Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O₂ concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 10⁵ cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O₂ uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.     

Kinetic modeling and implementation of superior process strategies for β-galactosidase production during submerged fermentation in a stirred tank bioreactor

This paper reports development and implementation of superior fermentation strategies for β-galactosidase production by Lactobacillus acidophilus in a stirred-tank bioreactor. Process parameters (aeration and agitation) were optimized for the process by application of Central Composite Design. Aeration rate of 0.5 vvm and agitation speed of 250 rpm were most suitable for β-galactosidase production (2001.2 U/L). Further improvement of the operation in pH controlled environment resulted in 2135 U/L of β-galactosidase with productivity of 142.39 U/L h. Kinetic modeling for biomass and enzyme production and substrate utilization were carried out at the aforementioned pH controlled conditions. The logistic regression model (X₀ = 0.01 g/L; X_max = 2.948 g/L; μ_max = 0.59/h; R² = 0.97) was used for mathematical interpretation of biomass production. Mercier's model proved to be better than Luedeking–Piret model in describing β-galactosidase production (P₀ = 0.7942 U/L; P_max = 2169.3 U/L; P_r = 0.696/h; R² = 0.99) whereas the latter was more efficient in mathematical illustration of lactose utilization (m = 0.187 g/g h; Y_x/s = 0.301 g/L; R² = 0.98) among the two used in this study. Strategies like fed-batch fermentation (3694.6 U/L) and semi-continuous fermentation (5551.9 U/L) further enhanced β-galactosidase production by 1.8 and 2.8 fold respectively.     

Modeling of gas–liquid mass transfer in a stirred tank bioreactor agitated by a Rushton turbine or a new pitched blade impeller

A combined computational fluid dynamics (CFD) and population balance model (PBM) approach has been applied to simulate hydrodynamics and mass transfer in a 0.18 m³ gas–liquid stirred bioreactor agitated by (1) a Rushton turbine, and (2) a new pitched blade geometry with rotating cartridges. The operating conditions chosen were motivated by typical settings used for culturing mammalian cells. The effects of turbulence, rotating flow, bubbles breakage and coalescence were simulated using the k–ε, multiple reference frame (MRF), Sliding mesh (SM) and PBM approaches, respectively. Considering the new pitched blade geometry with rotating aeration microspargers, $k_{\text{L}} a$ mass transfer was estimated to be 34 times higher than the conventional Rushton turbine set-up. Notably, the impeller power consumption was modeled to be about 50 % lower. Independent $k_{\text{L}} a$ measurements applying the same operational conditions confirmed this finding. Motivated by these simulated and experimental results, the new aeration and stirring device is qualified as a very promising tool especially useful for cell culture applications which are characterized by the challenging problem of achieving relatively high mass transfer conditions while inserting only low stirrer energy.     

The histopathology of a human mesenchymal stem cell experimental tumor model: support for an hMSC origin for Ewing's sarcoma?

Sarcomas display varied degrees of karyotypic abnormality, vascularity and mesenchymal differentiation. We have reported that a strain of telomerized adult human bone marrow mesenchymal stem cells (hMSC-TERT20) spontaneously evolved a tumorigenic phenotype after long-term continuous culture. We asked to what extent our hMSC-TERT20 derived tumors reflected events found in human sarcomas using routine histopathological procedures. Early versus late passage hMSC-TERT20 cultures persistently expressed mesenchymal lineage proteins e.g. CD105, CD44, CD99 and vimentin. However, late passage cultures, showed increased immunohistochemical staining for CyclinD1 and p21WAF1/Cip1, whereas p27Kip1 staining was reduced. Notably, spectral karyotyping showed that tumorigenic hMSC-TERT20 cells retained a normal diploid karyotype, with no detectable chromosome abnormalities. Consistent with the bone-forming potential of early passage hMSC-TERT20 cells, tumors derived from late passage cells expressed early biomarkers of osteogenesis. However, hMSC-TERT20 cells were heterogeneous for alpha smooth muscle actin (ASMA) expression and one out of six hMSC-TERT20 derived single cell clones was strongly ASMA positive. Tumors from this ASMA+ clone had distinctive vascular qualities with hot spots of high CD34+ murine endothelial cell density, together with CD34- regions with a branching periodic acid Schiff reaction pattern. Such clone-specific differences in host vascular response provide novel models to explore interactions between mesenchymal stem and endothelial cells. Despite the lack of a characteristic chromosomal translocation, the histomorphology, biomarkers and oncogenic changes were similar to those prevalent for Ewing's sarcomas. The phenotype and ontogenesis of hMSC-TERT20 tumors was consistent with the hypothesis that sarcomas may arise from hMSC, providing a unique diploid model for exploring human sarcoma biology.     

