Online monitoring of microcarrier based fibroblast cultivations with in situ microscopy

Animal cell culture is widely used in biotechnology for the production of many biological products. In situ microscopes acquire images directly from cell suspensions and analyze the images in matters of cell concentration, cell size distribution and cell morphology. Their applicability was already proven for yeast and suspended mammalian cell cultivations. In this work the in situ microscope was utilized to measure the level of colonization of fibroblasts on microcarrier surfaces during cultivation. For this study the murine cell line NIH-3T3 was used in combination with Cytodex 1 microcarriers. Cultivations were carried out in a 5 L stirred tank bioreactor equipped with the in situ microscope. Images were obtained sequentially with the in situ microscope over the whole cultivation time (900 images per sequence, 7.5 h per sequence on average). For the microcarrier analysis an image analysis algorithm based on a neural network was developed and implemented in the microscope analysis software.     

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.     

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.     

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.     

In situ dark field microscopy for on-line monitoring of yeast cultures

A new-type in situ probe has been developed to acquire dark field images of yeast in bioreactors. It has been derived from an in situ bright field microscope that is able to measure cell density in bioreactors during fermentation processes. The illumination part of the probe has been replaced with a dark field device, in which an aspheric condenser is used, so that high contrast dark field images can be obtained. The technique of second imaging is implemented to improve the sharpness of the images by means of a relay lens. This new in situ probe is expected to enable the evaluation of the cell viability without staining owing to modern image processing.     

Development of an optical system for the non‐invasive tracking of stem cell growth on microcarriers

The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non‐invasive and automated monitoring of cell number, confluence, and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance. The in situ epi‐illumination of mouse embryonic fibroblasts and human mesenchymal stem cells attached to Cytodex 1 and 3 microcarriers was achieved using a bespoke microscope. Robust image processing techniques were developed to provide quantitative measurements of confluence, aggregate recognition, and cell number, without the need for fluorescent labeling or cell detachment. Large datasets of cells counted on individual microcarriers were statistically analyzed and compared with NucleoCounter measurements, with an average difference of less than 7% observed from days 0 to 6 of a 12‐day culture noted, prior to the onset of aggregation. The developed image acquisition system and post‐processing methodologies were successfully applied to dynamically moving colonized microcarriers. The proposed system offers a novel method of cell identification at the individual level, to consistently and accurately assess viable cell number, confluence, and cell distribution, while also minimizing the variability inherent in the current invasive means by which cells adhered to microcarriers are analyzed. Biotechnol. Bioeng. 2017;114: 2032–2042. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Microfibrous carriers for cell culture: A comparative study

Small patches of polyethylene terephthalate (PET) nonwoven microfibrous matrices have excellent properties and can be used as carriers for culturing cells in agitated bioreactors. The microfibrous carriers are highly porous and can provide large surface areas and three‐dimensional space for high‐density cell growth. In this work, the microfibrous carriers and several commercial microcarriers were used to study cell attachment kinetics, growth, and monoclonal antibody production with Chinese hamster ovary cells. Compared with commercial solid and macroporous microcarriers, the microfibrous carriers showed better or similar performances. In addition, the microfibrous carriers provided a wider operable range for agitation rate than commercial microcarriers, effectively protecting cells from shear stress and carrier collisions. In addition, the microfibrous carriers are available at a much lower cost than commercial microcarriers, providing an attractive alternative to microcarrier‐based large‐scale cell cultures. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

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     

A mechanistic analysis of the inoculum requirement for the cultivation of mammalian cells on microcarriers

For the cultivation of mammalian cells on microcarriers a minimum inoculum concentration is required to initiate cell attachment and subsequent cell growth. A critical cell number model has been proposed to elucidate the mechanism of the inoculum requirement. In this model it was hypothesized that after inoculation a critical number of cells per microcarrier is required for normal growth to occur; failure to acquire enough cells will impede cell growth. This critical cell number model was expressed mathematically and used to simulate cell distribution and growth on microcarriers under different cultivation conditions. By comparing the simulated growth kinetics with the experimental results, the actual critical cell number per microcarrier was identified. The critical number could be reduced by employing an improved medium for the cultivation.     

