Continuous flow cultures of a HeLa cell line as a basis for a steady supply of Rubella virus

A HeLa cell line was propagated in semicontinuous suspension culture, 85 liters final volume, and in continuous flow culture with a volume of 300 ml. or 5 liters in an autoclavable medium to which 8% calf serum had been added. A medium containing 0.1% Methocel and 2% calf serum was also tested. Maximum productivity was obtained at a dilution rate of 0.33 day^?1 with a cell density of about 1.0 × 10⁶ cells/ml. The same cell line was also infected with Rubella virus and the production of virus was followed at the 5-liter cultivation level.     

Variability of DNA Content in Individual Cells of <Emphasis Type="Italic">Bacillus</Emphasis>

THE average content of DNA in Bacillus spores is unaffected by the growth medium and is constant for each species¹. Within the Bacillus group, however, the average amount of DNA per spore varies from species to species¹. A cell of B. cereus contains on average twice as much DNA as the spore and during sporulation this DNA is divided equally between the spore and the sporangium². Estimates^3–5 of genome size vary from 1.3 × 10⁹ to 10 × 10⁹ daltons and the number of genomes per cell from 2 to 4^4–8. Some of these variations may be associated with differences within the cells rather than differences of methodolqgy; spores within a population vary not only in size and shape, but also in their content of stainable chromatin⁹. Moreover, in ‘Renografm’ density gradients¹⁰, spores band within a range of densities. If spores are taken from a narrow part of this range, regrown and rebanded, the original pattern of dispersal occurs, suggesting that spores in the same population normally show variation in density as well as in size (A. I. Aronson, personal communication).     

Modulation of one of three murine bone marrow stromal cell lines to adipose cells by serum and insulin

Adipose cells have been recognized as an integral component of the bone marrow hematopoietic microenvironment in vivo and as an essential cell type required for in vitro maintenance of stem cells. Four stromal cell lines obtained from the adherent cell population of murine bone marrow cultures have been enriched and purified by multiple trypsinizations. We noted that these cell lines exhibited an accumulation of vacuoles of lipid, the extent of which varied be-tween cell lines in response to a change from medium containing 10% fetal calf serum to medium containing 20% horse serum. The lipid was lost when the cell lines were transferred back into the medium supplemented with fetal calf serum. In light of the reported lipogenic and antilipolytic effects of insulin on fibroblasts and adipocytes, we investigated the ability of insulin to induce adipocyte transformation of these bone marrow stromal cell populations. Three cell lines were exposed to bovine insulin at concentrations ranging from 10^?9 to 10^?6 M. All three cell lines responded to the insulin by accumulating lipid, but the extent of accumulation and the insulin concentration at which maximum lipid content was attained were population specific. One cell line (MC₁) responded fully at physiological levels of insulin (10^?9 M), whereas the other two showed lipid accumulation only at pharmacological concentrations. The initial growth of MC₁ was inhibited in the presence of 10^?9 M insulin which is compatible with the observed differentiation to adipocytes. The growth of MC₃ was unaltered in the presence of physiological concentrations of insulin, whereas that of MC₄ was accelerated. Grafts of organ cultures of the cell lines under the kidney capsule of syngeneic mice developed specific characteristics rep-resentative of the different cell lines. In particular, the majority of the grafts of MC₁ consisted primarily of fat cells which were not observed in the grafts of MC₃ and MC₄. These data strongly suggest that these cell lines comprise cells with different potentialities and that the MC₁ line represents a preadipocyte stromal cell of bone marrow.     

Insulin binding in cultured Chinese hamster kidney epithelial cells: The effect of serum in the medium

