Microcarrier Culture Research Articles

Efficient expansion and osteogenic differentiation of mesenchymal stem cells in microcarrier culture

Efficient expansion and osteogenic differentiation of mesenchymal stem cells in microcarrier culture

Hepatic differentiation of human embryonic stem cells on microcarriers

Translation of stem cell research to industrial and clinical settings mostly requires large quantities of cells, especially those involving large organs such as the liver. A scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiated cells. To increase the culture efficiency in bioreactor system, high surface to volume ratio needs to be achieved. We employed a microcarrier culture system for the expansion of undifferentiated human embryonic stem cells (hESCs) as well as for directed differentiation of these cells to hepatocyte-like cells. Cells in single cell suspension were attached to the bead surface in even distribution and were expanded to 1×106cells/ml within 2 days of hESC culture with maintenance of the level of pluripotency markers. Directed differentiation into hepatocyte-like cells on microcarriers, both in static culture and stirred bioreactors, induced similar levels of hepatocyte-like cell differentiation as observed with cells cultured in conventional tissue culture plates. The cells expressed both immature and mature hepatocyte-lineage genes and proteins such as asialoglycoprotein receptor-1 (ASGPR-1) and albumin. Differentiated cells exhibited functional characteristics such as secretion of albumin and urea, and CYP3A4 activity could be detected. Microcarriers thus offer the potential for large-scale expansion and differentiation of hESCs induced hepatocyte-like cells in a more controllable bioreactor environment.

Evaluation of Macroporous and Microporous Carriers for CHO-K1 Cell Growth and Monoclonal Antibody Production

The emergence of microcarrier technology has brought a renewed interest in anchorage-dependent cell culture for high-yield processes. Well-known in vaccine production, microcarrier culture also has potential for application in other fields. In this work, two types of microcarriers were evaluated for small-scale monoclonal antibody (mAb) production by CHOK1 cells. Cultures (5 ml) of microporous Cytodex 3 and macroporous CultiSpher-S carriers were performed in vented conical tubes and subsequently scaled-up (20 ml) to shake-flasks, testing combinations of different culture conditions (cell concentration, microcarrier concentration, rocking methodology, rocking speed, and initial culture volume). Culture performance was evaluated by considering the mAb production and cell growth at the phases of initial adhesion and proliferation. The best culture performances were obtained with Cytodex 3, regarding cell proliferation (average 1.85 ± 0.11 × 10(6) cells/ml against 0.60 ± 0.08 × 10(6) cells/ ml for CultiSpher-S), mAb production (2.04 ± 0.41 μg/ml against 0.99 ± 0.35 μg/ml for CultiSpher-S), and culture longevity (30 days against 10-15 days for CultiSpher-S), probably due to the collagen-coated dextran matrix that potentiates adhesion and prevents detachment. The culture conditions of greater influence were rocking mechanism (Cytodex 3, pulse followed by continuous) and initial cell concentration (CultiSpher-S, 4 × 10(5) cells/ml). Microcarriers proved to be a viable and favorable alternative to standard adherent and suspended cultures for mAb production by CHO-K1 cells, with simple operation, easy scale-up, and significantly higher levels of mAb production. However, variations of microcarrier culture performance in different vessels reiterate the need for optimization at each step of the scale-up process.

Interview: Microcarrier culture in stem cell bioprocessing

Steve Oh is currently Principal Scientist and Director of the Bioprocess Internship Programme at the Bioprocessing Technology Institute, one of the biomedical research institutes under the Agency for Science Technology and Research. The focus of the Bioprocessing Technology Institute’s research is on developing simple yet rigorous and reproducible methods for producing high quality adherent stem cells (human pluripotent, multipotent stem cells and their differentiated derivatives, e.g., cardiomyocytes, neural cells and osteoblasts), for applications such as identifying drug candidates and tissue repair. Microcarriers have the versatility of different shapes, sizes, charges and the ability to present diverse extracellular matrices or peptides on their surfaces for cell expansion and differentiation. Microcarriers also enable adherent dependent stem cells to be cultured in suspension bioreactors instead of plasticware, which can readily be transferred to commercial manufacturing. They are also developing mo...

