Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.     

Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers

The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved.     

Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

Summary A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S₃ cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. This research was supported in part by the National Institutes of Health (GN 29127) and Ventrex Laboratories, Portland, Maine.     

The culture of human skin fibroblasts on microcarriers in the presence of serum substitute: Ultroser G

The effects of the substitution of serum by Ultroser G on human skin fibroblasts cultured on microcarriers were analysed. Cultures could not be established on microcarriers in the presence of Ultroser G. However, microcarrier cultures started in the presence of 10% foetal calf serum, and transferred to 2% Ultroser G after 7 days resulted in high cell densities.     

The culture of human skin fibroblasts on microcarriers in the presence of serum substitute: Ultroser G

The effects of the substitution of serum by Ultroser G on human skin fibroblasts cultured on microcarriers were analysed. Cultures could not be established on microcarriers in the presence of Ultroser G. However, microcarrier cultures started in the presence of 10% foetal calf serum, and transferred to 2% Ultroser G after 7 days resulted in high cell densities.     

Expansion of human mesenchymal stem cells on microcarriers

The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 microcarriers were not damaged when held at the minimum speed, N_JS, for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8–10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the microcarriers in the flasks were 3,000 microcarriers ml⁻¹ (ca. 1 g dry weight l⁻¹), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at N_JS (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov’s theory of isotropic turbulence.     

Selection of microcarrier diameter for the cultivation of mammalian cells on microcarriers

The kinetics of mammalian cell growth in a microcarrier culture are affected by the distribution of cells on microcarriers. It has been shown previously that a critical cell number per microcarrier is required for the growth of FS-4 cells on microcarriers. It is advantageous to alter the cell distribution on microcarriers to allow for a larger fraction of microcarriers to acquire enough cells to initiate normal growth. This can be achieved by selecting the diameter of the microcarriers employed. It has also been shown previously that the critical cell number could be reduced by choosing a better culture medium to support low density growth. However, even if all cells inoculated into a culture are capable of growing to confluence, it is still necessary to select the microcarrier diameter ration ally to improve the growth kinetics. The method of selecting the microcarrier diameter is discussed. By employing a improved medium as well as using microcarriers of selected diameter, the multiplication ratio was in creased to 15- to 16-fold for FS-4 cells, as opposed to 3- to 4-fold typically obtained in a batch culture.     

Optimization of Conditions for Production of Channel Catfish Ovary Cells and Channel Catfish Virus DNA

Medium supplements were examined for their effect on the growth of channel catfish ovary cells. It was found that the usual serum supplement of 10% fetal calf serum could be successfully replaced with a combination of 5% fetal calf serum and a mixture of insulin, transferrin, and selenous acid. It was also found that these cells could be grown in a more efficient manner on microcarrier beads. This type of culture produced 14 times the number of cells per milliliter of total medium used compared with the usual tissue culture flasks used for cell growth. The microcarrier system also provided for greater production efficiency of DNA from channel catfish virus, a virus that infects this cell line.     

Attachment characteristics of normal human cells and virus-infected cells on microcarriers

The attachment kinetics of normal and virus-infected LuMA cells were studied to improve the production of live attenuated varicella viruses in human embryonic lung (LuMA) cells. Normal LuMA cells and LuMA cells infected by varicella virus at various cytopathic effects (CPE) were grown on microcarriers. Ninety-three percent of suspended LuMA cells attached to the solid surface microcarriers within fifteen minutes and cell viability was greater than 95% when the cell suspension was stirred. Low serum levels did not affect the attachment rate of virus-infected cells in the microcarrier culture system. Kinetic studies showed that varicella infected cells had a lower attachment rate than normal LuMA cells. Virus inoculum (= infected cells) at low CPE showed a relatively better attachment rate on cell-laden microcarriers than virus inoculum at a higher CPE. Maximum titers were obtained at 2 days post-infection. Based on cell densities, the use of viral inoculum showing a 40% CPE led to an approximately 2- and 1.2-fold increase in the cell associated and in cell free viruses, respectively, than a virus inoculum with a CPE of 10%.However, the ratio of cell-free to cell-associated virus in a microcarrier culture was very low, approximately0.04–0.06. These studies demonstrate that the virus inoculum resulting in a high CPE yielded a high production of cell-associated and cell-free virus in microcarrier cultures because of the high cellular affinity of the varicella virus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Human diploid fibroblast growth on polystyrene microcarriers in aggregates

