Improvement of Storage Medium for Cultured Human Retinal Pigment Epithelial Cells Using Factorial Design.

Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

Effects of serum-free storage on morphology, phenotype, and viability of ex vivo cultured human conjunctival epithelium.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/µm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/µm; P=0.98 and 0.89±1.0 microvilli/µm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/µm; P=0.47 and 0.07±0.07 microvilli/µm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.

Trefoil factor family 3 expression in the oral cavity.

This study examined the expression, in oral keratinocytes and in the major and minor salivary glands, of Trefoil factor family 3 (TFF3) peptide. Trefoil factor family 3 messenger RNA (mRNA) and peptide were detected in cultures of normal oral keratinocytes by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. Trefoil factor family 3 was found, by immunohistochemical analyses, to be expressed in the basal layers of the oral mucosal epithelium. In salivary glands, immunohistochemical staining showed that TFF3 peptide expression was strongest in the mucous acini of the submandibular and the small salivary glands. Serous cells in the same glands showed weak staining. In the parotid gland, many serous acini showed weak positive staining, while other areas did not. In all glands examined, the intercalated, striated, and collecting ducts were moderately TFF3-positive. Double immunostaining confirmed that mucous (MUC5B positive) cells were moderately or strongly positive for TFF3 and that some serous (MUC7 positive) cells showed restricted TFF3 expression, mostly in a granular pattern. The prevalence of the TFF3 peptide in the salivary secretions of healthy volunteers was detected by western blotting of saliva from minor salivary glands (four of five) and the parotid gland (one of five) and of mixed submandibular/sublingual saliva (five of five). In conclusion, the submandibular and small salivary glands appear to be the major producers of oral TFF3, but duct cells of all glands and keratinocytes of the oral mucosa may also contribute as sources of TFF3 in the oral cavity.

The effects of low level laser treatment on recovery of nerve conduction and motor function after compression injury in the rat sciatic nerve.

An animal study is presented examining the effect of low level laser (LLL) treatment on nerve regeneration following axonotmesis. Twenty animals received a standardised injury to the right sciatic nerve using a time, load and length sequence (10 min, 150 N, 5 mm) known to cause extensive axonal degeneration of the rat sciatic nerve. The LLL treatment was administered using a hand-held laser probe in light contact with the skin on the dorsal aspect of the hind leg overlying the site of the axonotmesis injury to the sciatic nerve. A group of 10 animals were treated with 6J of LLL (GaAlAs 830 nm) daily for a period of 28 d. Ten more animals were treated daily with a sham exposure setting and served as controls. Nerve function was assessed by a recognised method of walking tract print analysis; the "Sciatic Functional Index" (SFI), and nerve regeneration was assessed by recording the evoked compound action potentials (cAP) in the common peroneal nerve. At 21 d post-injury, the laser-treated group had a significantly lower median SFI than the sham laser-treated group, indicating that the real laser treatment had improved functional recovery in the nerve. However, no differences were found between the evoked cAP parameters that were measured in the laser-treated and sham laser-treated groups. Histological examination reiterated the lack of difference between the two groups. Consequently, the effects of LLL on recovery must have occurred more peripherally to the point measured.

An in vivo model for the identification of serum proteins in the acquired subgingival pellicle.

The present study describes an in vivo model for the collection of the subgingival pellicle adsorbed to tooth surface, and the identification of some serum proteins within this layer. Clean dentin slabs were prepared from freshly extracted teeth, and then placed subgingivally for 2 h. The dentin slabs with their adsorbed pellicle layer were processed for transmission electron microscopy. Thin sections were made from the specimens, and treated with antisera to human immunoglobulins and albumin. The reactions were visualized by means of protein A-gold complex, which allowed semiquantification of the serum proteins. The indicator proteins were all identified within the pellicle material, but their amounts and distribution varied. Albumin demonstrated higher amounts in the pellicle layer than other proteins, followed by IgA, IgG, and IgM in descending order. The model described seems useful for studying the acquired subgingival pellicle under varying degrees of disease and health.

Effect of autonomic nerve stimulation on bleb formation in striated duct cells of the rat submandibular gland.

Several previous investigations have shown that blebs form on the apical surface of the striated duct cells of the rat submandibular gland on feeding after starvation. In the present report the influence of autonomic nerve stimulation on bleb formation was studied by electron microscopy. Both parasympathetic and sympathetic stimulation were performed, using electric nerve stimulation. In addition, sympathetic nerve stimulation in combination with alpha- or beta-adrenergic blockers was used. Massive bleb formation took place in response to sympathetic nerve stimulation. This response was almost completely abolished by the administration of alpha- but not by beta-adrenergic blocker. Bleb formation was not seen after parasympathetic nerve stimulation.

