Non‐Newtonian rheology in suspension cell cultures significantly impacts bioreactor shear stress quantification

The fields of regenerative medicine and tissue engineering require large‐scale manufacturing of stem cells for both therapy and recombinant protein production, which is often achieved by culturing cells in stirred suspension bioreactors. The rheology of cell suspensions cultured in stirred suspension bioreactors is critical to cell growth and protein production, as elevated exposure to shear stress has been linked to changes in growth kinetics and genetic expression for many common cell types. Currently, little is understood on the rheology of cell suspensions cultured in stirred suspension bioreactors. In this study, we present the impact of three common cell culture parameters, serum content, cell presence, and culture age, on the rheology of a model cell line cultured in stirred suspension bioreactors. The results reveal that cultures containing cells, serum, or combinations thereof are highly shear thinning, whereas conditioned and unconditioned culture medium without serum are both Newtonian. Non‐Newtonian viscosity was modeled using a Sisko model, which provided insight on structural mechanisms driving the rheological behavior of these cell suspensions. A comparison of shear stress estimated by using Newtonian and Sisko relationships demonstrated that assuming Newtonian viscosity underpredicts both mean and maximum shear stress in stirred suspension bioreactors. Non‐Newtonian viscosity models reported maximum shear stresses exceeding those required to induce changes in genetic expression in common cell types, whereas Newtonian models did not. These findings indicate that traditional shear stress quantification of cell or serum suspensions is inadequate and that shear stress quantification methods based on non‐Newtonian viscosity must be developed to accurately quantify shear stress.     

Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines

Data on viscous (eta') and elastic (eta') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 mum as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 mum mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, \documentclass{article}\pagestyle{empty}\begin{document}$ \eta = \eta _0 \exp [K\dot \gamma ;{ - \beta } \phi /(1 - K'\sigma \phi _c /D)] $\end{document} was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field. (c) 1993 John Wiley & Sons, Inc.     

Effects of cell motility and morphology on the rheology of algae suspensions

Algae have been proposed as a source of biofuels and high value chemical products, but if this potential is to be fully realised, it is crucial to understand the factors affecting the suspension rheology. Suspensions of three algae species, Tetraselmis chuii, Chlorella sp. and Phaeodactylum tricornutum, were sheared in a rotational rheometer in order to characterise their rheology and examine the effects of cell concentration, motility and morphology. The volume fraction ranged from 0.05 to 0.2, and the shear rate from 20 to 200 s^?1. The rheology measurements are fitted to the Herschel-Bulkley model, and the intrinsic viscosity is estimated using both Einstein’s equation and the Krieger-Dougherty model, which are found to perform well for low concentrations. The intrinsic viscosity of T. chuii suspensions is shown not to be constant, but decreases with strain rate, indicating that the suspension viscosity is less sensitive to the cell concentration at high strain rates. The rate of decline is constant for strain rates below approximately 100 s^?1, after which it continues to decline linearly, but at a slower rate. It is speculated that this transition at 100 s^?1 is related to the appearance of flocculation at low strain rates. The effect of the cell motility on the rheology of T. chuii suspensions is investigated by comparing the rheology of motile and passive cells. The shear-thinning behaviour is absent and the effective viscosity is considerably lower for the passive cell suspensions, indicating that the motility of the T. chuii cells causes them to align to resist the flow. In contrast, the Chlorella sp. suspensions exhibit shear-thickening behaviour, which has not previously been reported. Finally, the influence of the effective aspect ratio on the cell suspensions is examined by comparing the intrinsic viscosity of all three species. The algal species with the largest aspect ratio, P. tricornutum, has the largest intrinsic viscosity, while the smallest aspect ratio strain, Chlorella sp., has the smallest viscosity. However, it is shown that the increase in viscosity of motile compared to non-motile T. chuii suspensions cannot be attributed to a change in the effective aspect ratio of individual cells due to the motion of the flagella alone.     