Polystyrene microcarriers were prepared in four size ranges (53–63 m, 90–125 m, 150–180 m and 300–355 m) and examined for ability to support attachment and growth of human diploid fibroblasts. Cells attached rapidly to the microcarriers and there was a direct relationship between cell attachment and microcarrier aggregation. Phasecontrast and scanning electron microscopic studies revealed that while aggregation was extensive, most of the aggregate consisted of void volume. Cell growth studies demonstrated that human diploid fibroblasts proliferated well in microcarrier aggregates, reaching densities of 2.5–3×10⁶ cells per 2 ml dish after 6 days from an inoculum of 0.5×10⁶ cells per dish. When cells were added to the microcarriers at higher density (up to 5×10⁶ cells per 2-ml culture), there was little net growth but the cells remained viable over a 7-day period. In contrast, cells died when plated under the same conditions in monolayer culture. When the microcarriers were used in suspension culture, rapid cell attachment and rapid microcarrier aggregation also occurred. In 100-ml suspension culture, a cell density of 0.7×10⁶ cells per ml was reached after 7 days from an inoculum of 0.1×10⁶ cells. Based on these data, we conclude that microcarrier aggregation is not detrimental to fibroblast growth. These data also indicate that small microcarriers (53–63 m) (previously thought to be too small to support the growth of diploid fibroblasts) can support fibroblast growth and this occurs primarily because microcarriers in this size range efficiently form aggregates with the cells.     

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.     

Production of live attenuated varicella-zoster virus in human embryonic lung cells using microcarrier

Summary Attenuated varicella-zoster virus was propagated in human fetal embryonic lung cells grown on microcarriers to produce live attenuated varicella vaccine. We have investigated the characteristics of cell growth and virus production in microcarrier culture system at various culture conditions. The cell-associated and cell-free virus yields in microcarrier were comparable to those in the stationary tissue cultures.     

Cell growth on immobilized cell growth factor. 8. Protein-free cell culture on insulin-immobilized microcarriers

In order to develop a new protein-free cell culture system, microcarriers immobilized with insulin were synthesized. For the synthesis, glass and polyacrylamide beads were treated for the introduction of amino groups on the surface, and insulin was immobilized on the surface by using several method. Anchorage-dependent cells. mouse fibroblast cells STO and fibroic sarcoma cells HSDM(1)C(1), and the anchorage-independent cells, mouse hybridoma cells SJK132-20 and RDP 45/20 were cultivated on the microcarriers immobilized with insulin. The insulin-immobilized microcarriers did not have any effect on the proliferation of the anchorage independent cells but promoted the growth of anchorage-dependent cells remarkably. The activity of immobilized insulin was larger than that of free or adsorbed insulin. The repeated use of the insulin-immobilized microcarrier was possible, and the promotion activity in the the repeated use was greater than that in the use. (c) 1992 John Wiley & Sons, Inc.     

Growth of human vascular endothelial cells on various types of microcarriers

Various types of microcarriers were tested as growth substrate for the cultivation of either endothelial cells from human umbilical cord veins or of EA. hy926, an immortalized cell line of endothelial origin. Cell growth was tested on microcarriers in tissue culture flasks and spinner flasks. Solid (Cytodex type I, II, III, Gelibeads, Mica) and macroporous (Polyhipe, CultiSpher GL, PolyporE type I) microcarriers were tested. For the solid carriers the best results were obtained with Mica and for the macroporous carriers with CultiSpher GL.Abbreviations DAPI 4,6-diamidino-2-phenylindole-di-hydrochloride - DEAE diethylaminoethyl - EC vascular endothelial cells - FGF fibroblast growth factor - HUVEC vascular endothelial cells from human umbilical cord veins - IF 11 mixture of Iscove's MDM and F12 basal media - NCS newborn calf serum - PBS phosphate buffered saline - TE 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in PBS     

