Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol.

Modifications in the techniques for the induction of mammalian somatic cell hybridization by polyethylene glycol (PEG) have led to procedures that are rapid, simple, and effective. The basic improvements, for both monolayer and suspension fusions, are a short exposure to PEG and a rapid dilution of PEG following treatment. There is a marked effect of PEG concentration on cell hybridization, and there seem to be inherent differences between cells in terms of the extent of cell fusion induced by PEG.     

Comparison of different culture modes for long-term expansion of neural stem cells

Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. The cultured cells are different in both the ability to proliferate and heterogeneity. In order to find the appropriate methods for large-scale expansion of NSCs, we systematically compared the NSCs cultured in suspension with those cultured in monolayer. The forebrain tissue was removed from embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase and mechanical trituration. The cells were cultured in both suspension and monolayer. The NSCs cultured in suspension and in monolayer were compared on viability, ability to proliferate and heterogeneity by fluorescent dyes, immunofluorescence and flow cytometry on DIV21 (21 days in vitro), DIV56 and DIV112, respectively. The results indicated that the NSCs cultured in both suspension and monolayer represented good viability in long-term cultures. But they displayed a distinct ability to proliferate in long-term cultures. The NSCs cultured in monolayer preceded those cultured in suspension on the ability to proliferate on DIV21 and DIV56, but no obvious difference on DIV112. The NSCs population cultured in suspension displayed more nestin-positive cells than those in monolayer during the whole process of culture. The NSCs population cultured in monolayer, however, displayed more βIII tubulin-positive cells than those in suspension in the same period. The suspension culture mode excels the monolayer culture mode for large-scale expansion of NSCs.     

The effects of cell adhesion on the growth and protein productivity of animal cells

We investigated the effect of cell adhesion on cellgrowth and productivity of recombinant protein inChinese hamster ovary (CHO) cells. Cells cultured innormal tissue culture dishes attached to the dishsurfaces and grew as a monolayer, while cells culturedin non-treated dishes proliferated in suspension assingle cells without adhering to the dish surfaces. On an agarose-coated dish surface, cell aggregatesformed without attaching to the dish. Growth rates inboth suspension cultures were slightly lower thanthose in monolayer culture. Cell cycle analysisindicated that the duration of the G₁ phase insuspension cultures was longer than that in monolayerculture, suggesting that attachment to the substratummainly affected the transition from the G₁ to theS phase. Consistent with this, CDK inhibitor p27,that inhibits the G₁S transition, was induced inthe cells cultured in suspension.To assess the productivity of recombinant proteins,CHO cells were transfected with a plasmid containingmurine interferon (mIFN-) under thecontrol of the cytomegalovirus promoter. Insuspension culture, mIFN- productivity wasslightly lower than that in the monolayer culture. When protein kinase C was activated by phorbol ester,mIFN- production was enhanced in both themonolayer and suspension cultures. However, theproductivity in the suspension culture was lower thanthat in the adherent culture even in the presence ofhigh concentrations of phorbol ester. These resultssuggested that cell adhesion to the substratum affectsvarious features of CHO cells.     

Cytochemical properties of prematurely condensed chromosomes

Prematurely condensed chromosomes (PCC) have been obtained by polyethylene glycol (PEG) induced fusion in suspension of the Chinese hamster metaphase cultured cells with those in interphase. As alternative approach the PEG-fusion of the Chinese hamster asynchronous culture cells in monolayer with subsequent incubation in free medium was used. A comparative cytofluorimetric investigation of PCC and chromatin of the interphase nuclei of corresponding ploidy has shown some increase (up to 10%) of acridine orange and olivomycin binding with PCC chromatin. A similar slight increase in low molecular weight ligands binding with chromatin was also found in mitotic chromosomes. The data obtained confirm the opinion about the similarity of events taking place in chromatin during physiological mitosis and premature chromosome condensation. The cytochemical study of chromatin availability to low molecular weight ligands can be used as a criterion for judging on the properties of the artificially condensed chromatin.     

