Feasibility studies of oncornavirus production in microcarrier cultures

Summary Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis. For reprint requests address: Dr. Keith Jensen, Pfizer, Inc., Groton, CT 06340. This work was supported in part under contract N01-CP-33234 within the Virus Cancer Program of the National Cancer Institute.     

Anchorage-dependent cell expansion in fiber-shaped microcarrier aggregates

This article describes a three-dimensional culture system for the expansion of anchorage-dependent cells using fiber-shaped microcarrier (MC; Cytodex3) aggregates, termed “MC fibers.” The fiber encapsulates the cells, the MC aggregates, and collagen and is covered with a poly-l -lysine membrane. The thin structure of the fiber enables sufficient supply of O₂ and nutrients to the cell. Using the MC fiber, we demonstrated the efficient expansion of C2C12 cells with high viability through serial passaging. Therefore, our culture system is useful for various applications where large-scale cell expansion is required, such as in pharmaceutical technologies, regenerative medicine, and cultured meat production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2755, 2019.     

Oscillations in an epidemiological model based on asynchronous probabilistic cellular automaton

Consider a contagious disease affecting a host population composed of two groups with distinct habits. At each time step, each individual of this population can be in one of two states: susceptible (S) or infective (I). Here, a SIS epidemic model based on cellular automaton (CA) is proposed to study the disease spreading in such a population. In this model, the state transitions are described by probabilistic rules and each group has its own schedule to update the states of its individuals. We also propose a set of difference equations (DE) to analyze this population dynamics and we show how these two approaches (CA and DE) can be equivalent. We noticed that oscillations can be found in the composition of the group with more active social life, but not in the composition of the other group.     

Development of the optimal inoculation conditions for microcarrier cultures

The environmental conditions under which anchorage-dependent mammalian cells are grown are not necessarily those under which a culture should be initiated. Cell attachment is a physical process, and those factors which affect forces involved in cell attachment differ from the biological factors which affect cell growth. We have conducted an extensive experimental study to define clearly the optimal environmental conditions for MRC-5 cell attachment onto microcarriers. These inoculation conditions are particularly important when the serial propagation of mammalian cells on microcarriers is considered as in a human vaccine production process. The conditions which were investigated are: initial serum content (% v/v), initial pH, inoculation level (cells/bead), agitation rate (rpm), and the concentration of microcarriers (g/L). The initial distribution of attached cells was found to have a significant affect on the overall efficiency of anchorage-dependent cell cultures, and was used to evaluate attachment efficiency. Based on the experimental results, we propose an optimized protocol for the inoculation of microcarrier cultures.     

High-density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.     

High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures

A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 10⁷ cells per ml were obtained. The described system is scaleable.     

On the effects of the spatial distribution in an epidemic model based on cellular automaton

Several theoretical studies on disease propagation assume that individuals belonging to different groups regarding their health conditions are homogeneously distributed over the space. This is the well-known homogenous mixing assumption, which supports epidemiological models written in terms of ordinary differential or difference equations. Here, we consider that the host population infected by a contagious pathogen is composed by two groups with distinct traits and habits, which can be homogeneously mixed or not. The pathogen propagation is modeled by using an asynchronous probabilistic cellular automaton. Our main goal is to examine how a heterogeneous spatial distribution of these groups affects the endemic state. We noted that homogeneous distribution favors the occurrence of oscillations in the population composition. Surprisingly, we found out that the propagation dynamics of the heterogeneous distribution can also be described by a set of ordinary difference equations.     

A simple cellular automaton model for influenza A viral infections

Viral kinetics have been extensively studied in the past through the use of spatially homogeneous ordinary differential equations describing the time evolution of the diseased state. However, spatial characteristics such as localized populations of dead cells might adversely affect the spread of infection, similar to the manner in which a counter-fire can stop a forest fire from spreading. In order to investigate the influence of spatial heterogeneities on viral spread, a simple 2-D cellular automaton (CA) model of a viral infection has been developed. In this initial phase of the investigation, the CA model is validated against clinical immunological data for uncomplicated influenza A infections. Our results will be shown and discussed.     

A cellular automaton model for tumour growth in inhomogeneous environment

Most of the existing mathematical models for tumour growth and tumour-induced angiogenesis neglect blood flow. This is an important factor on which both nutrient and metabolite supply depend. In this paper we aim to address this shortcoming by developing a mathematical model which shows how blood flow and red blood cell heterogeneity influence the growth of systems of normal and cancerous cells. The model is developed in two stages. First we determine the distribution of oxygen in a native vascular network, incorporating into our model features of blood flow and vascular dynamics such as structural adaptation, complex rheology and red blood cell circulation. Once we have calculated the oxygen distribution, we then study the dynamics of a colony of normal and cancerous cells, placed in such a heterogeneous environment. During this second stage, we assume that the vascular network does not evolve and is independent of the dynamics of the surrounding tissue. The cells are considered as elements of a cellular automaton, whose evolution rules are inspired by the different behaviour of normal and cancer cells. Our aim is to show that blood flow and red blood cell heterogeneity play major roles in the development of such colonies, even when the red blood cells are flowing through the vasculature of normal, healthy tissue.     

