Neural Induction by Treatment with Ca2+-free Saline Solution in the Gastrula Ectoderm of Cynops pyrrhogaster and Inhibition of Neural Induction with Fetal Calf Serum

The relation between the inducing activity and the cell-dissociation effect of Ca²⁺-free (or Ca²⁺, Mg²⁺-free) saline solution (CF or CMF) on the early gastrula ectoderm was examined. In the culture medium containing no fetal calf serum (FCS), most ectoderm cells treated with CF or CMF died within a few days and only a few differentiated into epidermal cells. However, when the culture medium contained 2% FCS, ectoderm cells treated with CF or CMF differentiated into neural crest derivatives (NCDs), such as mesenchyme cells, pigment cells, and nerve cells. The frequency of the induction depended only on the duration of CF- or CMF-treatment. FCS alone had no inducing activity on ectoderm cells. On the contrary a high concentration of FCS gave an inhibitory effect on the induction. These results indicate that CF is a neuralizing factor and that CF-treated cells require FCS, not for induction, but for survival and differentiation. With CF, the maximum induction of NCDs required a longer duration than that necessary for complete cell-dissociation. This result suggests that the induction depends on some effects of CF other than cell-dissociation.     

Excitability changes of presumptive ectoderm following mesodermal induction

Dissociated ectodermal cells of the early newt gastrula which have been treated with CMF (Ca-Mg-free saline) for 5 hr differentiate into muscle cells when cultured in HFCS (heated fetal calf serum) for up to 9-12 days. Similarly dissociated cells placed into FCS (fetal calf serum) culture differentiate into epidermis. Differences in cell-cluster formation have been found between HFCS and FCS in early cell cultures (6 hr), and membrane excitability phenomena associated with the differentiation of these clusters into the muscle cells or epidermal cells have been investigated, respectively. The HFCS cultures consist of cell clusters which have few of microvilli at their surfaces and which form loose contacts by means of lamellipodia. FCS cultures consist of cell clusters which have numerous microvilli at their surfaces and which make tight contacts between cells by means of ridge-structure precursors. The different reaggregation pattern of dissociated ectoderm cells in HFCS reflects changes in the cell membrane surface induced by HFCS. The sequential genesis of action potentials in cells destined to form muscle cells in HFCS is very similar to those produced by somitic muscle cells in vivo and their ionic dependence for generating action potentials is related to epidermal action potentials in vitro (FCS).     

Studies on the development of melanophores in in vitro cultured amphibian neural plates

Various parts of neural plates of Japanese newt (Cynops pyrrhogaster) neurula embryos were cultured alone in drops of culture media (Niu-Twitty's balanced salt solution or modified Leibovitz L-15 medium) with or without fetal calf serum (FCS). Although none of the parts gave rise to melanophores in a medium without FCS, some produced melanophores in a medium with FCS. The localization of melanophore-producing areas in the neural plates corresponded to that of Tada's (1944) findings. The assumption that FCS affects survival and development of melanophores is excluded, because neural fold cells do not require FCS to develop into melanophores. Therefore, there may be in FCS some factor which acts on the specialization of neural plate cells into melanophores. The results of this experiment suggest that this factor may be heat labile. The findings also indicate that FCS does not induce melanophores in gastrula ectoderm, but only affects neurula neural plate cells so as to give rise to melanophores.     

Limb bud chondrogenesis in cell culture, with particular reference to serum concentration in the culture medium

When limb bud mesodermal cells of stages 23–24 chick embryos were plated at low cell density (2 × 10⁵ cells/cm²) and cultured in medium containing 10% fetal calf serum (FCS) (serum-rich medium), all cells became fibroblastic and no chondrocyte differentiation occurred in the culture. However, when cells of the same origin were cultured in a medium containing only 0.1% FCS (serum-poor medium), almost all the cells formed aggregates which developed further to form cartilage nodules. The loss of chondrogenic activity in serum-rich medium culture was irreversible: cultivation of the limb bud cells in serum-rich medium for 12 h abolished chondrogenic activity completely and these cells could not resume activity on re-cultivation in serum-poor medium. Calf, horse and chick serum at a concentration of 10% also induced the loss of chondrogenic activity in low cell density culture. Failure of chondrogenesis in serum-rich medium culture seemed to be due to the commitment of bipotential limb bud mesodermal cells to fibroblastic cells rather than to selective detachment of pre-committed chondroblasts.     

Effects of Inducers on Inner and Outer Gastrula Ectoderm Layers of Xenopus laevis

Abstract. Gastrula ectoderm, isolated from Xenopus laevis , was cultured in Holtfreter solution or modified Leibovitz medium (L-15) by the sandwich-method with or without inducer. The ectoderm (SD cell layers) consists of two cell sheets, representing a superficial (S) and a deep (D) layer. In the L-15 medium rather than in Holtfreter solution, the two cell layers separate out into distinct cell masses. This difference in cell affinity under certain experimental conditions could indicate that the deep layer contains endodermal cells. However, an endodermal character of the deep layer can be ruled out by induction experiments with vegetalizing factor or dorsal blastopore lip as inducers. Under the influence of vegetalizing factor the outer as well as the inner ectoderm layer differentiated into mesodermal derivatives such as notochord and somites. The results of the experiments with dorsal blastopore lip as inducer indicate that both inner and outer ectoderm layers are responsive to the neural stimulus. The lower neural competence of the outer ectoderm layer observed by several authors in normogenesis is discussed with regard to the hypothesis about short distance diffusion of the neuralizing factor and/or close cell-to-cell contact between inducing tissue and ectodermal target cells.     

Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum

The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium.     

Neural-inducing activity and glycoprotein synthesis of newly mesodermalized ectoderm in the newtCynops (Amphibia)

Summary An artificially mesodermalized ectoderm (mE) shows the same properties as the organizer: chordamesoderm formation and neural induction. The neural-inducing activity of the mE was inhibited by treatment with protein synthesis inhibitors (cycloheximide and puromycin) and a specific inhibitor of protein glycosylation (tunicamycin). These antibiotics also inhibited chordamesoderm differentiaton, especiallly that of notochord. Newly synthesized proteins of the mE were compared with those of presumptive ectoderm (pE) using two-dimensional PAGE. There were differences in relative amounts of many protein spots. These results suggest that neural-inducing activity is related to glycoproteins synthesized during the early phase of mesodermalization.     

INDUCTIVE CAPACITIES OF GLOMERULAR BASEMENT MEMBRANES AND DENTINE MATRIX

The inductive capacities of the basement membranes of calf kidney glomeruli and the dentine matrix of the incisors of 23-day rabbit fetuses were examined on the presumptive ectoderm of Triturus gastrulae. The basement membranes caused almost entirely neural induction and the dentine matrix caused mesodermal induction. These findings suggest that intercellular substances play an important role in the inductive effects of heterologous tissues.     

The ability of fetal calf serum, new-born calf serum and normal steer serum to promote the in vitro development of bovine morulae

The objective of this study was to compare fetal calf serum, new-born calf serum and normal steer serum as medium supplements in the development of bovine morulae in vitro . Bovine morulae were cultured in Hams F-10 tissue culture medium (HF-10) supplemented with 5% or 10% (v/v) fetal calf serum (FCS), new-born calf serum (NBCS) or normal steer serum (NSS). Embryos were recovered at slaughter from mixed bred donor cows of mixed breeding following estrus synchronization with prostaglandin and superovulation with follicle stimulating hormone. A total of 88 morulae were recovered, washed in HF-10 + 1% Bovine Serum Albumin and randomly assigned to treatments. Embryos were cultured in microdrops of medium under paraffin oil at 37 degrees C in a 5% CO(2) humidified atmosphere. Observations for stage of development were made every 24 hours. In vitro development was analyzed by assigning to each embryo a value of 0-5 based on the most advanced stage reached (0= no development, 5= development to a hatched blastocyst). Analysis of variance of these data revealed a significant treatment effect (P<.001) while no level effect or treatment x level interaction was apparent. Comparison of treatment means by Duncans new mulitple range test showed that NSS was superior to NBCS (P<.05) which was in turn superior to FCS (P<.05) as supplements of HF-10 in promoting the in vitro development of bovine morulae.     

Modulation of neural commitment by changes in target cell contacts in Pleurodeles waltl

In amphibian development, neural structures arise from the presumptive ectoderm at the gastrula stage by an inductive interaction with the chordamesoderm. It has been previously reported that early gastrula presumptive ectoderm can be neuralized when it is dissociated into single cells. A similar result is reported here with regard to Pleurodeles waltl presumptive ectoderm. Using this experimental model system we demonstrate: first, that neuronal and glial lineages can be specified from the presumptive ectoderm without any intervention of the natural inducing tissue; and second, that whereas rupture of cell-cell contacts evoked neural induction, dissociation immediately followed by reaggregation reduces the neuralizing response, pointing toward an active role played by cell-cell contacts of presumptive ectodermal cells in the modulation of neural commitment.     

Induction of unscheduled DNA synthesis in cultured human oral keratinocytes by sodium fluoride

The effect of treatment of cultured human oral keratinocytes with sodium fluoride (NaF) has been investigated with respect to induction of unscheduled DNA synthesis (UDS). Oral keratinocytes were isolated from excised buccal mucosa of normal individuals by trypsinization at 4 degrees C overnight, followed by separation of the epithelium of mucosa from lamina propria mucosae with forceps. Isolated cells were cultured in vitro and all experiments were performed with secondary cultures. For detection of UDS, the keratinocytes were cultivated with medium containing 1% fetal calf serum (FCS) for 2 days and then treated with 100-300 micrograms/ml NaF for 4 h in medium containing 1% FCS and 10 mM hydroxyurea (1% FCS-HU medium). Following treatment with NaF, UDS was measured by direct scintillation counting of [3H]thymidine incorporated into DNA of the cells in 1% FCS-HU medium. Significant levels of UDS were induced in a dose-related fashion by NaF treatment. The results suggest that NaF causes DNA damage in cultured human oral keratinocytes.     

