Sodium Citrate as a Substitute for Fetal Calf Serum in the Micronucleus Test

The micronucleus test developed recently by Schmid and coworkers (Boller and Schmid 1970, Ledebur and Schmid 1973, Schmid 1976) is a rapid, convenient, and sensitive procedure for the detection of induced chromosome aberrations in vivo. It is now widely used for evaluating the mutagenic potential of drugs and other chemicals. The test involves the demonstration of micronuclei which result from lagging of acentric chromosome fragments or even of whole chromosomes during mitosis due to spindle disruption in the anucleate young erythrocytes of bone marrow smears (for details see Schmid 1976). Success or failure of the technique largely depends on the quality of the smear. Cell clumping and cell damage render the smear valueless. Schmid (1976) recommends the use of fetal calf serum for preparing the best smears. However, as he also noted, fetal calf serum is very expensive. Moreover it is not readily available in certain countries, particularly developing ones. It is not easy to procure heat inactivated human AB serum either, which Schmid has suggested as a good substitute for fetal calf serum. Difficulty in obtaining these important elements of the procedure is overcome to a great extent by the brief use of 1% sodium citrate solution at 20-25 C as a substitute for fetal calf serum.     

Chromosome preparations in the field from mammals long after death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01% colchicine solution to 5 ml of medium-cell suspension. After 1/2-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KCl. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanol:acetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Giemsa, if required after pretreatment of the preparations for banding, e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Chromosome Preparations in the Field from Mammals Long After Death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01 % colchicine solution to 5 ml of medium-cell suspension. After Vi-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KC1. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanokacetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Gietnsa, if required after prclieatment of the preparations for banding; e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

A requirement for cholesterol for phorbol ester-induced adhesion of a human monocyte-like cell line

The human monocyte/macrophage-like cell line U937, which is a cholesterol auxotroph, is nonadherent. However, it becomes adherent after treatment with phorbol 12-myristate 13-acetate (phorbol ester). We investigated the effects of cellular cholesterol depletion and repletion on the effectiveness of phorbol ester to induce adhesion to substratum. Almost 70% of cellular cholesterol is depleted by incubation of the cells for 24 hrs in the growth medium in which delipidated fetal calf serum is substituted for fetal calf serum without affecting viability or the rate of growth. The use of delipidated fetal calf serum inhibited phorbol ester-induced adhesion by 40%. If the cells were preincubated in the medium containing delipidated fetal calf serum 6 hrs prior to addition of phorbol ester, adhesion was inhibited by 90%. Addition of cholesterol to the medium containing delipidated fetal calf serum, which replenishes cellular cholesterol, restored the ability of phorbol ester to induce adhesion to levels seen in cells cultured in the medium containing fetal calf serum. Epicholesterol was not as effective as cholesterol in supporting adhesion. Cholesterol depletion did not inhibit phorbol ester stimulation of superoxide anion production. These observations indicate a function for cholesterol in phorbol ester-induced adhesion that is independent of phorbol ester-induced superoxide anion production. It is proposed that cholesterol is required for synthesis and/or proper orientation and distribution, in the plasma membrane, of macromolecule(s) that mediate phorbol ester-induced adhesion.     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

Transplacental mutagenesis: The micronucleus test on fetal mouse blood

Summary The induction of cytogenetic damage (micronuclei) in mouse fetal blood was studied with four selected mutagens: cyclophosphamide, procarbazine, trenimon, and mitomycin-C. For comparison the standard micronucleus test on maternal bone marrow was also performed. In contrast to the results obtained from maternal bone marrow the changes in the cellular composition in fetal blood were only slight after treatment with mutagens. A significant and dosepdependent increase in the incidence of micronucleated fetal blood cells was found with all four mutagens. The inducibility of micronuclei by indirect mutagens was particularly interesting. The three mutagens other than mitomycin-C induced a higher frequency of micronucleated polychromatic erythrocytes in fetal blood cells than in maternal bone marrow. The results indicate that this modified micronucleus test is well suited and useful for mutagenicity screening of environmental chemicals and especially for assessment of risks to the fetus when pregnant females are exposed to environmental chemicals.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

