Growth of human normal erythroid progenitors in liquid culture: a comparison with colony growth in semisolid culture

Most studies of erythropoiesis in vitro have employed cloning methods in semisolid medium. We have recently described a two-step liquid culture procedure that supports the proliferation and differentiation of human erythroid progenitors. In the present study, we have modified the procedure to allow large-scale cultures of erythroid cells derived from normal donors. The culture is divided into two phases. In the first phase, which is erythropoietin (Epo) independent, the early erythroid progenitors multiply and differentiate. In the second, Epo-dependent phase, they mature into orthochromatic normoblasts and enucleated erythrocytes. Using this procedure, erythroid cell yield reached 7.5 x 10(6)/ml and a total of 7 x 10(8) cells could be harvested per blood unit. A comparison of the growth of erythroid cells in liquid culture to their colony growth in semisolid culture indicated that cell growth was superior: 1) in liquid culture in terms of cell yield per originally cultured mononuclear cell, 2) per ml culture and per culture surface area and in the purity of the resultant erythroid cell population. In addition, it permits easier manipulation of the culture condition and components and sampling of greater than 1 x 10(7) cells at each maturation stage subsequent to the proerythroblast stage. This liquid culture procedure might provide an important experimental tool for studying erythroid cell development.     

Long-term storage of tissue samples for cell culture

The establishment of cultured cell lines from skin biopsies stored at -196 degrees C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately.     

Transforming growth factors (TGFs): Properties and possible mechanisms of action

Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released hy murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells “autostimulate” their own growth by releasing factors that rebind at the cell surface. The term “autocrine secretion” has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.     

Penaeid (Penaeus japonicus) lymphoid cells replicate by cell division in vitro

Penaeid cell culture has gained much attention as a potential model to facilitate researches on the characterization of the virus and to develop more sophisticated and improved diagnostic procedures for use in the aquaculture industry. However, to date, cell division processes of cultured penaeid cells have not been found, which is suggested as one of the reasons that block the establishment of the continuous penaeid cell lines. We reported here the cell division processes of cultured lymphoid cells of Penaeus japonicus. The culture medium used was based on M199 and was modified by supplementing saline components. Cultures were incubated at 25 degrees C, and 5% CO2 was supplemented. In primary cultured lymphoid cells, dividing cells in different shapes were found. Cell division processes of 12 dividing lymphoid cells were tracked. After cell division, their daughter cells turned into fibroblast-like or epithelioid cells. These results proved that the culture conditions used were suitable for lymphoid cells of I japonicus to proliferate in vitro and that cultured lymphoid cells still had the ability to carry out cell division. These findings would give light to the establishment of continuous penaeid cell lines and would also provide us with the knowledge of cell division processes of the penaeid.     

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.     

Increased levels of apoptosis of leukocyte subsets in cultured PBMCs compared to whole blood as shown by Annexin V binding: relevance to cytokine production

Most of the investigatory studies of cytokine production by cells have been performed on purified cells or cell lines by measuring the secreted cytokine levels in the bulk culture supernatant. However, results of cytokine production from isolated peripheral blood mononuclear cells (PBMCs) cultivated in synthetic media, have been reported to be inaccurate and of low reproducibility. Isolation procedures have been shown to be toxic to certain cells. We hypothesised that purified cell culture techniques may result in increased levels of apoptosis of cells compared with whole blood culture techniques. To compare the effects on cell viability between PBMCs and whole blood techniques, an Annexin V binding assay was utilised. The effect of different cell concentration and serum/plasma concentrations on apoptosis levels in the various leukocyte subsets in PBMC and whole blood cultures following stimulation was investigated. There were significantly increased levels of apoptosis of cells in PBMC compared to whole culture at similar plasma concentrations, suggesting that cell viability was plasma concentration-dependent. There were significantly increased levels of apoptosis in PBMC cultures at the same cell concentration to whole blood techniques, suggesting that interaction between all cellular elements (as in whole blood techniques) is important in maintaining cell viability. These results suggest that whole blood culture techniques provide the best conditions for study of leukocyte cytokine production. If PBMC culture is performed, similar plasma and cell concentration to whole blood will best preserve cell viability.     

Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence.

BACKGROUND: Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. METHODS: Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. RESULTS: In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. CONCLUSION: Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.     