Characterization of a fetal calf serum-derived molecule reactive with human natural antibodies: its occurrence in tissue culture-grown type C RNA viruses.

By means of a sensitive radioimmunoprecipitation (RIP) assay, simian sarcoma virus-simian sarcoma-associated virus (SSV-SSAV), purified from culture fluids of infected normal rat kidney (NRK) cells, was shown to acquire a surface antigen from serum used in the tissue culture medium. This antigen, which was acquired when serum from either fetal calf, horse, swine, rabbit, or chicken origin was used, accounted for a substantial portion (but not all) of the total precipitating activity exhibited by natural human antibodies for membrane-associated antigens of these viruses. By 1) alcohol precipitation, concanavalin A chromatography, and Sephadex G-150 filtration of fetal calf serum (FCS) proteins or 2) chromatography of serum proteins over a human IgG-containing immunoaffinity column, a glycoprotein of approximately 55,000 daltons has been identified which is a minor constituent of FCS (less than 0.1% of total protein) and has the antigenic capacity of whole FCS.     

Role of the thymus in generation of lymphocyte functions: II. Serum-mediated inhibition of development of mitogen-reactive lymphocytes in embryonic mouse thymus organ cultures

The thymuses of 14-day-old mouse embryos could be grown in serum-free organ cultures for at least 14 days with development of relatively large numbers of lymphocytes. These also acquired a strong reactivity to the mitogens concanavalin A (Con A) and leucoagglutinin (LA). Supplementing the organ culture medium with serum from calf (CS), fetal calf (FCS), mouse (MS), or fetal mouse (FMS) gave a serum concentration-dependent inhibition of development of mitogen reactivity, without clearly altering the quantitative lymphoid development in the organ cultures. Adult sera were more suppressive than fetal sera. All of nine tested FCS lots were inhibitory and the inhibiting activity was mainly found in the albumin fraction upon Sephadex G-200 gel filtration. Although FCS prevented development of mitogen-reactive cells in organ cultures of thymuses of 14-day-old embryos, it had much less effect on cultures of 15-day-old thymuses. FCS present during the entire organ culture period most efficiently inhibited generation of mitogen reactivity. If present only during the first or second half of the 14-day culture period, the inhibition was still marked but less complete.     

Effects of chick brain extract on the proliferation of chick neuroblasts cultured in media supplemented with low and high serum concentrations

The proliferative activity of chick neuroblasts cultured in a medium containing a low (5%) or a high (20%) concentration of fetal calf serum (FCS) was analyzed and the influence of a chick brain extract was investigated. Morphological observations and tritiated thymidine incorporation measurements have shown that neuroblasts from 6 day-old chick embryo cerebral hemispheres proliferate more actively in the medium with 5% FCS compared to the medium with 20% FCS. The medium containing 5% FCS favoured the maintenance of neuronal cells in a neuroblast stage as shown by electron microscopy. The stimulatory effect of brain extract on the proliferation of neuroblasts is stronger in the low serum culture condition. These findings indicate that a low serum-containing medium is an adequate condition to study neuronal proliferation and effects of growth factors on these cells.     

Stimulatory and inhibitory effects of serum and conditioned medium on cell spreading

This work deals with the effects of coating culture dishes with chick embryo fibroblast conditioned medium (CM) and 1% fetal calf serum (FCS) on the rates of attachment and spreading of chick embryo fibroblasts. It appeared that a FCS coating over a CM precoating exerted a remarkable promoting cooperative effect on cell spreading whereas a CM coating over a FCS precoating had a very marked inhibitory effect. Precoating with cobalt-protoporphyrin IX (CoPP) was also performed to serve as a substrate for FCS or CM coating. This CoPP precoating was found to exert a promoting effect for FCS coating only.     

Effects of heat treatment and concentration of fish serum on cell growth in adhesion culture of Chinese hamster ovary cells

The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated. The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached 60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 °C and the addition of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately 50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low concentrations to medium may be useful for the growth of CHO cells without FCS.     

Cellular adhesion to collagen

BALB/3T3 cells were released from tissue culture plates with EGTA, and their rates of attachment to collagen gels polymerized on Millipore filters; were measured. Cell attachment in serum-free medium was 20–50% of that which occurred in medium containing 10% fetal calf serum (FCS). Cell attachment to gels pretreated with FCS and assayed in serum-free medium was identical with that of gels in FCS-containing medium. Thus, it seems there are two separate mechanisms of attachment to collagen; one involving direct attachment and a second mediated by a serum component(s) which binds to collagen.     

Utilization of whey fractions as a substitute for fetal calf serum in culture media

Summary A dairy by-product was used in cell line cultures as a replacement for fetal calf serum (FCS). Various fractions were prepared from whey. All fractions have the ability to stimulate DNA synthesis in human and mouse 3T3 fibroblast and hybridoma cultures. They also allow hybridoma proliferation in the long term using only 0.5% FCS. In these latter experiments, heated and ultrafiltered fractions were more effective than 10% fetal calf serum.     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.