Keratinocyte differentiation inversely regulates the expression of involucrin and transforming growth factor beta1

Extensive skin loss from a variety of conditions such as severe thermal injury is associated with significant functional morbidity and mortality. In recent years, the healing quality has been improved for patients who suffer burns due in part to the usage of skin replacement mainly prepared from multi-layered sheets of cultured keratinocytes. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-beta1 (TGF-beta1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor and has any functional influence on dermal fibroblasts. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms express different levels of TGF-beta1. To address this hypothesis, keratinocytes were grown in serum free medium (KSFM) supplemented with bovine pituitary extract (50 microg/ml) and EGF (5 microg/ml). When cells reached confluency, conditioned medium was removed and replaced with 50% KSFM with no additives and 50% DMEM without serum and cells were allowed to form multi-layers and differentiate. The conditioned medium was then collected every 48 h up to 24 days and the level of TGF-beta1 and the efficacy of a keratinocyte released fibroblast mitogenic factor were evaluated by ELISA and (3)H-thymidine incorporation, respectively. Northern analysis was also employed to evaluate the expression of TGF-beta1, involucrin, TIMP-1, and 18 S ribosomal RNA in keratinocytes at different times of the onset of differentiation. The microscopic morphology of keratinocytes at different times of induction of cell differentiation showed detachments (nodules) of many regions of keratinocyte sheet from culture substratum within 1-2 weeks. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. The results of TGF-beta1 evaluation revealed that mono-layers of cultured keratinocytes which were round, attached, and proliferating in KSFM + BPE and EGF containing medium released a significantly higher level of TGF-beta1 (196 +/- 58 pg /ml) relative to those grown in multi-layer forms (28 +/- 7.8 pg/ml). A longitudinal experiment was then conducted and the results showed that cells on the onset of differentiation released even greater level of TGF-beta1 (388 +/- 53 pg/ml) relative to those grown in KSFM + BPE and EGF. This finding was consistent with the expression of TGF-beta1 mRNA evaluated in keratinocytes grown in test medium for various duration. In general, the level of TGF-beta1 protein and mRNA gradually reduced to its lowest level within 12 days of growing cells in our test medium. When aliquots of the collected keratinocyte conditioned medium were added to dermal fibroblasts, the level of (3)H-thymidine incorporation increased only in those cells receiving aliquots of conditioned medium containing high levels of TGF-beta1. When involucrin was used as a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. In conclusion, high involucrin expressing differentiated keratinocytes seem to be quiescent in releasing both TGF-beta1 and a fibroblast mitogenic factor.     

Selective growth and serial passage of mouse melanocytes from neonatal epidermis in a medium supplemented with bovine pituitary extract

Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F-10 plus bovine pituitary extract (BPE), insulin, and transferrin. Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating. On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes. From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts. The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested. In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte-enriched cultures, namely, those at the early stages of the primary culture. FBS was added at a concentration of 1% for the first day. These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes.     

Evaluation of the effects of cosmetic or dermo-pharmaceutical products on cutaneous energy metabolism using the Episkin model of reconstructed epidermis

This study was implemented to test the Episkin model of reconstructed epidermis in the evaluation of the efficacy of cosmetic or dermopharmaceutical products on cutaneous energy metabolism. The energy metabolism is evaluated by measuring the concentration of intracellular ATP by a method using an ultrasensitive bioluminescent reaction. The work presented compares results obtained in reconstructed epithelium and monolayer primary cultures of human keratinocytes.After application of a hydrosoluble product, the increase in intracellular ATP is identical in a monolayer culture of keratinocytes (+239±18% versus control) and in Episkin (+248±21% versus control). An emulsion was also tested on the two models. It is only possible to test the emulsion at a dilution of under 0.05% on a keratinocyte culture, and this means that the real efficacy of the product is underestimated (+145±18% versus control). The three-dimensional model enables the application of the undiluted emulsion, and the results show an increase in intracellular ATP of +420±80% versus control: products in final formulation can be tested in normal conditions of use.Abbreviations BPE bovine pituitary extract - DMEM Dulbecco's modified Eagle's medium - EDTA ethylene diamine tetraacetic acid - EGF epidermal growth factor - K-SFM keratinocytes serum free medium - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide - O/W oil in water - PBS phosphate-buffered saline     

Bovine pituitary extract provides remarkable protection against oxidative stress in human prostate epithelial cells

