Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

Effects of medium alterations on in vitro development of Brugia malayi larvae in cultured mosquito thoraces.

A recent study showed that 1-day-old, intracellularly lodged larvae of Brugia species develop in vitro to the infective third-stage larvae (L3) in excised thoraces of susceptible mosquitoes in the diphasic insect tissue culture medium containing a nutrient agar base overlaid with a 1:1 mixture of Schneider's Drosophila medium and Grace's insect cell culture medium supplemented with 20% fetal bovine serum (FBS) and antimicrobial agents. In the present investigation, the diphasic culture medium was used to evaluate the effects of medium alterations on the development of 1-day-old, intracellularly lodged larvae of subperiodic Brugia malayi in excised thoraces of Aedes aegypti to the L3. One-day-old larvae developed to the L3 in medium without nutrient agar base, at pH 7.0 and pH 7.5, in Hanks' balanced salt solution (HBSS) and in HBSS supplemented with bovine albumin fraction-V (BAF-V). These larvae also developed in the absence of FBS in the overlay medium, in overlay medium containing 5-20% FBS, in medium components obtained from different sources, in serum free Sf-900 (GIBCO) medium, and when FBS is replaced by BAF-V in the overlay medium. The percentage of L3 was not increased substantially in infected excised thoraces of mosquitoes when nutrient supplements, such as folic acid, p-aminobenzoic acid, glucose, lipid concentrate, hemin, or reduced glutathione, were added to the overlay medium containing BAF-V. These results suggested that 1-day-old, intracellularly lodged larvae developed to the L3 in infected excised thoraces of mosquitoes at almost the same rate as in intact mosquito, when excised thoraces were maintained alive under optimal conditions in a culture medium.     

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H₂O₂ exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H₂O₂ to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 M) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.     

培养成年大鼠心肌细胞存活的形态标志

目的：通过探寻增加培养成年大鼠心肌细胞存活率以及防止再分化的方法,揭示培养成年大鼠心肌细胞存活的形态标志。方法：采用Langendorff系统灌流心脏,胶原酶消化法分离成年大鼠心肌细胞,分3组进行细胞培养：①基础培养液＋凋亡抑制剂;②基础培养液＋5%胎牛血清;③基础培养液＋5%胎牛血清＋凋亡抑制剂。结果：①培养前3天杆状心肌细胞比例逐渐降低,无血清培养组比血清培养组降低程度大。培养前3天凋亡率逐渐升高,无血清培养组比血清培养组凋亡率高,加入凋亡抑制剂对凋亡率无影响。②有血清培养2~3天的成年大鼠心肌细胞闰盘部位伸出伪足,促使细胞贴壁生长;当培养至第6天时,细胞侧面也伸展出贴壁的伪足,细胞丧失杆状形态,横纹消失。而无血清培养的细胞无伪足生成,随着培养时间增加,细胞末端变圆钝,横纹变模糊。凋亡抑制剂对伪足形成率无影响。③培养存活的成年大鼠心肌细胞骨架重排,发生再分化。④血清培养组细胞胞内核间距离随着培养时间的增加而减小,无血清培养组则保持不变。结论：成年大鼠心肌细胞培养至第2~3天时,闰盘部位形成伪足是细胞存活的形态标志,加入血清是伪足形成的必要条件。     

Characterization and fed-batch culture of hybridoma overexpressing apoptosis suppressing gene bcl-2