外源性线粒体DNA在细胞内的重建（简报）

To create human mitochondrial DNA (mtDNA)-transferred cells as cell model of mitochondria defects-related diseases, platelet of normal young and old subjects were isolated as donor of mtDNA, then fused with mtDNA-depleted cells (rho0 cells) under induction of PEG1500. Auxtrophy test, cytochrome c oxidase activity assay and PCR amplification of mtDNA were done to confirm the transferring of mtDNA. Cell clones were visible in the medium 10 to 15 days after fusion, which grew well in medium lacking uridine, had positive COX activity and contained objective fragment of mtDNA by PCR, opposite to rho0 cells. Transferring of mtDNA to rho0 cells was identified, and mtDNA-transferred cell models were successfully created.     

The intracellular pH and cell proliferation of the LS cell line and its derivative LSM adapted to monolayer growth]

The dynamics of intracellular pH (pHi) during proliferation of cells of LS line in bicarbonate-containing media and of its derivative LSM line adapted to grow in a monolayer has been studied. The contact of LS cells with a solid substrate was not accompanied by their spreading and by an increase in pHi. The pHi values of growing and resting LS cells were practically equal (7.03 and 6.97, respectively). The adhesion and spreading of LSM cells were accompanied by an increase in pHi. The proliferation of LSM cells occurred at different pHi values: at 7.32 on solid substrate with serum, at 7.18 on substrate without serum, at 7.13 in a serum-containing suspension, at 6.97 in a suspension without serum. The highest growth rate was observed at the increased pHi value. Cell proliferation on the substrate stopped at pHi values within 7.10 and 7.13 which were equal to or exceeded the pHi of growing cells in suspension. No difference was observed between LS and LSM cells in the activities of Na+/H+ exchange and transport of Cl- into cells that are involved in pHi regulation. Transport of HCO3- into the cytoplasm of LSM cells was more active than that of LS cells. The role of pHi in the anchorage dependence of cell proliferation is discussed.     

Control of rat C6 glioma cell proliferation: uncoupling of the inhibitory effects of hydrocortisone hormone in suspension and monolayer cultures

We undertook a comparative study of the effects of the hormone hydrocortisone (Hy) on C6 glioma cells grown in monolayer and in suspension in cultures. We found Hy reversibly renders C6 cells anchorage- and serum-dependent for their growth. In monolayer cultures, Hy was found to inhibit cell cycle traversing exclusively at G1 phase. In agarose suspension, Hy was found to block colony development. Hy-resistant variants were selected and isolated in agarose suspension. Examination of these variants showed that cells selected for Hy-resistance in suspension can be Hy sensitive when anchored to a solid substrate. We conclude that resistance to Hy in suspension and resistance to it in monolayer culture are two independent phenotypes.     

Shedding of mitotic cells from the surface of multicell spheroids during growth

During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exonential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytoflurometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell-to-cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis.     

Growth of the IB-RS-2 Pig Kidney Cell Line in Suspension Culture and Its Susceptibility to Foot-and-Mouth Disease Virus

The adaptation of the pig kidney cell line IB-RS-2, clone 60, to growth in suspension culture is described. When fully adapted, an approximate threefold increase in viable cells was obtained within 72 hr from initial cell concentrations of 5 x 10(5) per ml in culture volumes up to 1,500 ml. The monolayer cells (99th passage level) used to initiate the suspension cultures and the fully adapted suspension cells were shown to have an aneuploid chromosome karyotype, whereas earlier monolayer cultures (32nd passage level) had a pseudodiploid karyotype. Replicate virus titrations in monolayers prepared from suspension-adapted cells, IB-RS-2 monolayer cells, BHK monolayer cells, and in suckling mice showed that the suspension cells had retained sensitivity to foot-and-mouth disease virus. The geometric mean peak infectivity of seven strains of foot-and-mouth disease virus grown in IB-RS-2 suspension cells was 10(8.2) plaque-forming units per ml, with a mean complement-fixing activity of approximately 135 complement-fixing units per ml. These preliminary results indicate that submerged cultures of these cells on an industrial scale may be useful for commercial foot-and-mouth disease vaccine production.     

Tumorsphere assay provides more accurate prediction of in vivo responses to chemotherapeutics

Although the sphere culture system has been widely used in stem cell biology, its application for drug screening is limited due to lack of standardized, rapid analytical tools. To optimize sphere cultures for in vitro screening of drugs, we evaluated the properties of primary tumor cells growing as tumorspheres and compared their chemosensitivity to those of cells growing in monolayer. Most cells in tumorsphere cultures were quiescent whereas cells in monolayer culture had a high mitotic index. Moreover, doxorubicin showed better cytotoxicity than paclitaxel in the sphere cultures, but their efficacy was reversed in the monolayer cultures. Importantly, the response of cytotoxic outcomes for suspension cultures matched the in vivo response better than monolayer cultures, providing support for the use of short term suspension cultures of primary cells as a model for drug testing.     

Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer-derived mammary tumor cells in suspension

Summary The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY-beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incoerporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in susfpension resulted in 10- to 400-fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Incrases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 μM hydrocorticone further increased MMTV yield two-to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure. This work was supported by the T. J. Martell Foundation for Cancer and Leukemia Research and by USPHS grant 5P-30CA23102. F. M. is a trainee on MSTP grant GM07280 from the National Institute of Health. This work was submitted in partial fullfillment of the requirements for the Ph. D. degree (F. M.).     

DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this "contact effect" to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.     

Cell membrane stability and biochemical response of cultured cells of groundnut under polyethylene glycol-induced water stress

Cell membrane stability (CMS) in suspension cultures of two groundnut cultivars was studied under polyethylene glycol(PEG)-induced water stress. There was a negative relationship between PEG concentration in the medium and membrane stability measured as electrolyte leakage. The CMS values in the cell cultures correlated well with the whole plant tissue and permitted the differentiation of cultivars based on their known response to drought stress. The cell membrane stability was lower (more electrolyte leakage) when cells were grown in culture as compared to the intact plant tissue. Kadiri-3, the drought tolerant cultivar maintained higher CMS than JL-24, the drought susceptible one. With increasing PEG levels the concentration of Potassium in cultured cells declined in both cultivars. However, Kadiri-3 maintained higher K values than JL-24 accompanied with greater cell membrane stability. Total soluble sugars also increased with increasing stress in both cultivars; the increase being higher in Kadiri-3. There was no significant change in the total free amino acids but proline accumulated markedly in both varieties. However, no relationship was found between proline levels and CMS. The results demonstrated that CMS test can also be used under in vitro conditions to differentiate the drought tolerant and susceptible cultivars and the cellular K level has a positive relationship with membrane stability.     

PEG-tolerant cell clones of chili pepper: Growth,osmotic potentials and solute accumulation

Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated in MS liquid medium supplemented with 6.25 M 2,4-d and 0.44 M BA. Cell clones were isolated by plating the cell suspension on filter paper discs supported by polyurethane foam that were bathed with culture medium containing 15% PEG. The cell clones T6 and T7 were chosen based on their characteristics of growth and friability. These cell clones were established as cell suspensions in the presence of 15% PEG and subsequently subcultured in increasing concentrations of osmoticum. By this approach the cell clones T7 and T6 were capable of growing in the presence of 20 and 25% PEG, respectively. The cell clone T7 was found to grow better in the presence of 5–10% PEG after a period of subculturing in the absence of osmoticum indicating that the tolerance trait was stable. The tolerant cell clones exhibited a 3 to 3.5-fold decrease in the osmotic potentials in comparison with the nonselected cells suggesting that osmotic adjustment occurred. K⁺ was the major contributing solute to the osmotic potential in all the cell cultures among those tested and was found to be higher in concentration in the PEG-tolerant clones (1.3–3 times higher than nonselected cells). Proline and glycine betaine levels showed a positive correlation with the degree of tolerance to water deficit in the PEG-tolerant cell clones. The levels of proline in the cell clone T7 subcultured in the absence of PEG in the culture medium decreased to values similar to those of nonselected cells, whereas the contents of glycine betaine in the same conditions were maintained at high levels.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - PEG polyethylene glycol     

Anchorage-independent colony formation of SV40 transformed BALB/c-3T3 cells in serum-free medium: role of cell- and serum-derived factors

The colony-forming response of SV40 transformed BALB/c-3T3 cells in agarose suspension culture was studied in a serum-free medium (with insulin, transferrin and serum albumin as the only macromolecular supplements) that was optimized for colony formation of fibronectin-attached monolayer cultures. In this serum-free medium, the SV3T3 cells fail to form colonies in agarose suspension. However, they can be induced to anchorage-independent colony formation by the growth factors that are additionally required by their untransformed counterparts for proliferation in monolayer culture. The SV3T3 cells are also rendered anchorage-independent for colony formation in serum-free medium by conditioned medium from dense monolayer serum-free SV3T3 cultures. These experiments suggest that it is the cell-substrate interaction that is responsible for the growth factor autonomy of fibronectin-attached transformed cells.     

