Effects Of Fetal Bovine Serum Research Articles

Isolation and Characterization of the Primary Marmoset (Callithrix jacchus) Retinal Pigment Epithelial Cells.

Marmosets have emerged as a valuable primate model in ophthalmic research due to their similarity to the human visual system and their potential for generating transgenic models to advance the development of therapies. In this study, we isolated and cultured primary retinal pigment epithelium (RPE) cells from marmosets to investigate the mechanisms underlying RPE dysfunction in aging and age-related macular degeneration (AMD). We confirmed that our culture conditions and materials supported the formation of RPE monolayers with functional tight junctions that closely resembled the in vivo RPE. Since serum has been shown to induce epithelial-mesenchymal transition (EMT) in RPE cells, we compared the effects of fetal bovine serum (FBS) with serum-free supplements B27 on transepithelial electrical resistance (TER), cell proliferation, and morphological characteristics. Additionally, we assessed the age-related morphological changes of in vivo and primary RPE cells. Our results indicate that primary marmoset RPE cells exhibit in vivo-like characteristics, while cells obtained from an older donor show evidence of aging, including a failure to form a polarized monolayer, low TER, and delayed cell cycle. In conclusion, our primary marmoset RPE cells provide a reliable in vitro model for developing novel therapeutics for visual-threatening disorders such as AMD, which can be used before animal experiments using marmosets.

Effects of fetal bovine serum on trophectoderm and primitive endoderm cell allocation of in vitro-produced bovine embryos.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

Xeno-free protocol for GMP-compliant manufacturing of human fetal pancreas-derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) have been suggested as an appropriate source for diabetes cell-based therapies. The high proliferation and differentiation capacity of fetal MSCs and the role of fetal pancreatic-derived MSCs (FPMSCs) in islet generation make them good candidates for diabetes treatment. To manufacture clinical-grade MSCs, animal-free culture protocols are preferred. The current study aimed to establish a xeno-free/GMP-compliant protocol for FPMSCs manufacturing. The focus was on the effects of fetal bovine serum (FBS) replacement with pooled human serum (HS).Material and methodsFPMSCs were isolated and expanded from the pancreas of legally aborted fetuses with few modifications in our previously established protocol. The cells were expanded in two different culture media, including DMEM supplemented with 10% FBS or 10% pooled HS. A side-by-side comparison was made to evaluate the effect of each serum on proliferation rate, cell cycle, senescence, multi-lineage differentiation capacity, immunophenotype, and tumorigenesis of FPMSCs.ResultsFlow cytometry analysis and three-lineage differentiation ability demonstrated that fibroblast-like cells obtained from primary culture had MSCs’ characteristics. The FPMSCs displayed similar morphology and CD markers expression in both sera. HS had a higher proliferative effect on FPMSCs than FBS. In FBS, the cells reached senescence earlier. In addition to normal karyotypes and anchorage-dependent growth, in vivo tumor formation was not seen.ConclusionOur results demonstrated that HS was a better serum alternative than FBS for in vitro expansion of FPMSCs. Compared with FBS, HS increased FPMSCs’ proliferation rate and decreased their senescence. In conclusion, HS can effectively replace FBS for clinical-grade FPMSCs manufacturing.Graphical abstract

Premature cellular senescence in isolated chondrocytes from polydactyl cartilage in FBS containing serum in comparison to human serum

