Hydrodynamic damage to animal cells.

Animal cells are affected by hydrodynamic forces that occur in culture vessel, transfer piping, and recovery operations such as microfiltration. Depending on the type, intensity, and duration of the force, and the specifics of the cell, the force may induce various kinds of responses in the subject cells. Both biochemical and physiological responses are observed, including apoptosis and purely mechanical destruction of the cell. This review examines the kinds of hydrodynamic forces encountered in bioprocessing equipment and the impact of those forces on cells. Methods are given for quantifying the magnitude of the specific forces, and the response thresholds are noted for the common types of cells cultured in free suspension, supported on microcarriers, and anchored to stationary surfaces.     

Influence of hydrodynamic shear stress on microcarrier-attached cell growth: Cell line dependency and surfactant protection

Animal cell injuries due to fluid-mechanical forces generated by hydrodynamic mixing in bioreactors could become more detrimental for microcarrier systems. In this work, the effects of cell protective surfactants such as pluronic F68 (F68) and polyvinyl alcohol (PVA) were investigated on microcarrier-grown cells under various hydrodynamic stress conditions. Experimental results indicated that adding 0.2% F68 or 0.2% PVA in media enhanced Vero Cell growth against fluid-mechanical forces, but not CHO-K1 and BHK-21 cells. Although affecting the specific growth rate of Vero cells, hydrodynamic shear forces only slightly influenced CHO-K1 and BHK-21 cells. The cellular sensitivity against fluid-mechanical forces for these three cell lines had the following order: Vero cells > CHO-K1 cells > BHK-21 cells. It seems that, the hydrodynamic effects on microcarrier-grown cells and the effectiveness of surfactant protection are heavily dependent on the culture cell type.     

The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding

Suspension animal cell culture is now routinely scaled up to bioreactors on the order of 10,000 L, and greater, to meet commercial demand. However, the concern of the ‘shear sensitivity’ of animal cells still remains, not only within the bioreactor, but also in the downstream processing. As the productivities continue to increase, titer of ~10 g/L are now reported with cell densities greater than 2 × 10⁷ cells/mL. Such high, and potentially higher cell densities will inevitably translate to increased demand in mass transfer and mixing. In addition, achieving productivity gains in both the upstream stage and downstream processes can subject the cells to aggressive environments such as those involving hydrodynamic stresses. The perception of ‘shear sensitivity’ has historically put an arbitrary upper limit on agitation and aeration in bioreactor operation; however, as cell densities and productivities continue to increase, mass transfer requirements can exceed those imposed by these arbitrary low limits. Therefore, a better understanding of how animal cells, used to produce therapeutic products, respond to hydrodynamic forces in both qualitative and quantitative ways will allow an experimentally based, higher, “upper limit” to be created to guide the design and operation of future commercial, large scale bioreactors. With respect to downstream hydrodynamic conditions, situations have already been achieved in which practical limits with respect to hydrodynamic forces have been experienced. This review mainly focuses on publications from both the academy and industry regarding the effect of hydrodynamic forces on industrially relevant animal cells, and not on the actual scale-up of bioreactors. A summary of implications and remaining challenges will also be presented.     

Death of stoma guard cells in leaf epidermis under disturbance of energy provision

Cyanide is an apoptosis inducer in stoma guard cells from pea leaf epidermis. Unlike CN-, the uncoupler of oxidative and photosynthetic phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), the combination of CCCP, 3-(3 ,4 -dichlorophenyl)-1,1-dimethylurea (DCMU), benzylhydroxamate (BH), myxothiazol, antimycin A, and a glycolysis inhibitor 2-deoxyglucose (DG) did not induce destruction of guard cell nuclei for 20 h of incubation of epidermal peels in the light. DCMU prevented the effect of CN- as a programmed cell death (PCD) inducer. CCCP, the combination of DCMU and CCCP, or the combination of DCMU, CCCP, BH, myxothiazol, antimycin A, and DG supplemented by CN- caused destruction of cell nuclei; the number of the cells lacking nuclei in this case was higher than with CN- alone. DG and CCCP caused cell destruction after longer incubation of the isolated epidermis - after 2 days and to a greater degree after 4 days. The effect of DG and CCCP was reduced by illumination. Cell destruction during long-term incubation was prevented by the combination of DG and CCCP. From data of electron microscopy, DCMU and dinitrophenyl ester of iodonitrothymol (DNP-INT) prevented apoptotic changes of the nuclear ultrastructure induced by CN-. The suppression of the destruction of the guard cell nuclei under combined action of DG and CCCP was apparently caused by switching of cell death from PCD to necrosis. Thus, the type of cell death - via apoptosis or necrosis - is controlled by the level of energy provision.     

