Microcarrier Cell Culture System Research Articles

Developing efficient bioreactor microcarrier cell culture system for large scale production of mesenchymal stem cells (MSCs)

Background & Aim A large-scale production of clinical-grade MSCs and their derived products is essential due to their immense therapeutic potential. Even though 3D bioreactors are cost efficient in scale up, the plastic adherence of MSCs, makes expansion in suspension cultures challenging. A variety of microcarriers (MCs) allow plastic adherent cells to grow on their surface while maintaining cells in suspension within a bioreactor for expansion. However, this leads to loss of cells, particularly during separation of cells from the carriers. To overcome this, we identified “dissolvable microcarriers” such as Corning Synthemax II dissolvable MCs, which removes the filtration step. Our aim was to develop an efficient bioreactor microcarrier cell culture system to improve the yield of MSCs. Methods, Results & Conclusion Cells from primary human Adipose derived cell line at passage 4 were seeded into a T175 flask (media: aMEM + 10% Human platelet lysate). The cells were expanded for 3 days, harvested by TryplE dissociation and seeded onto a Corning Dissolvable MCs and the control solid core MCs at 8000 cells/cm2, in two separate PBS mini 0.5L MagDrive bioreactors containing 350ml of pre-warmed media. Bioreactors were operated at 16 rpm for the first 12 hours, and 22 rpm for the remainder, in an incubator (37C, 5%CO2). Cells were harvested at 7 days, dissociated from the control MCs using TryplE (15-20 min) followed by passing through a 100um filter, to separate the cells from MCs. The Corning dissolvable MCs were digested with a manufacturer recommended Pectinase/EDTA/TryplE solution, without filtration. Cells from both cultures were washed in DPBS and analysed. A total of 3 replicate runs were performed. The post-harvest cell count (median± range) for dissolvable MCs was 104.6±39.6 million cells and for the control MCs was 76.04±6.28 million cells. The culture supernatant was assayed for pH and osmolality and were found to be similar for both MCs. Analysis of vascular endothelial growth factor(VEGF), showed that dissolvable MCs had a lower level of secreted VEGF (5690±476.4 pg/ml/million cells) compared to control MCs (10103±5384 pg/ml/million cells). Similar trend was noted for macrophage chemoattractant protein-1; 6302±2094 pg/ml/million cells in dissolvable MCs vs 9047±2621 pg/ml/million cells in control MCs. These results indicate that dissolvable MCs could lead to a higher yield from bioreactors (36% increase). However, it is important to further characterise cells and assess their therapeutic potential. A large-scale production of clinical-grade MSCs and their derived products is essential due to their immense therapeutic potential. Even though 3D bioreactors are cost efficient in scale up, the plastic adherence of MSCs, makes expansion in suspension cultures challenging. A variety of microcarriers (MCs) allow plastic adherent cells to grow on their surface while maintaining cells in suspension within a bioreactor for expansion. However, this leads to loss of cells, particularly during separation of cells from the carriers. To overcome this, we identified “dissolvable microcarriers” such as Corning Synthemax II dissolvable MCs, which removes the filtration step. Our aim was to develop an efficient bioreactor microcarrier cell culture system to improve the yield of MSCs. Cells from primary human Adipose derived cell line at passage 4 were seeded into a T175 flask (media: aMEM + 10% Human platelet lysate). The cells were expanded for 3 days, harvested by TryplE dissociation and seeded onto a Corning Dissolvable MCs and the control solid core MCs at 8000 cells/cm2, in two separate PBS mini 0.5L MagDrive bioreactors containing 350ml of pre-warmed media. Bioreactors were operated at 16 rpm for the first 12 hours, and 22 rpm for the remainder, in an incubator (37C, 5%CO2). Cells were harvested at 7 days, dissociated from the control MCs using TryplE (15-20 min) followed by passing through a 100um filter, to separate the cells from MCs. The Corning dissolvable MCs were digested with a manufacturer recommended Pectinase/EDTA/TryplE solution, without filtration. Cells from both cultures were washed in DPBS and analysed. A total of 3 replicate runs were performed.