Background. Transplantation of chondrocytes is one of the methods for treatment of articular cartilage defects. For this purpose, it is necessary to isolate these chondrocytes and expand in serum containing culture medium. Sera contain growth factors that are added to medium. Fetal bovine serum (FBS) is used for standard cell culture. However bovine proteins of FBS are accompanied with risk of host immune system reactions. Therefore, it is necessary to replace FBS with human serum for clinical cases. In addition, infants have chondrocytes with high proliferation potency versus adults. In this study, the effects of FBS and human placental serum was investigated on morphology and cellular senescence related genes expression of chondrocytes of infants. Methods. Chondrocytes after isolation of from polydactyly cartilage tissue samples, were cultured in 10% FBS and human serum up to passage 5. Human placental serum was obtained from venous blood of embryonic surface of human placenta. Chondrocytes in two culture medium were compared morphologically by inverted microscope. For cellular senescence evaluation, relative expression of IGF1 and P16 genes were assessed by quantitative real-time RT PCR. Results. Proliferation of chondrocytes is markedly decreased in FBS groups in comparison with human serum. In FBS containing medium, phenotypes of cells were changed from spindle shape to flat appearance after five passages. The expression IGF1 and P16 genes are increased in chondrocytes grown in FBS containing medium and it was significant about P16. Morphologic results were in agreement with Real-time RT PCR results. Conclusion. Human placental serum has advantages such as high proliferation rate on human chondrocytes, prevention from cellular senescence and immunological problems in using cultured chondrocytes for cartilage tissue engineering.

Developmental regulation of Dirofilaria immitis microfilariae and evaluation of ecdysone signaling pathway transcript level using droplet digital PCR

BackgroundCurrent measures for the prevention of dirofilariasis, caused by the dog heartworm, Dirofilaria immitis, rely on macrocyclic lactones, but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, the aim of this study was to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development regulation.MethodsDirofilaria immitis microfilariae extracted from dog blood were incubated under various culture conditions to identify those most suitable for in vitro culture and development of the microfilariae, and to determine the effects of fetal bovine serum (FBS), mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript levels of the ecdysone signaling pathway components were measured with droplet digital PCR (ddPCR).ResultsIn vitro conditions that best promote early development of D. immitis microfilariae to the “late sausage stage” have been identified, although shedding of the cuticle was not observed. FBS had inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to microfilarial growth. The transcript level study using ddPCR also showed that ecdysone signaling system components were upregulated in developing microfilariae and that 20-hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro.ConclusionsThe arthropod host environment provides cues required for the rapid development of D. immitis microfilariae, and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding of the developmental process of D. immitis microfilariae.Graphical

Establishment and optimization of hepatocyte steatosis model

Objective: To establish a new model of hepatic steatosis cells by optimizing the original ethanol or high fat, the present study proposed an in vitro hepatocyte steatosis model for the study of fatty liver. Methods: Oil red O staining was used to observe the effects of fetal bovine serum, oleic acid and ethanol on lipid accumulation in human liver cell line L02 in a concentration- and time-dependent manner. RT-PCR was used to detect the mRNA expression levels of PPAR-γ and AP-2, and the suitable conditions for the establishment of hepatocyte steatosis model were screened out. A t-test was used for comparison between the two groups, and one-way Analysis of Variance (ANOVA) was used in more than three groups. Results: Oil red O staining showed the number of reddish-orange lipid droplets in L02 cells gradually increased with the increase of fetal bovine serum, oleic acid and ethanol in a concentration - and time-dependent manner. Compared with 0.00% oleic acid and 2% ethanol, the count value of red particle was 100.00% ± 17.63% at the beginning and after 24 h, 0.003% oleic acid and 2% ethanol jointly acted in L02 cells. After incubation for 48 hours with 2% ethanol and serum-free DMEM medium, the accumulation of lipid droplets was the highest with a count value of 802.38%+71.06%(t = 42.36, P < 0.001). RT-PCR analysis showed the lipid accumulation induced by this method was positively correlated with the mRNA expression of PPAR-γ and AP-2. Conclusion: L02 cells were successfully exposed to high fat and ethanol, and the hepatocyte steatosis model was established and optimized, suggesting that the occurrence of hepatic cell steatosis was related to the up-regulation of PPAR-γ and AP-2.

