大规模动物细胞培养技术研究进展

利用动物细胞大规模培养技术可生产多种生物制品,为提高细胞活力和表达水平及有利于表达产物的纯化,采用有多种添加成分的无血清培养基培养细胞,选择更有利于细胞生长又可提高培养细胞密度的微载体和条件温和、易操作、气体交换速度快的生物反应器,在线监控细胞生存环境和生理活动,减少培养过程培养基中的抑制因素,可创造更适合细胞生存的环境,提高表达水平,向细胞中导入抗凋亡基因,可提高细胞活性和蛋白产量。利用多也微载体以球转球方式大规模培养动物细胞有很好的发展前景。     

Culture of 293 cells in different culture systems: Cell growth and recombinant adenovirus production

Growth of 293 cells (human embryonic kidney) was compared in various cell culture systems including static flasks, cell aggregates and a variety of porous microcarriers. The best results were achieved with Fibra-Cel carriers and cell aggregates (1.1–1.4 × 10⁶ cells/ml). Virus production was compared using a recombinant replication-deficient adenovirus as a model. Virus yields of lysates from cells grown on Fibra-Cel carriers, as cell aggregates and in static flasks were comparable (1.7–1.9 × 10⁸ pfu/ml).     

Microcarrier culture of lepidopteran cell lines: implications for growth and recombinant protein production

Several microcarrier systems were screened with Sf-9 and High-Five cell lines as to their ability to support cell growth and recombinant (beta-galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex-1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore-1 and -2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra-Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 x 10(6) (Sf-9) and 2.7 x 10(6) (High-Five) cells mL(-1) (8 x 10(6) and 5.5 x 10(6) cells per cm(2) of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. Beta-Galactosidase (beta-gal) production of Sf-9 cells on Fibra-Cel disks (infected at 3.3 x 10(6) cells mL(-1)) was prolonged (192 h), and specific protein production was similar to that of high-density free cell infection. Cultispher-S microcarriers were found to be a very efficient system for the growth of High-Five cells, whereas no growth of Sf-9 cells took place for the same system. Concentrations of about 9 x 10(6) cells mL(-1) were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after beta-gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final beta-gal titers of 946, 1728, and 1484 U mL(-1) were obtained for infection densities of 3.4, 7.2, and 8.9 x 10(6) cells mL(-1), respectively.     

N-acetylglucosamine and adenosine derivatized surfaces for cell culture: 3T3 fibroblast and chicken hepatocyte response

3T3 fibroblasts and primary chicken hepatocytes were cultured on derivatized polystyrene surfaces to examine the effect of cell-specific ligands on cellular morphology and growth. Surfaces were prepared by derivatizing chloromethylated polystyrene with N-acetylglucosamine (GlcNAc; recognized by the chicken asialoglycoprotein receptor) and adenosine (not recognized by adult hepatocytes). These surfaces were compared with tissue culture polystyrene (TCPS), acid-cleaned glass, and the unmodified chloromethylated polystyrene. The spreading, cytoskeletal structure and growth of the fibroblasts following attachment to these surfaces were examined. The extent of attachment, total protein levels, and DNA contents for surfaces-attached chicken hepatocytes were also measured. Fibroblast spreading was greatest on polymer surfaces derivatized with GlcNAc, whereas cytoskeletal structure and growth rate were independent of surface chemistry. Although chicken hepatocytes attached most efficiently to the GlcNAc derivatized polymer, the total protein and DNA levels of the surface-attached cells were not affected. In anticipation of the application of these polymers for cell culture and hybrid artificial organ design, the GlcNAc-derivatized polystryrene was fabricated into porous microcarriers. Fibroblasts grew avidly on the microcarriers, whereas chicken hepactocytes adhered well to the formed large aggregates arounds the microcarriers.     

Changes in Morphology,Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine

Tissue engineering of chondrocytes on a Random Positioning Machine (RPM) is a new strategy for cartilage regeneration. Using a three-dimensional RPM, a device designed to simulate microgravity on Earth, we investigated the early effects of RPM exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of RPM exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis (TGF-β₁, osteopontin); and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours of RPM exposure disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours on the RPM, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β₁ was enhanced during RPM culture for 24 h. Taking these results together, we suggest that chondrocytes exposed to the RPM seem to change their extracellular matrix production behaviour while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.     

Encapsulation of osteoblast seeded microcarriers into injectable, photopolymerizable three-dimensional scaffolds based on d,l-lactide and epsilon-caprolactone

UMR-106 seeded microcarriers were encapsulated into in situ, photopolymerizable three-dimensional scaffolds based on d,l-lactide and epsilon-caprolactone. UMR-106 and rat bone marrow cells proliferated and differentiated well on the microcarriers. The microcarriers were completely colonized after 14 days in culture. The viscous polymer paste allowed to mix the UMR-106 seeded microcarriers and gelatin (porosigen) properly. After the photopolymerization process, microcarriers and gelatin were evenly distributed throughout the scaffold. Gelatin was leached out within 7 h, and a porous scaffold was obtained. The microcarriers remained in the scaffold even after 7 days which demonstrates that they were well entrapped in the polymer. Increasing the amount of entrapped microcarriers (20-50%) leads to scaffolds with a reduced cross-linking. Hence, the microcarriers leached out. The encapsulated UMR-106 cells did not show pyknotic nuclei which demonstrates that the photopolymerization and handling the viscous polymer/gelatin/microcarrier paste is not detrimental for the cells.     