Microcarrier culture enhances osteogenic potential of human periodontal ligament stromal cells

Regeneration of periodontal tissue represents a major challenge to modern tissue engineering, since cell-based therapies require large amounts of periodontal ligament stromal cells (PLSC), which can be obtained only by in vitro expansion. Ideally, the period of the in vitro expansion should be optimized for the generation of large enough numbers of pre-specified progenitor cells ready to contribute to the restoration of periodontal tissues.In the present study, we used a commercially available, three-dimensional culturing platform and alginate microcarrier cell culture system for the propagation of human PLSCs, which were derived using the explant outgrowth method. Induction of osteogenic differentiation resulted in rapid and robust mineralization of the extracellular matrix in PLSCs grown on microcarriers, but not in PLSCs grown under standard culture conditions. Gene expression studies revealed upregulation of osteogenesis-related genes, BMP2, ALP, RUNX2, MSX2, cementum protein 23, bone sialoprotein, osteopontin and periostin, in undifferentiated and differentiating microcarrier cultures of PLSCs. In addition, the microcarrier culture enhanced the expression of β-catenin, intermediate filament protein vimentin and focal adhesion proteins vinculin and paxillin.Our study shows that microcarrier culture allows rapid generation of large numbers of PLSCs pre-specified towards an osteogenic-like phenotype. This method may be useful for the development of new tissue engineering protocols for the reconstruction of periodontal tissues.

High quality in vitro expansion of human endothelial progenitor cells of human umbilical vein origin.

The limited availability of qualified endothelial progenitor cells (EPCs) is a major challenge for regenerative medicine. In the present study, we isolated human EPCs from human umbilical vein endothelial cells (HUVECs) by using magnetic micro-beads coated with an antibody against human CD34. Flow cytometric assay showed that majority of these cells expressed VEGFR2 (KDR), CD34 and CD133, three molecular markers for early EPCs. It was also found that a bioreactor micro-carrier cell culture system (bio-MCCS) was superior to dish culture for in vitro expansion of EPCs. It expanded more EPCs which were in the early stage, as shown by the expression of characteristic molecular markers and had better angiogenic potential, as shown by matrix-gel based in vitro angiogenesis assay. These results suggest that HUVECs might be a novel promising resource of EPCs for regenerative medicine and that a bio-MCCS cell culture system might be broadly used for in vitro expansion of EPCs.

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.

Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension

3-dimensional microcarrier (3D-MC) cell culture systems are often used for expansion of stem cells including mesenchymal stem cells (MSCs) for high cell volumes required in clinical applications. However, compared to 2-dimensional (2D) cell culture, effects of 3D-MC systems on MSC differentiation have not been well studied. In this study, the behavior of various sources of MSCs from two species was observed and compared on 3D collagen I-coated-MCs (COL-MC) versus 2D culture. Proliferation of all MSCs cultured on 3D COL-MC was much decreased compared to 2D culture. Unexpectedly, COL-MC-cultured MSCs underwent spontaneous osteogenesis without exogenous addition of biochemical factors, as evidenced by increased osteogenic genes expression, ALP activity, calcium deposition, and collagen I secretion. Furthermore, MSCs cultured on 3D-MC alone without collagen I coating is sufficient to induce osteogenesis. The spontaneous lineage commitment induced by 3D-MC culture was mediated by increased cytoskeletal tension and actomyosin contraction of MSCs, which could be prevented by latrunculin B and blebbistatin, inhibitors of cytoskeletal tension and actomyosin contraction respectively. Our findings show that the combination of bioengineered MC and MSCs alone can induce specific lineage commitment very efficiently. These data have strong implications in simplifying tissue engineering strategies for therapeutic applications.

A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR

The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative PCR. The detection limit of this assay is 3 RCAs in 1 × 10 10 vector particles per 70 ml of microcarrier cell culture. The main advantage of the combination of cell culture and PCR detection is that replicated virus can be detected long before cytopathic effects become visible and therefore, it is much faster than conventional cell culture-based assays. This assay was validated by spiking replication-incompetent adenoviral vectors with wild-type adenovirus serotype 5 (wt Ad5) as a positive control for RCA. It was found that the replication of wt Ad5 is hampered above a vector particle per cell ratio of 50. However, if microcarrier beads are used, many cells can be grown in a small suspension culture and consequently a large number of vector particles can be tested for contamination with RCA.

A Novel Bioreactor Microcarrier Cell Culture System for High Yields of Proliferating Autologous Human Keratinocytes

Rapid and efficient resurfacing of various skin defects by autologous keratinocyte transplantation is significant in skin wound healing. We developed a novel bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous human keratinocytes on a large scale. In this Bio-MCCS we used porcine gelatin microbeads as microcarriers for autolgous keratinocytes and spinning bottles as fermentation tanks. First, the microbeads were modified by culturing them with autologous dermal fibroblasts that were subsequently killed when they proliferated to confluence on the microbeads. We then performed the Bio-MCCS by expanding ketatinocytes on the microbeads in spinning bottles at 37 degrees C, 5% CO2. Our results showed that keratinocytes rapidly attached to and actively proliferated on the modified microbeads in the Bio-MCCS, achieving high cell densities on the modified microbeads (MTT assay and PI staining). Keratinocytes cultured on the modified microbeads in the Bio-MCCS remained proliferating potentials as shown by positive PCNA staining and BrdU labeling. In contrast, keratinocytes cultured on nonmodified microbeads in the Bio-MCCS proliferated slowly, rapidly ceased to proliferate, and finally dislodged from the microbeads. When removed from the Bio-MCCS and cultured under static conditions, keratinocytes were able to leave the modified microbeads and formed a multilayered epidermal equivalent on the culture surfaces. While stored at room temperature, keratinocytes remained at higher viabilities on the modified microbeads when compared to those on nonmodified microbeads. The achievement of high yields of proliferating autologous keratinocytes by this Bio-MCCS offers a practical potential of resurfacing various skin defects by direct administration of autologous keratinocyte microbeads on various skin defects.

