Engineering challenges in high density cell culture systems

High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO₂, a significant accumulation of dissolved CO₂ is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.     

Theory of cell age distribution in suspension culture with feed and/or drain

A mixed problem for the M'Kendrick-von Foerster equation satisfied by the number density function in terms of the age of the viable cells in a suspension culture with feed and/or drain is solved, and a method of calculating the number density function and time-dependent generation time from observed data of cell number and cell mass is presented. This theory is adequate to analyze the growth of cells that undergo binary fission. The equation of mass balance follows as a natural consequence of this treatment. The equation of substrate balance in consideration of the effect of cell volume is derived rigorously.     

Direct measurements of cell number using computer-aided video microscopy

Quantitative studies in cell culture require accurate measurements of cell density and kinetics. We have developed a direct, rapid, and noninvasive method for measuring cell number in monolayer culture. Using computer-aided video microscopy, cell number was measured without detaching or chemically destroying the cells, thereby allowing sequential measurements in the same cell population. Cell number measured by computer-aided microscopy closely correlated with hemocytometer counts and determinations of total cell protein. For high-density monolayers of mesenchymal cells, however, staining was required for accurate counts. Unlike other techniques for measuring cell density, computer-aided microscopy was especially accurate in medium- to low-density cultures (less than 6000 cells/cm2). In addition, we applied this technique to the construction of separate proliferation curves for glomerular mesangial and vascular endothelial cells in coculture. These measurements by cell type in coculture are impossible using conventional methods for determining cell number.     

Influence of plating density on individual cell growth, cell division and differentiation of neonatal rat heart primary cultures

The influence of plating cell density of an originally enriched myocardial cell population has been studied in neonatal rat heart cells in culture. Low density (LDM) is defined as a density (24 h after plating) of 209 +/- 44 cells/mm2 (mean +/- SEM) and is compared with high density (HDM), 419 +/- 67 cells/mm2. Cell growth is evaluated by the total cell number, the percentage of myocardial cells (M) in culture (PAS method) and the protein content per cell. Some differentiation parameters such as beating rates, glycogen concentration, enzymatic activities (cytochrome C oxidase and glycogen phosphorylase) are studied with time in culture (48, 96 and 192 hr). High density was designed to yield a complete confluency of the cells within 24 hr after plating and to minimize cell division of the non-muscle cells (F). At high density, cell division of F cells is effectively limited, thus leading to a more stable model regarding the cell density per plate and the percentage of M cells: 85.7 +/- 4% and 33.4 +/- 6% in LDM cultures compared with 86.5 +/- 4.7% and 51.7 +/- 9.8% in HDM cultures at 24 and 192 hr (mean +/- SEM). Heart cells increase similarly in size with age in culture in both groups. In HDM cultures the spontaneous contractions begin sooner (24 hr) than in LDM cultures and are more rapidly synchronized. The beating rate is higher in HDM cultures between 48 and 96 hr; however, after this time it falls in HDM and does not fall in LDM. Thus the overgrowth of muscle cells by non-muscle cells is not responsible for loss of beating with time in culture but more likely high density could be a limiting factor for isotonic contraction. There is more glycogen per myocyte in LDM than in HDM cultures. The cell density influences the enzymatic activities of cytochrome C oxidase and glycogen phosphorylase. The cytochrome oxidase activity is higher in HDM cultures than in LDM cultures at 96 hr whereas glycogen phosphorylase activity is higher in LDM cultures at time 96 and 192 hr. In LDM cultures, the ratio cytochrome C oxidase/glycogen phosphorylase decreases with time in culture from 1.685 +/- 0.680 at 48 hr to 0.780 +/- 0.290 at 192 hr but not in HDM cultures (2.13 +/- 0.36 and 1.64 +/- 0.34 respectively). Thus plating density influences properties of heart cell cultures with regard to the overgrowth of the F-cell population and the differentiated state of M cells.     

Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment

Summary Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment. PUF has a macroporous structure giving a high surface area to volume ratio. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 × 10⁸ cells/ cm³ PUF and 3.5 × 10⁷ cells/ cm³ PUF, respectively) in PUF stationary cultures. In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture. A maximum cell density of 2.4 × 10⁷ cells/cm³ culture vessel volume was obtained in a packed-bed culture of Vero cells. Offprint requests to: K. Funatsu     

Regulation of mammalian cell growth by autocrine growth factors: analysis of consequences for inoculum cell density effects

Effects of inoculum cell density on mammalian cell growth in culture have been observed in a variety of experimental systems. Although these effects have been attributed generally to medium conditioning by the cells, there has previously been no quantitative theory proposed for this phenomenon based on developments in molecular and cell biology. In this article, we offer such a theory founded on the regulatory action of autocrine growth factors. A particularly relevant example of these is platelet- derived growth factor (PDGF), which is produced by fibroblastic cells in response to stimulation by transforming growth factor beta (TGFbeta), a common serum constituent, and provides a mitogenic signal for the same cells. A simple mathematical model for the production, diffusive transport, and binding of autocrine growth factors to cell surface receptors, coupled to a model for the dependence of cell proliferation on growth factor receptor binding allows prediction of initial cell population growth rate as a function of inoculum cell density. We focus on situations involving anchorage-dependent cell growth, in which the cells are attached to a surface. A number of clear results are obtained, most notably the following: 1) for cells cultured on spherical microcarrier bead surfaces, the inoculum cell density needed to produce a given growth rate is linearly proportional to the bead radius; and 2) all other factors being equal, the inoculum cell density on a unit surface area basis needed to produce a given growth rate is greater for spherical microcarrier surfaces than for flat culture dish surfaces. These two results are consistent with the experimental observations of Hu and coworkers(1,2) for fibroblast growth in minimal medium plus serum. The model also allows elucidation of the influence of other system parameters, both biological and physical, on initial cell proliferation rate and the inoculum cell density dependence.     

A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×10⁷ cells ml^–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.     

A new methodology of mesenchymal stem cell expansion using magnetic nanoparticles

Mesenchymal stem cells (MSCs), which can differentiate into multiple mesodermal tissues, may be useful for autologous cell transplantation, if MSCs, which are isolated from bone marrow in small numbers, can be expanded in vitro. We developed a combined methodological approach to enrich and proliferate MSCs in vitro using magnetic nanoparticles. Our magnetite cationic liposomes (MCLs), which have a positive surface charge in order to improve adsorption, accumulated in MSCs at a concentration of 20 pg of magnetite per cell. The MCLs exhibited no toxicity against MSCs in proliferation and differentiation to osteoblasts and adipocytes. The MSCs magnetically labeled by MCLs were enriched using magnets and then cultured, resulting in much higher density (seeding density, 1000 cells/cm²) than in ordinary culture (seeding density, 18 cells/cm²). When MSCs were seeded at high density using MCLs, there was a 5-fold increase in the number of cells, compared to culture prepared without MCLs. Our results suggest that this novel culture method using magnetic nanoparticles can be used to efficiently expand MSCs for clinical application.     

Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures

The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.     

Buoyant density fluctuations during the cell cycle of Bacillus subtilis

A simple rapid method for preparing synchronous cultures of Bacillus subtilis has been used to investigate changes in density during the cell cycle. Asynchronous cells separated on a stepped Percoll density gradient had a mean cell density of 1.117 g ml^-1±0.004. Samples from a synchronous culture exhibited variation (ca. 1.5%) in mean cell density which was greatest at the onset of cell division. An asynchronous control culture showed little variation in density. These results are discussed in relation to previous work on Escherichia coli.     