Cell growth optimization in microcarrier culture

Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm² of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.     

Growth behavior of number distributed adherent MDCK cells for optimization in microcarrier cultures

An assay for measuring the number of adherent cells on microcarriers that is independent from dilution errors in sample preparation was used to investigate attachment dynamics and cell growth. It could be shown that the recovery of seeded cells is a function of the specific rates of cell attachment and cell death, and finally a function of the initial cell‐to‐bead ratio. An unstructured, segregated population balance model was developed that considers individual classes of microcarriers covered by 1–220 cells/bead. The model describes the distribution of initially attached cells and their growth in a microcarrier system. The model distinguishes between subpopulations of dividing and nondividing cells and describes in a detailed way cell attachment, cell growth, density‐dependent growth inhibition, and basic metabolism of Madin‐Darby canine kidney cells used in influenza vaccine manufacturing. To obtain a model approach that is suitable for process control applications, a reduced growth model without cell subpopulations, but with a formulation of the specific cell growth rate as a function of the initial cell distribution on microcarriers after seeding was developed. With both model approaches, the fraction of growth‐inhibited cells could be predicted. Simulation results of two cultivations with a different number of initially seeded cells showed that the growth kinetics of adherent cells at the given cultivation conditions is mainly determined by the range of disparity in the initial distribution of cells on microcarriers after attachment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

MDCK microcarrier cultures: Seeding density effects and amino acid utilization

Summary The growth of Madin Darby canine kidney cells on microcarriers was studied at different cell seeding densities. Maximum growth was attained when a cell-to-bead ratio of 7∶1 was used. Under these conditions an initial concentration of above 3×10⁵ cells/ml resulted in a yield of over 2×10⁶ cells/ml in 2 d. The amino acid utilization of cells from Dulbecco's modified Eagle medium was studied under the above conditions. Eight amino acids (arg, cys, gln, ile, leu, met, ser, and val) showed rapid and near complete depletion from the medium. Five amino acids (his, lys, phe, thr, and tyr) showed limited depletion. Two amino acids (ala and gly) were released into the medium by the cells.     

Evaluation of a microcarrier process for large-scale cultivation of attenuated hepatitis A

Microcarrier culture was investigated for the propagation of attenuated hepatitis A vaccine in the anchorage-dependent human fibroblast cell line, MRC-5. Cells were cultivated at 37°C for one to two weeks, while virus accumulation was performed at 32°C over 21 to 28 days. The major development focus for the microcarrier process was the difference between the cell and virus growth phases. Virus antigen yields, growth kinetics, and cell layer/bead morphology were each examined and compared for both the microcarrier and stationary T-flask cultures. Overall, cell densities of 4–5×10⁶ cells/ml at 5–10 g/l beads were readily attained and could be maintained in the absence of infection at either 37°C or 32°C. Upon virus inoculation, however, substantial cell density decreases were observed as well as 2.5 to 10-fold lower per cell and per unit surface area antigen yields as compared to stationary cultures. The advantages as well as the problems presented by the microcarrier approach will be discussed.     

A probability cellular automaton model for hepatitis B viral infections

The existing models of hepatitis B virus (HBV) infection dynamics are based on the assumption that the populations of viruses and cells are uniformly mixed. However, the real virus infection system is actually not homogeneous and some spatial factors might play a nontrivial role in governing the development of HBV infection and its outcome. For instance, the localized populations of dead cells might adversely affect the spread of infection. To consider this kind of inhomogeneous feature, a simple 2D (dimensional) probability Cellular Automaton model was introduced to study the dynamic process of HBV infection. The model took into account the existence of different types of HBV infectious and non-infectious particles. The simulation results thus obtained showed that the Cellular Automaton model could successfully account for some important features of the disease, such as its wide variety in manifestation and its age dependency. Meanwhile, the effects of the model's parameters on the dynamical process of the infection were also investigated. It is anticipated that the Cellular Automaton model may be extended to serve as a useful vehicle for studying, among many other complicated dynamic biological systems, various persistent infections with replicating parasites.     