Best practices in cell culture: an overview

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.     

Differentiated cultures of primary hamster tracheal airway epithelial cells

Summary Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The, secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI as judged by the appearance of β tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions.     

Gp38k, a protein synthesized by vascular smooth muscle cells, stimulates directional migration of human umbilical vein endothelial cells.

Gp38k is a 383-amino-acid secreted glycoprotein expressed by cultured vascular smooth muscle cells during the time of transition from a proliferating monolayer culture to a nonproliferating multilayered (differentiated) culture. Expression continues as the cell culture forms multicellular nodules. Because this transition period involves active cell migration, we evaluated the effects of exogenously added gp38k on vascular endothelial cell (HUVEC) migration and chemotaxis. Here we demonstrate that gp38k acts as a chemoattractant for HUVECs and stimulates cell migration in Boyden chambers at a level comparable to that achieved with the known endothelial cell chemoattractant bFGF. The migration effect is neutralized by the presence of a polyclonal anti-gp38k antibody. Because gp38k expression is also correlated with changes in culture morphology, we also assessed its ability to act as an agonist of HUVEC morphology using cultures growing on Matrigel. We report that gp38k stimulates endothelial cell tubulogenesis in this assay system. These results provide the first evidence that gp38k may function in angiogenesis by stimulating the migration and reorganization of vascular endothelial cells.     

Cost-effective engineering of a small-scale bioreactor

Several methods exist for increasing the scale of cell culture in the laboratory. While these methods provide significant increases in biomass, they are often prohibitively expensive for many laboratories. We have engineered a small-scale bioreactor with a novel means of introducing oxygen through the catalytic breakdown of hydrogen peroxide using a manganese oxide catalyst. We have also adapted and modified an existing assay for dissolved oxygen to be compatible with culture conditions. In this system we have been able to culture CHO cells at densities of up to 10(7) cells/mL without the use of automated feedback systems.     

Enhanced growth of Sf-9 cells to a maximum density of 5.2 x 10(7) cells per mL and production of beta-galactosidase at high cell density by fed batch culture

Significant improvement in cell growth and protein production has been achieved in Sf-9 insect cell cultures using pulse additions of multicomponent nutrient feed concentrates (Bédard et al., 1994; Chan et al., 1998). The present work focuses on investigating an alternative feeding strategy wherein the nutrients are fed in a semi continuous manner. Fed batch culture experiments were carried out to compare the two different feeding strategies, pulse and semi continuous and a process developed to achieve a cell density of 5.2 x 10(7) cells/mL of Sf-9 cells in a 3.5 L bioreactor. Production of recombinant protein beta-galactosidase was carried out by infecting the cells with baculovirus at a MOI of 10 at cell densities of 17 x 10(6)cells/mL. Specific productivity could be maintained at cell densities as high as 14 x 10(6) cells/mL. The results presented indicate that the feeding method can provide significant improvements in the performance with a reduction in the amount of total nutrients added. On-line monitoring of the culture using the capacitance probe showed that the capacitance probe can be used successfully to monitor the biomass and infection process even at higher cell densities.     

In vitro generation of human activated lymphocyte killer cells. II. N-acetyl-D-galactosamine inhibits a distinct subpopulation of human activated lymphocyte killer cells generated in mixed lymphocyte culture

A range of monosaccharides was tested for its ability to inhibit the generation of cytotoxic cells during mixed lymphocyte culture. The most discriminatory effect was produced by N-acetyl-D-galactosamine (NADG). The presence of this sugar at the initiation of the coculture significantly inhibited in a dose-dependent manner the induction of a subset of nonspecific activated lymphocyte (ALK) cells preferentially able to lyse the K562 target cell (natural killer, NK-like cells) but had no effect on the generation of either specific cytotoxic T lymphocytes or another separate subset of ALK cells mediating lysis of an NK-insensitive melanoma cell line. The addition of conditioned medium containing interleukin 2 and interferon (IFN) at the start of culture reversed the inhibitory effect of the sugar. Under conditions of limiting dilution, the frequency of NK-like precursors ranged from 1/50 to 1/1200 with different mononuclear cells (MNC) and in all cases the presence of NADG from Day 0 of culture selectively decreased the frequency of these precursors. At the concentrations used NADG had no effect on NK-like cell cytolysis once generated. The addition of recombinant gamma-IFN did not abrogate the inhibitory effect of NADG and in MLC of some individuals decreased the frequencies of ALK cell precursors. These data provide further evidence for the heterogeneity of ALK cells and indicate that what is usually referred to as NK-like cell activity in in vitro culture is mediated by a subpopulation of MNC which are activated and induced to differentiate along a pathway independent of that of other ALK subsets.     