Integrin-associated protein and thrombospondin-1 as endothelial mechanosensitive death mediators

Recently, it was reported that the offset of hemodynamic forces induces an unusual pattern of apoptosis in vascular endothelium (1). Although the apoptotic trigger covers all cells and is maintained for a longer time period, only few cells become apoptotic. So, in contrast to common apoptosis inducers, the lack of hemodynamic forces initiates only a low basal level of apoptosis, however steadily increases with time, this way preventing the complete vessel destruction upon an only transient offset of blood flow. The molecular means by which the mechanical stimulus and apoptosis are smoothly coupled have now been identified as an autocrine loop of thrombospondin-1 (TSP-1) and the alpha(v)beta(3) integrin/integrin-associated protein (IAP) complex as its receptor. Vascular EC (EC) secrete TSP-1 only in postconfluent static monolayers and not under flow. This also holds true for the IAP whereas the alpha(v)beta(3) integrin is present under static conditions, as well as under flow, assigning the IAP an essential and new switch function in the receptor complex.     

In vivo and in vitro studies on apoptosis in OSE cells and inclusion cysts of pregnant heifers

Elevated progesterone concentration during pregnancy and use of progesterone-like contraceptives are known to reduce ovarian cancers. This study was undertaken to decipher whether or not there is any relationship between progesterone (also oestrogen)-mediated ovarian surface epithelium (OSE) apoptosis and expression of p53, a cell-cycle arresting protein and potential tumour suppressor. Immunohistochemical staining with cytokeratin confirmed epithelial nature of the cells in the OSE layer and inclusion cysts that invaginate inside stroma after ovulation takes place. The in situ apoptosis index was determined during oestrus, and at mid and late-pregnancy stages in heifers. Epithelia of both tissues exhibited significantly high nuclear staining, suggesting that these cells are aiming to apoptotic destruction. To further establish a role of progesterone, the OSE cells were exposed in vitro to two concentrations of oestrogen and progesterone. It was revealed that progesterone at both concentrations and oestrogen only at high concentration converted a large proportion of these cells apoptotic. The stimulatory effect of progesterone (and to some extent oestrogen) was also seen on p53 expression in the same cultivated OSE cells. The steroid dosage dependence for apoptosis and p53 expression was also somewhat similar. Assuming that progesterone action is mediated through p53-caused apoptosis as a mechanism to evade malignant transformation of OSE cells, p53 expression at mRNA and protein level was investigated in the OSE layer in proximity to stroma, antrum and corpus luteum (CL). In cycling animals CL produces a large amount of progesterone and also oestrogen to maintain the post-ovulatory cycle and to suppress the gonadotropin production. Hence, cells undergoing re-epithelialization and which are in contact with CL were expected to undergo maximum apoptotic modification. Indeed we got the maximum p53/p53 gene expression in these cells. We conclude that progesterone during cycling and pregnancy may reduce the risk of developing ovarian cancer by ceasing cell cycle and diverting damaged and mutagenized OSE cells for apoptosis, and the process may be mediated through elevated p53 synthesis. However, it is also possible that progesterone and p53-induced apoptosis may be entirely different cancer suppressive actions but coincidently happening together.     

Thermal and Hydrodynamic Environments Mediate Individual and Aggregative Feeding of a Functionally Important Omnivore in Reef Communities

In eastern Canada, the destruction of kelp beds by dense aggregations (fronts) of the omnivorous green sea urchin, Strongylocentrotus droebachiensis, is a key determinant of the structure and dynamics of shallow reef communities. Recent studies suggest that hydrodynamic forces, but not sea temperature, determine the strength of urchin-kelp interactions, which deviates from the tenets of the metabolic theory of ecology (MTE). We tested the hypothesis that water temperature can predict short-term kelp bed destruction by S. droebachiensis in calm hydrodynamic environments. Specifically, we experimentally determined relationships among water temperature, body size, and individual feeding in the absence of waves, as well as among wave velocity, season, and aggregative feeding. We quantified variation in kelp-bed boundary dynamics, sea temperature, and wave height over three months at one subtidal site in Newfoundland to test the validity of thermal tipping ranges and regression equations derived from laboratory results. Consistent with the MTE, individual feeding during early summer (June-July) obeyed a non-linear, size- and temperature-dependent relationship: feeding in large urchins was consistently highest and positively correlated with temperature <12°C and dropped within and above the 12–15°C tipping range. This relationship was more apparent in large than small urchins. Observed and expected rates of kelp loss based on sea temperature and urchin density and size structure at the front were highly correlated and differed by one order of magnitude. The present study speaks to the importance of considering body size and natural variation in sea temperature in studies of urchin-kelp interactions. It provides the first compelling evidence that sea temperature, and not only hydrodynamic forces, can predict kelp bed destruction by urchin fronts in shallow reef communities. Studying urchin-seaweed-predator interactions within the conceptual foundations of the MTE holds high potential for improving capacity to predict and manage shifts in marine food web structure and productivity.     