Summary [¹²⁵I]Insulin (porcine) binding to an epithelial cell line established from a Chinese hamster kidney, CHK-AC_E−100, showed an optimum at pH 8.0 and reached a maximum after 2.5 h incubation at 25°C. Dissociation of bound [¹²⁵I]insulin was facilitated by the addition of unlabeled insulin in the dilution buffer. Porcine insulin effectively competed for [¹²⁵I]insulin binding to the cultured cells and was 30 and 90 times as potent as guinea pig insulin and porcine proinsulin in causing 50% inhibition of [¹²⁵I]insulin binding; glucagon was completely ineffective. Scatchard analysis of the binding data yielded a curvilinear plot and a capacity of 0.6 ng/10⁶ cells; the average affinity of the empty receptor, , was calculated to be 1.78×10⁸ M ⁻¹ and that of the filled receptor, , 0.57×10⁸ M ⁻¹. Substitution of fetal bovine serum (FBS) in the culture medium with bovine calf, bovine newborn, or bobby calf serum altered insulin binding characteristics in the cells and reduced cell growth. Insulin binding characteristics of cells grown in hormone-supplemented medium containing 0 to 0.1% FBS were similar to those of cells grown in minimum essential medium (MEM) containing 2 to 5% FBS. The data indicated that the established Chinese hamster kidney epithelial cell line CHK-AC_E−100 possessed specific insulin receptors and the characteristics of the receptors could be manipulated by changing the serum in culture medium.     

Establishment and characterization of a new cell line (SSP‐9) derived from Atlantic salmon Salmo salar that expresses type I if n

In the present work, the establishment and biological characterization of a new cell line, SSP‐9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitz's (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub‐cultured over 100 passages to produce a continuous cell line with an epithelial‐like morphology. The SSP‐9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40–52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP‐9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up‐regulation in the expression of the genes that regulate immune responses, such as ifn and mx‐1. SSP‐9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc‐II) and interleukin 12b (il‐12b), and flow cytometry assays confirmed that SSP‐9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell–virus interactions and immune processes.     

The in vitro production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism $\text{in vivo}$.     

Antigenic Heterogeneity in Cell Populations of Cloned Polyoma,Virus-induced Tumour Lines

WALLS and Negroni¹ isolated in tissue culture two cell clones from a polyoma virus-induced fibrosarcoma of H₃B/Bi mice. One (malignant cell line 7₁) produced transplantable tumours in adult syngeneic mice; the other (transformed non-malignant line 7₂) was not tumorigenic but induced resistance to the growth of the malignant cells. We have now confirmed these results and extended them to show that other malignant cells (tissue culture clones C₄ and C₅) from a polyoma virus-induced parotid tumour of syngeneic mice are also inhibited by pre-immunization with a non-malignant line (7₂). The number of cells required to induce tumours in 50% of the animals is ten times higher in the preimmunized mice than in the controls, for both fibrosarcoma and parotid malignant lines (Table 1).     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

The production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism .     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity     

Selection of mouse macrophage-like sublines that differ in leukemogenic potential and characterization

The murine macrophage-like cell line (Mm-1), which is nonleukemogenic to syngeneic SL mice, was originally derived from spontaneously differentiated cells of a clonal line of mouse myeloid leukemia cells (M1). In the present experiment, variant cell lines with a high (Mm-A), moderate (Mm-P), and little or no (Mm-S1 and Mm-S2) leukemogenic potential were obtained from the Mm-1 cells. The mean survival times of syngeneic SL mice inoculated i.p. with 5 X 10(6) Mm-A and Mm-P cells were 17 and 33 days, respectively, whereas almost all the mice inoculated with Mm-S1 or Mm-S2 cells survived for more than 90 days. These variant cell lines did not lose their macrophage-like characteristics in vitro. These variant cell lines phagocytized latex beads and sensitized sheep erythrocytes, produced lysozyme, and adhered to culture dishes. The four variant cell lines showed no significant difference in proliferation rates in vitro in liquid medium containing 10% calf serum, but Mm-A cells could grow both in soft agar medium in the absence of ascitic fluid containing colony-stimulating factor (CSF) and in liquid medium containing 1% serum, whereas Mm-P cells could grow in the liquid medium but not in soft agar medium without ascitic fluid, and Mm-S1 and Mm-S2 cells could not grow in either medium. The ratio of the nuclear area to the cell area (NCR) of Mm-A cells was a high (51%) but those of Mm-S1 and Mm-S2 cells were low (40-41%), and that of Mm-P cells was intermediate (44%). The leukemogenicity of Mm-1 cell lines was roughly correlated with their NCR. The possibility that interactions between Mm-1 variant cells and host immune cells might be involved in the mechanisms of their different leukemogenicities was not supported by results on the in vitro susceptibilities of Mm-1 variant cells to the cytostatic actions by normal macrophages and spleen cells and on leukemogenicities of the Mm-1 variant cells in athymic nude mice. A possible method of control of the leukemogenicity of Mm-1 variant cells is discussed.     

