Alligator mandibular development during long term organ culture

Summary The development of the first branchial (mandibular) arch of the American alligator (Alligator mississippiensis) is studied in organ culture for the first time. There is extensive mandibular morphogenesis in vitro and the pattern of the differentiated elements, bones, muscles, and cartilage, is comparable to that found during normal development in ovo. In addition, we observe the development of paired lingual swellings and the formation of a small tongue consisting of fibrous, lipid containing, and muscular tissues, as found in the normal tongue. Several culture variables are examined: (a) although alligator embryos normally develop at 30°C, we successfully culture the mandibular rudiments with good short term (3 wk) results at 37°CC; (b) at 30° C, we are able to culture arches for long term periods of 6 wk; (c) the mandibular arches develop well in both serum containing medium and in a chemically defined medium free from added hormones. The reptilian mandibular arches exhibit excellent, patterned, development and may have less stringent culture requirements than their avian and mammalian counterparts. Collection and specimen export was performed under U.S. Fish and Wildlife Permit PRT2-2511 and Northern Ireland Permit B/WL2/80 issued to M.W.J.F. Collaborative study was made possible by the 1981 award of a Research Travelling Scholarship to M.W.J.F. from The Wellcome Trust, for which we are grateful. This work is supported by Grant 8113610 CB from the MRC of Great Britain, Grant EP109/74/75 from the Northern Ireland Eastern Health & Social Services Board, and Grants DE-02848 and DE-03569 from the National Institutes of Health, Bethesda, MD.     

Ploidy variation of pronamide-treated maize calli during long term culture

Summary Anther-derived calli of corn were treated with 10 M pronamide for 2, 3 and 4 days. The ploidy level of the calli was then evaluated using flow cytometry, at different times after the treatment. Untreated haploid calli did not change in ploidy level for 97 days but by 466 days, there were up to 50% diploid or higher ploidy cells thus showing that spontaneous doubling may occur during corn calli subculture with this genotype. Pronamide treatment did increase the percentage of diploid and tetraploid cells and by 466 days, all of the lines showed an additional change toward higher ploidy levels. This change may be due to spontaneous chromosome doubling or to differential cell cycle times of cells with different ploidy levels. The ploidy level of plants regenerated from the cultures was determined by counting the guard cell chloroplast numbers and the correlation with the ploidy level of the cultures was r²=0.84. These studies show that pronamide treatments can increase haploid maize callus chromosome numbers and that spontaneous chromosome doubling can occur with time in maize callus.     

Autoclavable low cost serum-free cell culture media. The growth of L Cells and BHK cells on peptones

The growth of L-929 cells on a series of peptones, and protein hydrolysates was examined, and it was found that when MEM was supplemented with any of a series of peptones, cell growth was about as good as when serum was used as a supplement. Protein hydrolysates did not support cell growth very well and at higher concentrations actually reduced cell growth. L-929 and L-60TM cells were grown both as monolayers or stationary suspensions and in agitated systems in MEM supplemented with 0.5—1% bactopeptone. The addition of macromolecular compounds, insulin or oleic acid had no effect on cell growth. BHK cells were also grown on media supplemented with bactopeptone but richer media (MEM-alpha, F-12, or RMPI1640) gave higher cell yields. The cells did not form the monolayers observed with fetal calf serum, but a partial suspension system. Addition of a detergent Darvan #2 gave a totally suspension culture in both stationary and agitated systems. The production of Sindbis virus in BHK cells grown in serum-free media was examined and the yield of virus was found to be about the same as that produced in serum-supplemented systems. It is estimated that the cost of cell production media could be reduced by about 90% by the replacement of serum supplement by peptones.     

BHK21 fibroblast aggregation inhibited by glycopeptides from the cell surface.