The Effects of Serum Proteins on Magnesium Alloy Degradation in Vitro

Magnesium (Mg) alloys are promising materials for biodegradable implants, but their clinical translation requires improved control over their degradation rates. Proteins may be a major contributing factor to Mg alloy degradation, but are not yet fully understood. This article reports the effects of fetal bovine serum (FBS), a physiologically relevant mixture of proteins, on Mg and Mg alloy degradation. FBS had little impact on mass loss of pure Mg during immersion degradation, regardless of whether or not a native oxide layer was present on the sample surface. FBS reduced the mass loss of Mg-Yttrium (MgY) alloy with an oxidized surface during immersion degradation, but increased the mass loss for the same alloy with a metallic surface (surface oxides were removed). FBS also influenced the mode of degradation by limiting the depth of pit formation during degradation processes on commercially pure Mg with metallic or oxidized surfaces and on MgY alloy with oxidized surfaces. The results demonstrated that serum proteins had significant interactions with Mg-based biodegradable metals, and these interactions may be modified by alloy composition and processing. Therefore, proteins should be taken into account when designing experiments to assess degradation of Mg-based implants.

FBS or BSA Inhibits EGCG Induced Cell Death through Covalent Binding and the Reduction of Intracellular ROS Production.

Previously we have shown that (−)-epigallocatechin gallate (EGCG) can induce nonapoptotic cell death in human hepatoma HepG2 cells only under serum-free condition. However, the underlying mechanism for serum in determining the cell fate remains to be answered. The effects of fetal bovine serum (FBS) and its major component bovine serum albumin (BSA) on EGCG-induced cell death were investigated in this study. It was found that BSA, just like FBS, can protect cells from EGCG-induced cell death in a dose-dependent manner. Detailed analysis revealed that both FBS and BSA inhibited generation of ROS to protect against toxicity of EGCG. Furthermore, EGCG was shown to bind to certain cellular proteins including caspase-3, PARP, and α-tubulin, but not LC3 nor β-actin, which formed EGCG-protein complexes that were inseparable by SDS-gel. On the other hand, addition of FBS or BSA to culture medium can block the binding of EGCG to these proteins. In silico docking analysis results suggested that BSA had a stronger affinity to EGCG than the other proteins. Taken together, these data indicated that the protective effect of FBS and BSA against EGCG-induced cell death could be due to (1) the decreased generation of ROS and (2) the competitive binding of BSA to EGCG.

Stimulatory Effects of Serum for Ex-Vivo Culture of Mesenchymal Stromal Cells Can be a Parameter for Heterogeneity in Hematopoietic Supporting Activity

Although hematopoietic supporting activity of mesenchymal stromal cells (MSCs) have been widely used in clinical trials to facilitate the hematopoietic recovery, variations have been observedin supporting effects. In the present study, we show that the niche activity of MSCs is regulated by stimulatory effects of fetal bovine serum during ex-vivo culture of MSCs.First, we observed extensive variations in the numbers of colonies (CFU-F)(up to 40-folds) differences in numbers when human bone marrow (BM)-derived MSCs were cultured under different batches of fetal bovine serum (FBS),and two batches with high (SS) and low (NSS) stimulating effects were selected. 5 independent BM-MSCs cultured with stimulating serum exhibitedsignificantly lower doubling times, higher numbers of CFU-F and higher % of CD146+ cells. When umbilical cord blood-derived CD34+ cells were co-cultured on each MSC group, significantly higher expansion of CD34+CD90+ cells and higher numbers of colonies were observed than those cultured with non-stimulatory serum. Similarly, numbers of primitive hematopoietic cells analyzed by long-term cultureinitiating cells (LTC-IC) were significantly higher in stimulating serum conditions than in non-stimulating condition. Furthermore, MSCs under stimulatory serum exhibited higher level expression of niche cross-talk molecules (CXCL-12 and Jagged-1) than MSCs under non-stimulatory condition, indicating higher support of HSC self-renewal. Whenexamined for murine model, the stimulatory serum caused similar difference in colonogenic activity (2.5-folds higher CFU-F) and phenotypic marker for mesenchymal progenitors (PDGFR-alpha+ cells). Moreover, when each group MSCs were co-transplanted with HSCs into congenic recipient, significantly higher reconstitution of donor-derived cells were observed without changes in the lympho-myeloid lineage distribution in the MSCs with stimulatory serum group than in MSCs with non-stimulatory group (51.2% vs 8.2%, respectively, p<0.005). In contrast, priming of HSCs with 2hr pre-incubation with each group MSCs before transplantation or co-transplantation of MSCs cultured under hypoxic conditions did not exhibit additional increase in the reconstitution level by donor-derived cells.Of note, the enhancing effects of stimulatory serum on MSCs or on the co-cultured HSCs were reversed when the MSC culture conditions were switched to non-stimulatory serum, indicating that the enhancing effects were caused by extrinsic stimulating effects rather than clonal difference of MSC subpopulation. To explore the differential signals between MSCs grown under stimulatory and non-stimulatory condition, microarray-based analysis of gene expression were analyzed. The gene ontology (GO) analysis showed that extensive signals related to cell growth and survival was enriched in the MSCs grown in stimulatory conditions compared to those in non-stimulatory conditions. Furthermore, upstream signal analysis showed that signals for P53, TRIB3, TGF-beta1, RABL6 or ATF4 were significantly up-regulated in the MSCs under stimulatory serum condition. Taken together, our study shows that MSCs can exhibit significant heterogeneity in the hematopoietic niche activity depending on the stimulatory effects of the serum during ex-vivo culture to cause difference in hematological recovery, and suggest that optimization of CFU-F-based screening of MSCs can produce more predictable outcomes in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

