Fetal Calf Serum Supplementation Research Articles

Quality Analysis of Minerals Formed by Jaw Periosteal Cells under Different Culture Conditions.

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties.

Stabilization of hyperforin dicyclohexylammonium salt with dissolved albumin and albumin nanoparticles for studying hyperforin effects on 2D cultivation of keratinocytes in vitro

Due to the limited chemical stability of the natural hyperforin molecule, a more stable form of hyperforin, i.e., the hyperforin dicyclohexylammonium salt (HYP-DCHA) has been used for ex vivo and in vitro experiments in recent years, but its actual stability under typical cell culture conditions has never been studied before.In this contribution the stability of HYP-DCHA was examined under typical cell culture conditions. Different cell culture media with and without fetal calf serum (FCS) supplementation were studied with regard to further stabilization of HYP-DCHA determined with HPLC analysis. Furthermore, albumin nanoparticles were examined as a stabilizing carrier system for HYP-DCHA. In this context, the interaction between HYP-DCHA and albumin nanoparticles (ANP) was examined with regard to size and loading with HYP . The effects of HYP-DCHA either supplied in cell culture medium or loaded on ANP on viability and cytotoxicity were studied in vitro on HaCaT monolayers (human keratinocyte cell line).HYP-DCHA supplied in FCS-containing medium was recovered completely after 24h of incubation. However, a lack of FCS caused a total loss of HYP-DCHA after less than 24h incubation time. Supplying HYP-DCHA loaded on ANP in an FCS-free medium resulted in a recovery of about 60% after 24h incubation. HYP-DCHA supplied in medium along with FCS showed a slow dose-dependent decrease in viability of HaCaT cells without any cytotoxic effects (antiproliferative effect). Treatment with HYP-DCHA with a lack of FCS resulted in a significantly faster decrease in viability which was mainly due to cytotoxicity. The latter was true for HYP-DHCA-loaded ANP where increased cytotoxicity was observed despite the presence of FCS.The results show that the stability of the widely used HYP-DCHA is rather limited under cell culture conditions. Especially a lack of FCS leads to degradation and/or oxidation of HYP-DCHA probably causing an increased cytotoxicity. In contrast, FCS supplementation fairly stabilizes HYP-DCHA under cell culture conditions while albumin nanoparticles may serve the same stabilization purpose despite increasing cytotoxic effects onto the cells themselves.

PP-027 - Maturing the oocyte: A comparison of maturation media and the effect of fetal calf serum supplementation

PP-027 - Maturing the oocyte: A comparison of maturation media and the effect of fetal calf serum supplementation

Successful re-endothelialization of a perfusable biological vascularized matrix (BioVaM) for the generation of 3D artificial cardiac tissue.

Generating cellularized 3D constructs with clinical relevant dimensions is challenged by nutrition supply. This is of utmost importance for cardiac tissue engineering, since cardiomyocytes are extremely sensitive to malnutrition and hypoxia in vitro and after implantation. To develop a perfusable myocardial patch, we have focused on seeding a decellularized biological vascularized matrix (BioVaM) with endothelial cells. BioVaM is produced by decellularization of porcine small intestinal segments with preserved arterial and venous pedicles, which can be connected to a perfusion system in vitro or the host vasculature in vivo. The BioVaM vessel bed was re-seeded with porcine primary endothelial cells (pCEC). Seeding efficiency was influenced by detergent composition used for decellularization (sodium dodecyl sulfate (SDS) and/or Triton X-100) and the medium composition used for re-seeding. After decellularization, residual SDS was detected in the matrix affecting the survival of pCEC which showed a low tolerance to SDS and Triton X-100. Sensitivity to detergents was attenuated by supplementation of the medium with bovine serum albumin (BSA) or fetal calf serum (FCS). Pre-conditioning of the BioVaM with 20% FCS was not sufficient to attain pCEC survival in the vascular bed. However, re-cellularization was achieved by prolonged FCS supplementation during cultivation, resulting in a perfusable, re-endothelialized matrix of 11 cm2 in size. This achievement represents a promising step towards engineering of perfusable, 3D cardiac constructs with clinically relevant dimensions.

The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen

The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72h at 5°C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37°C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40×106sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5mg/ml), FCS (10%) or no additive (control) at 37°C, cooled to 5°C and stored for up to 72h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72h storage period, compared to the control (P<0.01). The supplementation of BSA and FCS had a protective effect on motility (P<0.05), plasma membrane integrity (P<0.01) and acrosomal integrity (P<0.01) at 72h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72h during storage period, compared to the control (P<0.001). Although supplementation of BSA and FCS caused significant (P<0.01) decreases in malondialdehyde (MDA) level at 48h and 72h, they significantly (P<0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties.

