Current state and perspectives in modeling and control of human pluripotent stem cell expansion processes in stirred‐tank bioreactors

Implementation of model‐based practices for process development, control, automation, standardization, and validation are important factors for therapeutic and industrial applications of human pluripotent stem cells. As robust cultivation strategies for pluripotent stem cell expansion and differentiation have yet to be determined, process development could be enhanced by application of mathematical models and advanced control systems to optimize growth conditions. Therefore, it is important to understand both the potential of possible applications and the apparent limitations of existing mathematical models to improve pluripotent stem cell cultivation technologies. In the present review, the authors focus on these issues as they apply to stem cell expansion processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:355–364, 2017     

Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential

Objectives: For reasons of provision of highly‐specific surface area and three‐dimensional culture, microcarrier culture (MC) has garnered great interest for its potential to expand anchorage‐dependent stem cells. This study utilizes MC for in vitro expansion of human bone marrow mesenchymal stem cells (BMMSCs) and analyses its effects on BMMSC proliferation and differentiation. Materials and methods: Effects of semi‐continuous MC compared to control plate culture (PC) and serial bead‐to‐bead transfer MC (MC bead‐T) on human BMMSCs were investigated. Cell population growth kinetics, cell phenotypes and differentiation potential of cells were assayed. Results: Maximum cell density and overall fold increase in cell population growth were similar between PCs and MCs with similar starting conditions, but lag period of BMMSC growth differed substantially between the two; moreover, MC cells exhibited reduced granularity and higher CXCR4 expression. Differentiation of BMMSCs into osteogenic and adipogenic lineages was enhanced after 3 days in MC. However, MC bead‐T resulted in changes in cell granularity and lower osteogenic and adipogenic differentiation potential. Conclusions: In comparison to PC, MC supported expansion of BMMSCs in an up‐scalable three‐dimensional culture system using a semi‐continuous process, increasing potential for stem cell homing ability and osteogenic and adipogenic differentiation.     

Microcarrier expansion of mouse embryonic stem cell-derived neural stem cells in stirred bioreactors

Neural stem cells (NSCs) are self-renewing multipotent cells, able to differentiate into the phenotypes present in the central nervous system. Applications of NSCs may include toxicology, fundamental research, or cell therapies. The culture of floating cell clusters, called "neurospheres," is widely used for the propagation of NSC populations in vitro but shows several limitations, which may be circumvented by expansion under adherent conditions. In particular, the derivation of distinct populations of NSCs from embryonic stem cells capable of long-term culture under adherent conditions without losing differentiation potential was recently described. However, the expansion of these cells in agitated bioreactors has not been addressed until now and was the aim of this study. Selected microcarriers were tested under dynamic conditions in spinner flasks. Superior performance was observed with polystyrene beads coated with a recombinant peptide containing the Arg-Gly-Asp (RGD) motif (Pronectin F). After optimization of the culture, a 35-fold increase in cell number was achieved after 6 days. High cellular viability and multipotency were maintained throughout the culture. The study presented here may be the basis for the development of larger scale bioprocesses for expansion of these and other populations of adherent NSCs, either from mouse or human origin.     

Optimal in-vitro expansion of chondroprogenitor cells in monolayer culture

A continuous production of large quantities of chondroprogenitor cells for the manufacture of engineered cartilage tissue products is required. Expansion of the cell population in vitro has become an essential step in the process of tissue engineering of articular cartilage and the optimization of the culture conditions is a fundamental problem that needs to be addressed. The analysis of both seeding density and passage length was considered crucial in the optimization of expansion processes, and their correct selection should be taken as a requisite to establish culture conditions for monolayer systems. The determination of the optimal seeding density and the corresponding passage length for cell expansion in a serial passaging operation was found to be a compromise between growth kinetics and process time. This optimal determination was carried out using a mathematical approach that led to values of 10(4) cell/cm(2) for seeding density and 73 h for passage length. Additional considerations concerning the running cost of the process were introduced. Although the optimal passage length gave the desired expansion factor in a minimum process time, the selection of an alternative value of 120 h was shown to reduce the cost of the expansion process in more than 60%. The optimization approach presented will contribute to the development of feasible large scale expansion operations of chondroprogenitor cells required by the cartilage tissue engineering industry.     

Advances in cell culture: anchorage dependence

Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes.     