Plant cell suspension culture rheology

The results of rheological measurements on 10 different plant cell suspension cultures are presented. Nicotiana tabacum (tobacco) suspension cultures grown in serial batch subculture display high viscosity and power law rheology. This "undesirable" rheology is shown to be a result of elongated cell morphology. The rheology of Papaver somniferum (poppy) cell suspensions is quite different; poppy suspensions behave as Newtonian fluids and have relatively low viscosity (less than 15 cP) at fresh cell densities up to 250 g/L. This flow behavior can be attributed to a lack of elongation in batch-grown poppy cells. A simple correlation for the viscosity as a function of cell density is developed for poppy suspensions up to 300 g fresh weight (FW)/L. It is shown that tobacco cells do not elongate when grown in semicontinuous culture (daily media replacement). These semicontinuously cultured cells have rheological behavior that is indistinguishable from that of poppy, further confirming the dependence of rheology on plant cell morphology. The rheology of a wide variety of other plant suspensions at 200 g FW/L is presented. Most cell suspensions, including soybean, cotton, bindweed, and potato, display low viscosities similar to poppy suspensions. Only carrot and atriplex exhibit slight pseudoplastic behavior which corresponded to a slight degree of cellular elongation for these cultures. This demonstrates that complex rheology associated with elongated cell morphology is much less common than low-viscosity Newtonian behavior. High viscosity in plant cell culture is therefore not an intrinsic characteristic of plant cells but, instead, is a result of the ability to grow cultures to extremely high cell densities due to low biological oxygen demand. (c) 1993 John Wiley & Sons, Inc.     

Regenerative medicine bioprocessing: Concentration and behavior of adherent cell suspensions and pastes

Regenerative medicines based on human cells demand their harvesting, culture, and processing. Manufacturing processes are likely to include cell concentration and subsequent controlled dosing of concentrates, for example, to the patient or tissue construct. The integrity and functionality of the cells must be maintained during these processing stages. In this study the performance of two different cell concentration protocols (involving centrifugation and resuspension) are compared and consideration given to possible causes of cell loss. Further studies examine cell size and rheological behavior of anchorage‐dependent mammalian cell suspensions, and the effect of capillary flow stress (0.5–15 Pa, laminar flow regime) on cell number and membrane integrity as quantified by flow cytometry. The cell concentration protocols achieved maximum cell volume fraction of around 0.3 and the improved protocol exhibited intact cell yield of 80 ± 13%, demonstrating proof‐of principle for achieving tissue‐like cell concentrations by a process of centrifugation and orbital shaking. Volume mean cell diameter (cell diameter at the mean cell volume) for the rat aortic smooth muscle cells (CRL‐1444) used in this study was 22.4 µm. Concentrated cell suspension rheology approximated to power law behavior and exhibited similar trends to reports for plant and yeast cells. Capillary transfer at 2–15 Pa (wall shear stress) did not significantly affect cell number or membrane integrity while losses observed at low shear (0.5, 1.0 Pa) were probably due to surface attachment of cells in the apparatus. Biotechnol. Bioeng. 2009;103: 1236–1247. © 2009 Wiley Periodicals, Inc.     

Theoretical and experimental analysis of the sedimentation kinetics of concentrated red cell suspensions in a centrifugal field: determination of the aggregation and deformation of RBC by flux density and viscosity functions

The flow properties of blood are mostly determined using various viscometric approaches, and described in terms of a shear rate or shear stress dependent apparent viscosity. The interpretation of results are rather difficult, especially at low shear rates when particle sedimentation and migration within the viscometer gap are significant. By contrast, analysing the separation process in concentrated RBC suspensions in a centrifugal field also yields information about the viscosity function, including particle-particle interaction and deformation parameters. In this paper, the sedimentation process is approached by means of the theory of kinematic waves and theoretically described by solving the corresponding one-dimensional quasi-linear partial differential equation based on viscosity/flow function as a function of volume concentration. The sedimentation kinetics of rigid spherical RBC suspended in saline and normal RBC suspended in Dx-saline solutions were investigated by means of a separation analyser (LUMiFuge 114). The instrument detects the light transmission over the total length of the cell containing the suspension. During centrifugation the analyser automatically determines the position of the particle free fluid/suspension interface or the sediment by means of a special algorithm. The data obtained with sedimentation of rigid spherical RBC at different volume concentrations demonstrate that, in the case of suspensions rotated in containers of constant cross section, there is good agreement between the theory of kinematic waves developed by Anestis and Schneider (1983) and the results of the experiments. Such good agreement was obtained even though a restrictive one-dimensional model was used to obtain the theoretically derived sedimentation time course. In addition, we describe an algorithm enabling the experimental determination of the viscosity and related flux density function to be made for any suspension. Through this approach, we investigated in detail the rheological behavior of suspended rigid spheres at low Reynolds numbers ranging from 10(-6) to 10(-3). The method here introduced also enabled us to investigate RBC suspensions with respect to the deformability and interactions of the cells by means of the separation analysis. Normal, rigid as well as aggregating RBC exhibited marked differences in the sedimentation kinetics, which were quantified by means of the flux and viscosity functions based on the theory of kinematic waves.     