High Yields of Autologous Living Dermal Equivalents Using Porcine Gelatin Microbeads as Microcarriers for Autologous Fibroblasts

Permanent skin replacement requires a dermal component to ensure adequate long-term graft stability and to prevent wound contraction. This study was to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous living dermal equivalents on a large scale. Autologous fibroblasts were isolated from split-thickness skin biopsy from a leg ulcer patient, inoculated onto macroporous porcine gelatin microbeads, and incubated in a bioreactor (Cellspin) in serum-free fibroblast growth medium or in DMEM medium containing 10% fetal calf serum (FCS). Fibroblasts rapidly adhered to and actively proliferated on the microbeads in the bioreactor in both serum-free and serum-containing medium. MTT assay showed the number of fibroblasts on the microbeads reached up to 5.3- or 4.0-fold the cells seeded in DMEM medium containing 10% FCS or serum-free medium, respectively. When removed from Bio-MCCS and cultured under static conditions, fibroblasts were able to leave the microbeads and proliferate to confluence on the bottom of tissue culture flasks. When stored at room temperature in DMEM containing 10% FBS, fibroblast cultured on the microbeads retained highest viabilities for at least 3 weeks, up to 82% of originals. This Bio-MCCS using porcine gelatin microbeads as carriers for fibroblasts offers a new option of mass production of autologous living dermal equivalents.

Propagation kinetics of retrovirus transgene vector and replication-competent retrovirus in static and microcarrier cell culture systems using different medium exchange strategies

While retrovirus vector (RV) is the main virus vector used in human gene therapy trials, the biosafety issues that surround currently used RVs have become a matter of concern. Similar to the insertional mutagenesis in the therapeutic target cells, the generation of replication-competent retrovirus (RCR) must be minimized during the manufacture of the virus vector. This work investigated the kinetics of RV and RCR production in PA317-RCM1 producer cells in static and microcarrier cell culture systems. RCR in the progeny of transduced Mus dunni cells was detected by the PCR method and the titer of RCR was quantified by cell-based S+/L− assay. The specific rates of RCR production in microcarrier cultures were 271–462% higher than those in the static well-plate cultures. Increased medium exchange operations yielded higher specific rates of RCR production in both static and microcarrier cultures. The optimal medium exchange strategy was on an every 2-day schedule, yielding the highest RV/RCR ratio in static culture but not microcarrier culture. Results of this study presented the difficulty for gene therapy processes that together with the product RV also unwanted RCR produced in two different cell culture systems.

Bioreactor Microcarrier Cell Culture System (Bio-MCCS) for Large-Scale Production of Autologous Melanocytes

Restoration of cutaneous pigmentation can be achieved in stable vitiligo by autologous cultured melanocyte transplantation. It was the goal of this study to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous melanocytes in large scale. In this Bio-MCCS, porcine gelatin microbeads were used as microcarriers, spinning bottle as fermented tank. Autologous melanocytes were able to attach to and proliferate on the gelatin microbeads in serum-free melanocyte medium in the Bio-MCCS, reaching up to 24-fold the cells seeded on day 15 (MTT assay). These autologous melanocytes cultured on gelatin microbeads could leave the microbeads and proliferate on the bottom of tissue culture flasks. Although Pluronic F68 has been widely used to protect animal cells from hydrodynamic stress in animal cell bioreactors, Pluronic F68 at a concentration of 0.25-1.0% showed no significant protective effects on the autologous melanocytes cultured on the microbeads and subjected to mechanical stress in the Bio-MCCS. This Bio-MCCS using porcine gelatin microbeads as microcarriers enabled large-scale production of autologous melanocytes, offering a potential treatment for large-area stable vitiligo by direct administration of the melanocytes cultured on the gelatin microbeads to the vitiliginous site.