Physiological response dynamics of the brown tide organism Aureococcus anophagefferens treated with modified clay

On the basis of experiences in mitigating harmful algal blooms (HABs) with modified clay (MC), a bloom does not continue after the dispersal of the MC, even though the density of the residual cells in the water remains as high as 20–30% of the initial cell density. This interesting phenomenon indicates that in addition to flocculation, MC has additional mechanisms of HAB control. Here, Aureococcus anophagefferens was selected as a model organism to study the physiological response dynamics of residual cells treated with MC, and RT-qPCR was used to measure the differential expression of 40 genes involved in anti-oxidation, photosynthesis, phospholipid synthesis, programmed cell death and cell proliferation at five time points. The results showed that every functional gene category exhibited a "V" shaped pattern with a turning point. It was reflected that there were two processes for MC inhibiting the growth of residual cells. One is the oxidative stress process (OSP) caused by ineffective collision with MC, whose effect weakened gradually; another is the programmed cell death process (PCDP) caused by the lysis of damaged residual cells, whose effect enhanced two days after MC treatment. In addition, the scanning electron micrographs verified that some of the residual cells were deformed or even lysed. Combined with the effects of OSP and PCDP in dynamics, the growth of residual cells was inhibited and was followed by gradual bloom disappearance. This study further elucidates the mechanism of MC controlling HABs at the molecular level and enable a more comprehensive understanding of HAB mitigation using MC.     

Comparison of several techniques for the detection of apoptotic astrocytes in vitro

Implication of apoptosis in numerous physiological and pathological processes has resulted in the development of numerous methods to detect apoptosis, but none of them is adapted to all cell types. In this study, we induced apoptosis on murine immortalized astrocytes with urine from multiple sclerosis (MS) patients. Among techniques allowing the detection of apoptotic cells, only a few are adapted to adherent cells such as astrocytes. We compared several techniques (propidium iodide labelling and flow cytometry analysis, TUNEL and annexin V labelling in immunofluorescence, DNA ladder, ELISA tests to detect nucleosomes) in order to choose the method best adapted to our adherent cellular model and to discuss their practicability for the detection of apoptosis on adherent cells.
For technical course, propidium iodide labelling followed by flow cytometry analysis as a quantitative technique, and TUNEL in IF (easier and quicker than propidium iodide) as a semiquantitative test were both retained as best adapted to our case.
Moreover, in our model, we have observed that phosphatydilserine externalization and DNA fragmentation were concomittant after induction of apoptosis.
Techniques studied in this article would allow an enlarged study of the apoptotic mechanism in several pathologies by culture of adherent cells sensitive to apoptosis in vitro .     

Manually and automatically produced pellet cultures of human primary chondrocytes: A comparative analysis

Cartilage defects are often associated with restriction of the locomotor system. New methods are required to investigate cartilage tissue and for the repair of cartilage tissue. 3D cultures are promising due to better simulation of in vivo conditions. The aim of this study was to provide a model system for studying cartilage tissue. We solved this problem by automated production of pellet cultures of human primary chondrocytes in media with and without antibiotics using the Biomek® Cell Workstation and consequent automated bioscreening with a high‐throughput screening system, and compared with the regular manual processes. The Biomek® Cell Workstation allows the cultivation of different cell types (suspensions cells and adherent cells) and 3D cell cultures (pellet cultures, alginate beads and spheroid cultures). The proliferation was analyzed by DNA quantification and compared with the EZ4U proliferation assay as a new tool for pellet cultures. The toxicity was evaluated by the detection of ubiquitous adenylate kinases. The proliferation increased from day 14 until day 35 and was associated with a decrease in the cytotoxicity. The comparative analysis showed similar results for manual and automated processes. We concluded that the manual methods can be replaced by automated processes (pellet manufacturing and screening), which would allow large‐scale procedures to support studies on cartilage regeneration.     

Model-based analysis of Arabidopsis leaf epidermal cells reveals distinct division and expansion patterns for pavement and guard cells

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.     

Characterization of the hematopoietic target cells of AEV, MC29 and AMV avian leukemia viruses