Bovine pituitary extract (BPE) is routinely used as a mitogenic supplement in serum-free growth medium. In addition to its mitogenic activity, BPE contains a variety of growth factors and hormones with reported antioxidant activity. This study examines the antioxidant potential of BPE in nontumorigenic human prostate epithelial cells (RWPE-1). Treatment of RWPE-1 cells with BPE (50 microg/ml) provided significant protection against H(2)O(2)-induced cell death, deoxyribonucleic acid fragmentation, protein oxidation, and membrane damage. Treatment with heat (71 degrees C, 10 min) and proteolytic enzymes reduced the antioxidant activity of BPE, suggesting that proteins present in BPE may be responsible for the antioxidant activity. Residual catalase activity present in BPE was responsible for a portion (30%) of the antioxidant activity. Interestingly, RWPE-1 cells treated with BPE and H(2)O(2) rapidly accumulated intracellular reactive oxygen species (ROS) to a greater extent than cells receiving only H(2)O(2). Pretreatment of RWPE-1 cells with tyrosine kinase inhibitors (genistein, tyrphostin 47, and AG-1296) before the addition of H(2)O(2) diminished BPE protection against H(2)O(2)-induced cell death, whereas treatment with purified mitogens commonly found in BPE, growth hormone and basic fibroblast growth factor, did not protect against oxidative damage. Taken together, these data suggest that BPE contains proteins or protein complexes with remarkable antioxidant activity. These yet unidentified compounds appear to confer protection against H(2)O(2)-induced cell death by tyrosine kinase-dependent pathways that increase intracellular ROS generation. The antioxidant activity of BPE may represent a confounding variable when studying oxidative stress in cells maintained in BPE-supplemented serum-free medium.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

Use of sugarcane bagasse and grass hydrolysates as carbon sources for xylanase production by Bacillus circulans D1 in submerged fermentation

The use of sugarcane bagasse and grass as low cost raw material for xylanase production by Bacillus circulans D1 in submerged fermentation was investigated. The microorganism was cultivated in a mineral medium containing hydrolysate of bagasse or grass as carbon source. High production of enzyme was obtained during growth in media with bagasse hydrolysates (8.4 U/mL) and in media with grass hydrolysates (7.5 U/mL). Xylanase production in media with hydrolysates was very close to that obtained in xylan containing media (7.0 U/mL) and this fact confirm the feasibility of using this agro-industrial byproducts by B. circulans D1 as an alternative to save costs on the enzyme production process.     

Influence of the rapeseed protein hydrolysis process on CHO cell growth

Different protein hydrolysates were prepared from enzymatic hydrolyses of a rapeseed isolate (>90% protein content) using different commercial enzymes of non-animal origin. The extent of hydrolysis was controlled to produce hydrolysates corresponding to various degrees of hydrolysis (DH) from 5 to 30. These hydrolysates were characterized according to their solubility and size peptide pattern. Different growth behaviours of Chinese Hamster Ovary cells were observed when these various hydrolysates were added in serum-free medium containing transferrin, albumin and insulin. Hydrolysates from low degree of hydrolysis generally did not exhibit significant positive effect on cell growth; conversely hydrolysates from extensive hydrolysis, corresponding to a major low molecular size peptides content, usually allowed an increase of the maximal cell density. However, depending on the enzyme used, the supplementation with hydrolysates corresponding to a high degree of hydrolysis and composed of at least 70% peptides with a molecular size under 1kDa, led to different maximal cell density values, indicating the importance of enzyme specificity and consequently the nature of the released peptides. This result showed that the positive influence of the rapeseed hydrolysates on cell growth was not only due to a nutritional support tied to the addition of small peptides but may be related to the presence of peptides exhibiting growth or survival factor effects. Furthermore, total substitution of proteins (transferrin, albumin and insulin) in the cell culture medium by some rapeseed hydrolysates appeared to be a promising alternative to improve the cell growth in protein-free media.     

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.     

Application of bovine pituitary extract and polyglycolide/poly(lactide-co-glycolide) scaffold to the cultivation of bovine knee chondrocytes

In vitro cultivation of primary bovine knee chondrocytes (BKCs), using bovine pituitary extract (BPE) and porous scaffolds composed of polyglycolide (PGA) and 85/15 poly(lactide-co-glycolide) (PLGA), was investigated. Here, BPE was prepared from fresh bovine pituitaries, and cylindrical PGA/PLGA scaffolds with various chemical compositions were fabricated by solvent merging/particulate leaching method. Experimental results showed that in microcarrier systems, the rate of BKC growth on PGA surfaces is faster than that on PLGA surfaces, and the decrease in the medium pH value of BKCs-adsorbed PGA particles is faster than that of BKCs-adsorbed PLGA particles. After 28-day construct cultivation, the BKC amount and the content of glycosaminoglycans and collagen per construct increased with BPE protein concentration. For a constant BPE protein concentration, a higher PGA percentage in scaffold leads to a better biological environment for the growth of BKCs and the synthesis of extracellar matrices.     

Influence of media composition on the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis

Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x 10(6)/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x 10(6)/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced delta-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.     

Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.     