Regime analysis of a Baeyer–Villiger bioconversion in a three-phase (air–water–ionic liquid) stirred tank bioreactor

The aim of this work was to conduct a regime analysis on a three-phase (air–water–ionic liquid) stirred tank bioreactor of the Baeyer–Villiger bioconversion process, using [MeBuPyrr][BTA] ionic liquid as the dispersed phase. The regime analysis based on characteristic times of the different mechanisms involved (mixing, mass transfer, reaction) can yield a quantitative estimate of bioreactor performance. The characteristic time obtained for oxygen uptake rate (54 s⁻¹) was among the characteristic times determined for oxygen transfer (13–129 s⁻¹) under different operating conditions, suggesting that the oxygen transfer rate under certain operating conditions could be a limiting step in the bioconversion process. Further enhancement of oxygen transfer rates requires proper selection of the bioreactor operational conditions, and improved design of the ionic liquid used as oxygen transfer vector.     

Optimization of conditions for cell cultivation of Porphyra haitanensis conchocelis in a bubble-column bioreactor

Summary Process conditions for cell cultures derived from conchocelis of female red macroalga Porphyra haitanensis were optimized in an illuminated 0.3-l bubble-column photobioreactor, using CO₂ in air as the sole carbon source during a 20-day cultivation period. It reached the highest growth rate when the initial cell density was 700 mg l⁻¹ (dry weight), the optional aeration rate was 1.2 v/v/min, inorganic nitrate concentration was 15 mM and inorganic phosphate concentration was 0.6 mM. This is the first reported bioreactor cultivation study of cell cultures derived from conchocelis of Porphyra haitanensis.     

Mathematical modeling of mixing in a horizontal rotating tubular bioreactor: “Spiral flow” model

A horizontal rotating tubular bioreactor (HRTB) was designed as a combination of a thin-layer bioreactor and a biodisc reactor whose interior was divided by O-ring shaped partition walls. For the investigation of mixing in HRTB the temperature step method was applied. Temperature changes in the bioreactor were monitored by six Pt-100 sensors (t ₉₀ response time 0.08 s and resolution 0.002 °C) which were connected with an interface unit and a personal computer. In this work a modified tank in series concept was used to establish a mathematical model. The heat balance of the model compartments was established according to the physical model and the spiral flow pattern. Numerical integration was done by the Runge-Kutta-Fehlberg method. The mathematical mixing model called spiral flow model contained four adjustable parameters (N1, Ni, F _cr and F _p) and five parameters which characterized the plant and experimental conditions. The spiral flow model was capable to describe the mixing in HRTB properly, and its applicability was much better than with the simple flow model, presented earlier.List of Symbols A _ui m² ithpart of inner surface of bioreactor's wall - A _vi m² ith part of outlet surface of bioreactor's wall - C _p kJ kg^–1 K^–1 heat capacity of liquid - c _pr kJ kg^–1 K^–1 heat capacity of bioreactor's wall - D h^–1 dilution rate - E °C°C^–1h^–1 error of mathematical model - F _cr dm³ s^–1 circulation flow in the model - F _p dm³s^–1 back flow in the model - F _t dm³ s^–1 inlet flow in bioreactor - I °C intensity of temperature step, the difference in temperature between the temperature of the inlet liquid flow and the temperature of liquid in bioreactor before temperature step - K1 Wm^–2 K ^–1 heat transfer coefficient between the liquid and bioreactor's wall - K2 Wm^–2 K ^–1 heat transfer coefficient between the bioreactor's wall and air - L m length of bioreactor - m _s kg mass of bioreactor's wall - n min^–1 rotational speed of bioreactor - n _s number of temperature sensors - N1 number of cascades - Ni number of compartments inside cascade - r _u m inner diameter of bioreactor - r _v m outside diameter of bioreactor - s(t) step function - t s time - T °C temperature - T _c °C calculated temperature - T _m °C measured temperature - T _N 1,Ni°C temperature of liquid in defined compartments inside the cascade - T _N 1,S°C temperature of defined part of bioreactor's wall - T _N i,z°C temperature of surrounding air - V _t dm³ volume of liquid in the bioreactor     