Inhibition of ROCK–Myosin II Signaling Pathway Enables Culturing of Human Pluripotent Stem Cells on Microcarriers Without Extracellular Matrix Coating

Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free microcarriers. Cells propagated on the noncoated microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3×10⁶ cells/mL with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1±1.2×10⁶ cells/mL) with a cell yield of 412±77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91±1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.

An ovarian cell microcapsule system simulating follicle structure for providing endogenous female hormones

The aim of this study was to create a microcapsule system simulating native follicle structure by introducing microcarrier culture to microencapsulation for providing endogenous female hormones. Granulosa and theca cells of rat follicles were isolated. Granulosa cells were grown on microcarriers and enclosed together with theca cells in alginate-chitosan-alginate microcapsules. The cell viability and female hormone secretion were investigated in vitro. The microcapsules were transplanted to ovariectomized rats and the serum levels of estradiol and progesterone were measured for 60 days. The microencapsulated granulosa cells growing on microcarriers exhibited enhanced viability and promoted secreting ability of estradiol and progesterone compared with those without the microcarriers. Co-microencapsulation of granulosa cells and theca cells markedly elevated estradiol secretion in vitro. Transplantation of co-microencapsulated granulosa cells on microcarriers and theca cells maintained serum estradiol and progesterone at normal levels for 60 days. Microcarrier cell culture has been proved to be an effective method to enhance the viability of granulosa cells in microcapsules. Moreover, the transplantation of microcapsules enclosing granulosa cells on microcarriers and theca cells may be promising to provide endogenous female hormones for menopausal syndrome treatment.

Proteome analysis of virus–host cell interaction: rabies virus replication in Vero cells in two different media

The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection.

Microcarrier Culture for Efficient Expansion and Osteogenic Differentiation of Human Fetal Mesenchymal Stem Cells

Stirred microcarrier (MC) culture has been suggested as the method of choice for supplying large volumes of mesenchymal stem cells (MSCs) for bone tissue engineering. In this study, we show that in addition to the improvement in cell expansion capacity, MSCs propagated and harvested from MC culture also demonstrate higher osteogenic potency when differentiated in vivo or in vitro in three-dimensional (3D) scaffold cultures as compared with traditional monolayer (MNL) cultures. Cytodex 3 microcarrier-expanded human fetal MSC (hfMSC) cultures (MC-hfMSCs) achieved 12- to 16-fold expansion efficiency (6×105–8×105 cells/mL) compared to 4- to 6-fold (1.2×105–1.8×105 cells/mL) achieved by traditional MNL-expanded hfMSC culture (MNL-hfMSCs; p<0.05). Both MC-hfMSCs and MNL-hfMSCs maintained similar colony-forming capacity, doubling times, and immunophenotype postexpansion. However, when differentiated under in vitro two-dimensional (2D) osteogenic conditions, MC-hfMSCs exhibited a 45-fold reduction in alkaline phosphatase level and a 37.5% decrease in calcium deposition compared with MNL-hfMSCs (p<0.05). Surprisingly, when MC-hfMSCs and MNL-hfMSCs were seeded on 3D macroporous scaffold culture or subcutaneously implanted into nonobese diabetic/severe combined immunodeficient mice, MC-hfMSCs deposited 63.5% (p<0.05) more calcium and formed 47.2% (p<0.05) more bone volume, respectively. These results suggest that the mode of hfMSC growth in the expansion phase affects the osteogenic potential of hfMSCs differently in various differentiation platforms. In conclusion, MC cultures are advantageous over MNL cultures in bone tissue engineering because MC-hfMSCs have improved cell expansion capacity and exhibit higher osteogenic potential than MNL-hfMSCs when seeded in vitro into 3D scaffolds or implanted in vivo.

