Evaluation of the effect of chronic hydrodynamical stresses on cultures of suspensed CHO-6E6 cells.

The effect of hydrodynamic forces on animal cell cultures, while extensively studied, still lacks significant, fundamental understanding. A previous manuscript reported on the acute exposure of CHO cells to hydrodynamic forces in a second generation convergent-divergent microfluidic device (Mollet et al., 2007). In this study, the use of this device is extended in a proof of concept system in which suspended animal cells, grown in a typical bioreactor, are subjected to chronic exposure of moderately high levels of hydrodynamic forces by way of a continuous recycle loop between the bioreactor and the microfluidic device. A strain of CHO cells (CHO-6E6) was grown in a batch culture under controlled pH, temperature, and dissolved oxygen conditions. At mid exponential stage of growth in the bioreactor the recycle flow was initiated. The cells either stopped growing or started dying at EDR values that were significantly lower (one to two orders of magnitude) than those previously reported to kill cells from a single, acute exposure. These observations allow further refinement in the design of bioprocess equipment since it provides a more accurate threshold, above which one does not want to subject animal cells to continuous exposure to specific levels of hydrodynamic forces.

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.

Acute hydrodynamic forces and apoptosis: a complex question.

A second generation flow contraction device was developed and modeled which allows cells to be subjected to well-defined hydrodynamic forces. Studies were conducted with this system on wild-type Chinese Hamster Ovary cells (CHO-K1) and a strain of CHO cells which expresses the human Bcl-2 triangle gene (CHO-bcl-2). In this study, the following questions were asked: (1) Does an acute hydrodynamic force induce apoptosis in wild-type CHO and CHO-bcl-2 cells? (2) Does the type of culture media make a difference with respect to the induction of apoptosis or necrosis? and (3) Does culture history affect induction of apoptosis or necrosis? The results obtained with this new flow contraction device and corresponding computer simulations are consistent with previously published studies with respect to the level of energy dissipation rate (EDR) required to create significant cell lysis. Second, while detectable relative to the control in the T-flask experiments, only a small fraction of the cells become apoptotic when exposed to a sub-lysis level of EDR (<10(8) W x m(-3)). Third, cells cultured in suspension with serum free media do not exhibit any higher or lower sensitivity (with respect to apoptosis) to various levels of EDR when compared to control cultures grown in T-flask and serum containing media; on the other hand, necrosis is significantly increased in experiments performed on suspended cells without serum. Fourth, the addition of the Bcl-2 gene product might slightly reduce the occurrence of apoptosis in T-flask culture; however, the baseline response is so low that the difference is insignificant.

Bioprocess equipment: characterization of energy dissipation rate and its potential to damage cells.

A study was conducted in which analytical, computational, and experimental measurements combined with analysis were made to characterize the local energy dissipation rate in a variety of conditions, vessels, and geometries that animal cells would encounter in typical bioprocessing situations. With no gas-liquid interfaces present, as expected, the local energy dissipation rate is typically orders of magnitude lower than what has been experimentally demonstrated to catastrophically damage typically used, suspended animal cells. However, local energy dissipation rates shown to remove animal cells from microcarriers are achievable under some normal operating conditions and geometries. Whether local energy dissipation rates created under typical operating conditions can have nonlethal effects is still an open question and currently under investigation. Whether the sensitivity of other, nontypical, suspended animal cells such as cells obtained directly from tissue (primary cells) and clusters of cells, such as islets, are more sensitive than the typically used cells is also still under investigation.

Synthesis and antitubulin activity of N1- and N4-substituted 3,5-dinitro sulfanilamides against African trypanosomes and Leishmania.

Thirty analogues of N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-propylsulfanilamide (GB-II-5, compound 3), a new antikinetoplastid antimitotic agent, have been synthesized and evaluated. The addition of simple functional groups to the N1 aromatic ring generally decreases antiparasitic and antimitotic potency, but placement of a dibutyl substituent at the N4 nitrogen to give N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (compound 35) augments antitrypanosomal and antileishmanial activity. Compound 35 possesses IC(50) values of 0.12 and 2.6 microM against cultured T. brucei and L. donovani amastigote-like forms, surpassing the activity of compound 3 against these parasites by 3.4- and 1.9-fold, respectively. Compound 35 inhibits the assembly of leishmanial tubulin with an IC(50) of 6.9 microM and displays antimitotic effects in cultured T. brucei as assessed by flow cytometry, but shows little effect on purified mammalian tubulin, and displays 100-fold selectivity for trypanosomes over two mammalian cell lines. Although 3 and 35 were not effective in initial in vivo antitrypanosomal assays, the in vitro potency and selectivity of these compounds make N(1)-aryl-3,5-dinitro-N(4),N(4)-dialkylsulfanilamides a promising new class of antikinetoplastid agents that act on parasite tubulin.