In Vitro Effects of FBS and Ovarian Steroids on Osteoblast Viability

We evaluated the effects of different concentrations (0, 10‐8 M and 10‐7M) of progesterone (P4), testosterone (T) and estradiol (E2) on the osteoblast viability in vitro. Osteoblast lineages (OSTEO‐1) were cultured in 96‐well plates in DMEM medium a density of 5 x 103 cells/100 μL medium, with or without 10% fetal bovine serum (FBS). Cells were maintained at 37 ºC, with 5% CO2, for up to 48 hours. Cell viability was analyzed by MTT tetrazolium assay. The cells cultured with 10% FBS were more viable than those cultured without FBS. When all hormones were put together, the cell viability was reduced at the 10‐7M concentrations compared with the control and 10‐8 M. The viability of cells cultured without FBS was a) stimulated by E2 (10‐7 M and 10‐8 M (0.1048 ± 0.0227 and 0.1069 ± 0.0124, respectively)) compared with the control group (0.0566 ± 0.008); b) stimulated by P4 (10‐7 M (0.1088 ± 0.0124)) compared with the control group (0.0566 ± 0.008) and 10‐8 M (0.0924 ± 0.022); and c) stimulated by both concentrations of T (10‐7 M (0.0908 ± 0.0153) and 10‐8 M (0.0924 ± 0.0022)) compared with the control group (0.0566 ± 0.008). Our results demonstrated that FBS stimulated osteoblast viability, and P4 (10‐7 M), and both concentrations of T and E2 stimulated the viability of the cells cultured without FBS, suggesting that these steroid hormones may be important at the onset of osteoblast cells viability in vitro for up to 48 hours.

Effects of fetal bovine serum and estrus buffalo serum on maturation of buffalo (Bubalus bubalis) oocytes in vitro.

Aim:The aim was to assess the effects of fetal bovine serum (FBS) and estrus buffalo serum (EBS) on in vitro maturation rate of oocytes in buffalo.Materials and Methods:Maturation rate of oocytes was assessed in two maturation media supplemented with 20% FBS and EBS. Oocytes maturation rate was evaluated on the basis of cumulus cell expansion and extrusion of polar body after 24 h of in vitro culture in CO2 incubator.Results:The average percentage of in vitro matured oocytes in FBS was 83.80%, and EBS was 77.45%, respectively. The results revealed a significant (p<0.05) increase in maturation rate of oocytes in FBS than EBS.Conclusion:Buffalo oocytes were better in vitro matured in FBS than EBS.

Differentiation of adipose-derived stem cells into Schwann-like cells: fetal bovine serum or human serum?