A new photometric method for oxygen consumption measurements in cell suspensions

A new technique is described for measuring O2 consumption rates and O2 concentrations in suspensions of respiring cells. Aliquots of a cell suspension kept in a special thermostated precision syringe are injected into the measuring system in defined time intervals. The O2 content of these samples is determined photometrically, as reported previously. The O2 consumption per cellular wet weight and/or per single cell can be calculated from the cell volume fraction, the physical density, the cell concentration in the suspension, and the time-dependent decline of the O2 concentration in the precision syringe. The minimum detectable amount of O2 is 0.1 microliter O2, which corresponds to 0.001 (vol/vol) of O2 if a 100-microliters sample of suspended cells is analyzed. Reproducibility of the O2 consumption measurement is 9% of the measured value. The advantages offered by this method are the straightforward calibration in absolute terms, the short time required for one analysis (2-6 min), a high sensitivity, the simultaneous determination of overall O2 concentration and O2 consumption rates in cell suspensions, and the great variability in the application.     

Rheological properties of agar microgel suspensions prepared using water-in-oil emulsions

Agar microgel suspensions containing sugar fatty acid esters were prepared using a water-in-oil (w/o) emulsion system. The volume fraction of microgel particles was adjusted by centrifugation to obtain high volume fraction suspensions. The relationship between the apparent equilibrium shear modulus and the volume fraction was analyzed using a hard-sphere model. The rigidity of the microgel particles never affected the rheological property under the random close-packed-volume fraction, but it affected the fracture of the structure composed of packed microgel particles. The length of fatty acid emulsifiers also affected the rheological properties of the condensed suspensions. The stress growth under step shear flow was observed to study the lubrication effect of close-packed microgel particles containing ER-190 (C 22:1) or O-170 (C 18:1). Although both suspensions showed strain softening, the tendency was different in each. Such a w/o microgel suspension is a likely candidate for use in low-fat foods.     

Shear Flow Behavior of Aqueous Suspensions of Potato Parenchyma Powder

The shear flow behavior of potato powder suspensions prepared from two different particle sizes and with a range of solids volume fraction (Φ) was studied. A concentrated sucrose solution was used as the continuous phase to maintain particle buoyancy. The shear flow properties were measured at 20, 50 and 80 °C. The suspensions obeyed a power-law equation in the dilute regime while the Herschel-Bulkley equation was the best fit for almost all semi-dilute and more concentrated suspensions. With increasing Φ, particle size and temperature, a gradual development of shear-thinning behavior was evident which coincided with an increase in the consistency index and the development of a yield stress in the suspensions. Potato powder suspensions therefore behave very differently to potato starch suspensions, with flow properties dominated by the effect of intra- and inter-cellular components in the potato powder particles that are transferred to the continuous phase and that alter suspension properties.     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

Relationship between Salt Tolerance and Resistance to Polyethylene Glycol-Induced Water Stress in Cultured Citrus Cells

Salt-tolerant selected cells of Shamouti orange (Citrus sinensis) and Sour orange (Citrus aurantium) grew considerably better than nonselected cells at any NaCl concentration tested up to 200 millimolar. Also, the growth response of each treatment was identical in the two species. However, the performance of cells of the two species under osmotic stress induced by polyethylene glycol (PEG), which is presumably a nonabsorbed osmoticum, was significantly different. The nonselected Shamouti cell lines were significantly more sensitive to osmotic stress than the selected cells. The salt adapted Shamouti cells were apparently also adapted to osmotic stress induced by PEG. In Sour orange, however, the selected lines had no advantage over the nonselected line in response to osmotic stress induced by PEG. This response was also similar quantitatively to the response of the selected salt-tolerant Shamouti cell line. It seems that the tolerance to salt in Shamouti, a partial salt excluder, involves an osmotic adaptation, whereas in Sour orange, a salt accumulator, such an adaptation apparently does not occur. PEG-induced osmotic stress causes an increase in the percent dry weight of salt-sensitive and salt-tolerant cells of both species. No such increase was found under salt stress. The size of control and stressed cells is not significantly different.     