Radio-frequency plasma treatment improves the growth and attachment of endothelial cells on poly(methyl methacrylate) substrates: implications in tissue engineering

The aim of this study was to improve the biocompatibility of poly(methyl methacrylate) (PMMA) substrates for possible applications in corneal prostheses or in micro-carrier cell culture systems. PMMA substrates were exposed to radio-frequency (RF) argon and nitrogen plasmas for 5 and 10 min each. The PMMA films were examined by Fourier transform infrared (FT-IR) spectroscopy, to characterize the surface changes after plasma exposure. Plasma treatment in general was found to decrease the water contact angle of PMMA, thus increasing its hydrophilicity. There was also an associated increase in the work of adhesion of plasma-treated PMMA substrates. PMMA substrates exhibited differential properties towards endothelial cell (CPA-47) growth. The untreated PMMA surface did not support endothelial growth, compared with both polystyrene (TCPS) and plasma-treated PMMA, while plasma (PL):PMMA exhibited growth rates slightly lower than the TCPS control, as assessed by [3H]thymidine incorporation profiles. The compatibility and growth supportive properties of PL: PMMA were further confirmed by an MTT assay, which showed preserved cellular viability and mitochondrial activity of the cells. Confocal microscopic visualization of cells with fluorescence-labeled vimentin showed normal organization of the cytoskeletal fibers, indicating appropriate attachment to the substrate. Cells growing on PL:PMMA maintained their functionality, as seen from Factor VIII expression. Taken collectively, the findings of this study point out the suitability of RF plasma treatment in inducing desirable changes in PMMA substrates, so as to improve their ability to support the growth and attachment of endothelial cells.

The Comparative Release of FGF1 by Hypoxia and Temperature Stress

The signal peptide-less FGF gene family prototype, FGF1 is released in response to temperature stress in vitro as a latent reducing agent-sensitive homodimer non-covalently complexed with the extravesicular p40 domain of p65 synaptotagmin (Syt)1. Because FGF1 is well recognized as an angiogenesis factor in vivo and angiogenesis is known to be induced by hypoxia, we examined the release of FGF1 and p40 Syt1 under conditions of hypoxia and temperature stress using a chemostatic microcarrier cell culture system. We report that like the pathway used by FGF1 and p40 Syt1 release under temperature stress, hypoxia also induces the release of FGF1 and p40 Syt1 with similar kinetic and pharmacologic properties including the requirement for functional cysteine residues. Lastly, FGF1 and p40 Syt1 release in response to hypoxia and temperature stress is sensitive to lipoxygenase and cyclooxygenase inhibitors suggesting that arachidonic acid metabolism may play an important role in the mechanism of FGF1 release in vitro.

Characterization of a microcarrier cell culture system for23Na MR spectroscopy studies

A MR spectroscopy method is described for the simultaneous discrimination and observation of sodium from the three compartments created by an intact cell monolayer. Results are reported for Madin Darby Canine Kidney (MDCK) cells, an epithelial-like continuous cell line, cultured on Cytodex 1 microcarrier beads and perfused with medium containing 6 mM dysprosium (III) tripolyphosphate [Dy(TPP)2(7-)] as shift reagent. The sodium spectrum shows three resonances which are assigned to the shifted intrabead (basolateral) and extrabead (apical) pools and the unshifted intracellular pool. Ouabain inhibition of the Na(+)-K(+)-ATPase cellular pump mechanism was used to demonstrate the sensitivity of the method for monitoring intracellular sodium. The supported MDCK cells in this system remained viable after exposure for 5 h to medium containing Dy(TPP)2(7-) at a concentration of 6 mM, as determined by trypan blue dye exclusion and by comparison of the log growth rate and ability to form domes in subsequent generations of exposed cells vs unexposed controls.

Growth of a murine coronavirus in a microcarrier cell culture system

The growth of the murine coronavirus MHV-A59 on murine DBT cells adapted to dextran-made Cytodex 1 microcarriers was studied in comparison with cells grown on plastic dishes. With a microcarrier concentration of 5 g/l in spinner flasks, a density of 3 × 10 6 cells/ml was reached in 7 days. Under these conditions, cells supported virus growth to the same extent as when they were grown on the plastic substratum. This was shown by a similar development of virus-induced syncytia, the release of an equivalent number of infectious progeny virions per cell, similar recoveries observed after concentration and purification and an identical appearance of the purified virus under the electron microscope. On the other hand, the technical convenience of microcarriers and the ease of scale-up emphasize their potential for the growth of coronaviruses.

A microcarrier cell culture system for large scale production of hepatitis a virus

Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4). Infectious medium from passage 12 was used to inoculate a large (5000 cm 2) microcarrier cell culture maintained in suspension. The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells. Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm 2 plastic flasks. The results show that virus production per cell was similar in both systems. The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased. Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture. The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial. Weekly harvests of medium with HAV antigen titres around 10 −2 contained antigenic material sufficient for several thousands of anti-HAV IgM tests. Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.