Access to autologous Schwann cells is limited due to lack of donor site and its difficult isolation and culture. Therefore, one of the possible ways to obtain to Schwann cells is to differentiate mesenchymal stem cells into glial pathway using various materials and protocols. The aim of this study was to compare the effects of fetal bovine serum and human serum on Schwann cell differentiation of adipose-derived stem cells to choose the best serum for use in future research. For this purpose, after isolation of human adipose-derived stem cells, it was characterized and differentiated into Schwann cell lineage using two protocols which one of them contained fetal bovine serum and the other human serum. At the end, morphological evaluation declared an increased detachment of cells in response to human serum. On the other side, immunocytochemistry showed that there was a significant increase in the number of cells expressing glial fibrillary acidic proteins and S100 in fetal bovine serum-treated group when compared to human serum-treated one (P<0.05). It was concluded that fetal bovine serum was more effective than allogeneic human serum in Schwann cell differentiation of adipose-derived stem cells.

63 THE EFFECT OF A MODIFIED CRYOPRESERVATION METHOD ON THE VIABILITY OF FROZEN–THAWED PRIMORDIAL GERM CELL ON THE KOREAN NATIVE CHICKEN (Ogye)

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus, conservation of genetic material in chickens was attempted by preserving primordial germ cells (PGC) in LN2. This study established a method for preserving chicken PGC that enables long-term storage in LN2 for preservation of species. The purpose of this study was to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGC in the Korean native chicken (Ogye). Primordial germ cells were separated from a germinal gonad using a fine glass micropipette under a microscope and were suspended in a freezing medium containing freezing and protecting agents [e.g. dimethyl sulfoxide (DMSO) and ethylene glycol (EG)]. The PGC were then purified using the magnetic-activated cell sorting (MACS) method. The viability of the PGC in both groups was determined by the trypan blue exclusion method. The values of the 0, 5, 10, and 15% DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36%, respectively. The viability of PGC after freeze-thawing was significantly higher for the 10% EG plus FBS treatment than for the 10% EG plus CS treatment (P &lt; 0.05; 64.36 v. 50.66%). This study established a method for preserving chicken PGC that enables systematic storage and labelling of cryopreserved PGC in LN2 at a germplasm repository and ease of entry into a database. In the future, the importance of this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germ line chimeras.

한국재래닭 (오계) 원시생식세포에 있어 동결방법의 개선이 융해 후 생존율에 미치는 영향

This study was conducted to establish methods for preserving chicken primordial germ cells (PGCs) for long-term storage in liquid nitrogen and for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGCs from Korean Native Chicken (Ogye). PGCs separated from a germinal gonad of an early embryo at day 5.5-6 (stage 28) were suspended in a freezing medium containing freezing and protective agents (dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The values from 0, 5, 10, and 15 % DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36 %, respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% EG + FCS treatment (p

돼지 단위발생 배아의 발달과정에서 세포사멸에 관한 연구

이배체 단위발생 돼지 난자를 체외 배양시 배반포 형성 단계에서 FBS (우태아 혈청), BSA (우혈청 알부민), EGF(상피세포 성장인자)를 배양액에 첨가하였을 때 이배체 단위발생 에서 총세포수, 세포사멸 및 세포사멸에 관여하는 유전자의 발현 효과를 조사하고자 본 연구를 수행하였다. 0.4% BSA를 배양액에 첨가 하였을 때 2 세포기 단계 단위발생의 발달은 배반포 까지는 강화 되었다 (p<0.01). FBS 처리 시는 배반포의 세포 수는 감소시켰으나 세포 사멸률은 증가하였다(p<0.01). 하지만 EGF가 존재할 때 BSA 처리는 총 세포수를 증가 시켰다. RT-PCR의 결과에 의하면 EGF는 0.4% BSA가 존재하는 배양액에서는 Bcl-xL mRNA 발현을 증가시키고 BSA와 EGF 가 단독으로 존재 할 때는 효과가 없었다. 하지만 FBS 처리시 Bcl-xL 유전자 발현은 감소하고 Bak 유전자의 발현은 증가시킨다. 이러한 결과 세포사멸에 관여하는 유전자의 발현은 배양액의 첨가물에 따라 유의적으로 영향을 받으며, 돼지 배아의 체외 배양시 세포사멸과 초기발달에 관여함을 시사한다. This study was conducted to determine the effects of fetal bovine serum (FBS), bovine serum albumin (BSA) and epidermal growth factor (EGF) on blastocoel formation, total cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell stage parthenotes to the blastocysts stage (p<0.01). Treatment of FBS reduced cell numbers of blastocysts (p<0.01) and increased the percentage of apoptosis in blastocysts (p<0.001). However, BSA increased cell numbers, only in the presence of EGF reverse-transcriptase polymerase chain reaction revealed that EGF enhanced the mRNA expression of Bcl-xL in the presence of 0.4% BSA but BSA and EGF alone had no effect. However, Treatment of FBS reduced Bcl-xL mRNA expression (p<0.05) and enhanced Bak expression. This result suggests that apoptosis related gene expression is significantly affected by supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

