Mammalian Cells In Suspension Research Articles

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones.

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min -1 at 100 g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 x 10(6) cells ml -1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process. (Himmelfarb et al. 1969; Feder and Tolbert 1983) usually have the problem of filter clogging, and perfusion culture processes with gravitational cell settling (Tokashiki and Arai 1989) are limited in scale-up because of low separation efficiency. Recently centrifugal separation of cells from culture medium has been used for perfusion culture processes (Hamamoto et al. 1989; Tokashiki et al. 1990; Apelman 1992; Jäger 1992). This process essentially has a high separation efficiency and can avoid further operational problems, such as filter clogging, by the type of filtration. To investigate the scale-up feasibility of the centrifuge culture system, we have developed a new type of centrifuge with four layers of spiral cell-settling zones to increase the separation area, and a novel cell separation and withdrawal method using a perfluorocarbon.

Enhanced Settling of Mammalian Cells in Tanks with Inclined Plates/Simulation by Fluid Mechanical Model and Experiment.

In perfusion culture of mammalian cells, the cells should be separated from the spent medium and retained in the culture vessel. A possible cell separator for this purpose is a multiple inclined plate space in an agitated or air-circulated culture vessel. Local fluid velocity and cell concentration contours in an assumed hybridoma cell suspension, which was held stationary in such a gravitational cell settler consisting of multiple open-ended spaces partitioned by inclined plates, were estimated by numerically solving the momentum and continuity equations. The simulated maximum convection flow velocity was 20 times the Stokes’ velocity of the cells. The flow penetrated into the space below the lower ends of the inclined plates. The model estimated a larger cell settling rate than the Ponder-Nakamura-Kuroda (PNK) formula. In our experiment, the cell suspension of hybridoma 2E3 was held stationary in a vessel containing multiple inclined plates, and the position of the interface between the clear and turbid fluids was observed. The falling rate of the interface was larger than that predicted by the PNK formula. The model can be utilized for designing such cell separators for perfusion culture of suspended mammalian cells, including optimization of the size and interval of the plates.

Allografts of CNS tissue possess a blood‐brain barrier: III. Neuropathological, methodological, and immunological considerations

Development of a blood-brain barrier (BBB) within mammalian CNS grafts, placed either intracerebrally or peripherally, has been controversial. Published data from this laboratory have emphasized the presence or the absence of a BBB within solid mammalian tissue or cell suspension grafts is determined intrinsically by the graft and not by the surrounding host parenchyma (e.g., brain, kidney, testis, etc.). Nevertheless, correctly interpreting whether or not a BBB exists within brain grafts is manifested by methodologies employed to answer the question and by ensuing neuropathological and immunological consequences of intracerebral grafting. The present study addresses these issues and suggests misinterpretation for the absence of a BBB in brain grafts can be attributed to: (1) rupture of interendothelial tight junctional complexes in vessels of CNS grafts fixed by perfusion of the host; (2) damage to host vessels and BBB during the intracerebral grafting procedure; (3) graft placement in proximity to inherently permeable vessels (e.g., CNS sites lying outside the BBB) supplying the subarachnoid space/pial surface and circumventricular organs such as the median eminence, area postrema, and choroid plexus; and (4) graft rejection associated with antigen presenting cells and the host immune response. The latter is prevalent in xenogeneic grafts and exists in allogeneic grafts with donor-host mismatch in the major and/or minor histocompatibility complex. CNS grafts between non-immunosuppressed outbred donor and host rats of the same strain (e.g., Sprague Dawley or Wistar rats) can be rejected by the host; these grafts exhibit populations of immunohistochemically identifiable major histocompatibility complex class I+ and class EE+ cells (microglia, macrophages, etc.) and CD4+ T-helper and CD8+ T-cytotoxic lymphocytes. PC12 cell suspension grafts placed within the CNS of non-immunosuppressed Sprague Dawley rats are rejected similarly. Donor cells from solid CNS grafts placed intracerebrally and stained immunohistochemically for donor major histocompatibility complex (MHC) class I expression are identified within the host spleen and lymph nodes; these donor MHC expressing cells may initiate the host immune response subsequent to the cells entering the general circulation through host cerebral vessels damaged during graft placement. Rapid healing of damaged cerebral vessels is stimulated with exogenously applied basic fibroblast growth factor, which may prove helpful in reducing the potential entry of donor cells to the host circulation. These results have implication clinically for the intracerebral grafting of human fetal CNS cell suspensions.

Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant Chinese hamster ovary cells.