Identification of media supplements that improve the viability of primarily cell cultures ofCrassostrea gigas oysters

Media supplements have been investigated for their influence on the viability of primary cell cultures from the heart ofCrassostrea gigas oysters. Soluble factors of vertebrate origin were tested, belonging to five families of supplements that had proven to increase the viability of insect and mammal cell cultures. Using two-level complete factorial assays, factors and mutual interactions were screened within each family with a MTT reduction assay. Results pointed out the positive influence of hormones, growth factor, antioxidants and lipids on the mitochondrial metabolism of oyter's heart cells. Consequently, a new concentrated complex supplement was developed. At 10% (v/v) final concentration in modified Leibovitz L-15 medium, it increases by 30% the cellular viability of one-week old cultures as compared with non-supplemented medium, a similar improvement as the one obtained with 10% (v/v) fetal calf serum. Combined with fetal calf serum, this new supplement doubles the cellular viability of one-week old cultures and allows networks of cardiomuscular cells to be maintained functional over three monthsin vitro.Abbreviations MTT-3 (4,5-dimethylthiazol;-2-yl)2,5-diphenyl-tetrazolium bromide - FCS fetal calf serum     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

5-Methylthioribose. Its effects and function in mammalian cells

The growth responses of 5-deoxy-5-methylthioribose on a 5'-deoxy-5'-methylthioadenosine phosphorylase containing cell line (BW5147) and the methylthioadenosine phosphorylase-deficient cell line (L1210D) were examined. Methylthioribose was shown to dramatically affect these cells, increasing their growth rate, saturation density, and viability. It was also found that methylthioribose could satisfy the methylthio dependence of the enzyme-deficient cell line, L1210D. A model is proposed to explain the selective growth of methylthioadenosine phosphorylase-deficient cells in medium lacking a methylthio donor but containing fetal calf serum. It is hypothesized that cellularly exported methylthioadenosine is degraded to methylthioribose in the presence of medium containing serum of high methylthioadenosine phosphorylase activity (i.e. fetal calf serum). The resultant methylthioribose can then be used to satisfy the methylthio requirement of these cells. To test this theory, various purified preparations of bovine liver methylthioadenosine phosphorylase were used to artificially increase the specific activity of methylthioadenosine phosphorylase in horse serum. In each case, it was demonstrated that only medium containing serum of enzyme activity nearly equal to that of the glutathione-stimulated fetal calf serum activity, supported the growth of methylthio-dependent cells in the absence of methylthio compounds. The data suggest that the degradation of methylthioadenosine and subsequent formation of methylthioribose represents an essential process in the growth of mammalian cells.     

A plant extract which enhances the plating efficiency of lymphoid cell lines and enhances the survival of normal lymphoid cells in vitro

A water soluble extract from the bark of the Samoan medicinal plantAlphitonia zizyphoides A. Gray (Rhamnaceae), enhances the plating efficiencyin vitro of lymphoid cell lines as well as the survival of bone marrow cells and normal T and B lymphocytes. Furthermore, the inclusion of bark-extract into culture media enhances the cloning efficiency of a T-hybridoma cell line by more than 30 times at otherwise unsuitably low serum concentrations, but does not completely substitute for serum. The enhanced growth of a B-cell hybridoma is also paralleled by an increased production of monoclonal antibodies in cultures containing low cell densities.Abbreviations AZH/w water extract ofAlphitonia zizyphoides - AZH/c sugar-free fraction of the same extract - CM complete medium - FCS fetal calf serum - NMR nuclear magnetic resonance - PBS phosphate buffered saline - SEM standard error of the mean - TLC thin layer chromatography     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Subpopulations of human T lymphocytes. III. Distribution and quantitation in peripheral blood, cord blood, tonsils, bone marrow, thymus, lymph nodes, and spleen.