Effects of retinoic acid on cell proliferation and cell differentiation in a rat tracheal epithelial cell line

Abstract. The effects of 3.3 times 10-⁷ M to 3.3 times 10^-5 M all- trans -retinoic acid (vitamin-A acid) on the total cell population dynamics of 165 S, a keratinizing epithelial cell line from carcinogen-exposed rat trachea, were studied. During the first 6 days of culture, cells accumulated on the dish in the presence of the vitamin to twice the density of controls. [³H]thymidine incorporation into DNA and percentage of [³H]thymidine-labelled cells in autoradiographs were stimulated in a dose-dependent fashion to a maximum of 25- and 34-fold, respectively. Exfoliation of cells from the cultures was also enhanced 2–3-fold, resulting in nearly twice the total number of cells (attached plus exfoliated) in the presence of the vitamin.
During 19 days of culture, retinoic acid maintained a higher level of [³H]thymidine incorporation and cell exfoliation in 165 S cells so that by day 19, total cell production was more than three times that seen in controls. At this time, vitamin-treated cultures showed a reduced cell saturation density compared to controls. The higher final cell density in the control cultures was a result of multilayering and papillary formation which did not occur in the presence of retinoic acid. The papillae in control cultures stained specifically with Rhodamine B or with the eosin and orange G components of the Papanicolaou method. A count of the number of eosin and orange G positive cells in the attached and exfoliated cell compartments showed an 8-fold reduction of keratinization in retinoic acid-exposed cultures. Our results show that retinoic acid is a growth stimulant in these cell cultures, causing increased cell proliferation and exfoliation accompanied by inhibition of keratinization.     

Low tumor cell density environment yields survival advantage of tumor cells exposed to MTX in vitro

Stable resistance to methotrexate has been well characterized after prolonged treatment of the HT-29 colon cancer cell line, but the mechanism of cell survival at the early stages of the drug resistance process still remains unclear. Here, we demonstrate that human cancer cells in vitro are sensitive to methotrexate only above a critical cell culture density, which specifically coincides with their ability to deplete the extracellular nucleosides from a fully supplemented culture medium. At lower cell densities, extracellular nucleosides remain intact and allow salvage nucleotide synthesis that renders cells insensitive to the drug. Consistently, medium conditioned by cells seeded at standard cell densities sensitizes low cell density cultures. Extracellular nucleosides are the determinants of sensitivity because the latter effect can be mimicked with the use of inhibitors of nucleoside cellular import and reversed by supplying exogenous thymidine and hypoxanthine. Interestingly, treatment at a sensitizing cell density does not preclude the survival of less than 1% of the cells--which have no intrinsic resistance--owing to the inability of the dying cell population to condition the culture medium; this population thus survives indefinitely to continuous treatment by keeping adapted to a low cell number. This cell density-dependent adaptive process accounts for the initial steps of in vitro resistance to methotrexate (MTX) and provides a novel mechanistic insight into the cell population dynamics of cell survival and cell death during drug treatment.     

Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.     

Analysis of ARD1 function in hypoxia response using retroviral RNA interference

Cellular hypoxia response is regulated at the level of hypoxia-inducible factor (HIF) activity. A number of recently identified oxygen sensors are HIF-modifying enzymes that respond to low oxygen by altering HIF modification and thus lead to its activation. In addition to the HIF proline hydroxylases and asparagine hydroxylases, ARD1 is recently described as a HIF-1alpha acetylase that regulates its stability. We found that ARD1 is down-regulated in a number of cell lines in response to hypoxia and hypoxia mimic compounds. After surveying these lines for erythropoietin production and retroviral transfection efficiency, we chose to use HepG2 cells to study the function of ARD1. ARD1 short hairpin RNA delivered by a retroviral vector caused >80% reduction in ARD1 message. We observed decreases in erythropoietin and vascular endothelial growth factor protein production, whereas there was no change in the HIF-1alpha protein level. A gene chip analysis of HepG2 cells transduced with virus expressing ARD1 short hairpin RNA under normoxia and hypoxia conditions or with virus overexpressing recombinant ARD1 confirmed that inhibition of ARD1 does not cause activation of HIF and downstream target genes. However, this analysis revealed that ARD1 is involved in cell proliferation and in regulating a series of cellular metabolic pathways that are regulated during hypoxia response. The role of ARD1 in cell proliferation is confirmed using fluorescence labeling analysis of cell division. From these studies we conclude that ARD1 is not required to suppress HIF but is required to maintain cell proliferation in mammalian cells.     