Bead-to-bead transfer of Vero cells in microcarrier culture

Cell harvesting technique is of considerable importance in the scale-up of microcarrier cultures of anchorage-dependent cells. The traditional methods are often time- and labor-consuming and cause physiological damage to the cells. Bead-to-bead cell transfer provides an attractive solution to the scale up process. By intermittent agitation, successful cell transfer was achieved. Significant cell growth was observed where bare beads contacted with confluent ones. Most of the fresh microcarriers reached near confluence four days after addition into the culture medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microsupport with two-dimensional geometry (2D-MS). In situ determination of the growth kinetics of anchorage-dependent cells by Laser Diffraction Particle Sizing (LDPS)

The procedure used to establish in situ (without cell trypsinization) the growth kinetics characteristics of anchorage-dependent cells propagated on microcarriers by Aperture Impedance Pulse Spectroscopy can be replaced by a novel method based on the time-dependent shifts of the size distribution histograms of cell-laden microcarriers. This we have named Laser Diffraction Particle Sizing. This revised version was published online in August 2006 with corrections to the Cover Date.     

Method for the passaging of microcarrier cultures to a production scale for producing high titre Disabled Infectious Single Cycle-Herpes Simplex Virus Type-2

A complementary cell line CR2 is currently used to propagate the Disabled Infectious Single Cycle Herpes Simplex Virus Type 2 (DISC HSV-2) on a small laboratory scale upto 15 L. These cultures are initiated by passaging the cells from roller bottle cultures. Whilst this is suitable for the laboratory scale it is totally impractical for use in seeding an industrial manufacturing scaled version of the culture. It is paramount to have a robust system for passaging cells from a small microcarrier culture system to a larger one by a serial subculturing regime. Here we report on the successes we have had in our laboratory in scaling up our production system for the DISC HSV-2 from small 1-L cultures to a 50-L vessel with the maintenance of the viral productivity. Ease of use, reproducibility and the need to minimise overall production times were factors which were taken into consideration whilst developing our procedures. We were aware of the need to keep a production train simple and as short as possible as this was the small scale study for an envisaged manfacturing process.     

Agitation induced cell injury in microcarrier cultures. Protective effect of viscosity is agitation intensity dependent: Experiments and modeling

The effect of medium viscosity on the specific death rate of bovine embryonic kidney (BEK) cells cultured in spinner flask microcarrier cultures has been examined for various impeller speeds. Two types of media were used, a serum-containing growth medium and a serum-free maintenance medium. The latter does not support cell growth. We found that increasing medium viscosity suppresses cell death rates in both growth and maintenance medium cultures in an agitation-intensity-dependent fashion; the beneficial effect of medium viscosity in reducing the specific death rate is amplified as the agitation rate is increased. Furthermore, increasing medium viscosity has no effect on the specific death rate of the cells when the agitation rate is below a critical level. A model based on the turbulent energy content of eddies in the dissipation spectrum of turbulence of length scales on the order of magnitude of the microcarrier diameter and lower has been developed to account for cell death due to both bead-to-bead and bead-to-eddy interactions. The model constitutes a significant departure from previous efforts first because both types of interactions are accounted for simultaneously and second because the properties of a spectrum of eddies instead of the Kolmogorov-scale eddy size alone are used in the model. The model explains the functional dependence of the specific death rates on the medium viscosity at varying agitation intensities.     

Pilot production of u-PA with porous microcarrier cell culture

A recombinant DNA CHO cell line secretingurokinase-type plasminogen activator (u-PA) wascultivated with Cytopore cellulose porousmicrocarriers in a 30l Biostat UC stirred tankreactor. After 26 days of culture, using a spinfilter toretain cells in bioreactor, the cell density couldreach 1.33 × 10⁷ ml^-1. The maximal u-PAactivity in supernatant was 7335 IU·ml^-1, and204l supernatant containing 7.1 g u-PA was harvested.After 100 days of culture with 0.1% fetal bovineserum medium, a modified cell retention system whichcan be washed-out backward, substituted thespinfilter to prevent filter clogging. The maximalcell density was over 10⁷ ml^-1, the maximalu-PA activity in supernatant reached 6250IU·ml^-1, and 1604l supernatant containing about51 g u-PA was harvested. Compared to perfusionculture, batch medium-replaced culture could raiseutilizing efficiency of the medium, increase cell specificproductivity and improve the quality of the product which wasnot steady in a 37 °C environment. Cells can movefrom seed porous microcarriers occupied by cells tovacant microcarriers spontaneously, withouttrypsinization, and continue to grow until all microcarriers contained cells. It shows that Cytoporeporous microcarriers are very useful and convenient toscale up cultivation step by step.     

Perfusion culture using microcarrier for the production of Varicella-Zoster virus in human embryonic lung cells

Perfusion culture with microcarriers was conducted to produce cell-associated and cell-free Varicella-Zoster virus (VZV) with human embryonic lung cells. After the cells were infected with VZV infected cells, glucose in the medium decreased rapidly, suggesting that VZV propagation was related closely to the use of glucose. While the yield of cell-associated VZV in microcarriers was 9,350 PFU/cm2, almost two-thirds of that in T-80 flask and cell factory, the yield of cell-free VZV in microcarriers was only about 10% of that in T-80 flask and cell factory.