Dependence of the gametogenesis induction,zygote formation and their germination on the culture density of the homothallic algaChlamydomonas geitleri Ettl

The induction of gametogenesis has its beginning in the most diluted cell suspension after the transfer of the cells to a nitrogen-less medium. Here, the highest percentage frequencies of zygotes are formed within the same period of time in comparison with the less diluted cell suspensions. The zygotes formed in the mostly diluted cell suspensions mature relatively very slowly and germinate very irregularly. The induction of gametogenesis retards in the denser cultures probably due to the strongest homeostatic forces trying to return the cell population to its initial stage. In our experiments up to now, the effects of the changing irradiance of the cells inside the suspension were not separated from the effects of the changing number of the cells in the given volume of the culture. Neither were the ratios of distilled water to the amount of the zygotes, nor to the vegetative cells constant. Thus, it is necessary to consider the mentioned effects as the result of an interaction of both factors under consideration. Each of the cell populations behaved as an autonomous whole. Also the populations, starting after the transfer to a nitrogen-less medium of the same culture density, may sometimes differ significantly in the observed characteristic features at various phases of their growth. The discontinued supply of nitrogen obviously causes a change of the cell metabolism in favour of the nitrogen-less substances, especially in the more diluted cell suspension. This work completes and explains some earlier results obtained from the study of the life cycle inChlamydomonas geitleri.     

Increased capsaicin content in PFP-resistant cells of chili pepper (Capsicum annuum L.)

Cell suspensions of chili pepper (Capsicum annuum L.) were subjected to a selection process on semisolid medium containing the amino acid analog p-fluorophenylalanine (PFP). Four cell lines with different degrees of resistance were selected and suspension cultures were established from each of them. Resistance was retained even after 75 days of culture in the absence of PFP. PFP-resistant cell lines accumu lated higher levels of capsaicin than sensitive lines even after prolonged culture in PFP-free medium. Capsaicin production in non-selected cells was only 26.8% of that found in one cell line resistant to 500 M PFP. The capsaicin content in the non-selected cell suspension and in one of the resis tant cell lines was 6.7% and 24.9% respectively, that of fruits.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - PFP p-fluorophenylalanine - d. wt. dry weight - f. wt. fresh weight     

An elasto-visco-plastic model of cell aggregates

Concentrated cell suspensions exhibit different mechanical behavior depending on the mechanical stress or deformation they undergo. They have a mixed rheological nature: cells behave elastically or viscoelastically, they can adhere to each other whereas the carrying fluid is usually Newtonian. We report here on a new elasto-visco-plastic model which is able to describe the mechanical properties of a concentrated cell suspension or aggregate. It is based on the idea that the rearrangement of adhesion bonds during the deformation of the aggregate is related to the existence of a yield stress in the macroscopic constitutive equation. We compare the predictions of this new model with five experimental tests: steady shear rate, oscillatory shearing tests, stress relaxation, elastic recovery after steady prescribed deformation, and uniaxial compression tests. All of the predictions of the model are shown to agree with these experiments.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Disintegration of Microorganisms and Preparation of Yeast Cell Walls in a New Type of Disintegrator

Microbial cells were disintegrated in a new type of rotary disintegrator with a disc stirrer by a combination of shear force layers, collisions, and rolling of glass beads which were brought into motion by the stirrer. The rate of disintegration at a given dry bed volume of Ballotini beads and a given volume of cell suspension is proportional to the peripheral velocity of the stirrer up to 18 m/sec. Horizontal arrangement of the stirrer increases the effectiveness about five times; 100% disintegration of yeast cells was achieved under optimal conditions within 72 sec at a concentration of 3.5g (dry weight)/100 ml of suspension, and within 96 sec at a concentration of 10.5g (dry weight)/100ml. At 17.5 g (dry weight)/100 ml, the stirrer began to slip. The cell walls of yeast were obtained at the desired degree of crushing and the course of purification was determined by infrared spectral analysis.     