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.     

Flow Characterization of a Spinner Flask for Induced Pluripotent Stem Cell Culture Application

We present detailed quantitative measurement analyses for flow in a spinner flask with spinning rates between 20 to 45 RPM, utilizing the optical velocimetry measurement technique of Particle Image Velocimetry (PIV). A partial section of the impeller was immersed in the working fluid to reduce the shear forces induced on the cells cultured on microcarriers. Higher rotational speeds improved the mixing effect in the medium at the expense of a higher shear environment. It was found that the mouse induced pluripotent stem (iPS) cells achieved the optimum number of cells over 7 days in 25 RPM suspension culture. This condition translates to 0.0984 Pa of maximum shear stress caused by the interaction of the fluid flow with the bottom surface. However, inverse cell growth was obtained at 28 RPM culture condition. Such a narrow margin demonstrated that mouse iPS cells cultured on microcarriers are very sensitive to mechanical forces. This study provides insight to biomechanical parameters, specifically the shear stress distribution, for a commercially available spinner flask over a wide range of Reynolds number.     

Three-dimensional adipose tissue model using low shear bioreactors

Summary Presented here are techniques developed to culture and analyze three-dimensional (3-D) adipose-like tissues as a means to bridge the gap between current liminations in culturing preadipocytes (PAs) and that of providing clinically relevant volumes of adipose tissue useful for soft tissue engineering stratgies in reconstructive surgery. Pilot studies were performed to determine techniques to visualize and analyze 3-D PA-like tissues as well as to develop successful strategies to culture 3T3-L1 cells in a high aspect ratio vessel rotating-wall bioreactor both with and without microcarriers. Next, a series of cultures were accessed to verify these techniques as well as to compare the culture of the cells with and without microcarriers. Finally, a perfused rotating-wall bioreactor was used to further investigate the nature of the aggregates or tissues being generated. The aggregates that formed in the perfused system were analyzed via histology and in vivo animal studies. PA-like tissues as large as 4–5 mm in diameter without microcarriers that were capable of lipid-loading and composed of viable cells were achieved. We have successfully demonstrated that large tissue aggregates can be grown in bioreactor culture systems.     

Hydrodynamic effects on BHK cells grown as suspended natural aggregates

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John Wiley & Sons, Inc.     

Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γ production in suspension and inside microcarriers in protein-free media

We have recently developed a protein-free medium (PFS) able to support the growth of Chinese hamster ovary (CHO) cells in suspension. Upon further supplementation with some plant protein hydrolysates, medium performances reached what could be observed in serum-containing media [Burteau et al. In Vitro Cell. Dev. Biol.-Anim. 39 (2003) 291]. Now, we describe the use of rice and wheat protein hydrolysates, as non-nutritional additives to the culture medium to support productivity and cell growth in suspension or in microcarriers. When CHO-320 cells secreting recombinant interferon-gamma (IFN-γ) were cultivated in suspension in a bioreactor with our PFS supplemented with wheat hydrolysates, the maximum cell density increased by 25% and the IFN-γ secretion by 60% compared to the control PFS. A small-scale perfusion system consisting of CHO-320 cells growing on and inside fibrous microcarriers under discontinuous operation was first developed. Under these conditions, rice protein hydrolysates stimulated recombinant IFN-γ secretion by 30% compared to the control PFS. At the bioreactorscale, similar results were obtained but when compared to shake-flasks studies, nutrients, oxygen or toxic by-products gradients inside the microcarriers seemed to be the main limitation of the system. An increase of the perfusion rate to maintain glucose concentration over 5.5 mM and dissolved oxygen (DO) at 60% was able to stimulate the production of IFN-γ to a level of 6.6 μg h⁻¹ g⁻¹ of microcarriers after 160 h when a cellular density of about 4 × 10⁸ cell g⁻¹ of carriers was reached.     

Induction of osteogenic differentiation of osteoblast-like cells MG-63 during cultivation on fibroin microcarriers

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.     

Three‐dimensional culture systems for the expansion of pluripotent embryonic stem cells

Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two‐dimensional (2D) static culturing techniques are inadequate for large‐scale production. The culture of mammalian cells in three‐dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher‐S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher‐S out‐performed the Solohill microcarriers. When cultured for successive passages on Cultispher‐S microcarriers, mESCs maintained their pluripotency, demonstrated by self‐renewal, expression of pluripotency markers and the ability to undergo multi‐lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher‐S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra‐1–60, NANOG, and OCT‐4. Our study highlights the importance of optimization of initial seeding parameters and provides proof‐of‐concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683–695. © 2010 Wiley Periodicals, Inc.     