Identification of L-altrose in the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3

Abstract Butyrivibrio fibrisolvens strain CF3, a stricyly anaerobic bacterial isolate from the ovine cecum, produces extracellular polysaccharides (EPS) when grown on a defined medium containing glucose as the carbon source. EPS were purified from culture supernatants and their monosaccharide composition was determined by two different procedures. Analysis of EPS hydrolysates by thin-layer chromatography (TLC) yielded spots coincident with standard glucose, altrose, and 1,6-anhydroaltrose. These results were corroborated by both gas-liquid chromatography (GLC) and GLC-mass spectroscopy (GLC-MS) of alditol acetates prepared from EPS hydrolysates. Purification of the altrose from EPS hydrolysates was accomplished by preparative paper and column chromatography. Polarimetry demonstrated the isolated altrose to have the L -configuration. The occurrence of this hexose in nature has not yet been reported.     

Fermentation of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing

This work aims to evaluate the fermentability of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing using Candida guilliermondii FTI 20037 yeast. The inoculum was obtained from yeast culture in a medium containing glucose as a carbon source supplemented with rice bran extract, CaCl₂·2H₂O and (NH₄)₂SO₄ in 50 mL Erlenmeyer flasks, containing 20 mL of medium, initial 5.5 pH under agitation of an orbital shaker (200 rpm) at 30°C for 24 h. The cellulosic hydrolysates, prior to being used as a fermentation medium, were autoclaved for 15 min at 0.5 atm and supplemented with the same nutrients employed for the inoculum, except the glucose, using the same conditions for the inoculum, but with a period of 48 h. Preliminary results showed the highest consumption of glucose (97%) for all the hydrolysates, at 28 h of fermentation. The highest concentration of ethanol (20.5 g/L) was found in the procedure of sugarcane bagasse pretreated by hydrothermal processing (195°C/10 min in 20 L reactor) and delignificated with NaOH 1.0% (w/v), 100°C, 1 h in 500 mL stainless steel ampoules immersed in an oil bath.     

Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.     

Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth

The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special^® enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N^® enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.     

Immortalization of rat keratinocytes by transfection with polyomavirus large T gene

Transfection of primary rat keratinocytes with the polyomavirus large T gene promotes the establishment of cell lines. The keratinocytes express the large T protein and can be continuously cultured in medium containing a low concentration of calcium. The immortalized keratinocytes retain the ability to differentiate when the calcium concentration is increased to normal levels.     

The production of extracellular polysaccharides by a hydrocarbon assimilating yeast.

Investigations were made on the extracellular polysaccharides production by a hydrocarbon assimilating yeast. The yeast Candida lipolytica was grown on two different media containing n-hexadecane as the sole carbon source. Polymeric materials precipitated from the culture medium with ethanol were determined gravimetrically at various growth periods. The hydrolysis of the precipitated material and their chromatographic analysis revealed the presence of mannose, glucose and galactose in the yeast extract-containing medium. The proportions of these sugars differed at various growth periods. The hydrolysates of the polymers of the yeast extract-free medium contained more xylose than those of the yeast extract containing medium.     

Isolation and culture of amelanotic melanocytes from human hair follicles

We report a method to establish amelanotic melanocytes (AMMC) in culture and we investigate the effects of various components in the culture medium. Normal human scalp from cadaver donors was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. The individual hair follicles were washed exhaustively and suspensions of hair follicle cells were prepared and cultured in Eagle's minimum essential medium supplemented with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), cholera toxin and keratinocyte serum-free medium (K-SFM). Geneticin was used to eliminate contaminating fibroblasts. Proliferation of AMMC was observed after addition of TPA and K-SFM including bovine pituitary extract (BPE) into the culture medium. Cell type was determined by staining with monoclonal antibodies, NKI/beteb and HMB-45, which recognize premelanosomal and melanosomal antigens, respectively. The AMMC were also examined using transmission electron microscopy. Treatment with geneticin eliminates the majority of fibroblasts and does not impair the growth of keratinocytes or AMMC. After contaminating fibroblasts and keratinocytes were removed, two distinct cell morphologies remained: (1) large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and (2) small bipolar cells, which initially were unpigmented and proliferated very rapidly. We observed that TPA at various concentrations stimulated the proliferation of the cells, and at high concentrations could induce the formation of multiple dendrites. K-SFM including BPE accelerated the proliferation of the cells in a dose-dependent manner. After passage 3, almost all cells expressed premelanosomal and melanosomal antigens, recognized by NKI/beteb and HMB-45, respectively. Active mitochondria, abundant rough endoplasmic reticulum, Golgi complexes, ribosomes and melannosomes (predominantly in stages I, II or III with some at stage IV in some AMMC) were observed ultrastructurally in the cytoplasm of the cultured cells.     

Development of an animal‐component free medium for vero cells culture

This work describes the development of an animal‐component free medium (IPT‐AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum‐free medium (SFM) referred as IPT‐SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT‐SFM was further improved to obtain an animal‐component free medium named IPT‐AFM. IPT‐AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue‐culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non‐animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT‐AFM were investigated in T‐flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 ± 0.18 and a specific growth rate μ 0.019 ± 0.003 h^?1 were achieved in IPT‐AFM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009