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.     

Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.     

The hydrodynamic components in joints: proof of a membrane-cistern system within the calcified zone of the femur head cartilage

The theoretical postulate of a hydrodynamic system for the transmission of forces at the hip joint led to the discovery of a two-membrane system. Within the calcified cartilaginous zone of the femoral head there exists a system of small spaces and cisterns which can only be understood in the postulated sense as an overall acting hydrodynamic system. A correlation between the destruction of the calcified zone, i.e. the hydrodynamic system, and arthrosis is indicated. Ongoing studies and deliberations will help to discover the true model of the transmission of forces and impulses and to solve the problems of treating arthrosis.     

Hydrodynamic flow-mediated protein sorting on the cell surface of trypanosomes

The unicellular parasite Trypanosoma brucei rapidly removes host-derived immunoglobulin (Ig) from its cell surface, which is dominated by a single type of glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG). We have determined the mechanism of antibody clearance and found that Ig-VSG immune complexes are passively sorted to the posterior cell pole, where they are endocytosed. The backward movement of immune complexes requires forward cellular motility but is independent of endocytosis and of actin function. We suggest that the hydrodynamic flow acting on swimming trypanosomes causes directional movement of Ig-VSG immune complexes in the plane of the plasma membrane, that is, immunoglobulins attached to VSG function as molecular sails. Protein sorting by hydrodynamic forces helps to protect trypanosomes against complement-mediated immune destruction in culture and possibly in infected mammals but likewise may be of functional significance at the surface of other cell types such as epithelial cells lining blood vessels.     

Physical disturbance and community organization on a subtidal cobble bed

This study examines the role of physical disturbance in regulating the development and maintenance of macroalgal community structure in a subtidal cobble bed on the Atlantic coast of Nova Scotia, Canada, after a mass mortality of sea urchins. Displacement by hydrodynamic forces of cobbles with and without attached macroalgae was measured in mark-recovery experiments. Cobbles without macroalgae experienced low levels of overturning or displacement by waves. In contrast, cobbles with large fleshy algae (such as kelps and fucoids) were displaced by drag forces along the seabed during periods of moderately strong wave action. Displacement of these “drifters” occurred once the attached alga reached a critical mass (relative to the weight of the anchoring cobble), which varied among species of differing morphology. The rate of displacement of drifters was an exponential function of the ratio of attached algal biomass to rock weight. The loss of larger erect forms from the cobble bed results in a patchwork of low-lying turfs. Clusters of small boulders, experimentally transplanted to the cobble bed, accumulated a greater biomass of large fleshy algae than surrounding cobbles after one year, likely because of the greater stability of boulders. Algal-mediated hydrodynamic displacement of cobbles appears to be an important form of physical disturbance in the cobble habitat, restricting the types and sizes of algae that can grow on cobbles following an urchin die-off.     

Quantification of damage to suspended insect cells as a result of bubble rupture

It is proposed that when cells are either attached to, or very near, a rupturing bubble, the hydrodynamic forces associated with the rupture are sufficient to kill the cells. Four types of experiments were conducted to quantify the number and location of these killed cells. We determined: (1) the number of cells killed as a result of a single, 3.5-mm bubble rupture; (2) the number and viability of cells in the upward jet that results when a bubble ruptures; (3) the number of cells on the bubble film; and (4) the fate of cells attached to the bubble film after film rupture. All experiments were conducted with Spodoptera frugiperda (SF-9) insect cells, in TNM-FH and SFML medium, with and without Pluronic F-68. Experiments indicate that approximately 1050 cells are killed per single, 3.5-mm bubble rupture in TNM-FH medium and approximately the same number of dead cells are present in the upward jet. It was also observed that the concentration of cells in this upward jet is higher than the cell suspension in TNM-FH medium without Pluronic F-68 by a factor of two. It is believed that this higher concentration is the result of cells adhering to the bubble interface. These cells are swept up into the upward jet during the bubble rupture process. Finally, it is suggested that a thin layer around the bubble containing these absorbed cells is the "hypothetical killing volume" presented by other researchers. (c) 1994 John Wiley & Sons, Inc.     

