaerobic conditions. Scanning electron microscopy revealed that C. albicans produced a compact multilayered biofilm embedded in noticeably higher quantity of extracellular polymeric matrix in aerobic/dynamic conditions compared with anaerobic/static conditions. Our data indicate that biofilm formation of C. albicans and non-albicans Candida species is modulated by hydrodynamic conditions and ambient oxygen gradients. However, further work is required to fully elucidate how Candida biofilms persist within the oral milieu under such challenging ecological pressures.

Effect of oral bacteria on growth and survival of Candida albicans biofilms.

The purpose of this study was to evaluate the effect of eight aerobic and anaerobic oral commensal bacterial species on in vitro Candida albicans biofilm development. A single isolate of C. albicans 2560 g, and eight different species of oral bacteria comprising, Actinomyces israelii, Lactobacillus acidophilus, Prevotella nigrescens, Porphyromonas gingivalis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, and Streptococcus intermedius were studied using an in vitro biofilm assay. Biofilm formation was quantified in terms of the ability of Candida to grow on polystyrene plastic surfaces co-cultured with the foregoing bacteria. A viable cell count was used to quantify the sessile yeast growth and scanning electron microscopy was employed to confirm and visualize biofilm formation. Co-culture with differing concentrations of bacteria had variable effects on Candida biofilm formation. Co-culture with the highest concentrations of each of the foregoing bacteria resulted in a consistent reduction in the yeast counts in the candidal biofilm (P<0.05), except for L. acidophilus, S. mutans, and, S. intermedius co-cultures. Further, on regression analysis a significant negative correlation between the co-culture concentration of either P. gingivalis or E. coli and viable yeast counts in the biofilm was noted (P<0.05) although this was not evident for the other bacterial species. Taken together, our data indicate that, quantitative and qualitative nature of the bacteria modulate C. albicans biofilm formation in mixed species environments such as the oral cavity.