Glycopeptides were removed by trypsinization from the surface of baby hamster kidney cells (line BHK21-C13), digested by pronase and separated into 2 fractions by exclusion chromatography. The addition of small amounts of either glycopeptide fraction to shaken suspensions of lightly trypsinzied cells inhibited their rapid aggregation, but one fraction was more active than the other and in higher concentrations it was able to inhibit aggregation completely. After this fraction was purified by high-voltage electrophoresis one subfraction also inhibited aggregation. The effect of the glycopeptides increased following their pretreatment with neuraminidase, but preincubation with periodiate or galactose oxidase destroyed all activity. Galactose oxidase also inhibited cell aggregation directly. Similar glycopeptides from virus-transformed BHK21 cells, oligosaccharides and intact and desialysed human urinary glycoproteins had comparatively little or no effect on BHK21 cell aggregation. The results suggest terminal beta-galactosides and possible alpha-galactosides, and to some extent a particular substructure of cell surface heteroglycans are necessary for their inhibitory activity. The parent, plasma membrane of glycoproteins might serve as adhesive binding sites in cell cohesion, but some evidence indicates cell surface sialyl- and galactosyltransferases may not ordinarily act as their complementary binding receptors.     

The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium.

Although glutamine is a major carbon source for mammalian cells in culture, its chemical decomposition or cellular metabolism leads to an undesirable excess of ammonia. This limits the shelf-life of glutamine-supplemented media and may reduce the cell yield under certain conditions. We have attempted to develop a less ammoniagenic medium for the growth of BHK-21 cells by a mole-to-mole substitution of glutamine by glutamate. This results in a medium that is thermally stable but unable to support an equivalent growth yield. However, supplementation of the glutamate-based medium with asparagine (3 mM) and a minimal level of glutamine (0.5 mM) restored the original growth capacity of the cultures. Substitution of the low level of glutamine with the glutamine dipeptides, ala-gln (1 mM), or gly-gln (3 mM) resulted in an equivalent cell yield and in a thermally stable medium. The ammonia accumulation in cultures with glutamate-based medium was reduced significantly (>60%). Factors mediating growth and adaptation in medium substituted with glutamate were also investigated. The maximum growth capacity of the BHK-21 cells in glutamate-based medium (without glutamine) was achieved after a period of adaptation of 5 culture passages from growth in glutamine-based cultures. Adaptation was not influenced by increases in glutamate uptake which was constitutively high in BHK cells. Adaptation was associated with changes in the activities of enzymes involved in glutamate or glutamine metabolism. The activities of glutamine synthetase (GS) and alanine aminotransferase (ALT) increased significantly and the activity of phosphate-activated glutaminase (PAG) decreased significantly. The activity of glutamate dehydrogenase (GDH) showed no significant change after adaptation to glutamate. These changes resulted in an altered metabolic profile which included a reduced ammonia production but an increased alanine production. Alanine production is suspected of being an alternative route for removal of excess nitrogen.     

Two-dimensional versus three-dimensional culture systems: Effects on growth and productivity of BHK cells

The influence of surface growth (two-dimensional microcarriers) and three-dimensional growth (aggregates and macroporous supports) in agitated, suspended batch culture systems upon growth and productivity of BHK was compared. Cultures using three porous microcarriers (CultiSpher G, Cellsnow EX, and Cytocell), one nonporous microcarrier (Cytodex 3) and natural aggregates were performed in stirred tanks using two different agitation rates (60 and 100 RPM). With the exception of Cytocell, cell growth, viability, and productivity were similar when three-dimensional structures (porous microcarriers and aggregates) were used. Nonporous microcarriers only compared well at 60 RPM as growth ceased under overagitation. These results suggest that cultures less susceptible to fluid shear are advantageous for scale-up. (c) 1996 John Wiley & Sons, Inc.     

Radiosensitivity of human T-lymphocytes proliferating in long term culture

A method is described for determining the radiation sensitivity of dividing human T-lymphocytes in long-term culture. The results are fitted to a single-hit multi-target model. For cobalt-60 gamma radiation Do values of cells from five normal individuals range from 0.99 to 1.37 Gy with an overall Do of 1.09 Gy, and the extrapolation numbers range from 1.20 to 1.71 with an overall extrapolation number of 1.42.     