Mouse hybridoma 2E3 transfected with human bcl-2 gene survived longer with increasing expression level of bcl-2 when cultured in DME medium supplemented with 9% serum. One of the transfectants, 2E3BCMGbcl-2, overexpressed bcl-2 and could maintain viable cell density higher than the initial density for more than four days at a low 0.5% serum concentration. In comparison a mock transfectant 2E3BCMG remained viable for only one day. However, both hybridomas died out within a day in serum-free medium. These results suggested that bcl-2 needed a small amount of some serum components to suppress apoptosis of the hybridoma. Overexpression of bcl-2 also suppressed apoptosis of the hybridoma induced by glutamine deprivation. When hybridoma 2E3BCMGbcl-2 was inoculated in DME medium supplemented with 9% serum and cultured for 10 d with additional 2% serum feed at day 4 of the culture, viable cell density increased 2-fold and antibody produced 3-fold, in comparison with mock transfected 2E3 cultured in the same manner. The mock transfectant with additional feed of serum at day 4 of the culture showed no difference in viable cell density and antibody production. These results suggested that the mock transfectant committed to apoptosis before day 4 of the culture and the additional serum at day 4 could not reverse the commitment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation

A continuous cell line, the flounder embryonic cell line (FEC), was established from gastrula-stage embryos of a marine cultured fish, the Japanese flounder Paralichthys olivaceus and cultured for more than 200 d with more than 60 passages. FEC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), and basic fibroblast growth factor (bFGF). The cells were small and round, and grew actively and stably in culture. The effect of temperature, FBS concentration and bFGF on FEC cell growth was examined. Cells grew well between 24 and 30 degrees C, but had a reduced growth rate below 18 degrees C. The growth rate of FEC cells in medium containing 15% FBS was higher than that in medium containing 7.5% FBS. Addition of bFGF to the medium also significantly increased the growth rate. Chromosome analysis revealed that FEC cells have a normal diploid karyotype with 2n = 48. High survival rate was obtained after cryopreservation of cell cultures. The susceptibility of the cell line to piscine viruses was examined. Two viruses tested were shown to induce CPE (cytopathic effect) on FEC cells. FEC cell culture infected with fish iridovirus was further elucidated by electron microscopy. Many virus particles were found in the cytoplasm of the virus-infected FEC cells. These results indicated that the FEC cell line could be potentially used to isolate and study fish viruses.     

实用淋巴细胞培养技术

采自人体的外周静脉血液,用淋巴细胞分离液进行分析,收集分离得到淋巴细胞,同时回收血液的血清成分,此血清不经灭活可直接用于培养。本实验对影响淋巴体外激少在、增殖的相关因子进行了详细的统计分析,实验结果表明,在基础培养液ＲＰＭＩ１６４０中添加１００μｇ／ｍｌ的ＰＨＡ、１００ｉｕ／ｍｌ的ＩＬ－２和１０％的自体或胎血清,可以有产地激活淋巴细胞并在营养条件允许的情况下长期处于增鱼的状态。     

Effects of arachidonic acid and other unsaturated fatty acids on mitogenesis in human lymphocytes.

The effect of fatty acids and other lipids on mitogenic responses in cultured human peripheral blood lymphocytes was studied. Several-fold enhancement of tritiated thymidine incorporation was observed at 0.1 to 5.0 micrograms/ml concentrations of arachidonic acid. Other unsaturated fatty acids produced less marked changes. Increased responsiveness was demonstrable in a variety of media including RPMI 1640 supplemented with 10% fetal calf serum. Changes were also observed in uridine incorporation, total cell number, and blast transformation, indicating that the effect was not on thymidine transport or pool size per se. Arachidonic acid failed to affect PHA binding, indicating that the lectin-cell interaction was not altered. Higher concentrations of fatty acids were inhibitory.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs

Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum‐free media (K‐M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum‐containing media (FBS‐M) with cells cultured in four different serum‐free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS‐M. Additional proliferation assays were performed using pre‐coated fibronectin (FN) tissue culture plates and of the four serum‐free medium, only K‐M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS‐M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K‐M or FBS‐M, and, additionally, cells retained their multipotency in K‐M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K‐M or FBS‐M. Taken together, the data suggest that K‐M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency. J. Cell. Physiol. 226: 66–73, 2010. © 2010 Wiley‐Liss, Inc.     