Influence of X-ray irradiation on the ultrastructure of rat submandibular gland striated-duct cells.

Previous investigations have indicated that striated-duct cells react to stimulation with an apocrine secretion, morphologically demonstrated by bleb-like projections of the apical cytoplasm. Since bleb formation as an ultrastructural feature also has been debated and sometimes interpreted as a fixation artifact, it was considered essential to extend the studies by exposing the submandibular gland to X rays to establish whether such treatment would have any influence on the formation of blebs. The material used in the present study consisted of rat submandibular glands exposed to X rays in the range of 200-1800 rad. The glands were examined by both SEM and TEM. The duct cells exposed to 200 rad appeared normal, with no sign of alteration in their ability to produce blebs, whereas duct cells exposed to 750 rad showed no sign of bleb formation. Some of the duct cells exposed to 1800 rad showed considerable morphological changes, consistent with oncotic transformation. The results support the conclusion that bleb formation is a normal morphological feature and not an artifact. This study also indicates that the functional activity of the cells is reduced after exposure to X rays.

Ultrastructural studies on the bleb formation in seal and rat submandibular gland striated ducts.

For the present study Seal (Phoca vitulina) and rat submandibular gland striated ducts were investigated by electron microscopy. Both normal and stimulated animals (starved for 24 h and then fed 2 h before the tissues were removed) were examined. Basal invaginations of the cell membrane with areas heavily loaded with mitochondria were typical features of both animals. Secretory granules were especially numerous in the apical part of stimulated duct cells. The granules were separated from the luminal membrane of the cells by a condensed area called the spearating zone. Apical protrusions or blebs, which were frequently occurring in striated ducts of both animals, are interpreted as manifestations of apocrine secretion. It was concluded that this way of apocrine secretion is a fundamental function, since seals, which have salivary glands of a rather simple composition with acini which are functionally reduced, have retained the ability to form blebs.

Ultrastructural studies on apical blebs of striated ducts in the rat submandibular gland.

Apical blebs of rat submandibular gland striated ducts were studied by electron microscopy. The glands were stimulated by starvation of the animals for 24 h whereafter they were fed 15 min before death and tissue removal. After stimulation most striated duct cells developed apical blebs, with various shapes and usually without granules and vesicles. A semi-separating filamentous structure separates the cell from the content of the bleb. Apparently only the vesicular and granular content can pass through this structure and enter the bleb. Following the appearance of small ruptures in the bleb membrane, the bleb is discarded into the lumen of the duct where it disintegrates. Typical wall-like protrusions on the luminal surface of the duct cell, are probably, rudiments of the ruptured bleb membrane. It is suggested that the apical blebs are manifestations of apocrine secretion.

Ultrastructure of the "separating zone" of rat submandibular gland striated duct cells.

The separating zone of rat submandibular gland striated duct cells were studied in detail by electron microscopy. In order to get optimal stimulation of the glands the animals were starved for 24 h followed by feeding 15 min before sacrifice, and tissue removal. The separating zone is morphologically defined by numerous filaments which are located within the apical part of cell. Some of the filaments had a parallel arrangement, running horizontally and being attached to belt desmosomes at the cell periphery. Following the shedding of apical blebs, these filaments appeared to constitute the luminal boundary of striated duct cells. It was observed that the separating zones showed different degrees of density. This is supposed to reflect different stages in the reconstruction of the apical cell surface. It is thus considered that the separating zone, with numerous filaments, is of importance both of the apical secretion per se, and the formation of the new apical cell membrane.

Lactate dehydrogenase (LDH) isoenzyme pattern in normal and inflamed human dental pulp.

Enzyme variants may serve an adaptive role in providing the correct vectorial properties for the metabolism of a tissue, or broadening its environmental tolerance range. To determine if the LDH isoenzyme pattern of human dental pulp changes during inflammation, supernatants from normal and inflamed dental pulp homogenates were separated by polyacrylamide gel electrophoresis. Inflamed pulps had a higher M subunit content and a markedly increased enzyme activity. These results might reflect adaptive changes at the enzyme level associated with a partial shift towards anaerobic metabolism during inflammation of the pulp.

Differential effects of temperature on activity of LDH from seal and sheep skin.

The homeothermic seal possesses a heterothermic skin, while the skin of the sheep behaves as a truly homeothermic tissue. A new method for skin homogenization is described. The effect of temperature on the catalytic behaviour of seal and sheep skin LDH has been investigated by fluorimetric activity measurements, with results presented as Arrhenius plots. The seal skin enzyme had ten times higher activity at low temperatures as compared to the sheep skin. The mechanisms responsible for this different behaviour are discussed.