Developmental competence of ovine oocyte following vitrification: effect of oocyte developmental stage, cumulus cells, cytoskeleton stabiliser, FBS concentration, and equilibration time

The aim of the present study was to examine the effects of fetal bovine serum (FBS) concentration, equilibration time, and oocyte pre-treatment with cytochalasin B (CCB) on subsequent development of vitrified-warmed ovine immature (GVCOCs) and matured (MII) oocytes with (MIICOCs) or without cumulus cells (MIIDOs). In Experiment 1, the effects of FBS concentrations (10 and 20%) during the vitrification-warming procedure were examined. Survival rates after warming were not different between GVCOCs, MIICOCs and MIIDOs oocytes. After in vitro fertilization, rate of cleaved embryos in MIICOCs group at the presence of 20%FBS was higher than MIIDOs and GVCOCs groups. In Experiment 2, the effects of equilibration times (5, 7, and 10 min) were examined. There was no difference in survival rate of vitrified-warmed oocytes equilibrated at different times. Although, the rate of cleavage in MIICOCs and MIIDOs oocytes equilibrated for 10 and 7 min, respectively, was higher than 5 min equilibrated MIIDOs and 7 and 10 min equilibrated GVCOCs oocytes. In Experiment 3, the effects of oocyte pre-treatment with CCB were examined. Despite the insignificant difference in survival rate of vitrified-warmed ovine immature and matured oocytes, the rates of cleavage in CCB pretreated groups were significantly lower than untreated groups. Moreover, the blastocysts were only derived from those cumulus enclosed vitrified-warmed germinal vesicle (GV) and MII oocytes that had been exposed to 10% FBS in the absence of CCB. In conclusion, the presence of cumulus cells, 10% FBS, and the omission of CCB were beneficial for post-warming development of vitrified ovine oocytes.

Progesterone Receptor Inhibits Proliferation of Human Breast Cancer Cells via Induction of MAPK Phosphatase 1 (MKP-1/DUSP1)

The roles of progesterone (P(4)) and of progesterone receptor (PR) in development and pathogenesis of breast cancer remain unclear. In this study, we observed that treatment of T47D breast cancer cells with progestin antagonized effects of fetal bovine serum (FBS) to stimulate cell proliferation, whereas siRNA-mediated knockdown of endogenous PR abrogated progestin-mediated anti-proliferative effects. To begin to define mechanisms for the anti-proliferative action of P(4)/PR, we considered the role of MAPK phosphatase 1 (MKP-1/DUSP1), which catalyzes dephosphorylation and inactivation of MAPKs. Progestin treatment of T47D cells rapidly induced MKP-1 expression in a PR-dependent manner. Importantly, P(4) induction of MKP-1 was associated with reduced levels of phosphorylated ERK1/2, whereas siRNA knockdown of MKP-1 blocked progestin-mediated ERK1/2 dephosphorylation and repression of FBS-induced cell proliferation. The importance of PR in MKP-1 expression was supported by findings that MKP-1 and PR mRNA levels were significantly correlated in 30 human breast cancer cell lines. By contrast, no correlation was observed with the glucocorticoid receptor, a known regulator of MKP-1 in other cell types. ChIP and luciferase reporter assay findings suggest that PR acts in a ligand-dependent manner through binding to two progesterone response elements downstream of the MKP-1 transcription start site to up-regulate MKP-1 promoter activity. PR also interacts with two Sp1 sites just downstream of the transcription start site to increase MKP-1 expression. Collectively, these findings suggest that MKP-1 is a critical mediator of anti-proliferative and anti-inflammatory actions of PR in the breast.