Adaptation of cholesterol-requiring NS0 mouse myeloma cells to high density growth in a fully defined protein-free and cholesterol-free culture medium

NS0 has been used as a fusion partner for the production of hybridomas and has more recently been engineered to produce recombinant protein. A protein-free culture medium, designated W38 medium, has previously been developed which supported high density growth of rat myeloma and hybridoma cell lines. NS0 cells failed to grow in W38 medium and in a number of protein-free culture media which support the growth of other myeloma cell lines. NS0 cells are derived from the NS-1 cell line, which is known to require exogencus cholesterol. It was found that NS0 cells grew in W38 medium supplemented with phosphatidylcholine, cholesterol, and albumin and that NS0 were auxotrophic for cholesterol. Protein-free growth of NS0 cells was achieved by using -cyclodextrin to replace albumin as a lipid carrier. The maximal cell density reached in this protein-free medium was in excess of 1.5×10⁶ cell ml^–1. The lipid supplements in the medium precipitated after a few days storage at +4°C. In order to overcome this problem a protocol was developed which allowed NS0 cells to be adapted to cholesterol-independent growth in W38 medium. NS0.CF (cholesterol-independent NS0 cells) were cultured continuously in W38 medium for several months. In shake flask culture a cell density of 2.4×10⁶ cells ml^–1 was achieved in W38 medium compared with 1.41×10⁶ cells ml^–1 in RPMI 1640 medium containing 10% foetal bovine serum. NS0.CF cells readily grew in a 1 litre stirred bioreactor using W38 medium supplemented with Pluronic F68 reaching a density of 3.24×10⁶ cells ml^–1. NS0.CF were cloned protein-free by limiting dilution in W38 medium, giving colonies in wells that were seeded at an average density of 0.32 cells per 200 l. This study has demonstrated for the first time the growth of a cholesterol-requiring mouse myeloma cell line in a completely defined protein-free medium and its subsequent adaptation to cholesterol-independence.Abbreviations BSA bovine serum albumin - C cholesterol - CD cyclodextrin - F68 Pluronic F68 - GS glutamine synthetase - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - MSX methionine sulphoximine     

Embryonic Serum Factors Required for Transdifferentiation of Chick Embryo Neuroretinal Cells in Culture

The accumulation of δ crystallin (chick lens marker) in cultures of 9 day chick embryo neuroretinal cells is strongly promoted by chick embryo extract (CEE) or foetal calf serum (FCS), but much less so by adult sera (horse, chicken and newborn bovine serum). The "transdifferentiation-promoting" (TP) activity of FCS is absent from dialysed FCS but is largely recovered in the initial dialysis medium (F_DM). Similarly, the initial dialysis medium from CEE (E_DM) shows strong TP activity, whereas that from chicken or from horse serum does not. We conclude that the proposed TP factor(s) is (are) of relatively low molecular weight. By contrast, horse serum contains macromolecular factor(s) able to inhibit the TP activity of E_DM or F_DM. Rapid loss of neuronal cells (including those expressing choline acetyltransferase activity) is also observed in media based on F_DM, though whether this effect is mediated by the proposed TP factor(s) has not been determined. The TP activity is not directly related to growth rate or cell density, since cultures in F_DM alone grow poorly yet still accumulate δ crystallin.     

The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.     

Maximal packet size for serotonin in storage vesicles of intact human platelets

A procedure has been developed to measure maximal packet size for serotonin in vesicles of intact, washed human platelets. Vesicles were trace-labelled with H³-5HT and subsequently permitted to accumulate larger amounts of C¹⁴-5HT from the extracellular medium. Parallel platelet aliquots were incubated with unlabelled 5HT, and total and thrombin-releasable 5HT were measured by an enzymatic assay. Prior to incubation, 5HT packet size ranged from 0.05 to 0.53 × 10^?17 moles/platelet. No net addition of 5HT to vesicles occurred when this compartment contained more than 2–3 × 10^?17 moles/platelet of 5HT, suggesting that maximal packet size was 2.8 × 10^?17 moles/platelet. Under these conditions, a typical vesicle would contain 4 × 10^?18 molesof 5 HT, at an estimated concentration of 0.6 M. Nevertheless, when vesicles were full, they continued to exchange C¹⁴-5HT from the extracellular medium with H³-5HT sequestered in vesicles.     