Suspension culture on microcarriers and as aggregates enables expansion and differentiation of pluripotent stem cells (PSCs)

Background aimsHuman pluripotent stem cells (PSCs) hold a great promise for promoting regenerative medical therapies due to their ability to generate multiple mature cell types and for their high expansion potential. However, cell therapies require large numbers of cells to achieve desired therapeutic effects, and traditional two-dimensional static culture methods cannot meet the required production demand for cellular therapies. One solution to this problem is scaling up expansion of PSCs in bioreactors using culture strategies such as growing cells on microcarriers or as aggregates in suspension culture.MethodsIn this study, we directly compared PSC expansion and quality parameters in microcarrier- and aggregate-cultures grown in single-use vertical-wheel bioreactors.ResultsWe showed comparable expansion of cells on microcarriers and as aggregates by day 6 with a cell density reaching 2.2 × 10⁶ cells/mL and 1.8 × 10⁶ cells/mL and a fold-expansion of 22- and 18-fold, respectively. PSCs cultured on microcarriers and as aggregates were comparable with parallel two-dimensional cultures and with each other in terms of pluripotency marker expression and retention of other pluripotency characteristics as well as differentiation potential into three germ layers, neural precursor cells and cardiomyocytes.ConclusionsOur study did not demonstrate a clear advantage between the two three-dimensional methods for the quality parameters assessed. This analysis adds support to the use of bioreactor systems for large scale expansion of PSCs, demonstrating that the cells retain key characteristics of PSCs and differentiation potential in suspension culture.     

Three-dimensional culture of bovine chondrocytes in rotating-wall vessels

Summary The Rotating-Wall Vessel (RWV) was used to culture chondrocytes for 36 d to observe the influence of low-shear and quiescent culture conditions allowing three-dimensional freedom on growth, differentiation, and extracellular matrix formation. Chondrocytes were freshly isolated from bovine cartilage and placed into the RWV with Cytodex-3 microcarriers. Nonadherent petri dishes were initiated with microcarriers as representative of standard culture conditions. In the RWV, large three-dimensional aggregates (5–7 mm) were formed in suspension. In addition, a large sheet of matrix adhered to the oxygenator core and vessel endcaps. Petri dish culture resulted in the formation of sheets of chondrocytes with no matrix production. Immunocytochemical analyses on histologic sections of tissue obtained from the RWV and the petri dish controls were performed with antibodies against fibronectin, collagen II, chondroitin-4-sulfate, chondroitin-6-sulfate, and vimentin. Results demonstrated increased signal in the RWV material while the petri dishes demonstrated a slight decrease in signal. In addition, differentiated chondrocytes were observed in sections of RWV material through 36 d, while few were observed in the sections of petri dish material. These results indicate that the unique conditions provided by the RWV afford access to cellular processes that signify the initiation of differentiation as well as production of normal matrix material.     

Enhanced Production of Human Recombinant Proteins from CHO cells Grown to High Densities in Macroporous Microcarriers

Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q _P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q _P with increasing cell densities up to a threshold of >1 × 10⁸ cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q _P any further. A positive linear correlation (r ² = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 10⁶ to 3 × 10⁸ cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.     

A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells

Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of porcine bone-marrow-derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 x 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three-dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two-dimensional monolayer cultivation method.     

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.     

Production and glycosylation of recombinant beta-interferon in suspension and cytopore microcarrier cultures of CHO cells

Microcarriers are suitable for high-density cultures of cells requiring surface attachment and also offer the advantage of easy media removal for product recovery. We have used the macroporous microcarriers Cytopore 1 and 2 for the growth of CHO cells producing recombinant human beta-interferon (beta-IFN) in stirred batch cultures. Although these cells may grow in suspension, in the presence of Cytopore microcarriers they become entrapped in the inner bead matrix where they can be maintained at high densities. Cell growth rates were reduced in microcarrier cultures compared to suspension cultures. However, the beta-IFN yield was up to 3-fold greater as a result of an almost 5-fold higher specific productivity. Maximum productivity was found in cultures containing 1.0 mg/mL of Cytopore 1 or 0.5 mg/mL of Cytopore 2 with a cell/bead ratio of 1029 and 822, respectively. Beta-IFN molecules aggregated in the later stages of all cultures, causing a decrease in response by ELISA. However, the degree of aggregation was significantly less in the microcarrier cultures. The N-linked glycans from beta-IFN were isolated and analyzed by normal phase HPLC. There was no apparent difference in the profile of glycans obtained from each of the suspension and Cytopore culture systems. This suggests that Cytopore microcarriers may be useful in bioprocess development for enhanced recombinant glycoprotein production without affecting the glycosylation profile of the protein.     

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