Human T cell subpopulations (Tμ and Tγ) were examined for their distribution in the peripheral blood, cord blood, bone marrow, tonsils, thymus, lymph nodes and spleen. The proportions of Tμ and Tγ cells are comparable in the peripheral blood, tonsils and bone marrow. The proportions of Tγ cells in cord blood are significantly higher than those in the peripheral blood. Almost complete lack of Tγ cells was observed in lymph nodes. Spleen has very high proportions of Tγ cells. Thymuses have very low proportions of both Tμ and Tγ cells when compared with peripheral blood, cord blood, tonsils, and bone marrow. The receptors for IgM on Tμ cells appear to be masked by passively absorbed IgM and require prior in vitro incubation in medium containing fetal calf serum for the full expression of this marker.     

Physiological Saline Solutions as a Useful Tool in Micronucleus and Metaphase Slide Preparations

The micronucleus test (Schmid 1975) is widely used as an indicator of cytogenetic damage induced in vivo by clastogens and spindle poisons. Yamamoto and Kikuchi (1980) have recently showed that by comparing the relative size of micronuclei it is possible to determine whether an agent acts as a clastogen or a spindle poison. This finding will undoubtedly increase the use of the technique in routine protocols for the testing of chemicals. Besides, the test is quite sensitive and much simpler and faster than chromosome analysis. One obstacle, however, hinders several laboratories: the increasing unavailability of fetal calf serum. Recently, Das and Kar (1980) have proposed sodium citrate as a substitute for fetal calf serum. However, 1% sodium citrate solution is hypotonic, which fact may pose difficulties for an experimenter having to sacrifice several animals within a short time, a common situation in routine tests. We were able to overcome the problem of hypotonia by replacing fetal calf serum with the isotonic solutions 0.9% NaCl and Ringer's saline for mammals (9.00 g NaCl, 0.42 g KCl, 0.24 g CaCl₂, 0.20 g NaHCO₃, 1000 ml distilled. H₂O) at room temperature.     

Murine islet cryopreservation and corticosteroids: functional studies

Knowledge of protective effects of corticosteroids on traumatized cells prompted us to test the potential benefit of islet cryopreservation in the presence of hydrocortisone. Neonatal murine islets were isolated by collagenase, followed by 2- to 3-day tissue culture. Precryopreservation glucose-stimulated (50-500 mg/dl) insulin release was 25-388% above basal (mean = 113%) in 18/20 fresh islet preparations. Subsequent freezing was done in RPMI 1640 medium plus 10% (v/v) heat-inactivated fetal calf serum and 10% (v/v) Me2SO with or without 1 mg/ml hydrocortisone at 0.25 degrees C per minute in a programmed freezing system, to -80 degrees C, and stored for greater than 60 days at -196 degrees C. Thawing, by transfer to room air, was followed by dilution, 4x (v/v), in 4 degrees C RPMI plus 10% protein, after which glucose-stimulated insulin release was reassessed, showing 56-280% response over basal in 3/8 steroid-treated preparation and 20-220% response in 3/10 control preparations. Basal insulin release was 0.72 ng/microgram protein/hr in fresh islets (N = 20) and 0.22 ng/microgram protein/hr after freeze-thawing. We conclude that functional islet survival by this method is approximately 30% and that hydrocortisone did not improve viability.     

In vitro Proliferation of Human Bone Marrow Mesenchymal Stem Cells Employing Donor Serum and Basic Fibroblast Growth Factor

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10⁵ nucleated cells/cm²) with DMEM medium containing 10% serum (FCS or donor serum) and basic fibroblast growth factor, and incubated at 37 °C under a 5% CO₂ atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic fibroblast growth factor (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45^- CD105⁺ cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic fibroblast growth factor was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to 17 population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.     

