Leibovitz Medium Research Articles

An analysis of the aggregation and morphogenesis of area opaca endoderm cells from the primitivestreak chick embryo

ABSTRACT The aggregative behaviour and subsequent morphogenesis of extra-embryonic endoderm cells from primitive-streak chick embryos have been investigated. A relatively pure population of area opaca endoderm cells was obtained by differential dissociation, which involves partial separation of epiblast and endoderm cell clumps by sieving through Nitex mesh. For aggregation studies cells were cultured in rotating flasks in Leibovitz (L-15) medium, in saline or in saline supplemented with glucose (1 mg/ml). Aggregation was monitored using the Coulter Counter. In these three media aggregation is rapid; by 10 min an average of 61% of the population had aggregated, to reach a plateau at 30 min when an average percent adhesion value of 83 % was obtained. The aggregates in L-15 medium were large and compact. After several days in culture, they cavitated and formed smooth hollow vesicles with thin walls composed of one or a few cell layers. Aggregates formed in PCS were smaller and looser in appearance; the addition of glucose resulted in a certain degree of compaction. Some morphogenesis occurred under these conditions with the aggregates developing numerous irregular cavities. These experiments suggest that some of the factors that affect cell adhesion in early embryonic cells can be studied in vitro. The results also indicate that the ability to cavitate is an intrinsic property of the endoderm cells of the area opaca since this occurs in the absence of epi blast or mesoderm.

Organ culture of adult amphibian heart

A long-term culture method of beating heart explants of adult newt has been developed recently in our laboratory. This method has enabled us to study the ultrastructure and contractile activities of beating adult heart explants at selected intervals. Adult newt heart ventricle was excised and washed with amphibian balanced salt solution and cut into several pieces of approximately 0. 5-1. 0 mm. Heart pieces were cultured for 2 months at 25°C in a medium containing 90% modified Leibovitz medium (L-15), 10% fetal calf serum and 1% penicillin-Streptomycin. In first week of culture, more than 37% of heart explants were attached to the substrate, and more than 33% established pulsation rates. Some explants had a rate of 3-12 beat/min., while others had a rate of 28-67 beat/min. A few cells migrated from the explants and were attached to the substrate around the explants. These migrated cells did not show pulsation.

Fish Cell Culture: Properties of a Cell Line from the Sheepshead, Archosargus probatocephalus

A cell line designated SHF-1 was established from fin tissue of the sheepshead, Archosargus probatocephalus. Cells were fibroblastlike and grew best at 26 °C in Leibovitz (L-15) medium containing serum supplements and a final sodium chloride concentration of 0.150 M. The 2N number of primary sheepshead cells was 48 but the SHF-1 line at passage levels above 11 was heteroploid. SHF-1 cells did not replicate any of seven viruses tested. Species confirmation of SHF-1 was accomplished by the cytotoxic antibody test and was attempted with isoelectric focusing of lactic dehydrogenase isoenzymes. Plating efficiency and freeze viability studies were also performed. Key words: fish cell culture, sheepshead, Archosargus probatocephalus, microbiology

Fish cell culture: Characteristics of a cell line from the silver perch,Bairdiella chrysura

A cell designated SP-1 was established from tissue of the silver perch, Bairdiella chrysura. Cells were fibroblast-like and grew best at 26 degrees C in Leibovitz medium (L-15) containing 15% fetal bovine serum and 0.150 M sodium chloride. Passage 1 to passage 9 SP-1 cells contained a chromosome number of 48; at passages 27 and 50 the modal numbers were 51 and 54, respectively. Confirmation of the origin of SP-1 cells was made by the cytotoxic antibody dye-exclusion test. This cell line supported the growth of lymphocystis virus from the silver perch but was not found to replicate various other fish and mammalian viruses.

BILE ACID CONJUGATION IN HUMAN FETAL LIVER IN ORGAN 207 CULTURE

An organ culture system for prolonged maintenance of human fetal liver in vitro has been developed. Using this system, bile acid metabolism was investigated. Multiple liver specimens were obtained from human abortuses and stillbirths ranging from 9-30 wks. gestation(gest.). Within 3hrs. of hysterotomy, 1-2mm pieces of liver were placed in scored petri dishes and incubated in modified Leibovitz medium under air on a rocker panel at 37°C. Morphological integrity was demonstrated by LM and EM for periods up to 10 days in vitro. Functional viability was established by adding 14C-cholic acid to the medium and assaying the conjugates formed after extraction and TLC. Cholic acid(C)was taken up by the tissues and conjugated to form taurocholate(TC)and glycocholate(GC) which were then secreted into the medium at a constant rate during 10 days in vitro and in constant increments during a selected 24hr. period. TC is the predominant conjugate formed by fetuses < 20wks. gest. and with increasing gest. age the proportion of GC synthesized increases. Taurine added to the medium in concentrations equimolar to glycine enhanced TC synthesis at all gest. ages (p<0.001), increasing total conjugate synthesis. The addition of 2×10−4mM hydrocortisone(HC)to the medium increased the proportion of GC synthesized during early gest.(p=.01). The results establish that in the human fetus taurine is preferentially conjugated with primary bile acid and suggest that HC has a maturation effect on human fetal hepatocytes in organ culture.