Effect of inositol hexaphosphate on the transient behavior of red cells following a DMSO-induced osmotic pulse

An osmotic pulse can be used to incorporate inositol hexaphosphate (IHP) into red cells. The pulse is induced by equilibrating a red cell suspension with DMSO and then rapidly diluting with an isotonic IHP solution. Since IHP binds to hemoglobin and lowers the affinity for oxygen, this method may find application in the preparation of low-affinity cells for experimental and clinical use. The experiments reported here examined the dynamic changes of several red cell variables immediately following the osmotic pulse. The effect of IHP, which has been shown to dissociate red cell cytoskeletons, was evaluated by comparison with a matched phosphate-buffered saline (PBS) diluent. Red cell morphology, volume, and hemoglobin permeability were studied by fixing the cells at times ranging from 0.06 to 300 sec after dilution. Mechanical fragility was measured by subjecting the cells to a short period of shear stress at the same times after dilution. With both diluents, the cells underwent a rapid increase in volume followed by a return towards normal volume with a maximum at less than 250 msec. With IHP diluent, the period of hemoglobin permeability immediately followed the size peak and was completed by about 1 sec after dilution. PBS also induced a second leakage at longer times (10-120 sec), which resulted in a morphological dichotomy with ghosts and intact cells. The choice of diluent also affected sensitivity to shear stress. The IHP-treated cells had a mechanical fragility maximum at about 1 sec. The PBS-treated cells exhibited no enhanced mechanical fragility. An unexpected result was the inhibition of the second phase of lysis in PBS-treated cells by a properly timed shear stress.     

Power Laws in the Elasticity and Yielding of Plant Particle Suspensions

The yielding and flow behaviour of plant suspensions are perhaps the most important rheological properties in process and product design for applications in paper, biofuel and food industries. Studies are reported here on the yield properties and flow behaviour of suspensions of plant particles with different shapes (clusters of cells, individual cells and cell fragments). Carrot and tomato were selected as model plant systems to prepare suspensions at particle dry mass concentrations ranging from 0.010 to 0.065. The flow behaviour was characterised by an apparent yield stress and shear thinning. The Herschel-Bulkley yield stress obtained from up and return flow curves was compared to the yield stress calculated from oscillatory measurements. The dependence of the yield stress values on particle dry mass concentration is approximately a power-law, with a fitted exponent of 3 ± 0.5 for all the suspensions, independently of the plant origin and particle shape. This same power-law behaviour was found for the elastic modulus G′, and in this case the exponent was 3 for carrot and 4 for the tomato suspensions. The yield strain, calculated from oscillatory measurements, decreased slightly with dry mass fraction, but did not follow a power-law. We discuss possible explanations for power law behaviour, and provide a model for G′ based on folded elastic sheets, which predicts an exponent of 3, similar to the values obtained for these suspensions.     

High-frequency transformation of undeveloped plastids in tobacco suspension cells

Although leaf chloroplast transformation technology was developed more than a decade ago, no reports exist of stable transformation of undeveloped plastids or other specialized plastid types, such as proplastids, etioplasts, or amyloplasts. In this work we report development of a dark-grown tobacco suspension cell model system to investigate the transformation potential of undeveloped plastids. Electron microscope analysis confirmed that the suspension cells carry plastids that are significantly smaller (approximately 50-fold less in volume) and have a very different subcellular localization and developmental state than leaf cell chloroplasts. Using antibiotic selection in the light, we demonstrated that both plastid and nuclear transformation of these cell suspensions is efficient and reproducible, with plastid transformation frequency at least equal to that of leaf chloroplast transformation. Homoplasmic plastid transformants are readily obtained in cell colonies, or in regenerated plants, providing a more consistent and versatile model than the leaf transformation system. Because of the uniformity of the cell suspension model, we could further show that growth rate, selection scheme, particle size, and DNA amount influence the frequency of transformation. Our results indicate that the rate-limiting steps for nuclear and plastid transformation are different, and each must be optimized separately. The suspension cell system will be useful as a model for understanding transformation in those plant species that utilize dark-grown embryogenic cultures and for characterizing the steps that lead to homoplasmic plastid transformation.