Giant eosinophil colonies from cultures of bone marrow cells

Summary To date, the small size and slow growth of eosinophil colonies in vitro has hampered study of cloned eosinophils. We found enhanced eosinophil colony size and numbers in methylcellulose cultures of bone marrow cells utilizing defined supplemented bovine calf serum (DSBCS) in combination with EL4 conditioned medium (EL4-CM). At days 9, 16 and 23 significantly more eosinophil colonies and more cells/colony were present in cultures incubated with DSBCS/EL4-CM than in cultures incubated with fetal calf serum/EL4-CM. The ability to generate large numbers of eosinophils in vitro should facilitate study of cloned eosinophils. Supported in part by a grant from the National Institutes of Health, AI 20416, and by the Mayo Foundation. Editor's statement Previous approaches to in vitro culture of eosinophils generally have achieved, at best, mixed cultures of colonies of these cells and granulocyte-macrophage colonies. The improved culture methods described in this report produce more homogeneous eosinophil cultures and larger colonies of these cells. The procedure employs EL4 murine thymoma-conditioned medium, which apparently contains eosinophil colony-stimulating activity in the absence of granulocyte-macrophage colony-stimulating activity. David W. Barnes     

Cell growth stimulating effect of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> spores and their potential application for Chinese hamster ovary K1 cell cultivation

In this work, water-soluble extracts of Ganoderma lucidum spores (Gls), a Chinese medicinal herb that possesses cell growth stimulating function, were found to be an effective growth factor for Chinese hamster ovary (CHO) cell cultivation. The Gls extract was prepared and supplemented to CHO K1 cell culture media with various serum levels. Our results obtained from both the static culture and the spinner-flask suspension culture showed that use of small-amount Gls extract effectively promoted cell growth and suppressed cell apoptosis induced by serum deprivation with normal cell cycle maintained in a low-serum medium. The low-serum medium containing 1 % (v/v) fetal bovine serum (FBS) and 0.01 % (w/v) Gls extract showed a comparable performance on both cell growth and fusion protein productivity with the conventional CHO culture medium containing 10 % (v/v) FBS and a commercial serum-free medium. This is the first study of the potential of Gls extracts for use as an alternative cell growth factor and nutrient for CHO cells. The findings have presented a new approach to economic cultivation of CHO cells for therapeutic protein production.     

Fetal liver micronucleus assay in mice of 5-fluorouracil and related compounds

The cytogenetic effects of 5-fluorouracil (5-FU), 1-hexyl-carbamoyl-5-fluorouracil (HCFU) and 1-(2-tetrahydrofuryl)-5-fluorouracil (TF) were examined with the fetal liver micronucleus assay in mice. The frequencies of micronucleated polychromatic erythrocytes (MNPCEs) in fetal liver peaked at 27, 24 and 27 h, respectively, after single intraperitonealinjections into pregnant mice on day 13 of gestation. The highest frequency of MNPCEs by 5-FU treatment in fetal liver was 13.6%, whereas the frequency in maternal bone marrow was only 0.4%. The micronucleus frequency and the number of micronuclei per individual polychromatic erythrocyte were clearly dose-dependent.These results suggest that the micronucleus test in fetal liver has particular advantages compared to maternal bone marrow for evaluating the cytogenetic effects of 5-FU and related compounds after a single treatment. The cytogenetic effect was ranked 5-FU = HCFU > TF, in both a time-course study and a dose-response study of micronucleus distribution.     

IL 1 requirement for B cell activation revealed by use of adult serum

Fetal calf serum is an essential component of the culture medium developed by Mishell and Dutton for the immunization of murine spleen cells in vitro. Serum from adult donors (mouse, human, rabbit) does not support antibody synthesis in this system. This "deficiency" of adult serum can be overcome with IL 1. Adult serum in the presence of IL 1 is as effective in stimulating a B cell response against xenogeneic red cells as fetal calf serum. We attribute the capacity of fetal calf serum to support an immune response in the absence of exogenous IL 1 to serum factors that cause macrophages to release IL 1 endogenously. Our findings strengthen the notion that IL 1 plays an essential role in the process of B cell activation and suggests that the use of fetal calf serum should be avoided in studies concerned with the function of interleukin 1.