In Vitro最新文献

Synchronization of Plasmodium falciparum cultured in vitro results in a one-step growth pattern that allows the study of stage-specific metabolic activities of the parasites. Lactic acid (LA) was selected as a metabolic marker, and the concentration of this end product found in spent media was correlated with the different erythrocytic stages of the parasites. When the medium was changed at 12 h intervals, cultures containing predominantly trophozoites produced 3.66 +/- 0.55 mumol LA per 12 h per 10(7) parasitized cells (n = 26), an amount of LA that is about 8 to 20 times higher than that found in corresponding cultures containing predominantly ring forms. Depending on the stage of development, parasitized red blood cells produced between 5 and 100 times more LA than uninfected erythrocytes (3.72 +/- 0.62 mumol LA per 12 hours per 10(9) red blood cells) (n = 41) when cultured under identical conditions. The intraerythrocytic development of the parasites was not impaired by exposure to extracellular concentrations of LA up to 12 mM over a 12 h period. The growth resulting in such cultures was described as uninhibited and was characterized by a multiplication index of 10 or higher. Above the threshold of 12 mM of LA, progressive inhibition of parasite development occurred. The stage-specific LA production reported can be used to predict the amount of LA that will have accumulated at the end of a subsequent 12 h incubation period during synchronized in vitro growth of Plasmodium falciparum. Using these values, it is possible to establish an optimal medium exchange schedule, thereby assuring uninhibited growth and a correspondingly high parasite yield.

In lining epithelium of mammals certain recurrent architectural patterns are recognized that may be critical for epithelial organization in culture. Among these structural imperatives are three dimensional growth, restricted migration of cells, histophysiologic gradients, and continuity of epithelial membranes. Histophysiologic gradient culture procedures have been developed to comply with requirements suggested by normal tissue architecture. In a small chamber, 5 mm diam, epithelium grows attached to a thin permeable transparent collagen membrane or sandwiched between two apposed collagen membranes. The chamber is held in a plastic capsule so that metabolic exchange is limited to substances that diffuse across the collagen membranes to the adherent basal layer of epithelium. On a single membrane after 2 wk of growth, normal urothelium appears as a diffusely hypercellular urothelium, 6 to 10 cells thick. As the culture period is extended by 2 or more wk, multiple nodules of urothelium extend from the basal surface into the subepithelial space between the epithelium and the collagen membrane. Normal bladder, cultured between two apposed collagen membranes, gives rise in a few days to confluent epithelium that contains many extracellular cysts. Through an apparent merging of cysts, after 2 wk the urothelium appears as a highly organoid structure, a flattened cyst lined by completely stratified polarized urothelium. Such microbladders consist of a stratified epithelium without interruption of continuity. With histophysiologic gradient culture, processes in carcinoma and precursor lesions are accessible to study at the level of tissue organization.

Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm2) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment to 93% (P less than 0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occurring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfold increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to plastic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium.

Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.

The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5 X 10(3)/cm2 in the presence of 2 X 10(4)/cm2 lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10(-10) M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size. Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.

When HeLa cells are detached from their support by trypsin, trypsin-EDTA, or by mechanical means large zeiotic blebs are formed. After reseeding the cells onto glass these blebs shrink. Those blebs near the support collapse completely to form lobes in the neighborhood of lobopodia. By scanning and transmission electron microscopy we could show the transformation of these zeiotic blebs into true lobopodia. This implies a repair of cell surface structures and components as well as of the subcortical cytoskeleton.

A rapid and technically simple method for cloning both adhesive and nonadhesive mammalian cells is described. The procedure employs (a) honeycomb cloning plates and (b) nonlethal vital stains. Instead of placing cloning rings around colonies, cells are initially seeded at clonal density directly into a plate containing an array of cloning rings (the honeycomb plate). Hence, the time involved in placing cloning rings around colonies is eliminated. Second, clone-containing wells of the honeycomb plate are easily identified by staining plates with the nonlethal vital stains, MTT or INT tetrazolium. Vital staining eliminates the time involved in searching for clones. Last, clones are transferred with a cotton-tipped swab thereby eliminating the time involved in trypsinization of cells. In this fashion, one can pick and transfer clones of substrate adherent mammalian cells at a rate of one clone/10 to 15 s. Thus, mammalian cells can be cloned as rapidly as cloning can be carried out in microbial systems.

A serum-free culture medium was employed to evaluate both qualitative and quantitative aspects of secreted proteins derived from human bronchial explants over a period of 26 to 50 d. It was found that protein and hexose were secreted at a reasonably constant rate during this period. Viability of explants was assessed by histological examination, attachment to scored grids of the petri dish, incorporation of labeled precursors into tissue proteins, and by fluorographic analysis of the sodium dodecyl sulfate-polyacrylamide gel pattern of the secreted material. This culture system is useful as a model for the study of secretory products and processes and how they are affected by various stimuli.

The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a) N-nitrosomethylurea (NMU)-induced and (b) 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGE2, PGE1, and PGF2 alpha (6.7, 4.7, and 1.7 ng/10(6) cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF1 alpha and TXB2, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE2 (1 and 4 ng/10(6) cells per 48 h, respectively). PGE2 production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1.14.99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE2 levels increased at low concentrations of ibuprofen and then decreased at higher concentrations. At 100 micrograms/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respectively. There was no obvious dose-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiologically relevant amounts of PGS.

Certain functional analogs of amino acids were examined for their capacity to inhibit chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus transformed BALB/c (K-BALB) mouse cells. Partially synchronized cells cultured with aminoethylcysteine (AEC), parafluorophenylalanine (PFA), or valinol, and subsequently induced with either 5-iododeoxyuridine (IUdR), cycloheximide, or histidinol, showed inhibition of virus activation. Inhibition was concentration- and time-dependent (1 to 4 h) and not a consequence of cytotoxicity. Inhibition was competed out by the analogous amino acid and was specific to the induction process. After a 4 h analog treatment, heteronuclear RNA synthesis was reduced 24, 38, and 35% by PFA, AEC, and valinol, respectively, whereas cycloheximide or actinomycin D reduced synthesis by 60 and 90%, respectively; therefore, the analogs did not seem to inhibit induction through a general transcriptional block. Culture of cells in the presence of the analogs resulted in an abrupt reduction (70 to 90%) in DNA synthesis. Using synchronized cells, it was found that 0.1 mM AEC added in G1 phase and followed by IUdR induction almost totally inhibited virus expression. No inhibition was observed when AEC was added during S phase concomitantly with the inducer. AEC added to synchronous cells in G1 phase inhibited the progression of cells into S phase and the onset of DNA synthesis. The results show that K-BALB cells have an AEC-sensitive restriction point in G1 phase that might relate to the effects amino acid analogs have on cell replication and S phase dependent gene expression, as well as subsequent differentiation.