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.     

The sensitivity of the dinoflagellate Crypthecodinium cohnii to transient hydrodynamic forces and cell-bubble interactions

The increased interest in the benefits of omega-3 fatty acids for human health has resulted in the commercial development of the dinoflagellate Crypthecodinium cohnii for production of docosahexaenoic acid (DHA). The growing market demand for DHA requires highly efficient, very large scale cultures of DHA. While the effects of hydrodynamic forces on dinoflagellates have been investigated for several decades, the majority of the work focused on the negative effects of oceanic turbulence on the population growth of environmentally important dinoflagellates. In contrast, significantly less is known on the effect of hydrodynamic forces encountered by algae in bioprocesses. Unlike other studies conducted on algae, this study employed a microfluidic, flow contraction device to evaluate the effect of transient hydrodynamic forces on C. cohnii cells. It was found that C. cohnii cells can sustain the energy dissipation rate of 5.8 x 10(7) W/m3 without lysis. However, an obvious sublethal effect, the loss of flagella, was observed at a lower level of 1.6 x 10(7) W/m3. Finally the cell-bubble interaction and the effect of bubble rupture were also explored to simulate the conditions of sparged bioreactors.     

Anion Transport or Nucleotide Binding by Ucp2 Is Indispensable for Ucp2-Mediated Efferocytosis

Rapid and efficient engulfment of apoptotic cells is an essential property of phagocytes for removal of the large number of apoptotic cells generated in multicellular organisms. To achieve this, phagocytes need to be able to continuously uptake apoptotic cells. It was recently reported that uncoupling protein 2 (Ucp2) promotes engulfment of apoptotic cells by increasing the phagocytic capacity, thereby allowing cells to continuously ingest apoptotic cells. However, the functions of Ucp2, beyond its possible role in dissipating the mitochondrial membrane potential, that contribute to elevation of the phagocytic capacity have not been determined. Here, we report that the anion transfer or nucleotide binding activity of Ucp2, as well as its dissipation of the mitochondrial membrane potential, is necessary for Ucp2-mediated engulfment of apoptotic cells. To study these properties, we generated Ucp2 mutations that affected three different functions of Ucp2, namely, dissipation of the mitochondrial membrane potential, transfer of anions, and binding of purine nucleotides. Mutations of Ucp2 that affected the proton leak did not enhance the engulfment of apoptotic cells. Although anion transfer and nucleotide binding mutations did not affect the mitochondrial membrane potential, they exerted a dominant-negative effect on Ucp2-mediated engulfment. Furthermore, none of our Ucp2 mutations increased the phagocytic capacity. We conclude that dissipation of the proton gradient by Ucp2 is not the only determinant of the phagocytic capacity and that anion transfer or nucleotide binding by Ucp2 is also essential for Ucp2-mediated engulfment of apoptotic cells.     

Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces

A microfluidic device was fabricated via photolithographic techniques which can create transient elongational and shear forces ranging over three orders of magnitude while still maintaining laminar flow conditions. The contractional fluid flow inside the microfluidic device was simulated with FLUENT (a computational fluid dynamics computer program) and the local deformation forces were characterized with the scalar quantity, local energy dissipation rate. The sensitivities of four cell lines (CHO, HB-24, Sf-9, and MCF7) were tested in the device. The results indicate that all four cell lines are able to withstand relatively intense energy dissipation rates (up to 10(4)-10(5) kW/m(3)), which is orders of magnitude higher than the maximum local energy dissipation rates generated by impellers in bioreactors, but comparable to that associated with small bursting bubbles. While the concept that suspended animal cells are relatively robust with respect to purely hydrodynamic forces in bioprocess equipment is well known, these results quantitatively demonstrate these observations.     

斜纹夜蛾核型多角体病毒DNA诱导同源昆虫细胞的凋亡

发现野生型斜纹夜蛾核型多角体病毒（Spodoptera litura nuclear polyhedrosis virus,SpltNPV）DNA转染SL-1细胞能诱导细胞凋亡.SpltNPV-DNA转染其同源细胞系斜纹夜蛾核SL-1细胞6 h后,光镜下即可见细胞膜表面突出或形成小泡,细胞碎裂成凋亡小体,18 h后,细胞100%碎裂成凋亡小体.DAPI荧光染色显示感染细胞核渐呈半月形,直至碎裂被凋亡小体包裹.被转染的SL-1细胞DNA琼脂糖凝胶电泳呈典型梯形谱带.野生型SpltNPV病毒粒子感染的SL-1细胞既无多角体的出现,也无凋亡现象的发生.