A novel composition for the culture of human adipose stem cells which includes complement C3

Adipose tissue is an easily accessible and abundant source of stem cells. Adipose stem cells (ASCs) are currently being researched as treatment options for repair and regeneration of damaged tissues. The standard culture conditions used for expansion of ASCs contain fetal bovine serum (FBS) which is undefined, could transmit known and unknown adventitious agents, and may cause adverse immune reactions. We have described a novel culture condition which excludes the use of FBS and characterised the resulting culture. Human ASCs were cultured in the novel culture medium, which included complement protein C3. These cultures, called C-ASCs, were compared with ASCs cultured in medium supplemented with FBS. Analysis of ASCs for surface marker profile, proliferation characteristics and differentiation potential indicated that the C-ASCs were similar to ASCs cultured in medium containing FBS. Using a specific inhibitor, we show that C3 is required for the survival of C-ASCs. This novel composition lends itself to being developed into a defined condition for the routine culture of ASCs for basic and clinical applications.     

Alteration of the antigenicity of human lymphocytes by plant lectins and short-term tissue culture.

Human lymphocytes studied after being placed in culture for 1–6 wk progressively lost stimulating ability, i.e., lymphocyte defined antigens, when tested in one way mixed lymphocyte culture (MLC) but retained several other identifiable membrane components as well as the capacity to respond to mitogenic stimuli. Lymphocytes placed in culture with motogenic doses of PHA and Con-A after 1 and 2 wk strongly stimulated autologous responding fresh lymphocytes, but the MLC response of allogeneic fresh lymphocytes to stimulating lectin treated cells was even lower than the response to stimulating allogeneic cultured lymphocytes. The HL-A antigens on lectin treated cells or on lymphocytes through 6 wk in culture were clearly identifiable. Assays for T cell rosettes and B cell surface immunoglobulin showed both cell types to be present in numbers equal to fresh lymphocytes for up to 5 wk after culturing. However, the Fc receptor site on B cells was lost from cultured lymphocytes at the same time that MLC stimulation was lost. It is concluded that plant lectins can unmask new mitogenic sites on the cell surface as well as mask or delete existing sites, and that culturing lymphocytes for 1–6 wk will produce somewhat similar modulations. Modulation of surface membrane components by tissue culture or lectins may, therefore, have a profound effect in altering transplantation immunogenicity.     

无血清培养昆虫细胞(BTI-Tn-5B1-4)的适应过程

昆虫细胞培养是近年来迅速发展起来的动物细胞培养工程中的一个新领域。人们可以利用杆状病毒在昆虫细胞内的感染、复制,来大量生产昆虫病毒作为生物杀虫剂[1]。而昆虫细胞杆状病毒表达载体系统的建立,则可通过昆虫细胞的体外培养大量表达病毒携带的外源基因。实践证明,这…     

Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated rat lymphocytes

2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes culturedin vitro. In this study, we have examined the effects of 2-ME on cell growth and on SCE induction in cultures of unstimulated and phytohemagglutinin (PHA)-stimulated Fischer 344 rat lymphocytes. There were virtually no metaphases detected in cells cultured without PHA. In PHA-stimulated cultures, 2-ME decreased SCE-frequency but it enhanced SCE frequency in the presence of S to 12.5 µM bromodeoxyuridine (BRd U). Both mitotic and replication indices were increased in the PHA/2-ME system. The levels of incorporated exogenous thymidine, in the presence of 2-ME, were relatively low in unstimulated cells, suggesting that 2-ME is not mitogenic for T-cells. However, 2-ME enhanced PHA-induced response of T-cells as evidenced by increased levels of thymidine incorporation into cellular DNA. The growth promoting effects and the decrease in SCE frequency caused by 2-ME upon PHA stimulation indicate that 2-ME may alter the nature of interaction between PHA and cellular activating properties or the replicative processes.Abbreviations BRdU bromodeoxyuridine - FBS fetal bovine serum - SCE sister-chromatid exchanges - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - PBS phosphate buffered saline - PHA phytohemagglutinin - MI mitotic index - RI replication index - NADH nicotinamide adenine dinucleotide (reduced form)     