Mathematical modeling of mixing in a horizontal rotating tubular bioreactor: “Simple flow” model

The construction of the horizontal rotating tubular bioreactor (HRTB) represents a combination of a thin-layer bioreactor and a biodisc reactor. The bioreactor was made of a plastic tube whose interior was divided by the O-ring shaped partition walls. For the investigation of mixing properties in HRTB the temperature step method was applied. The temperature change in the bioreactor as a response to a temperature step in the inlet flow was monitored by six Pt-100 sensors (t ₉₀ response time 0.08 s and resolution 0.002 °C) which were connected with an interface unit and personal computer. Mixing properties of the bioreactor were modeled using the modified tank in series concept which divided the bioreactor into ideally mixed compartments. A mathematical mixing model with simple flow was developed according to the physical model of the compartments network and corresponding heat balances. Numerical integration of an established set of differential equations was done by the Runge-Kutt-Fehlberg method. The final mathematical model with simple flow contained four adjustable parameters (N1,Ni, F _cr andF _p ) and five fixed parameters.List of Symbols A _u m² inner surface of bioreactor's wall - A _ui m² i-th part of inner surface of bioreactor's wall - A _v m² outlet surface of bioreactor's wall - A _vi m² i-th part of outlet surface of bioreactor's wall - C _p kJ kg^–1 K^–1 heat capacity of liquid - C _pr kJ kg^–1 K^–1 heat capacity of bioreactor's wall - D h^–1 dilution rate - E °C °C^–1 h^–1 error of mathematical model - F _cr dm³s^–1 circulation flow in the model - F _p dm³ s^–1 back flow in the model - F _t dm³s^–1 inlet flow in the bioreactor - I °C intensity of temperature step, the difference in temperature between the temperature of the inlet liquid flow and the temperature of liquid in the bioreactor before the temperature step - K1 Wm^–2K^–1 heat transfer coefficient between the liquid and bioreactor's wall - K2 Wm^–2K^–1 heat transfer coefficient between the bioreactor's wall and air - m _s kg mass of bioreactor's wall - L m length of bioreactor - L _k m wetted perimeter of bioreactor - n min^–1 rotational speed of bioreactor - n _s number of temperature sensors - N1 number of cascades - Ni number of compartments inside the cascade - Nu Nusselt number - Pr Prandtl number - r _u m inner diameter of bioreactor - r _v m outside diameter of bioreactor - Re Reynolds number - s(t) step function - t s time - T °C temperature - T _c °C calculated temperature - T _m °C measured temperature - T _N1,Ni °C temperature of liquid in a defined compartment inside cascade - T _N1,S °C temperature of defined part of bioreactor's wall - T _S °C temperature of bioreactor's wall - T _v °C temperature of liquid in bioreactor - T _z °C temperature of surrounding air - V _t dm³ volume of liquid in the bioreactor Greek Symbols kJm^–1s^–1 K^–1 thermal conductivity of liquid in the bioreactor - kgm^–3 density of liquid in the bioreactor - m²s^–1 kinematic viscosity of liquid in the bioreactor Matrix Coefficient B - C - D - E B+C+D - G1 - G2 - G3 - A _ui - A _vi - Q ₁ - Q ₂ - Q ₃      

Bioprocess parameters of cell growth and human μ opioid receptor expression in recombinant Drosophila S2 cell cultures in a bioreactor

The paper describes a recombinant Schneider 2 (rS2) cell culture and protein expression in a bioreactor. S2 cells were transfected with a plasmid containing a fusion protein (human μ opioid receptor, hMOR, and green fluorescent protein, EGFP) under the control of inducible metallothionein promoter. A bioprocess in a bioreactor with 5% dissolved oxygen, 27°C and 120 rpm enabled the cell culture to attain 5.3×10⁷ viable cells/mL at 96 h. The induction decreased the cell multiplication (2.5×10⁷ viable cells/mL at 72 h). Glutamine and glucose and low levels of lactate were consumed. A fast recombinant protein synthesis took place and, at 6 h of induction, 2×10⁴ receptors/cell could be detected by a functional binding assay. Fluorescence measurements showed a progressive increase of recombinant protein expression with a maximal value of 1.26×10⁵ fluo counts/s at 24 h of induction. The data shown in this paper indicate a practical and scaleable cell culture bioprocess procedure for the preparation of recombinant proteins expressed in S2 cells.     