THE EFFECT OF FETAL CALF SERUM ON HUMAN DENTAL PULP STEM CELLS

Authors studied potential side effects of fetal calf serum (FCS) in cultivation media on human dental pulp stem cells (DPSC) during long-term cultivation. Two lines of DPSC obtained healthy donors (male 22 years, female 23 years) were used. Both lines were cultivated under standard cultivation conditions in four different media containing 10% or 2% FCS and substituted with growth factors. During long-term cultivation proliferation ability, karyotype and phenotype of DPSC were measured. Both lines of DPSC cultivated in a media containing 2% FCS and ITS supplement showed the highest number of population doublings. On the other hand the proliferation rate of DPSC cultivated in a media with 2% FCS without ITS supplement was slowest. Proliferation rate of DPSC cultivated in 10% FCS media with or without FGF-2 was comparable. DPSC cultivated in a media with 10% FCS showed a significantly higher amount of chromosomal aberrations. These chromosomal aberrations do not seem to be clonal but surprisingly we found large amounts of tetraploid cells in the 9th passage in both media containing 10% FCS. Our study proved that cultivation of DPSC in media containing higher concentration of FCS has critical side effects on cell chromosomal stability.

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.

137 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECTS ON THE DEVELOPMENT AND APOPTOSIS OF IN VITRO PRODUCED BOVINE EMBRYOS

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitro produced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N = 4320) were matured and fertilized in vitro (Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3 μM PES from Day 4 (after 96 h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N = 60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivo control (n = 15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means ± SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7 ± 1.7, 82.3 ± 1.6, 86.3 ± 1.4, 87.0 ± 1.5, P &gt; 0.05) and augmented blastocyst production (30.5 ± 2.5a, 41.8 ± 2.4b, 40.5 ± 2.6b, 47.2 ± 2.8b, P &lt; 0.05), respectively, for 0, 2.5, 5, and 10%. Additionally, increasing FCS concentration increased apoptosis in blastocysts (13.8 ± 1.2b, 19.1 ± 1.8b, 20.7 ± 1.9bc, 28.4 ± 2.3c, P &lt; 0.05, respectively, for 0, 2.5, 5, and 10%). The addition of PES from Day 4 in the culture medium did not affect (P &gt; 0.05) cleavage (87.0 ± 1.3 and 84.4 ± 1.3), blastocyst production (42.0 ± 2.8 and 43.0 ± 2.0), and apoptosis in blastocysts (20.7 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control group presented the lowest apoptosis rate (6.3 ± 1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality. São Paulo Research Foundation – FAPESP.

Serum‐free medium with osteogenic supplements induces adipogenesis in rat bone marrow stromal cells

Adult BMSCs (bone marrow stromal cells) contain MSCs (mesenchymal stem cells). MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and myoblasts and are thus considered useful in tissue engineering for therapeutic and clinical purposes. FCS (fetal calf serum) is usually included in the differentiation medium, but the clinical application of FCS may pose a problem for some patients. To improve the efficiency and safety of BMSC cultivation, the effect of SF (serum-free) conditions on the osteogenic differentiation of rat BMSCs was examined. In the presence of 10% FCS and osteogenic supplements, the cells formed mineralized von Kossa-positive deposits. Under SF conditions with osteogenic supplementation, however, the cells possessed cytosolic lipid vacuoles that could be stained with Oil Red O. The mRNA expression of PPARgamma (peroxisome proliferator-activated receptor gamma), an adipogenic marker, increased under the SF condition with osteogenic supplements. These data indicate that rat BMSCs differentiate into adipocytes under SF conditions even with osteogenic supplementation and that FCS is needed to induce proper osteogenic differentiation in rat BMSCs.