Mechanisms underlying impairment of endothelium-dependent relaxation by fetal bovine serum in organ-cultured rat mesenteric artery

Organ culture of blood vessels provides a useful technique to investigate long-term effects of drugs because tissue architecture and function are well preserved. Various growth factors are responsible for structural and functional changes during vascular diseases. We investigated long-term effects of fetal bovine serum (FBS) which contains such factors on endothelium-dependent relaxation using organ-culture method. Rat isolated mesenteric arteries with endothelium were cultured for 3 days without or with 10% FBS (FBS). Acetylcholine- and bradykinin-induced endothelium-dependent relaxations were significantly impaired in FBS, whereas sodium nitroprusside-induced relaxation of endothelium-removed artery was unchanged. Morphological examination revealed that endothelium was intact in FBS. Acetylcholine-induced nitric oxide (NO) release as detected by 4, 5-diaminofluorescein significantly decreased in FBS, whereas endothelial NO synthase expression was unchanged. A Ca 2+ ionophore, A23187-induced relaxation was unchanged in FBS. A phospholipase C activator, m-3M3FBS-induced relaxation of FBS was unchanged in either Ca 2+-containing or -free solution. Total expressions of transient receptor potential canonical channels (TRPCs: TRPC-1, -4, -5) were similar in FBS. These data suggest that FBS impairs endothelium-dependent relaxation by inhibiting events upstream of phospholipase C activation including phospholipase C, G-protein, and receptors in endothelium.

High-throughput 3-D cell-based proliferation and cytotoxicity assays for drug screening and bioprocess development

We have designed, built and tested a three-dimensional (3-D) cell culture system on modified microplates for high-throughput, real-time, proliferation and cytotoxicity assays. In this 3-D culture system, cells expressing the enhanced green fluorescent protein (EGFP) were cultured in nonwoven polyethylene terephthalate (PET) fibrous scaffolds. Compared to 2-D cultures in conventional microplates, 3-D cultures gave more than 10-fold higher fluorescence signals with significantly increased signal-to-noise ratio (SNR), thus extending the application of conventional fluorescence microplate readers for online monitoring of culture fluorescence. The 3-D system was successfully used to demonstrate the effects of fetal bovine serum, fibronectin coating of PET fibers, and cytotoxicity of dexamethasone on recombinant murine embryonic stem D3 cells. The dosage effects of 5-fluorouracil and gemcitabine on high-density colon cancer HT-29 cells were also tested. These studies demonstrated that the 3-D culture microplate system with EGFP expressing cells can be used as a high-throughput system in drug discovery and bioprocess development.

Effects of protein inter-layers on cell–diamond FET characteristics

Diamond is recognized as an attractive material for merging solid-state and biological systems. The advantage of diamond field-effect transistors (FET) is that they are chemically resistant, bio-compatible, and can operate without gate oxides. Solution-gated FETs based on H-terminated nanocrystalline diamond films exhibiting surface conductivity are employed here for studying effects of fetal bovine serum (FBS) proteins and osteoblastic SAOS-2 cells on diamond electronic properties. FBS proteins adsorbed on the diamond FETs permanently decrease diamond conductivity as reflected by the −45 mV shift of the FET transfer characteristics. Cell cultivation for 2 days results in a further shift by another −78 mV. We attribute it to a change of diamond material properties rather than purely to the field-effect. Increase in gate leakage currents (by a factor of 4) indicates that the FBS proteins also decrease the diamond–electrolyte electronic barrier induced by C–H surface dipoles. We propose a model where the proteins replace ions in the very vicinity of the H-terminated diamond surface.