果糖修饰壳聚糖微载体的制备及其原代大鼠肝细胞培养

利用果糖修饰的壳聚糖为材料,液体石蜡作分散介质,戊二醛作交联剂,通过反相悬浮法制备了性能优良的微米级果糖修饰壳聚糖微载体。用其进行原代大鼠肝细胞培养,利用相差显微镜和扫描电镜对细胞形态进行观察,并测定了细胞的代谢活性。结果显示果糖修饰的壳聚糖微载体是一种优良的肝细胞培养支架。     

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.     

Substrate-dependent differences in growth and biological properties of fibroblasts and epithelial cells grown in microcarrier culture

Normal diploid human fibroblasts and first passage monkey kidney epithelial cells were examined for growth and metabolic activity on microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells cm2 of surface area) on the glass microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells/cm2 of surface area) on the glass microcarriers than they did on the DEAE-dextran microcarriers and morphological differences were observed between the cells growing on the two substrates. On the DEAE-dextran microcarriers, the cells were much more resistant to protease-mediated detachment than were the cells on the glass microcarriers. In these respects, the cells grown on the glass microcarriers were similar to cells grown in conventional monolayer culture. Interestingly, the cells grown on the DEAE-dextran microcarriers expressed higher levels of proteolytic enzyme activity than the cells grown on the glass microcarriers. Substrate-dependent differences in prostaglandin production also occurred--both in unstimulated cells and in cells stimulated with 12-0-tetradecanoyl phorbol acetate. The unstimulated cells on the glass microcarriers produced slightly higher levels of three different prostaglandins than did the cells on the DEAE-dextran microcarriers. However, after stimulation the levels were much higher in the DEAE-dextran microcarrier cultures than in the glass microcarrier cultures. In contrast to these results, there was no significant, substrate-dependent difference in the production of infectious herpes simplex virus. Taken together, these findings suggest that when commercially-useful cells such as normal fibroblasts and epithelial cells are grown in large quantities on microcarriers, the nature of the substrate may have a profound effect on the growth and physiology of the cells. They also suggest that when microcarriers are used, unexpected results based on preliminary work in conventional monolayer culture may be obtained.     

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.     

Some Aspects of Melanin Formation of Melanocytes Cultured on Collagen-Coated Microcarrier Beads

Melanocytes cultured on collagen-coated Cytodex 3 microcarrier Sephadex beads caused remarkable pigmentation of the beads during the period of culture when optimal density was reached. Electron microscopy of melanocytes on the microcarriers revealed that the cells and their dendrites invaginate into the microcarrier surface layer. Removal of the cells by trypsinization showed that some pigment granules were left on the carrier surface and within the cavities present on the microcarrier surface. In order to investigate whether the pigmentation of the microcarriers could be a result of indole intermediates of melanogenesis present in the culture medium, extracts were studied by gas chromatography/mass spectrometry for the presence of these compounds. Two compounds (5,6-dihydroxyindole-2-carboxylic acid and 6-hydroxy-5-methoxyindole-2-carboxylic acid) so far have been identified in the medium extracts. Results indicate that microcarrier culture of melanocytes can serve as an interesting model for electron microscopy studies of melanocytes with regard to pigmentation and cell attachment.     

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.     

A cellular automaton model for microcarrier cultures

In order to achieve high cell densities anchoragedependent cells are commonly cultured on microcarriers, where spatial restrictions to cell growth complicates the determination of the growth kinetics. To design and operate large-scale bioreactors for microcarrier cultures, the effect of this spatial restriction to growth, referred to as contact inhibition, must be decoupled from the growth kinetics. In this article, a cellular automaton approach is recommended to model the growth of anchorage-dependent cells on microcarriers. The proposed model is simple to apply yet provides an accurate representation of contact-inhibited cell growth on microcarriers. The distribution of the number of neighboring cells per cell, microcarrier surface areas, and inoculation densities are taken into account with this model. When compared with experimental data for Vero and MRC-5 microcarrier cultures, the cellular automaton predictions were very good. Furthermore, the model can be used to generate contact-inhibition growth curves to decouple the effect of contact-inhibition from growth kinetics. With this information, the accurate determination of kinetic parameters, such as nutrient uptake rates, and the effects of other environmental factors, such as toxin levels, may be determined. (c) 1994 John Wiley & Sons, Inc.     