112. FETAL CALF SERUM AFFECTS hESC METABOLISM AND GENE EXPRESSION LEADING TO DIFFERENTIATION IN CULTURE

Fetal calf serum (FCS) has conventionally been used to support the growth and maintenance of human embryonic stem cells (hESCs). FCS however, is an undefined complex mixture containing factors which potentially alter the functionality of hESCs. Inclusion of FCS during embryo culture negatively impacts embryo metabolism and viability but comparative studies on hESCs have been hindered by the lack of serum and feeder independent culture systems. Using a recently available defined culture system, the effects of FCS on hESC metabolism and pluripotentcy were investigated. Mel2 hESCs were grown at 37°C in 5% CO2 for 3 days on matrigel (hESC-qualified) coated tissue culture wells in mTeSR1 medium. hESCs were then cultured in mTeSR1 (control) or in mTeSR1 supplemented with 20% FCS from different manufacturers or knockout serum replacement (KOSR), for 96 hours. Media was renewed daily. At the end of the culture period, spent media was collected and cells were trypsinised and counted and/or collected for gene expression analysis. FCS decreased cell survival and altered hESC morphology from densely packed colonies with distinct borders into non-uniform heterogeneous populations comprising of hESC-like cells and fibroblastic-like cells with high cytoplasmic to nuclear ratios. Media analysis revealed altered cell metabolism with increased glucose consumption rates per cell (P &lt; 0.01) with FCS supplementation, compared to cells cultured in mTeSR1 alone. Gene expression analysis revealed that FCS, regardless of its manufacturer, decreased the expression of some pluripotent markers and increased differentiation markers. A decrease in pluripotent gene expression was also observed in hESCs cultured with KOSR compared to mTeSR1 alone. Maintenance of homogeneity in hESC populations is crucial for the advancement of hESC clinical therapies. This study demonstrates that FCS promotes heterogeneity and impacts the metabolic function and gene expression of hESCs thereby supporting the need for serum-free culture systems as standard practice in hESC culture.

118 ARE LIPID CONTENTS AND APOPTOSIS DETERMINANTS OF IN VITRO-PRODUCED BOVINE EMBRYO SUSCEPTIBILITY TO VITRIFICATION?

Supplementation of fetal calf serum (FCS) during culture of bovine in vitro-produced embryos (IVPE) has been correlated with lipid accumulation, representing a limiting factor on embryo cryotolerance. In the present experiment we studied lipid content and apoptosis in Nelore IVPE cultured in different concentrations of FCS. The experimental design was a 4 × 2 × 2 factorial, in which embryos, exposed or not to the metabolic regulator, phenazine ethosulfate (PES), were cultured in 4 concentrations of FCS. In 16 replicates, 560 IVPE were vitrified after cultured in SOFaa supplemented with 0, 2.5, 5 or 10% FCS. On Day 2.5 of culture, one-half of the embryos in each group were treated with 0.3 μM of PES and then returned to standard culture conditions (5% O2, 5% CO2 and 90% N2 atmosphere at 38.5°C). All embryos were vitrified on Day 7 (Campos-Chillòn LF et al. 2006 Theriogenology 65, 1200-1214). After warming, embryos were placed into culture for 12 h under standard conditions. A sample of fresh embryos was stained with Sudan Black B for quantification of small (&lt;2 μm), medium (2-6 μm), and large (&gt;6 μm) cytoplasmic lipid droplets. Apoptosis was analyzed, before and after vitrification, using TUNEL. As a positive control (CV), in vivo-produced embryos were collected from a superovulated Nelore cow. Data were analyzed using ANOVA followed by Tukey’s test; and Pearson’s linear correlation (P &lt; 0.05). The results indicate that FCS concentration and PES treatment had no influence (P &gt; 0.05) on cleavage and blastocyst formation rates (mean of 85.7 ± 2 and 36.6 ± 5, respectively). The total number of cells in fresh embryos was also similar (P &gt; 0.05) among groups (mean of 134.1 ± 28). Additional results are shown in Table 1. Elevated concentrations of FCS increased lipid accumulation (r = 0.9) and the percentage of apoptotic cells in fresh and vitrified embryos, reducing the re-expansion rate after warming. Moreover, the number of apoptotic cells observed in fresh embryos was strongly correlated with the apoptosis observed after vitrification (r = 0.95). Although PES was able to reduce lipid accumulation, it was not efficient at preventing apoptosis in vitrified embryos. In summary, the results of this experiment indicate that the concentration of FCS during culture did not influence cleavage and blastocyst formation in bovine IVPE. We conclude that the apoptosis rate in fresh embryos is a key factor affecting survival after vitrification. Table 1.Lipid droplets, re-expansion, apoptosis of in vivo and IVPE Acknowledgements: FAPESP (07/57766-4).

Interconversion of biological characteristics of small cell lung cancer depending on culture conditions.