The in vivo assessment of a novel scaffold containing heparan sulfate for tissue engineering with human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and mature tissue macrophages, respectively. It was found that the survival of the implanted human cells was affected by the host response to the implant regardless of the presence of HS, highlighting the importance of controlling the host response to tissue engineering devices.     

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.     

EF1α and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells

Background  

RT-qPCR analysis is a widely used method for the analysis of mRNA expression throughout the field of mesenchymal stromal cell (MSC) research. Comparison between MSC studies, both in vitro and in vivo, are challenging due to the varied methods of RT-qPCR data normalization and analysis. Therefore, this study focuses on putative housekeeping genes for the normalization of RT-qPCR data between heterogeneous commercially available human MSC, compared with more homogeneous populations of MSC such as MIAMI and RS-1 cells.     

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.     

Stem cell-based therapy for Parkinson’s disease with a focus on human endometrium-derived mesenchymal stem cells

Parkinson’s disease (PD) as an increasing clinical syndrome is a multifunctional impairment with systemic involvement. At present, therapeutic approaches such as l -3,4-dihydroxy-phenylalanine replacement therapy, dopaminergic agonist administration, and neurosurgical treatment intend to relieve PD symptoms which are palliative and incompetent in counteracting PD progression. These mentioned therapies have not been able to replace the lost cells and they could not effectively slow down the relentless neurodegenerative process. Till now, there is a lack of eligible treatment for PD, and stem cells therapy recently has been considered for PD treatment. In this review, we demonstrate how human stem cell technology especially human endometrium-derived stem cells have made advancement as a therapeutic source for PD compared with other treatments.     

The transforming growth factor-beta (TGF-β) mediates acquisition of a mesenchymal stem cell-like phenotype in human liver cells

Transforming growth factor-beta (TGF-β) mediates several and sometime opposite effects in epithelial cells, inducing growth inhibition, and apoptosis but also promoting an epithelial to mesenchymal transition (EMT) process, which enhances cell migration and invasion. TGF-β plays relevant roles in different liver pathologies; however, very few is known about its specific signaling and cellular effects in human primary hepatocytes. Here we show that TGF-β inhibits proliferation and induces pro-apoptotic genes (such as BMF or BIM) in primary cultures of human fetal hepatocytes (HFH), but also up-regulates anti-apoptotic genes, such as BCL-XL and XIAP. Inhibition of the epidermal growth factor receptor (EGFR), using gefitinib, abrogates the increase in the expression of the anti-apoptotic genes and significantly enhances cell death. Simultaneously, TGF-β is able to induce an EMT process in HFH, coincident with Snail up-regulation and a decrease in E-cadherin levels, cells showing mesenchymal proteins and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic genes and increased expression of stem cell markers. Chronic treatment with TGF-β allows selection of a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. Process is reversible and the mesenchymal stem-like cells re-differentiate to hepatocytes under controlled experimental conditions. In summary, we show for the first time that human hepatocytes may respond to TGF-β inducing different signals, some of them might contribute to tumor suppression (growth inhibition and apoptosis), but others should mediate liver tumor progression and invasion (EMT and acquisition of a stem-like phenotype).     

Proteomic identification of ADAM12 as a regulator for TGF-β1-induced differentiation of human mesenchymal stem cells to smooth muscle cells

Background

Transforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.

Methods and Results

Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells.

Conclusions

These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.     

Preclinical assessment of thrombin-preconditioned human Wharton’s jelly-derived mesenchymal stem cells for neonatal hypoxic-ischaemic brain injury

Hypoxic-ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin-preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th-hWJMSCs. MSCs were administered the maximum feasible dose (1 × 10⁵ cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th-hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague-Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th-hWJMSCs on the growth of newborn babies. Our results suggest that th-hWJMSCs are non-toxic and non-tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.