Rapid and stable propagation ofOrnithogalum umbellatum L. in long term culture

Callus cultures were raised from bulb scale segments ofOrinthogalum umbellatum L. (Liliaceae), on a Murashige and Skoog (1962) medium (MS) with 8 mg/l naphthaleneacetic acid (NAA). Bulbous shoots developed from calli after 2 months using MS medium with 2 mg/l NAA and 0.5 mg/l N⁶ - benzyladenine (BA). Shoots were also induced directly from scales of regenerated bulb used as secondary explants on MS medium supplemented with 0.5 mg/l BA. Shoots developed roots in 1/2 - strength MS medium. Regenerants multiplied rapidly in 1/2-MS liquid medium. Chromosome instability was reduced in callus grown on 2 mg/l NAA compared to callus grown on 8 mg/l NAA. Callus retained regeneration potential for 5 years in this modified MS medium. The chromosome analysis of regenerants dervied from callus, even from long term culture of 5 years, revealed only diploid cells with normal karyotype comprising 2n=46 chromosomes. Stable nature of callus and regenerants were further confirmed by cytophotometry. This procedure can be applied for securing stable regenerants on a mass scale inO. umbellatum.     

A high throughput system for long term application of intermittent cyclic hydrostatic pressure on cells in culture

The process of bone remodeling is governed by mechanical stresses and strains. Studies on the effects of mechanical stimulation on cell response are often difficult to compare as the nature of the stimuli and differences in parameters applied vary greatly. Experimental systems for the investigation of mechanical stimuli are mostly limited in throughput or flexibility and often the sum of several stimuli is applied. In this work, a flexible system that allows the investigation of cell response to isolated intermittent cyclic hydrostatic pressure (icHP) on a high throughput level is shown. Human bone derived cells were cultivated with or without mechanical stimulus in the presence or absence of chemical cues triggering osteogenesis for 7-10 days. Cell proliferation and osteogenic differentiation were evaluated by cell counting and immunohistochemical staining for bone alkaline phosphatase as well as collagen 1, respectively. In either medium, both cell proliferation and level of differentiation were increased when the cultures were mechanically stimulated. These initial results therefore qualify the present system for studies on the effects of isolated icHP on cell fate and encourage further investigations on the details behind the observed effects.     

Increased expression of keratin polypeptides in long term culture of adult rat hepatocytes.

Adult rat hepatocyte cultures showed the presence of albumin after 1 week of seeding. As the cultures aged, the cells commenced expressing alpha-foetoprotein and later keratin polypeptides 55 and 52 kD but ceased expressing albumin and alpha-foetoprotein. Enhanced expression of keratin polypeptides was confirmed by Western blot analysis in long term cultures. A possible mechanism for dedifferentiation of hepatocytes in culture is discussed.     

Glucose tolerance during long term treatment with a somatostatin analogue.

Seven patients with active acromegaly were treated with SMS 201-995, an analogue of somatostatin, for one year, the maximum dose being 100 micrograms three times a day. Three patients had impaired glucose tolerance before treatment, due to insulin resistance in two and insulin deficiency in one. In all patients treatment with the analogue slightly increased postprandial glucose concentrations and suppressed insulin concentrations for two to two and a half hours after each injection; growth hormone concentrations decreased progressively with treatment. The patient with impaired glucose tolerance due to insulin deficiency developed diabetes mellitus after four months'' treatment; concomitant treatment with glibenclamide resulted in a decreased glucose concentration and increased insulin concentration. This analogue of somatostatin had only minor side effects on glucose tolerance in patients with acromegaly and may be used in patients with impaired glucose tolerance provided that glucose concentrations are monitored closely.     

Active cell aggregation by immature rat Sertoli cells in primary culture: a role for cell surface glycoproteins.

Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10-day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH-stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0- to 18-hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose-specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of ³H-aminoacid incorporation as well as cell aggregation in 24-hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease ¹²⁵I-FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.     

Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from the known alteration in assembly of N-linked glycans affecting the carbohydrate chains on the proteoglycan or some other combination of factors is discussed.