The perfusion culture of suspended mammalian cells requires a cell retention device, the best of which will retain all viable cells and reject all nonviable cells and debris. The inclined settler is a passive, simple, inexpensive, and easy-to-maintain device that has been shown in the past to selectively remove single nonviable cells of hybridoma cultures. In this work, we have demonstrated the preferential return of viable recombinant Chinese hamster ovary (CHO) cells through the use of a three-port settler maintained at lower temperatures and vibrated to reduce cell attachment and enhance cell return to the bioreactor. The residence time of CHO cells in the cooled, vibrated settler was determined by flow-cytometric discrimination of tracer recombinant CHO cells. Cells returning to the bioreactor through the underflow had an average residence time of 1.46 h in the settler. During perfusion cultures with cell densities above 10(6) cells/mL, cells seen to be stalled within the settler were easily dislodged by periodic air bubbling using a simple back-flushing procedure in which headspace gas was brought through the settler underflow port. The resuspended cells were returned to the bioreactor within an average of 32 min after bubbling. This study demonstrates that inclined sedimentation technology can be utilized to selectively recycle viable recombinant CHO cells with only a short retention time in an inclined settler.

High-density culture of FM-3A cells using a bioreactor with an external tangential-flow filtration device

A novel bioreactor system developed for high-density cultures of suspended mammalian cells is described using a tangential-flow filtration device outside the culture vessel to separate viable cells from spent medium. The filtration device is based on thin porous microfiltration membranes with a pore size of 0.20-0.65 microns. Because cells have a diameter of about 10-20 microns, they cannot permeate these membranes with the spent medium. So, allowing a perfusion culture to be created using this system. In most membrane filtration systems, clogging of the membranes has made long-term operation difficult. In this system, however, high pressure is not applied directly to the membrane, thus minimizing clogging. Also, clogging of the membrane was prevented by washing the membrane surface once a day, and increasing the membrane surface area. With this system, FM-3A cells were cultured and maintained at a high density of 3.0 x 10(7) cells/ml for two weeks, and a continuous culture was supported for as long as 34 days.

Light-induced photon emission by mammalian cells

In this work, the light-induced photon emission (IPE) by suspensions of mammalian cells was examined. IPE is extremely low and for detection a single photon counting device with a cooled EMI 9558QB photomultiplier tube was used. The mammalian cells in this study were from different tissues and different mammalian species including cat, Chinese hamster, cow, dog, human, monkey, mouse and rat. The IPE was detected in all mammalian cells tested, but was different for the various cell types, ranging from 4 to 100 photons per 10 4 cells. Although our data agree with previous studies in that the IPE of non-fibroblastic normal cells is distinct from that of malignant cells our results reveal that cells of fibroblastic origin show the highest IPE values.

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 microm 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 microm 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, eta = eta(0) exp[K gamma(.)(-beta)phi(c)(1 - K'' sigmaphi(c) /D)] 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.

Constant flowrate filtration of hybridoma cells suspensions

The fouling mechanism of rotating sieves (also called spinfilters) in suspended mammalian cells reactors is still poorly understood, and their design is consequently largely empirical. A laboratory set-up enabling constant flowrate filtration experiments is reported and its use described in order to study the fouling kinetics of 10 microns opening sieves by suspended hybridomas. Significant differences were obtained depending upon cell type and cell viability, under similar operating conditions. A roughly linear pressure drop increase, as predicted by constant flowrate incompressible cake filtration models, was obtained for most experiments. However, the influences of pumping flowrate and cell concentration on pressure drop evolution do not obey incompressible cake constant flowrate filtration laws; a surface colonization approach could possibly lead to a better description in that case.

Perfusion culture apparatus for suspended mammalian cells

A variety of processes have been proposed for mammalian cell culture in the commercial production of useful substances (e.g., monoclonal antibodies, therapeutic and diagnostic proteins). Among them, the perfusion culture of suspended non-immobilized cells is the most advantageous. Perfusion culture can be classified by the separation process of suspended cells from the culture mixture into three types, namely filtration, gravitational settling and centrifugation. From a commercial point of view, the present situation and technical problems of suspended cell perfusion culture will be reviewed based on the three types. The recent development of perfusion culture has been carried out mainly on the filtration separation process, but the centrifugation process seems to have a promising future because of operation stability and scale-up feasibility. The reasons will be explained in details.