Ventricular and atrial myocytes of newborn rats synthesise and secrete atrial natriuretic peptide in culture: Light-and electron-microscopical localisation and chromatographic examination of stored and secreted molecular forms

Summary We have demonstrated that atrial natriuretic peptide-like immunoreactivity is stored and secreted by ventricular and atrial myocytes in dissociated cell culture preparations from the heart of newborn rat. Culture preparations were maintained in either foetal calf serum-supplemented medium 199 or in hormone-supplemented, serum-free medium 199. The presence of atrial natriuretic peptidelike immunoreactivity in the cultured myocytes was demonstrated at both light-and electron-microscopical levels. Release of atrial natriuretic peptide-like immunoreactivity into the culture medium was measured by radioimmunoassay; molecular forms of the stored and secreted peptide were determined by gel column chromatography. The atrial natriuretic peptide-like immunoreactivity of cultured atrial and ventricular myocytes was concentrated in the perinuclear cytoplasm and was localised to electron-dense secretory granules. The number of immunoreactive ventricular myocytes and the intensity of their immunofluorescence changed with time in culture and was higher in cultures in foetal calf serum-supplemented medium than in serum-free medium. Gamma-atrial natriuretic peptide was stored and released by cultured atrial and ventricular myocytes, but was broken down to alpha-atrial natriuretic peptide in the growth medium. This process was foetal calf serum-independent, since it occurred in both the media used, indicating that cardiac myocytes in culture may release a factor that cleaves gamma-atrial natriuretic peptide to form alphaatrial natriuretic peptide.     

The effect of 20-hydroxyecdysone and protein on granule formation in the in vitro cultured fat body of Drosophila

The larval fat body of Drosophila melanogaster when cultured in a medium containing 20-hydroxyecdysone and foetal calf serum produces protein granules in the cytoplasm in a region-specific manner similar to that found in vivo. If ecdysteroid is omitted from this medium, the tissue continues to produce the granules at the same time, in the same region-specific manner, but in lower amounts. Only the high molecular weight fraction of the calf serum has the granule-inducing effect. Bovine serum albumin, herring protamine and bovine haemoglobin will also induce the granules to form. The degree of granule formation is directly proportional to the concentration of protein in the medium. Protein-free medium produces no granules, and protein concentrations in the medium above 3 mg/ml produce no further increase in granule formation. Although the medium containing foetal calf serum and 20-hydroxyecdysone induces more granule formation than medium containing only serum, the extent of granule formation does not differ at concentrations of hormone above 10^?6 M with any given concentration of serum. A minimal amount of serum (3.75%) permits measuring the effects of 20-hydroxyecdysone at concentrations below 10^?6 M. At this serum level the inducing effects of the hormone could be detected at concentrations of 10^?7 M.     

LOSS OF IRON FROM MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF [55FE]FERRITIN AND [55FE]FERRITIN RABBIT ANTIFERRITIN COMPLEXES

Mouse peritoneal macrophages in culture for 24 h were exposed to horse [⁵⁵Fe]ferritin and rabbit antihorse [⁵⁵Fe]ferritin antibody complex and the amount of ⁵⁵Fe in the medium was assayed up to 2 days after the pulse uptake. Cell survival was assayed by photographing the same areas of the tissue culture Petri dish on successive days and by counting cell numbers per unit area. In experiments in which quantitative assay for cell death is negligible, about 10–20% of the iron ingested by pinocytosis or phagocytosis is released to iron-free medium containing either freshly dialyzed or deironized newborn calf serum (10%). Over the 2-day postpulse period, iron loss is linear. This loss of iron to the medium is significantly reduced by adding iron-saturated newborn calf serum in the postpulse recovery period. A significant portion of the iron released to the medium is bound to transferrin. When human serum is used in the tissue culture system, similar quantities (10–25%) of the ingested iron are lost to the medium 2 days after the pulse.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.     

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10⁹ CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10⁹ CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.