siRNA cell arrays for high-content screening microscopy

RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNAs (siRNAs) into living cultured cells and reliable quality control of siRNA function must be in place. Here we describe the production of cell arrays for reverse transfection of tissue culture cells with siRNA and plasmid DNA suitable for subsequent high-content screening microscopy applications. All the necessary transfection components are mixed prior to the robotic spotting on noncoated chambered coverglass tissue culture dishes, which are ideally suited for time-lapse microscopy applications in living cells. The addition of fibronectin to the spotting solution improves cell adherence. After cell seeding, no further cell culture manipulations, such as medium changes or the addition of 7 serum, are needed. Adaptation of the cell density improves autofocus performance for high-quality data acquisition and cell recognition. The co-transfection of a nonspecific fluorescently labeled DNA oligomer with the specific siRNA helps to mark each successfully transfected cell and cell cluster. We demonstrate such an siRNA cell array in a microscope-based functional assay in living cells to determine the effect of various siRNA oligonucleotides against endogenous targets on cellular secretion.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.     

Quantitation of cell surface molecules on a differentiating, Ly-1+ B cell lymphoma

The number of molecules expressed on the B cell membrane is known to influence the level of immune responses. However, a careful study of the changes in numbers of cell surface molecules during B cell differentiation has not been undertaken. We have addressed this question by using an inducible B cell lymphoma, CH12. Scatchard analysis was used to quantitate the levels of expression of surface immunoglobulin, major histocompatibility complex-encoded class I and class II molecules, and Ly-1 molecules on these cells during their differentiation in response to lipopolysaccharide (LPS). We found that the density of most molecules on the initial population of CH12 cells was comparable to their densities on small splenic B cells. Upon culture, we could classify the molecules into two groups based on their change in expression. One group, represented by surface immunoglobulin and class II molecules, decreased (surface immunoglobulin) or did not change (class II) in number after LPS stimulation, but increased during culture in the absence of LPS. The second set, represented by class I and Ly-1 molecules, increased after LPS stimulation, but did not change as a result of culture. Although the characteristic behavior of class I and class II molecules was different, concomitant changes were observed in both class I (K and D) molecules, and in both class II (I-A and I-E) molecules.     

Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration

The adhesion molecule, CD44, interacts with ankyrin within its cytoplasmic domain and binds to hyaluronic acid (HA) at its extracellular domain. In this study, we focused on the functional domain in ankyrin (in particular, the ankyrin repeat domain [ARD]) responsible for CD44 binding and its role in regulating HA-mediated ovarian tumor cell function. Using recombinant fragments of ankyrin (e.g., ARD and subdomain 1 [S1, aa1-aa217], subdomain 2 [S2, aa218-aa381], subdomain 3 [S3, aa382-aa612], and subdomain 4 [S4, aa613-aa834]) and in vitro binding assays, we determined that the S2 but not S1, S3, or S4 of ARD is the primary ankyrin binding region for CD44. Microinjection of antiglutathione S-transferase (GST)-tagged S2 or GST-tagged ARD fusion protein into CD44-positive ovarian tumor cells (e.g., SKOV3 cell line) promotes ankyrin association with CD44 in plaque-like structures and membrane projections. Additionally, we demonstrated that transfection of SKOV3 cells with S2cDNA or ARD cDNA results in an upregulation of HA-mediated tumor cell migration. Taken together, we believe that the S2 of the ARD plays a pivotal role in the direct binding to CD44 and promotes the cytoskeleton activation required for HA-mediated function such as ovarian tumor cell migration.     

The kinetics of chick cell population aging in vitro.

We have examined the kinetics of chick cell population aging in vitro using the percentage of labeled nuclei, the number of colonies formed from a low density inoculum and the number of cells/colony to monitor culture age. The results from these studies showed a gradual age-associated decline in each of the parameters which was first detected early in the culture lifespan and well in advance of changes in total cell number at confluency. Our results also indicated that each of the above parameters, in addition to the calendar time cells had been in culture, could be used to estimate the percentage of lifespan completed by the culture. A comparison of the methods used to estimate the remaining culture lifespan indicated that the percentage of labeled nuclei was the most accurate in describing cell age.