Extracellular matrix free microcarrier cultures of human pluripotent stem cells induced by inhibition of rock-myosin II signaling

Extracellular matrix free microcarrier cultures of human pluripotent stem cells induced by inhibition of rock-myosin II signaling

Assessment of long-term effects of nanoparticles in a microcarrier cell culture system.

Nano-sized materials could find multiple applications in medical diagnosis and therapy. One main concern is that engineered nanoparticles, similar to combustion-derived nanoparticles, may cause adverse effects on human health by accumulation of entire particles or their degradation products. Chronic cytotoxicity must therefore be evaluated. In order to perform chronic cytotoxicity testing of plain polystyrene nanoparticles on the endothelial cell line EAhy 926, we established a microcarrier cell culture system for anchorage-dependent cells (BioLevitatorTM). Cells were cultured for four weeks and exposed to doses, which were not cytotoxic upon 24 hours of exposure. For comparison, these particles were also studied in regularly sub-cultured cells, a method that has traditionally been used to assess chronic cellular effects. Culturing on basal membrane coated microcarriers produced very high cell densities. Fluorescent particles were mainly localized in the lysosomes of the exposed cells. After four weeks of exposure, the number of cells exposed to 20 nm polystyrene particles decreased by 60% as compared to untreated controls. When tested in sub-cultured cells, the same particles decreased cell numbers to 80% of the untreated controls. Dose-dependent decreases in cell numbers were also noted after exposure of microcarrier cultured cells to 50 nm short multi-walled carbon nanotubes. Our findings support that necrosis, but not apoptosis, contributed to cell death of the exposed cells in the microcarrier culture system. In conclusion, the established microcarrier model appears to be more sensitive for the identification of cellular effects upon prolonged and repeated exposure to nanoparticles than traditional sub-culturing.

The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody.

Microcarriers are widely used for the large-scale culture of attachment-dependent cells with increased cell densities and, ultimately, higher product yield. In these processes, the specific culture conditions can affect the quality of the product, which is closely related to its glycosylation pattern. Furthermore, the lack of studies in the area reinforces the need to better understand the effects of microcarrier culture in product glycosylation. Consequently, in this work, the glycosylation profile of a monoclonal antibody (mAb) produced by adherent CHO-K1 cells grown in Cytodex 3 was evaluated under different conditions, and compared to that obtained of typical adherent cultures. It was found that microcarrier cultures result in a glycosylation profile with different characteristics from T-flask cultures, with a general increase in galactosylation and decrease in fucosylation levels, both with a potentially positive impact on mAb activity. Sialylation also varied but without a general tendency. This study then showed that the specific culture conditions used in microcarrier culture influence the mAb glycan profile, and each functional element (galactose, core fucose, sialic acid) is independently affected by these conditions. In particular, great reductions of fucosylation (from 79 to 55%) were obtained when using half volume at inoculation, and notable decreases in sialylation (from 23 to 2%) and glycoform heterogeneity (from 20 to 11 glycoforms) were observed for shake flask culture, potentially associated with the improved cell densities achieved in these culture vessels.

Biological characteristics of adult degenerative nucleus pulposus cells in a three-dimensional microcarrier stirring culture system

A major problem in reconstructing degenerative intervertebral discs is to obtain sufficient nucleus pulposus (NP) seeding cells with normal physiologic functions. The current study adopted a three-dimensional microcarrier culture system for massive cell expansion and evaluated the biological characteristics and physiological functions of the propagated adult degenerative NP cells. Isolated adult NP cells were cultured in either microcarrier stirring culturing system or traditional monolayer cultivation. The growth characteristics, proliferation, extracellular matrix secretion, and apoptosis potential were examined to evaluate the different features of the two cultivation methods. Compared to the monolayer cultivation system, the adhesion time of NP cells in the three-dimensional microcarrier culture system appeared longer with relatively transient stable growth period. MTT and (3)H-TdR assays suggested significantly elevated proliferation and higher thymidine incorporation rates in cells from microcarrier system compare to cells in the monolayer system at the exponential growth phase (p < 0.05). Western blot data complimented the immunostaining results that the NP cells in the microcarrier system expressed significantly more protein levels of both type collagens at the exponential growth phase than that in the monolayer system (p < 0.05). Further, significantly more (35)S labeled proteoglycan incorporation was noticed in the cells on the microcarriers at both the stable growth and the exponential growth phases (p < 0.05 and p < 0.01). In conclusion, the three-dimensional microcarrier stirring culture system provides a means of fast and massive propagation of NP seeding cells which maintain their normal physiological characteristics and functions.