Cell configuration-related control of vimentin biosynthesis and phosphorylation in cultured mammalian cells

The cell configuration-related control of a cytoskeletal protein (vimentin) expression was examined by varying cell shape between flat and spherical. Cultivation of cells in monolayer or in a spherical configuration on poly-2-hydroxyethylmethacrylate-coated plates revealed a preferential down regulation of vimentin synthesis during suspension culture. The mechanism(s) regulating the decrease in the expression of vimentin in spherical cells appears to be at the level of translation, because mRNAs extracted from monolayer and suspension-cultured cells were equally active in directing vimentin synthesis in the rabbit reticulocyte cell-free system. When after prolonged suspension culture, the cells were allowed to reattach and spread, vimentin synthesis recovered rapidly to the control monolayer rate. The phosphorylation of vimentin was also reduced dramatically during suspension culture. However, unlike the rapid recovery of vimentin biosynthesis upon reattachment (less than 6 h), the recovery in the rate of vimentin phosphorylation was much slower (greater than 20 h) and paralleled the recovery to the monolayer growth rate. Although the control of vimentin biosynthesis in suspension culture is a cell configuration-related process, the decrease in the rate of vimentin phosphorylation in suspension culture appears to be the result of the slower growth rate and may reflect the reported correlation between the rate of vimentin phosphorylation and the accumulation of cells in mitosis.     

A new dimension in suspension fusion techniques with polyethylene glycol.

Polyethylene glycol (PEG) induces the hybridization of mammalian cells at a much higher frequency when the cells are attached to a substrate during treatment than when the cells are treated in suspension. Since many cell types, e.g., lymphocytes, cannot attach to a substrate, a new technique for the PEG-induced fusion of cells in suspension was developed. This technique, referred to as "pancake fusion," is based on the centrifugation of suspended cells onto a coverslip and the PEG treatment of the cells on the coverslip as if they were attached to a substrate. With this technique, the frequency of hybridization of human white blood cells, which are incapable of attaching to a substrate, can be greatly increased.     

Human natural cell-mediated cytotoxicity against fetal fibroblasts. II. Isolation and target cell specificity of the effector cells.

Human peripheral Wood lymphocytes were depleted of natural killer cells cytotoxic against human fetal fibroblasts by allowing them to attack the fibroblast targets grown on plastic beads followed by gravity sedimentation under conditions in which single cells floated but the attacker cells sedimented with the carrier beads. The attacker cells could be released from the bead-grown targets and shown to be greatly enriched in natural cytotoxic activity. The effector cells depleted by fibroblast adsorption were also depleted of cytotoxic activity against other monolayer targets whereas suspension grown lymphoma and leukemia cells (MOLT-4, RAJI, and K-562) were killed as effectively as by non-depleted effector cells. In competition assays other monolayer cells inhibited the natural cytotoxicity against fetal fibroblasts but the suspension-grown cells were unable to compete. The results suggested that different effector cell populations were probably involved when monolayer vs suspension targets were used in assays for human natural cell-mediated cytotoxicity. The separation was not, however, functionally complete since in competition assays with suspension-grown target cells also monolayer cells were able to compete. Preliminary morphological characterization of the natural killer cells against fetal fibroblasts is also presented.     

Growth of Fish Cell Lines on Microcarriers

Microcarrier beads were evaluated as substrates for the propagation of five anchorage-dependent fish cell lines. Growth of rainbow trout gonad (RTG-2) and Atlantic salmon cells was limited on microcarriers maintained in suspension. However, stationary microcarriers were suitable substrates for the growth of RTG-2, AS, Chinook salmon embryo (CHSE-214), and fathead minnow cells. Cell yields ranged from 2 × 10⁶ to 2.9 × 10⁶ cells per ml, representing 7- to 10-fold increases over the initial cell concentrations. The yield of new RTG-2 cells per unit volume of growth medium was 2.8 times greater in microcarrier cultures than in standard monolayer cultures. Northern pike cells failed to grow on microcarriers. Yields of infectious pancreatic necrosis virus propagated in microcarrier cultures of RTG-2 cells were more than twice the yields in standard monolayer cultures. The greater economy of microcarrier cultures in terms of growth vessel and medium requirements holds great promise for the large-scale production of anchorage-dependent fish cell cultures and fish viruses.