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self‐renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large‐scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor‐intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel‐coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF‐microcarriers was less than that on MEF‐plates, the doubling time of hESCs on Matrigel‐microcarriers was indistinguishable from that of hESCs expanded on Matrigel‐coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier‐based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cell-microcarrier adhesion to gas-liquid interfaces and foam

The interaction of microcarriers, both with and without cells attached, with gas bubbles was studied. These studies consisted of qualitative microscopic observations of microcarriers with bubbles, quantitative measurements of microcarrier entrapment in foam, and quantitative measurements of the effect of bubble rupture at gas-medium interfaces. Ten different "protective additives" were evaluated for their ability to change the dynamic surface tension of the culture media and to prevent microcarrier adhesion to air bubbles during gas sparging and to prevent entrapment in the foam layer. These studies indicate that microcarriers, with and without cells, readily attach to gas-medium interfaces; yet unlike suspended cells, cells attached to microcarriers are not damaged by bubble ruptures at gas-medium interfaces. Only one surfactant was found to substantially prevent microcarrier entrapment in the foam layer; however, this surfactant was toxic to cells. No correlation was observed between surface tension and the prevention of microcarrier adhesion to gas-liquid interfaces. It is suggested that cell damage as a result of sparging in microcarrier cultures is the result of cells, attached to microcarriers, attaching to rising bubbles and then detaching from the microcarrier as this combination rises through the medium. It is further suggested that the hydrodynamic drag force of the rising microcarrier is sufficiently high to remove the bubble-attached cell from the microcarrier.     

WAVETM反应器和搅拌瓶中微载体球转球放大培养的比较

目的:哺乳动物细胞目前已广泛用于生物工程药物如单抗和疫苗的生产.而用于贴壁细胞规模化培养的微载体,也应时应需得以开发并应用于生物制药.贴壁细胞微载体培养在搅拌罐和WAVETM反应器中都能进行.而如要进行进一步的放大培养,球转球工艺不可或缺.为了发展球转球这一新的放大技术,以及考量WAVETM反应器这种新型大规模培养设备的应用性,大量的细胞培养和球转球实验在WAVETM反应器和搅拌瓶中进行.收集到的数据得以分析比较.方法:将Vero细胞分别接入WAVETM反应器和搅拌瓶中用微载体Cytodex 1进行培养.适当补充营养并控制温度、pH等培养条件使细胞增殖.长满微载体的细胞用清洗、消化等球转球工艺的一系列步骤而分离,并放大接种到新的培养体系.球转球工艺的有效性通过记录并统计分析细胞消化分离的回收率,以及细胞重新接种生长的存活力来评估.结果:统计学分析比较WAVETM反应器和搅拌瓶中得到的细胞分离回收率分别是67.56％和39.39％,数理统计P值小于0.0003;细胞重新接种存活率分别是95.17％和78.45％,P值等于0.0107.结论:在WAVETM反应器中进行的球转球放大工艺,其总体表现和有效性远高于在搅拌瓶中得到的结果.在WAVETM反应器中培养的Vero细胞有很好的细胞状态,作为种子链和生产用罐相比搅拌型反应罐均有很大的优越性.     

Collagen microcarrier spinner culture promotes osteoblast proliferation and synthesis of matrix proteins

In vitro propagation of osteoblasts in three-dimensional culture has been explored as a means of cell line expansion and tissue engineering purposes. Studies investigating optimal culture conditions are being conducted to produce bone-like material. This study demonstrates the use of collagen microcarrier beads as a substrate for three-dimensional cell culture. We have earlier reported that microcarriers consisting of cross-linked type I collagen support chondrocyte proliferation and synthesis of extracellular matrix. In this study, we investigated the use of collagen microcarriers to propagate human trabecular bone-derived osteoblasts. Aggregation of cell-seeded microcarriers and production of extracellular matrix-like material were observed after 5 d in culture. Expression of extracellular matrix proteins osteocalcin, osteopontin, and type I collagen was confirmed by messenger ribonucleic acid analysis, radioimmunoassay, and Western blot analysis. The efficient recovery of viable cells was achieved by collagenase digestion of the cell-seeded microcarriers. The collagen microcarrier spinner culture system provides an efficient method to amplify large numbers of healthy functional cells that can be subsequently used for further in vitro or transplantation studies.