Non-Viral Engineering of Skin Precursor-Derived Schwann Cells for Enhanced NT-3 Production in Adherent and Microcarrier Culture

Genetic engineering of stem cells and their derivatives has the potential to enhance their regenerative capabilities. Here, dendrimer- and lipofection-based approaches were used for non-viral neurotrophin-3 (NT-3) over-expression in Schwann cells differentiated from skin precursors (SKP-SCs). A variety of dendrimers were first tested for transfection efficiency on HEK 293T cells, with PAMAMNH2 G4 found most effective and used subsequently for SKP-SCs transfection. Plasmid-based expression resulted in increased NT-3 release from SKP-SCs in both adherent and microcarrier-based culture. In a proof-of-concept study, the microcarrier/SKP-SCs were implanted into the injured nerve, and transfected cells were shown to detach, integrate into the nerve tissue and associate with regenerating axons. Virus-free systems for transient neurotrophin expression are a feasible and biologically safe option to increase the therapeutic value of stem cells and stem cell-derived cells in nerve repair strategies. Further work to develop bioprocesses for expansion of SKP-SCs on microcarriers in bioreactors is still needed.

Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×10⁶ cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×10⁶ cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes, showing that cells maintained their multilineage differentiation potential.

Optimization of culture of mesenchymal stem cells: a comparison of conventional plate and microcarrier cultures

There has been increasing interest in mesenchymal stem cells (MSCs) because of their potential use for regenerative therapy; however, there is no well-defined protocol for MSCs culture. This study compares techniques of conventional plate and microcarrier culturing of MSCs. Here, different conditions for isolation and expansion of rat MSCs have been examined and it was found that plating density and plating time in primary culture played important roles for culture of these rat MSCs. When plated at 10(8) /cm(2) density for 72 h, in primary culture, recycling stem cells (RS cells) predominated, and characteristics of rat MSCs (including morphology, growth rate, phenotype and differentiation potentials) remained stable during expansion until passage 14. For subculture of the cells, it was found that their growth rate when incubated at 33 °C was higher than those incubated at 37 °C, and maximal increase was 10- and 6-fold respectively. When cultured using microcarriers, at a density of 1 × 10(5) /mg beads, growth kinetics, phenotype and differentiation potentials also remained constant for cells between passage 2nd and 14th; their maximal number increased 16-fold. Compared to conventional plate culture, culture using gelatine porous microcarrier Cultispher-S was superior for large-scale production of rat MSCs.

Differentiation of Human Embryonic Stem Cells to Cardiomyocytes on Microcarrier Cultures

We have developed an improved cardiomyocyte differentiation protocol where we stabilized embryoid bodies (EB) in serum- and insulin-free medium (bSFS) supplemented with p38 MAP kinase inhibitor (SB203580) by addition of 10 µm laminin-coated positively charged (protamine sulfate derivatized TSKgel Tresyl-5PW) microcarriers. This protocol achieved a maximum 3-fold cell expansion, differentiation efficiency of 20%, and an overall cardiomyocyte yield of 3 × 10⁵ CM/ml in static conditions. In comparison, EB cultures achieved 1.5-fold cell expansion, differentiation efficiency of 15%, and an overall cardiomyocyte yield of 1.1 × 10⁵ CM/ml. The scalability of this platform was demonstrated in suspended spinner cultures, producing a maximum of 2.14 × 10⁵ CM/ml in 50-ml cultures. This yield is two-fold higher than the control static EB-based platform (1.1 × 10⁵ CM/ml), and seven-fold higher than yields reported in literature, 3.1-9 × 10⁴ CM/ml. The robustness of this protocol was tested with HES-3 and H1 cell lines.