Two distinct cell lines were obtained from a single heterotransplanted tumor which had originated from a primary focus of small cell carcinoma of the lung (SCCL). They were maintained separately from the beginning in culture media with and without fetal calf serum supplementation. Cells in the serum-free medium grew mostly floating in loose aggregates and showed poor cell cohesiveness, scanty cytoplasm and a few intracytoplasmic small dense-cored granules; all of these features are characteristics of oat cell type SCCL. On the other hand, cells in the serum-supplemented medium grew mostly floating in flatter and more closely associated clumps, were larger, and showed increased cell cohesiveness, occasional tubular structures, better developed organelles including dense-cored granules, and an increased number of cell attachments; these features are characteristics of intermediate cell type SCCL. The modal number of chromosomes differed from each other. Neuron-specific enolase (gamma enolase) and aromatic L-amino acid decarboxylase (ADC) activities in cell pellets were significantly higher in both lines than in control non-small cell lung cancer cell lines. The alpha/gamma type enolase ratio was lower, as was the ADC activity, in serum-free cultures than in serum-supplemented cultures. Interchange of the culture medium induced changes of the growth pattern and cell type from "oat cell type" to "intermediate cell type" and vice versa. The chromosomal number also partially changed. These findings suggest that cultured cells of SCCL alter their growth pattern and cell type depending on the culture conditions and that the selective growth of one cell type might then take place.

IL‐1β is required for TLR‐mediated antimicrobial activity

Toll‐like receptor 2/1 activation on human monocytes induces vitamin D receptor (VDR) dependent expression of the antimicrobial peptide (AMP) cathelicidin, which is the only known TLR‐regulated AMP expressed in monocytes. We hypothesize that DEFB4, a VDR‐dependent AMP, will also be regulated by TLR2/1. Here we demonstrate that TLR2/1 activated monocytes in vitamin D sufficient human serum (HS) but not fetal calf serum (FCS) expresses DEFB4. Knockdown of either DEFB4 or cathelicidin ablates the TLR‐mediated antimicrobial activity against M. tb, indicating that both peptides are required. Supplementation of FCS with the vitamin D prohormone (25D3) rescues TLR‐induction of cathelicidin but not DEFB4. Induction of DEFB4 in HS is dependent on the VDR, but the active vitamin D (1,25D3) alone was not sufficient for DEFB4 expression, suggesting another factor is required, such as IL‐1β. TLR2/1 activated monocytes cultured in HS secrete significantly higher levels of IL‐1β than cells in FCS. TLR‐induced DEFB4 but not cathelicidin expression was synergistically increased with addition of IL‐1β or ablated when IL‐1β activity was inhibited. These data suggest that autocrine production of IL‐1β is required for the TLR‐induced antimicrobial activity.

Effect ofTheobroma cacaoflavonoids on immune activation of a lymphoid cell line

We analysed the effect of (-)-epicatechin and cocoa extract on the activation of a lymphoid cell line. Particularly the expression of IL-2 receptor alpha (IL-2Ralpha or CD25) and, the secretion of IL-2 and IL-4 were established after flavonoid treatment. Two media culture conditions (1 and 10 % of fetal calf serum supplementation) and the different moments of flavonoid addition (simultaneously or 2 h before cell-activation) were compared. IL-2Ralpha (CD25) expression on activated cells was significantly reduced by epicatechin and cocoa extract in a dose-dependent manner, achieving the highest inhibition of about 50 % when flavonoids were added 2 h before stimulation. IL-2 secretion was also inhibited by the presence of both epicatechin and cocoa extract, displaying 60 and 75 % of inhibition, respectively. Cocoa flavonoids were also able to enhance 3-4.5-fold IL-4 release. In summary, cocoa extract down-modulated T lymphocyte activation and therefore the acquired immune response. This fact could be important in some states of the immune system hyperactivity such as autoimmune or chronic inflammatory diseases.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), (2)-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription-polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro.

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.

Culture medium enhances semicarbazide-sensitive amine oxidase activity.

Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells. Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro. Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC). In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO. In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media. Semicarbazide-sensitive amine oxidase activity in FCS (5-20 microl) was significantly enhanced (approximately 1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement. Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages. Activity in rat plasma was reduced by approximately 25% in the presence of DMEM. The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine.HCl, and L-glutamine, were not correlated with enhancement. This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.

Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonists

Objective To examine whether gonadotrophin-releasing hormone agonist or oestradiol can directly affect DNA in leiomyoma cells. Design In vitro explant culture of leiomyoma cells. Setting University research group. Sample Leiomyoma cells were cultured from the specimens of four premenopausal women at myomectomy. Methods The presence of gonadotrophin-releasing hormone receptor in leiomyoma cells was determined by reverse transcriptase–olymerase chain reaction. Leiomyoma cells were treated with gonadotrophin-releasing hormone agonist or cultured in different concentrations of oestrogen, progesterone or fetal calf serum for one, four or seven days. Main outcome measures Cell number, expression of proliferating cell nuclear antigen, and DNA damage after one, four or seven days of treatment. Results Gonadotrophin-releasing hormone receptor messenger ribonucleic acid was detected on cultured leiomyoma cells. Leiomyoma cell growth was not affected by the addition of gonadotrophin-releasing hormone agonist or progesterone, but increased with oestrogen or fetal calf serum supplementation. Overexpression of proliferating cell nuclear antigen was prevented in cultures added with oestrogen or fetal calf serum, but not related to gonadotrophin-releasing hormone agonist treatment. Significant decreases in DNA damage as indicated by decreased comet number were found in the leiomyoma cultures treated with oestrogen or fetal calf serum for four and seven days but not with gonadotrophin-releasing hormone agonist or progesterone. Furthermore, 5% fetal calf serum supplementation was more growth supporting and more significantly reduced the comet number than 250 pM 17 β-oestradiol. Conclusion Cell growth, proliferating cell nuclear antigen expression and DNA damage are dependent on oestrogen or fetal calf serum, but independent of gonadotrophin-releasing hormone agonist or progesterone. Our findings suggest that gonadotrophin-releasing hormone agonist-induced leiomyoma shrinkage may be due in part to a mechanism involving DNA damage, and support the hypothesis that gonadotrophin-releasing hormone agonist exerts its action indirectly through oestrogen action on the tumour level.

Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonists.

To examine whether gonadotrophin-releasing hormone agonist or oestradiol can directly affect DNA in leiomyoma cells. In vitro explant culture of leiomyoma cells. University research group. Leiomyoma cells were cultured from the specimens of four premenopausal women at myomectomy. The presence of gonadotrophin-releasing hormone receptor in leiomyoma cells was determined by reverse transcriptase-olymerase chain reaction. Leiomyoma cells were treated with gonadotrophin-releasing hormone agonist or cultured in different concentrations of oestrogen, progesterone or fetal calf serum for one, four or seven days. Cell number, expression of proliferating cell nuclear antigen, and DNA damage after one, four or seven days of treatment. Gonadotrophin-releasing hormone receptor messenger ribonucleic acid was detected on cultured leiomyoma cells. Leiomyoma cell growth was not affected by the addition of gonadotrophin-releasing hormone agonist or progesterone, but increased with oestrogen or fetal calf serum supplementation. Overexpression of proliferating cell nuclear antigen was prevented in cultures added with oestrogen or fetal calf serum, but not related to gonadotrophin-releasing hormone agonist treatment. Significant decreases in DNA damage as indicated by decreased comet number were found in the leiomyoma cultures treated with oestrogen or fetal calf serum for four and seven days but not with gonadotrophin-releasing hormone agonist or progesterone. Furthermore, 5% fetal calf serum supplementation was more growth supporting and more significantly reduced the comet number than 250 pM 17 beta-oestradiol. Cell growth, proliferating cell nuclear antigen expression and DNA damage are dependent on oestrogen or fetal calf serum, but independent of gonadotrophin-releasing hormone agonist or progesterone. Our findings suggest that gonadotrophin-releasing hormone agonist-induced leiomyoma shrinkage may be due in part to a mechanism involving DNA damage, and support the hypothesis that gonadotrophin-releasing hormone agonist exerts its action indirectly through oestrogen action on the tumour level.

Modification of cell perifusion for extended study of hormone release in the rat pituitary.

We use a flow-through cell perifusion system allowing continuous sampling of Luteinizing Hormone (LH) release from excised pituitary tissue. With this system, we are able to sample from viable tissue in primary culture for three days as opposed to a more typical of primary culture of 6-12 hours. Rat pituitary tissue was perifused and challenged with a physiological dose of LHRH. Samples were collected for 3.5 hours on each of 3 consecutive days in a refrigerated fraction collector and assayed for LH concentration by radioimmunoassay. In these experiments we tested two types of media, each with or without fetal calf serum supplementation, for their ability to support pituitary tissue for extended lengths of perifusion. In addition, we broadened the use of aseptic technique, sterile media and equipment to limit microbial contamination. Our results indicate that perifusion of pituitary tissue can be done successfully for 3 or more days at a modest cost, greatly increasing the amount of information that can be collected from each piece of tissue harvested.