Exogeneous substances affecting development of in vitro-derived bovine embryos before and after embryonic genome activation

Three experiments were conducted to evaluate how exogenous substances [fetal bovine serum (FBS), arachidonic acid (AA), glutathione (GSH), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF), fibroblast growth factor (FGF), insulin, transferrin and selenium (ITS)] affect preimplantation bovine embryo development. Cumulus-oocyte complexes (COC) were matured and fertilized in vitro, and their development was monitored up to 192 h post insemination in a two-step culture system. In Experiment 1, inseminated oocytes were cultured in modified bovine embryo culture medium (mBECM) supplemented with FBS or BSA for up to 60 h post insemination, and the resultant 8-cell embryos were then cultured singly in mBECM supplemented with AA+GSH+PDGF+TGF. More (P<0.005) blastocysts were derived from 8-cell embryos produced in media containing FBS than BSA. In Experiment 2, the 8-cell embryos produced in mBECM supplemented with FBS were cultured singly in mBECM as follows: 1) no supplementation; 2) AA and GSH or 3) AA, GSH, PDGF and TGF. Compared with no supplementation, a significant (P<0.05) increase in the proportion of 16-cell embryos and morulae was obtained after the addition of either AA+GSH or AA+GSH+PDGF+TGF. In Experiment 3, oocytes were cultured singly in mBECM as follows: 1) no supplementation; 2) AA+GSH+PDGF+TGF; 3) AA+GSH+PDGF+TGF and FGF; 4) AA+GSH+PDGF+TGF and ITS; 5) AA+GSH+PDGF+TGF, FGF and ITS or 6) FBS. Eight-cell embryos grown in each system were subsequently cultured singly in mBECM with AA+GSH+PDGF+TGF. More (P<0.05) 16-cell embryos were obtained in medium supplemented with either AA+GSH+PDGF+TGF and ITS or FBS than in unsupplemented medium. Fewer (P<0.05) oocytes developed to the 8-cell stage with the addition of AA+GSH+PDGF+TGF and FGF than without. In conclusion, embryo development to the blastocyst stage is regulated by exogenous AA, GSH, PDGF, FGF and ITS in a stage-specific manner.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Effects of defined medium,fetal bovine serum,and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays

Summary We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS. In all cases examined, defined medium and medium containing HuS were superior to medium containing FBS in supporting growth of both normal and tumor cell cultures. However, cultures in defined medium showed an initial cell loss. Cells from the same tumor cultured in different media varied in their responses to chemotherapeutic agents. In light of these results, medium supplemented with HuS, which promoted attachment of these cells in culture and stimulated their growth, should be the most appropriate nutrient environment for determining the effects of therapeutic agents on cells as it most closely resembles the in vivo situation. Because there were also variations in growth rates and chemosensitivities of tumor cells cultured in different human serum samples, we suggest that optimal conditions in which to culture these cells include the serum of the patient whose tumor is removed. This serum may provide host factors that influence cell growth and interact with exogenous factors. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia. J. T. Emerman is a research scholar of the National Cancer Institute of Canada.     

Callus Induction and Plant Regeneration of Leaf Explants in Orychophragmus violaccus(L.) O. E.Schulz

Explants of Orychophragmus violaceus (L.) O. E. Schulz were acquired from young leaves which lower epidermis was stripped, Differentiation of calli in high frequency is in the case of that calli grown on B5 culture medium supplemented with 1 mg/l 2, 4-D should be transferred onto MS culture medium supplemented with 0.2 mg/l NAA. Effect of basic culture medium on the differentiation was discussed. In addition, protoplasts derived from calli of Orychophragmus violaceus were cultured, and small calli consisted of more than hundred cells had been obtained.