Effect of cyclophosphamide in vitro and on vaccinia virus replication in tissue culture

The effect of cyclophosphamide on the growth of Vero, BSC-1, and HeLa cells in monolayer cultures was studied. By using hemocytometer counts and tritiated thymidine uptake as indicators of growth, it was found that cyclophosphamide significantly interfered with the metabolism of Vero and BSC-1 cells when sustained in Leibovitz medium. Vero cells and HeLa cells grown in Eagle medium were not affected by exposure to cyclophosphamide. Vaccinia virus replication in Vero cell monolayer cultures incubated with cyclophosphamide was markedly augmented, and this enhanced growth was reflected by virus quantitation techniques and metabolic studies using tritiated thymidine uptake. No difference in the distribution of infectious particles was found when cyclophosphamide-treated and control infected cultures were compared. Pathways other than through hepatic enzymes appear available to activate cyclophosphamide in vitro. These effects are dependent on both the cell type and the medium in which the cells are grown. Cyclophosphamide can facilitate vaccinia virus replication in vitro through metabolic interactions at the cellular level. The precise mechanisms underlying this effect require further study.

Amino acid metabolism of myeloma cells in culture

The growth of myeloma cells in Leibovitz medium supplemented with 20% serum was limited by the depletion of glutamine. A simple modification of the Leibovitz medium by increasing the concentrations of glutamine, lysine, isoleucine, leucine, sodium pyruvate, galactose, and vitamins resulted in over 100% increase in cell growth yield. The total myeloma protein produced by the cells was increased by approximately 90% in modified Leibovitz media. Analysis of spent culture media for 19 amino acids showed that the concentrations of 8 amino acids were reduced; those of 5 amino acids were increased and the other 6 did not change significantly.

Plaque assay of rickettsiae in a mammalian cell line.

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.

Plaque Assay of Rickettsiae in a Mammalian Cell Line

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.

Intracellular Collagen Fibers in Cultured Human Synovium

In the search for the etiologic agent in rheumatoid arthritis we have engaged in a long-range study characterizing the differences of normal and rheumatoid synovial cells. In one experiment the normal fetal synovium of six newborn infants who died of various causes shortly after birth were cultured. Pieces of synovium from the knee were removed at autopsy under sterile conditions, and 2 mm fragments grown in Leibovitz medium supplemented with 5% calf serum, 0.1mM glutamine and 500 units of penicillin G. At 4 day intervals tissues were washed with buffer, fixed in 3% glutaraldehyde in cacodylic acid, and later post-fixed with 1% osmium tetroxide. Pieces of rheumatoid synovium removed from patients undergoing synovectomies were similarly treated, cultured and fixed.

Miniculture System を用いたポリオウイルスの血清中和試験に関する研究

1) A miniculture system has been devised using a continuous Vero cell line in the Leibovitz-15 (L-15) medium supplemented with bovine albumin fraction V. A highly humid atmosphere required for the incubation was obtained in a desicator by placing a small vial filled with water.2) The above described system was applied to the titration of human sera for the poliovirus neutralization. A good correlation was obtained between this and the conventional tube method. Further more poliovirus serum neutralization by our system had a tendency of showing a little higher titer in the lower serum dilution area, and a little lower titer in the higher dilution area in comparison to the conventional tube method.

Factors influencing fusion of rat palates grown in vitro.

AbstractA technique is described for growth in vitro of embryonic rat palatal tissue on defined and semi‐defined media. Palatal fusion closely resembling that occurring in vivo is found in these preparations. Fusion occurs in a large number of cases even in the absence of an animal extract. However, development is improved if fetal calf serum is added to the synthetic medium. Using Leibovitz Medium L‐15 with 20% fetal calf serum, complete fusion was obtained in 87% of the palates, partial fusion in 9% and no fusion was found in only 4% of the palates cultured.Incorporation of galactoflavin, a riboflavin antagonist in a riboflavin‐deficient diet of the mother or in the culture medium results in a decreased number of complete fusions and corresponding increase in partial fusions. A suggestion is made as to the manner in which galactoflavin acts in vivo to produce palatal cleft, based on the incidence and type of clefts and the histological appearance of the palatal tissues.A method is described in which the tissue sections are sliced prior to culture so that the behavior of individual palatal shelves can be studied during the fusion process. Fusion occurs between shelves if the medial margins are apposed, regardless of whether the two shelves come from the same embryo, from littermate embryos, or from non‐littermate embryos. Some possibilities for application of this technique are suggested.

Studies on Japanese B Encephalitis Virus Vaccines from Tissue Culture

Summary The growth curve of the OCT-attenuated 35-24°C line of JBE virus in HKC culture and factors resulting in increase of the virus yield were investigated for the purpose of vaccine production. Comparing 199, Eagle's basal, and Leibovitz media with the addition of 2% H.Al. there was no advantage of one over the other in respect to the titer of the virus yield. In medium 199 containing 2% H.Al. of pH 7.0, the titer of the complete harvest was always more than 1.0 log higher than that of the fluid harvest. When a medium of pH 8.0 was used, the titer of the fluid harvest was comparable to or slightly higher than that of the complete harvest at pH 7.0 and almost equal to that of complete harvest at pH 8.0. In medium 199 containing H.Al., a direct correlation between infectivity and hemagglutinin was noticed. Within the range tested, reduction of the amount of medium employed for the final harvest resulted in a proportional increase in yield of virus. It appears that titers of infectivity between 109 and 1010 per ml and of HA between 1:256 and 1:512 can be readily obtained without any concentration procedures and with no foreign protein present except that of the disintegrated tissue cells used as a substrate.