Comparisons of microcarrier cell culture processes in one hundred mini-liter spinner flask and fifteen-liter bioreactor cultures

Microcarrier cell culture process can be used to culture anchorange-dependent cells in large bioreactor vessels. The process performance in large bioreactors is usually less prominent than that in spinner flask vessels and bench scale reactors. In this study we investigated the microcarrier cell culture processes in 100?ml spinner flask and 15-liter bioreactor cultures, including the kinetics for cell attachment, cell growth and the production of Japanese encephaltilis vaccine strain (Beijing-1) virus. Under a fixed concentration of microcarrier and cell density used in inoculations, the attachment kinetics of Vero cells on Cytodex 1 microcarrier in a 15-liter bioreactor vessel was 2 folds slower than with 100?ml spinner flask culture. Virus replication in 15-liter bioreactor culture also revealed an approximately one day lag-time compared to 100?ml spinner flask culture. Findings presented herein provide valuable information for designing and operating microcarrier cell culture processes in large bioreactor vessels.     

Long-term storage of tissue samples for cell culture

Summary The establishment of cultured cell lines from skin biopsies stored at −196°C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately. This project was supported by the South Carolina Department of Mental Retardation and the Self Foundation.     

Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate

Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.     

K562 cell growth activity and metabolism characteristics in APA microencapsulated culture and modeling study

Cell microencapsulation is likely to play a major role in cell and transplantation therapies in the next decade. The microcapsules provide a special microenvironment in which cells always have different behaviors compared with free non-encapsulated culture. In this work, the behaviors of K562 leukemia cells were studied once entrapped in solid and liquefied APA microcapsules as well as in free non-encapsulated culture. Glucose pulse culture was employed to characterize the growth and metabolism of microencapsulated K562 cells. And mathematical modeling was presented to develop a basis for the deeper understanding of cells responses to different culture environments. Based on the results of experiments and modeling, it was found that cells presented a better growing pattern and maintain the activity at a higher level for extending time. The concentration of lactate was higher in solid microcapsules culture than that of liquefied microcapsules culture, but the cell number was lower. And the lactate yield coefficients (lactate/glucose) were 0.8129, 0.6978 and 0.601 for free non-encapsulated, solid microcapsules and liquefied microcapsules culture, respectively. An increase of glucose concentration led a decrease of cell activity, The glucose consumption ratio were 99.9%, 86.8%, 49.4% and 28.6% with the decrease in its concentration from 2 to 4, 6, 10 g/L, however, the lactate yield coefficient were 0.7184, 0.6654, 0.8239 and 0.9693, respectively.     

Propagation of fetal human RPE cells: preservation of original culture morphology after serial passage

The permissive effects of extracellular matrix (ECM) on in vitro growth and differentiation of fetal human retinal pigment epithelial (RPE) cells have been studied. Factors which enhanced the effect of ECM to support cell division were also examined, including growth factors, culture media, and serum requirement. Under the specific culture conditions we have defined, it is possible to propagate these RPE cells at low density (less than 20 cells/mm2) with excellent growth properties for greater than 72 doublings (fourteen passages) in serial culture. Later-passaged cells maintained the morphological appearance of early-passaged cultures. ECM produced by bovine corneal endothelial cells was by far the most predominant factor in promoting rapid cell proliferation and viability over repeated passaging. Basic fibroblast growth factor (bFGF) exerted a substantial effect on the rate of cell division at different serum concentrations on plastic dishes. In addition, this factor showed profound synergistic effect when RPE cells were maintained on ECM, both in the preservation of cell morphology and also in long term viability. Other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-beta (TGF-B), were also tested, but EGF effects were less prominent than those observed with bFGF, and TGF-B had an inhibitory effect at high concentrations. The ability to obtain a relatively large number of human RPE cells in vitro which preserve the appearance of early passage cells may provide useful opportunities to study the physiological properties and pathological alterations involving this important cell type.