The hematopoietic target cells of the three prototype strains of replication defective avian leukemia viruses (DLVs) were studied, using a newly developed, quantitative in vitro transformation assay. Our results show that the target cells of avian erythroblastosis virus (AEV) belong to the erythroid lineage while those of myelocytomatosis virus 29 (MC29) and avian myeloblastosis virus (AMV) belong to the myeloid lineage. As judged from suicide experiments using BUdR incorporation and irradiation with visible light, a higher proportion of AEV- and AMV-target cells are in cycle than MC29-target cells. Using differentiation specific antisera directed against cell surface antigens, we could demonstrate that the target cells of AEV express erythroblast-specific antigen(s) and less intensively erythrocyte-specific antigen(s), while those of MC29 and AMV express myeloblast-specific antigen(s). In addition, MC29-target cells express macrophage-specific antigen(s). None of the AEV-target cells are adherent or phagocytic, while a small proportion of the AMV-target cells are adherent and about half of the MC29-target cells are both adherent and phagocytic. Our results support the concept that DLVs specifically transform certain types of committed erythroid and myeloid progenitor cells. The target cells of AEV and AMV appear to resemble the respective transformed cells in their state of differentiation, whereas those of MC29 appear to be more immature than the corresponding transformed cells.     

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.     

Optimization of a MRC-5 Cell Culture Process for the Production of a Smallpox Vaccine

A cell culture process adaptable to produce smallpox vaccine at large scale has been developed. To achieve this, Design of Experiments (DOE) was applied to identify and optimize critical cell culture process parameters for MRC-5 cell growth and recovery during cell expansion. For cell growth, a 2^5?1 partial factorial (two level, five factor, 16 conditions) study was designed to evaluate the effects of basal media, seeding density, culture volume, feeding frequency and serum concentration on population doubling level (PDL) after 6–7 days in adherent T-flask cultures. Results indicated that lowering the cell density to 1×10⁴ cells/cm², increasing the culture volume to 0.5 ml/cm² and increasing serum concentration to 20% significantly improved cell expansion. These findings correlated with PDLs above 2.0 and cell densities above 1×10⁵ cells/cm² at the end of the study period. For cell recovery at passaging, a similar DOE was used to evaluate the effect of trypsin concentration, solution temperature, duration of treatment, incubation temperature and duration of standing time between quenching and reseeding. By increasing the trypsin treatment duration to 60 min and lowering the standing time between quenching and reseeding to within 1 h, the recovery of the MRC-5 cells was greatly improved. By using these newly defined conditions, a two-fold improvement in cell expansion was consistently achieved in both roller bottles and 10 layer Nunc^® Cell Factories (Cell Factories). Application of these new conditions for current Good Manufacturing Practices (cGMP) production of MRC-5 cell banks and clinical material demonstrated predictably high cell expansion as well as significantly higher production of vaccinia virus, thus providing the basis for manufacturing vaccinia virus at large scale. These findings demonstrate the need for cell culture optimization and the effectiveness of DOE to rapidly define processes suitable for cGMP manufacturing of a smallpox vaccine or other viral vaccine products.     

Internalization of ferromagnetic nanowires by different living cells

The ability of living cells, either adherent or suspended, to internalize nickel nanowires is demonstrated for MC3T3-E1, UMR106-tumour and Marrow-Stromal cells. Nanowires were produced by electrodeposition, 20 μm long and 200 nm in diameter. Cell separation and manipulation was achieved for the three cell types. Applied magnetic field successfully oriented the internalized nanowires but no clear anisotropy is induced on the adherent cells. Nanowires tend to bind to cytoplasm metalloproteins and trigger lysosome reorganization around the nucleus. This work demonstrates the applications of nanowires in adherent and suspended cells for cell separation and manipulation, and further explore into their role in nanobiotechnology.     

Co-culture of stromal and erythroleukemia cells in a perfused hollow fiber bioreactor system as an in vitro bone marrow model for myeloid leukemia

We have developed a hematopoietic co-culture system using the hollow fiber bioreactor (HFBR) as a potential in vitro bone marrow model for evaluating leukemia. Supporting stroma using HS-5 cells was established in HFBR system and the current bioprocess configuration yielded an average glucose consumption of 640 mg/day and an average protein concentration of 6.40 mg/mL in the extracapillary space over 28 days. Co-culture with erythroleukemia K562 cells was used as a model for myelo-leukemic cell proliferation and differentiation. Two distinct localizations of K562 cells (loosely adhered and adherent cells) were identified and characterized after 2 weeks. The HFBR co-culture resulted in greater leukemic cell expansion (3,130 fold vs. 43 fold) compared to a standard tissue culture polystyrene (TCP) culture. Majority of expanded cells (68%) in HFBR culture were the adherent population, highlighting the importance of cell-cell contact for myelo-leukemic proliferation. Differentiation tendencies in TCP favored maturation toward monocyte and erythrocyte lineages but maintained a pool of myeloid progenitors. In contrast, HFBR co-culture exhibited greater lineage diversity, stimulating monocytic and megakaryocytic differentiation while inhibiting erythroid maturation. With the extensive stromal expansion capacity on hollow fiber surfaces, the HFBR system is able to achieve high cell densities and 3D cell-cell contacts mimicking the bone marrow microenvironment. The proposed in vitro system represents a dynamic and highly scalable 3D co-culture platform for the study of cell-stroma dependent hematopoietic/leukemic cell functions and ex vivo expansion.     

Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.     

Mononuclear macrophage (M-CFC) colony formation in semisolid media in the absence of exogenous GM-CSF

Human fetal bone marrow (FBM) cells were examined for the ability to form colonies in the absence of exogenous colony-stimulating factor (CSF) in double layer agar, methylcellulose (MC), and in agar-MC (agar underlayer, MC overlayer) culture systems. Without exogenous CSF, macrophage colonies (M-CFC) were formed in a combined culture of agar and MC. Aggregates of 5-40 cells were observed on day 7. Gradually, large compact colonies which survived for 10-12 weeks of cultivation, were formed. They were composed of mononuclear monocytes and multinucleated cells. M-CFC progenitors were nonadherent, but their progeny became adherent during differentiation within the colony. Colony formation was cell-dose-dependent. Depletion of monocytes increased the number of colonies in agar-MC cultures and stimulated the development of some macrophage colonies in MC. Survival of monocyte progenitors was not dependent on CSF. Neither was their proliferation nor partial differentiation in agar-MC cultures. CSF increased M-CFC colony efficiency, however, if it was present when cultures were initiated. Addition of CSF to M-CFC growing for 2-5 weeks in CSF-deprived medium stimulated monocytes proliferation and transformation into macrophages. Epithelioid cells, an increase in the number of giant multinucleated cells, and granulocyte multiplication were also observed. The absolute dependence of macrophage colony formation on CSF described by others might be a result of inadequate culture conditions due to agar rather than an intrinsic physiological requirement.     

In vitro hemopoiesis within a microenvironment created by MC3T3-G2/PA6 preadipocytes

The clonal preadipose cell line, MC3T3-G2/PA6, has the capacity to differentiate into adipocytes in response to glucocorticoids and to support in vitro growth of hemopoietic stem cells (CFU-S). To study the relationship between these capacities, we precultured the MC3T3-G2/PA6 cells for varying days in the presence or absence of dexamethasone and then cocultured them with mouse bone marrow cells. Logarithmically growing cultures contained no detectable adipocytes and showed the highest growth-supporting activity for CFU-S, whereas cultures containing the largest number of adipocytes showed the lowest activity. When bone marrow cells were seeded onto 3-day-old MC3T3-G2/PA6 preadipocyte layers at 1 X 10(5) cells/35-mm dish, day 12 CFU-S grew with a population doubling time of about 37 hr, and at least 75% of them were associated with the cell layer between days 2 and 7. In the absence of the preadipocytes, CFU-S were not detected in the adherent cell fraction and decreased with a half-life of about 18 hr. More than 80% of CFU-C were also found to be associated with the preadipocyte layer, and they increased about 24-fold in number during 7 days in culture. Morphologically, hemopoietic cells developing into mature granulocytes and macrophages were distributed between the layers of preadipocytes. Dendritic processes of preadipocytes were frequently in close alignment with the hemopoietic cells. However, adipocytes failed to show such an intimate association with hemopoietic cells. These results indicate that MC3T3-G2/PA6 cells in the preadipocyte stage, but not in the adipocyte stage, have the capacity to support CFU-S growth, and that hemopoiesis in our cocultivation system proceed within the microenvironmental milieu provided by MC3T3-G2/PA6 preadipocytes.     

3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads

Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek^® Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.     

A model for hepatocarcinogenesis with clonal expansion of three successive phenotypes of preneoplastic cells

The two-stage model with clonal expansion of intermediate cells has often been used to describe the carcinogenesis process. The model hypothesizes that cells have to undergo two mutations on their way from the normal to the malignant stage. Biological experiments indicate the existence of three types of preneoplastic cells in hepatocarcinogenesis representing three successive intermediate stages in the development of malignant cells from normal cells. This finding suggests that hepatocarcinogenesis should be described by a multi-stage model with three intermediate stages, leading to a four-stage mutation model with clonal expansion of all types of intermediate cells. This model is presented and mathematical approximations for the number and size of nonextinct premalignant clones of the different cell types are derived. The model is applied to focal lesion data from a rat hepatocarcinogenesis experiment.     

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.