Fluorescent markers for hypoxic cells: A study of novel heterocyclic compounds that undergo bio-reductive binding

The bioreductive metabolism and binding of nitroaromatic compounds has been suggested as a method for the identification of hypoxic tumour cells. Bound metabolites of suitable nitroaryl compounds (and some other reducible aromatic compounds) may fluoresce, offering an alternative to radiolabelling or NMR etc. as a diagnostic method. In this paper, the synthesis of some heteroaromatic nitro-compounds is given together with the results obtained from testing of these and other mainly nitroaromatic compounds in vitro as potential bioreductive fluorescent probes for hypoxic cells in tumours. Compounds were incubated with oxygenated or hypoxic mammalian cell suspensions for various times before evaluation of the cellular fluorescence from bioreductive metabolites by fluorescence microscopy and flow cytometry. Among those compounds yielding fluorescent metabolites in cells, considerable variation in hypoxic: oxic differential fluorescence was observed. The in vitro mammalian cell test system showed several of the compounds to be sufficiently promising to merit further investigation in vivo.

Microencapsulation of mammalian cells in a HEMA‐MMA copolymer: Effects on capsule morphology and permeability

A new process for preparing uniform microcapsules with a hydroxyethyl methacrylate-methyl methacrylate copolymer (HEMA-MMA) has been devised. Capsule diameters were 900-1000 microns in diameter, (+/- 10-20 microns, +/- SD) depending on the precipitation conditions. The process involved the coextrusion of polymer solution (in PEG 200) and the mammalian cell suspension (here erythrocytes) through a needle assembly which is submerged in a layer of hexadecane which is in turn sitting above a stirred isotonic aqueous solution in a volumetric flask. The needle is repeatedly withdrawn from the hexadecane overlayer shearing a droplet from the needle tip which falls into the water, where the solvent is extracted to precipitate the polymer around the cells to yield the capsules. The morphology of the capsule wall was altered by changing the precipitation bath from phosphate buffered saline (PBS) to 0.3 M glycerol. This resulted in greater macroporosity in the wall, presumably because of the faster precipitation due to the higher solvent/precipitant compatibility with 0.3 M glycerol. The permeability to a series of test solutes (glucose, inulin, albumin, and alcohol dehydrogenase, ADH) increased by a factor of approximately 2, presumably because of the increased macroporosity. Addition of 15% water to the polymer solvent enhanced the macroporosity, presumably by bringing the system closer to the cloud point; however, there was no corresponding increase in permeability. There was a significant decrease in permeability between that of albumin (approximately 69,000 D) and ADH (approximately 150,000 D) suggesting that the molecular weight cut-off of these capsules was on the order of 100,000 D as desired. This process is now being evaluated for the encapsulation of pancreatic islets and other cells of potential clinical interest.

Ultrasonic propagation in mammalian cell suspensions based on a shell model

The interaction of ultrasonic waves with individual cells has been modelled on the basis that the cells can be represented by viscous liquid spheres surround by a viscoelastic shell (the membrane) immersed in a viscous fluid. The computational model includes thermal waves and requires 22 input parameters. Many of the parameters are not available in the literature and a detailed discussion is given on the procedures by which the values used in the model calculations were chosen. In spite of the arbitrariness of the choice of many of the parameter values, the computations show surprisingly good agreement with experimental measurements of ultrasonic attenuation in animal cell suspensions. The model has been used here to investigate different aspects of the interaction of ultrasound with the cells. It is found that the membrane is important only between 0.5 and 30 MHz and contributes less than 15% to the attenuation. Absorption is shown to be an important feature to include, while the scattering contribution to the attenuation is less than 1% at 3 MHz. The thermal effects are important at frequencies below 1 MHz and contribute some 65% to the attenuation at 100 MHz.

Influence of serum level, cell line, flow type and viscosity on flow-induced lysis of suspended mammalian cells

An investigation was conducted of the parametric dependence of cell lysis observed when mammalian cells growing in suspension are subjected intermittently to intense hydrodynamic forces. Two flow devices were tested: one consisting of a sudden contraction into a short length of capillary tubing, in which turbulent flow is obtained, and another consisting of a smoothly converging and diverging tube, in which laminar flow is obtained. Changes in the cell line and the serum level in which the cells were grown and subjected to flow trauma both affected the specific lysis rate (fraction of cells lysed per pass through the flow device) in the turbulent flow device. The threshold value of the average wall shear stress level was approximately the same in the turbulent and laminar flow devices (1500–1800 dyn/cm2). Increasing the viscosity of the medium with 70,000 MW dextran had no effect on the specific lysis rate in either flow device.

