Limiting cell aggregation during mesenchymal stem cell expansion on microcarriers

Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T‐flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex‐1^®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50 × 10⁶ cell g^?1 Cytodex‐1^®). Then, the MSC aggregates were put into contact with additional available surfaces (T‐flask, fresh and used Cytodex‐1^®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T‐flasks or fresh Cytodex‐1^® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex‐1^® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex‐1^® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Cell growth on microcarriers: comparison of proliferation on and recovery from various substrates

Three commercially-important types of cell were grown on four different microcarrier substrates. The cells, which included normal human diploid fibroblasts (MRC-5), primary chick embryo cells and Madin-Darby bovine kidney cells (MDBK), were compared with regard to proliferation on the substrates and with regard to recovery of viable cells from the same substrates. The substrates used included glass-coated microcarriers (Biosil), collagen microcarriers (Ventregel), DEAE-dextran microcarriers (Cytodex I) and collagen-linked DEAE-dextran microcarriers (Cytodex III). The established cell line (MDBK) grew well on all of the substrates and a high percentage of viable cells could be harvested from each substrate. The MRC-5 cells also grew well on all four substrates but high recovery rates were achieved only with cells grown on the glass-coated microcarriers or collagen microcarriers. In contrast, the primary chick embryo cells grew well only on the glass microcarriers and the recovery rate of cells harvested from this substrate was high. In some industrial operations, the re-utilization of cells after removal from the substrate is necessary. In these situations the appropriate choice of microcarriers for the cultivation of the cells may be critical.     

Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.     

Effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of human mesenchymal stem cells

With the aim to utilize human mesenchymal stem cells (hMSCs) grown in large scale for regenerative medicine, effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of hMSCs were studied. hMSCs could attach and grew on surface-type microcarriers of Cytodex 1, whereas almost no cell elongation and growth were observed on porous type microcarriers of Cytopores. The percentages of aggregated Cytodex 1 microcarriers at an agitation rate of 60 and 90 rpm were lower than that at 30 rpm, which was the lowest agitation rate necessary for the suspension of Cytodex 1 microcarriers, and the cells grew fastest at 60 rpm. hMSC could be subcultivated on Cytodex 1 by the beads-to-beads method at both 30 and 60 rpm without trypsinization. However, agitation at 60 rpm resulted in a markedly lower percentage of aggregated microcarriers not only before but also after subcultivation. The percentages of CD90- and CD166-positive cells among cells grown on Cytodex 1 at 60 rpm (91.5 and 87.6 %) were comparable to those of cells grown in the pre-culture on dishes. In conclusion, hMSCs could be subcultivated on Cytodex 1 by beads-to-beads method maintaining the expressions of the cell surface antigens CD90 and CD166, while adjusting agitation rate could decrease the microcarrier aggregation.     

A microcarrier-based cell culture process for the production of a bovine respiratory syncytial virus vaccine

Veterinary viral vaccines generally comprise either attenuated or chemically inactivated viruses which have been propagated on mammalian cell substrates or specific pathogen free (SPF) eggs. New generation vaccines include chemically inactivated virally-infected whole cell vaccines. The NM57 cell line is a bovine nasal turbinate persistently infected (non-lytic infection) with a strain of the respiratory syncytial virus (RSV). The potential of microcarrier technology for the cultivation in bioreactors of this anchorage dependent cell line for RSV vaccine production has been investigated. Both Cytodex 3 and Cultispher S microcarriers proved most suitable from a selection of microcarriers as growth substrates for this NM57 cell line. Maximum cell densities of 4.12×105 cells ml-1and 5.52×105 cells ml-1 respectively were obtained using Cytodex 3 (3 g l-1) and and Cultispher S (1 g l-1) in 5 l bioreactor cultures. The fact that cell growth was less sensitive to agitation rate when cultured on Cultispher S microcarriers, and that cells were efficiently harvested from this microcarrier by an enzymatic method, suggested Cultispher S is suitable for further evaluation at larger bioreactor scales (>5 l) than that described here. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative investigation of the use of various commercial microcarriers as a substrate for culturing mammalian cells

Microcarriers provide large adhesion area allowing high cell densities in bioreactor systems. This study focused on the investigation of cell adhesion and cell growth characteristics of both anchorage-dependent CHO-K1 and anchorage-independent Ag8 myeloma cell lines cultivated on four different microcarriers (Biosilon®, Microhex®, Cytodex 3®, Cytoline 2®) by considering the cell kinetics and physiological data. Experiments were performed in both static and agitated cell culture systems by using 24-well tissue culture plates and then 50-ml spinner flasks. In agitated cultures, the highest specific growth rates (0.026 h for CHO-K1 and 0.061 h for Ag8 cell line) were obtained with Cytodex 3® and Cytoline 2® microcarriers for CHO-K1 and Ag8 cell line, respectively. Metabolic characteristics showed some variation among the cultures with the four microcarriers. The most significant being the higher production of lactate with microcarriers with CHO-K1 cells relative to the Ag8 cells. SEM analyses revealed the differences in the morphology of the cells along with microcarriers. On Cytodex 3® and Cytoline 2®, CHO-K1 cells attached to the substratum through long, slender filopodia, whereas the cells showed a flat morphology by covering the substratum on the Biosilon® and Microhex®. Ag8 cells maintained their spherical shapes throughout the culture for all types of microcarriers. In an attempt to scale-up, productions were carried out in 50-ml spinner flasks. Cytodex 3® (for CHO-K1 cells) and Cytoline 2® (for Ag8 cells) were evaluated. The results demonstrate that high yield of biomass could be achieved through the immobilization of the cells in each culture system. And cell cultures on microcarriers, especially on Cytodex 3® and Cytoline 2®, represented a good potential as microcarriers for larger scale cultures of CHO-K1 and Ag8, respectively. Moreover, owing to the fact that the cell lines and culture media are specific, outcomes will be applicable for other clones derived from the same host cell lines.     