Cell growth and protein formation on various microcarriers

A large number of microcarriers are commercially available. The capability of cells to successfully proliferate on microcarriers varies with cell lines and media. Choosing the right microcarrier for a particular cell line is more than a choice of a microcarrier. It is part of an integrated process design. A detailed picture of cell growth and product formation will not only be essential in identifying the kind of microcarrier, but also in determining other parts of the process, such as operation mode and media. Our initial screening on thirteen microcarriers showed that cultures on some microcarriers reached a low cell density but high cell-specific productivity, and high density microcarrier cultures have a low specific productivity. The result is a similar product output per unit volume and time for these two types of cultures. An ideal culture system shall have increased volumetric productivity at elevated cell density. This requires the process goal to be incorporated as early as cell line construction and screening. A high output process can then be realized through high density culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

The extended serial subculture of human diploid fibroblasts on microcarriers using a new medium supplement formulation

Human diploid fibroblasts serially passaged on microcarriers exhibit a decrease in their proliferative capacity with each transfer from microcarrier-to-microcarrier. This phenomenon, which does not occur in the same time scale with cells cultured in T-flasks, has been a serious barrier to the systematic utilization of microcarriers in the scale-up of anchorage-dependent human diploid cell cultures. This decreases in cell growth with each passage is shown to be related to the serum content of the medium, with high serum concentrations resulting in a more rapid decrease in cell growth with each serial transfer. As a result, methods for reducing the serum requirement of the cells were investigated. A new medium supplement mixture, PPRF92, has been developed, which allows the serial passaging of MRC5 cells on Cytodex 1 microcarriers through as many as 13 microcarrier-to-microcarrier tranfers, and at a serum levels as low as 1%, with no decrease in the proliferative capacity of the cells until they approach their reported population doubling limit. This new supplement mixture is a significant improvement to microcarrier technology in that it enables the use of microcarriers in the early stages of inocculum build-up for the production purposes. (c) 1992 John Wiley & Sons, Inc.     

Human interferon production with diploid fibroblast cells grown on microcarriers.

A variety of diploid human fibroblast lines have been successfully grown to high densities (greater than 10(6) cell/ml) on recently developed microcarriers. Interferon induction using poly I.poly C and a superinduction procedure resulted in yields greater than 10,000 units/ml with one cell line. A direct comparison of microcarrier cultures to roller bottle cultures showed equivalent interferon yields on a per cell basis and some apparent differences relating to optimum inducer concentrations and kinetics of interferon accumulation.     

A modified matrix perfusion-microcarrier bead cell culture system. I. Adaptation of the matrix perfusion system for growth of human foreskin fibroblasts

Two strains of human foreskin fibroblast cells were incapable of sustained growth in a matrix perfusion culture system, possibly because of their inability to attach to the fiber surfaces. Addition of microcarrier beads to the extracapillary space allowed attaining high cell densities in excess of 10(7) cells per culture unit. Microcarrier beads were tested in hollow fiber culture devices containing membranes of 10(4) or 10(5) D nominal porosities. Best results were obtained when initial cell densities of at least (2-3) x 10(6) cells were used in units with 10(5) D pore size membranes and DEAE-Sephadex or polyacryl-amide microcarrier beads in the extracapillary space. This extension of the matrix perfusion system should be useful for growing other anchorage dependent cells while retaining the advantages of perfusion culture.     

Production of human immune interferon by recombinant mammalian cells cultivated on microcarriers

Recombinant Chinese hamster ovary (rCHO) cells were cultivated on microcarriers for the production of human immune (Gamma) interferon. The effect of basal medium, serum, and microcarrier concentration on interferon production was investigated. The specific interferon productivity in the post-confluent stage was similar to that in the growth stage. Control of the pH results in a significant improvement in the volumetric interferon production. The volumetric production rate of interferon by these rCHO cells did not decrease after one month of cultivation on microcarriers.     

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive,negative or collagen coated

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen, was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins adsorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.