Effect of gas—liquid interfaces on the growth of suspended mammalian cells: mechanisms of cell damage by bubbles

The hypothesis is examined that cell death in sparged systems for suspended mammalian cell culture is associated only with the region of bubble disengagement at the medium surface. The effects of bubble diameter, superficial gas velocities, and the nonionic surfactant Pluronic F-68 on hybridoma viability have been reported previously. The work reported here extends these experimental studies to a range of bubble columns and for the culture of not only hybridoma but also myeloma, BHK-21, and lymphoblastoid (RAJI) cells lines. In short bubble columns, small bubbles and high bubble frequencies are more detrimental to hybridoma cells than BHK-21 suspension cells. Both fetal calf serum (FCS) and Pluronic F-68 have concentration-dependent protective effects on cells. In defined serum-free media, gross mechanical disruption of hybridoma cells and loss of cells and medium components at the medium surface occur on sparging. With increasing bubble column height, a progressively increasing retention of cell viability is observed for all cell types. By implication, this is because the proportion of time spent by cells in the “destructive zone” at the medium surface is reduced as the column height increases. Finally, two mechanisms of cell damage are proposed: damage due to rapid oscillations caused by bursting bubbles, and damage due to shearing in draining liquid films (or lamellae) in foams. It is proposed that Pluronic (and other polymers such as polyethylene glycol and polyvinyl pyrollidone) act as cell protective agents by stabilizing foams and thereby reducing film drainage and bubble bursting in the vicinity of cells.

14] Measurement of cytosolic free Ca2+ with quin2

Publisher Summary The fluorescent Ca 2+ indicator dye—quin2—is the basis of one of the most popular methods for measuring cytosolic free Ca 2+ [(Ca 2+ ] i ], especially in suspensions of mammalian cells. Quin2 has several advantages over other methods for measuring cytosolic free Ca 2+ : (1) it can be loaded into the cytosol of millions of cells simultaneously without any micromanipulations or any disruption of the plasma membrane, (2) it is monitored with a conventional cuvette spectrofluorimeter, (3) its readings are usually easily calibrated at typical resting levels of [Ca 2+ ] i (∼10 −7 M ) and below (to 10 −8 M ), whereas all other techniques do their best at detecting activated levels (∼10 −6 M ) and are nearly at their detection limits at resting levels, and (4) it avoids many of the drawbacks particular to other [Ca 2+ ] i measurement techniques. The chapter compares quin2 with newer fluorescent indicators—fura-2 and indo-1—as they are much brighter in fluorescence than quin2. Because fura-2 and indo-1 are sensitive enough to give large calibratable signals from single cells or regions of cells, they allow a direct measurement of compartmentalization and heterogeneity.

Flow effects on the viability and lysis of suspended mammalian cells.

A mouse myeloma cell line growing in suspension was subjected intermittently to flow through a sudden contraction and turbulent flow in a capillary tube. The probability of lysis per pass through the capillary tube increased with average wall shear stress level and with residence time per pass in the tube. Lysis was first observed at a threshold average wall shear stress level of 1800 dyn/cm2. Although the flow caused lysis, it had no effect on cell viability.

Oxygen-Dependent Metabolism of Potential Magnetic Resonance Contrast Agents

Contrast agents have been investigated in an effort to increase the differences between the proton relaxation times of normal and pathologic tissue, and thus improve the ability to detect regions of pathology. Data on the oxygen-dependent metabolism of three nitroxides that are potential MRI contrast agents are presented. The amount of active, paramagnetic nitroxide remaining in suspensions of mammalian cells was followed as a function of time using electron-spin resonance spectroscopy. Data on the rates of nitroxide reduction over a wide range of oxygen concentrations are presented and show these rates as a function of the intracellular oxygen concentration. When intracellular oxygen concentrations were measured, only cells that were severely hypoxic reduced these potential contrast agents at increased rates. Because regions of some types of pathology are often hypoxic, the oxygen-dependent metabolism of nitroxides should allow contrast to be achieved on the basis of differences in metabolism between normal and diseased tissues. Further, the data suggest that future studies of nitroxide metabolism need to take into account the O2 concentration in order to understand the observed reduction rates and to predict the rates under other conditions.

Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension.

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.

High-efficiency preparation of DNA from limiting quantities of eukaryotic cells for hybridization analysis

This report describes a DNA preparation method which allows the detection of single-copy genes in samples of as few as 6000 eukaryotic cells. The technique uses proteinase K digestion in detergent and low-gelationtemperature agarose followed by solidification of the agarose and removal of the detergent by diffusion. RNase and restriction enzyme digestion are carried out in solution after remelting the agarose. The procedure can be performed successfully with mammalian cells in suspension, with parasitic protozoa and with pieces of mammalian tissue weighing less than l mg. Numerous samples can be processed simultaneously using frozen as well as fresh material.