Low-temperature storage of surface-attached living cell cultures

A method was described for low-temperature storage of surface attached living cell cultures so that when the cultures are revived most of the cells remain attached to the surfaces of the vessels in which they had grown. Revived cells begin increasing in numbers about 5 hr after warming and can be used immediately for inoculation of at least two viruses, herpes simplex virus and an adenovirus.     

Taxol transport in Taxus baccata cell suspension cultures

Taxol transport in Taxus baccata L. cell suspension cultures was studied using 〚¹⁴C〛-taxol as a tracer. The time course of uptake showed a saturable absorption that reached a maximum within 20 min. The uptake depended on its exogenous concentration and its accumulation was highly stimulated in the presence of 10–15 μM exogenous taxol. The absorbed molecule was found to localise both in the cell walls (20 %) and in the cell protoplasts (80 %), suggesting an accumulation within the vacuoles. Taxol uptake was strongly inhibited by Na-orthovanadate and verapamil, while Ca²⁺ was found to be one of the factors required for the active absorption of the molecule, since in the absence of this cation, the uptake was reduced by about 40 % and occurred mainly through a non-energy dependent mechanism. Taxol release into the culture medium was demonstrated not to depend on cell lysis, occurred through a mechanism that reached its maximum after 10–15 min and was strongly enhanced by treatment with Na-orthovanadate and verapamil, although the effect was found to be transient.     

A method for growing stationary plant suspension cell cultures

Summary Stationary culture of plant cell suspensions has been achieved. Slurries, produced when small amounts of agar (0.1–0.4%) were added to culture media, were used to suspend plant cells. Growth proceeded more slowly than in standard shake culture, but cells remained viable for months of culture. This method of growing plant cells in stationary culture should be useful for general applications including long-term cell culture, shipment of cultures, and physiological, molecular biological, and pathological studies. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Editor’s Statement This procedure for growing stationary suspension cultures in an agar slurry should be useful for shipping suspensions and for long-term maintenance of little used or back-up cultures.     

Simple, inexpensive method for controlled freezing of cell cultures

[This corrects the article on p. 616 in vol. 27.].     

Simple, inexpensive method for controlled freezing of cell cultures

We have devised a system for controlled cooling of living cells which eliminates the need for complicated, expensive equipment but allows excellent recovery of viable cells after storage in liquid nitrogen.     

A mechanical dissociation method for preparation of muscle cell cultures

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.     

Improved method for separation on CM-cellulose of the trypomastigote form of Trypanosoma cruzi from forms grown in fibroblast cell cultures

Trypomastigotes grown in fibroblast cell (L-cell) culture were more effectively and more rapidly separated from other cells on a CM-cellulose column with culture medium (MEM with 10% calf serum) for elution instead of phosphate-saline-glucose buffer (PSG). This effective separation was shown to be due to the presence of serum. Trypomastigotes weakly adhered to CM-cellulose resin by the tip of the body (mainly the flagellar tip) when they were suspended with CM-cellulose resin in PSG. Serum seemed to disturb the adhesion of trypomastigotes to the resin, but not the adhesion of amastigotes and fibroblast cells. Therefore, only trypomastigotes were rapidly eluted from a CM-cellulose column in the presence of serum.     

A new fluorescent analogue for the studies of anandamide transport in cell cultures

A new fluorescent analogue of anandamide bearing a BODIPY®-FL-fluorophore and linked to arachidonic acid via a 2,2′-(ethylenedioxy)-bis(ethylenediamine) residue was prepared. The fluorescent analogue was demonstrated to be a substrate of the cell anandamide uptake system (K _m 4.5 ± 0.9 μM, V _max 20 ± 1 amol/(min cell)) in rat glioma C6 cells.     

Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures

Glucose and lactate metabolic rates were evaluated for cultures of cord blood (CB) mononuclear cell (MNC), peripheral blood (PB) MNC, and PB CD34(+) cell cultures carried out in spinner flasks and in T-flasks in both serum-containing and serum-free media. Specific glucose uptake rates (q(gluc), in micromoles per cell per hour) and lactate generation rates (q(lac)) correlated with the percentage of colony-forming cells (CFC) present in the culture for a broad range of culture conditions. Specifically, the time of maximum CFC percentage in each culture coincided with the time of maximum q(gluc) and q(lac) in cultures with different seeding densities and cytokine combinations. A two-population model (Q(lac) = alpha[CFC] + beta([TC] - [CFC ]), where [TC] is total cell concentration; Q(lac) is volumetric lactate production rate in micromoles per milliliter per hour; alpha is q(lac) for an average CFC; and beta is q(lac) for an average non-CFC) was developed to describe lactate production. The model described lactate production well for cultures carried out in both T-flasks and spinner flasks and inoculated with either PB or CB MNC or PB CD34(+) cells. The values for alpha and beta that were derived from the model varied with both the inoculum density and the cytokine combination. However, preliminary results indicate that cultures carried out under the same conditions from different samples with similar initial CD34(+) cell content have similar values for beta and beta. These findings suggest that it should be possible to use lactate production data to predict the harvest time that corresponds to the maximum number of CFC in culture. The ability to harvest ex vivo hematopoietic cultures for transplantation when CFC are at a maximum has the potential to speed the rate at which immunocompromised patients recover. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 693-700, 1997.     

Characterization of cytokinin and adenine transport in Arabidopsis cell cultures

Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction. Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra- or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin (tZ) was used for uptake studies in Arabidopsis (Arabidopsis thaliana) cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore carbonyl cyanide m-chlorophenylhydrazone is an effective inhibitor of cytokinin uptake, consistent with H(+)-mediated uptake. Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.     

Rapid method for the assessment of cell lysis in microalgae cultures

A solid understanding of the effect of hydrodynamic forces encountered by microalgae in bioprocesses would benefit existing bioprocesses, eventually allowing an increase in their productivity. For this purpose, a sensitive method able to quantify cell lysis is crucial. Most of the available protocols and methods intended for this purpose were developed for animal or insect cells. In the case of microalgae, the commercial kits tested were unable to determine the cell lysis extension. The method proposed here was developed to relate the release of a cytoplasmic component (enzyme lactate dehydrogenase (LDH)) with cell lysis by measuring the NADH (reduced form of nicotinamide cofactor adenine dinucleotide) produced by LDH. Although different commercial kits based on similar processes are available, they are more complicated to use and not applicable to microalgae nor when longer-term tests are to be performed.