Establishment of a microcarrier culture system with serial sub-cultivation for functionally active human endothelial cells

A microcarrier culture system was established for a large-scale production of functional human endothelial cells. It has been difficult to cultivate human endothelial cells in large quantities for the reasons that specific growth factor and extracellular matrix are required for the survival and proliferation of the cells and the life span of the primary cells are limited. A lot of studies have reported that the shear stress gives significant influences on the structure, growth rate and biological functions of endothelial cells. We aimed to develop a convenient microcarrier culture system for human endothelial cells which can reproduce the flow effects experienced in vivo or in vitro. In 200 mL volume culture, human umbilical vein endothelial cells (HUVEC) could be serially sub-cultivated by optimizing the culture conditions such as shear strength, growth factor, beads and seeding cell concentration, serum concentration, and passage timing. The growth rate was enhanced depending on the shear strength and the life span of the cells was elongated until over 43PDL which is much longer than those of monolayer cultures. The cells maintained the diploidy of over 80% without obvious abnormal changes in the chromosomes. The serially sub-cultured microcarrier cells maintained various endothelial cell functions such as the syntheses of von Willebrand factor (vWf), prostacyclin and other biological substances, the expression of CD31, and the VEGF(165) dependent growth characteristic. The synthesis of biological products was affected by shear strength. In the case of prostacyclin, a different synthesis response was observed between steady flow and transiently reduced shear strength. The synthesis of endothelin-1 (ET-1) was down-regulated by increase of shear strength different from those of other products. The culture system was scaled up until 2 L volume under the optimum DO control. The cells synthesized IL-6 in response to shear strength. These results indicate that the established microcarrier system might be able to contribute to the supply of functional human endothelial cells for various medical applications such as the reconstruction of injured blood vessels caused by atherosclerosis or restenosis of coronary arteries after angioplasty, and the construction of an anti-coagulable artificial blood vessel or an artificial skin with good transplant-ability.

Translating Human Embryonic Stem Cells from 2-Dimensional to 3-Dimensional Cultures in a Defined Medium on Laminin- and Vitronectin-Coated Surfaces

While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.

Large-scale Expansion and Exploitation of Pluripotent Stem Cells for Regenerative Medicine Purposes: Beyond the T Flask

Human pluripotent stem cells will likely be a significant part of the regenerative medicine-driven healthcare revolution. In order to realize this potential, culture processes must be standardized, scalable and able to produce clinically relevant cell numbers, whilst maintaining critical biological functionality. This review comprises a broad overview of important bioprocess considerations, referencing the development of biopharmaceutical processes in an effort to learn from current best practice in the field. Particular focus is given to the recent efforts to grow human pluripotent stem cells in microcarrier or aggregate suspension culture, which would allow geometric expansion of productive capacity were it to be fully realized. The potential of these approaches is compared with automation of traditional T-flask culture, which may provide a cost-effective platform for low-dose, low-incidence conditions or autologous therapies. This represents the first step in defining the full extent of the challenges facing bioprocess engineers in the exploitation of large-scale human pluripotent stem cell manufacture.

Preliminary evaluation of microcarrier culture for growth and monoclonal antibody production of CHO-K1 cells

Background The large-scale production of biopharmaceuticals commonly relies on the use of suspension cell cultures, since they provide higher yields than adherent cultures. However, most mammalian cells grow in adherent mode and therefore need to go through a process of adaptation to suspended growth, which is not always simple or feasible. In this context, microcarrier cultures have introduced new possibilities, allowing the practical culture of anchorage-dependent cells, in suspension systems, achieving high yields. In these systems, cells grow as monolayers on the surface of small spheres – the microcarriers, which are usually kept in suspension in the culture medium by gentle rocking. The aim of the present study was to evaluate, compare and optimize the use of microcarrier culture for the growth and monoclonal antibody (mAb) production of CHO-K1 cells.