Human fibroblastic cells attach to controlled-charge and gelatin-coated microcarriers at different rates

Human embryonic fibroblastic cells, FS-4, attach to Cytodex 1 and Cytodex 3 microcarriers at different rates. The first order rate constant for attachment to Cytodex 1 is two-fold higher than that to Cytodex 3. After attachment no difference in growth kinetics on the two types of microcarriers was observed.     

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.     

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.     

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     

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.     

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.     

Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system

Embryonic stem (ES) cells have the ability to differentiate in vitro into a wide variety of cell types with potential applications for tissue regeneration. However, a large number of cells are required, thus strengthening the need to develop large-scale systems using chemically defined media for ES cell production and/or controlled differentiation. In the present studies, a stirred culture system (i.e. spinner flask) was used to scale-up mouse ES (mES) cell expansion in serum-containing (DMEM/FBS) or serum-free medium, both supplemented with leukemia inhibitory factor (LIF), using either Cytodex 3 or Cultispher S microcarriers. After 8 days, maximal cell densities achieved were (1.9+/-0.1), (2.6+/-0.7) and 3.5x10(6)cells/mL for Cytodex 3 in DMEM/FBS, Cultispher S in DMEM/FBS and Cultispher S in serum-free cultures, respectively, with fold increases of 38+/-2, 50+/-15 and 70. Both microcarriers were suitable to sustain mES cell expansion, though the macroporous Cultispher S seemed to be advantageous in providing a more protective environment against shear stress forces, which harmful effects are exacerbated in serum-free conditions. Importantly, mES cells expanded under stirred conditions using serum-free medium retained their pluripotency and the ability to commit to the neural lineage.     

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     

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.     

Attachment and growth kinetics of anchorage-dependent BHK cells on microcarriers

Anchorage-dependent Baby Hamster Kidney (BHK) cells were cultivated on polyhydroxyethylmethacrylate (PHEMA), polystyrene (PS), and Cytodex microcarriers. Analysis of the experimental data indicated that there were a finite number of sites on the microcarrier surfaces, available for anchorage. The number of these sites was determined by the chemical and physical structure of the surface. A small fraction of these sites were suitable for attachment of the cells before proliferation. A larger fraction of these sites did not support attachment but the cells could proliferate on them by the help of previously attached mother cells. The attachment and proliferation of the BHK cells on these microcarriers were satisfactorily modeled by surface saturation type of mathematical expressions.     

Attachment of marine sponge cells of Hymeniacidon perleve on microcarriers

Toward the development of an in vitro cultivation of marine sponge cells for sustainable production of bioactive metabolites, the attachment characteristics of marine sponge cells of Hymeniacidon perleve on three types of microcarriers, Hillex, Cytodex 3, and glass beads, were studied. Mixed cell population and enriched cell fractions of specific cell types by Ficoll gradient centrifugation (6%/8%/15%/20%) were also assessed. Cell attachment ratio (defined as the ratio of cells attached on microcarrier to the total number of cells in the culture) on glass beads is much higher than that on Cytodex 3 and Hillex for both mixed cell population and cell fraction at Ficoll 15-20% interface. The highest attachment ratio of 41% was obtained for the cell fraction at Ficoll 15-20% interface on glass beads, which was significantly higher than that of a mixed cell population (18%). The attachment kinetics on glass beads indicated that the attachment was completed within 1 h. Cell attachment ratio decreases with increase in cell-to-microcarrier ratio (3-30 cells/bead) and pH (7.6-9.0). The addition of serum and BSA (bovine serum albumin) reduced the cell attachment on glass beads.     

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.     

Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture

In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors.     

Engineering stem cell fate with biochemical and biomechanical properties of microcarriers

Microcarriers have been widely used for various biotechnology applications because of their high scale‐up potential, high reproducibility in regulating cellular behavior, and well‐documented compliance with current Good Manufacturing Practices (cGMP). Recently, microcarriers have been emerging as a novel approach for stem cell expansion and differentiation, enabling potential scale‐up of stem cell‐derived products in large bioreactors. This review summarizes recent advances of using microcarriers in mesenchymal stem cell (MSC) and pluripotent stem cell (PSC) cultures. From the reported data, efficient expansion and differentiation of stem cells on microcarriers rely on their ability to modulate cell shape (i.e. round or spreading) and cell organization (i.e. aggregate size). Nonetheless, current screening of microcarriers remains empirical, and accurate understanding of how stem cells interact with microcarriers still remains unknown. This review suggests that accurate characterization of biochemical and biomechanical properties of microcarriers is required to fully exploit their potential in regulating stem cell fate decision. Due to the variety of microcarriers, such detailed analyses should lead to the rational design of application‐specific microcarriers, enabling the exploitation of reproducible effects for large scale biomedical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1354–1366, 2013