Research Containing: Bioreactors

Enhanced neurotrophin synthesis and molecular differentiation in non-transformed human retinal progenitor cells cultured in a rotating bioreactor

by cfynanon 9 June 2015in Biology & Biotechnology No comment

One approach to the treatment of retinal diseases, such as retinitis pigmentosa, is to replace diseased or degenerating cells with healthy cells. Even if all of the problems associated with tissue transplant were to be resolved, the availability of tissue would remain an ongoing problem. We have previously shown that transformed human retinal cells can be grown in a NASA-developed horizontally rotating culture vessel (bioreactor) to form three-dimensional-like structures with the expression of several retinal specific proteins. In this study, we have investigated growth of non-transformed human retinal progenitors (retinal stem cells) in a rotating bioreactor. This rotating culture vessel promotes cell-cell interaction between similar and dissimilar cells. We cultured retinal progenitors (Ret 1-4) alone or as a co-culture with human retinal pigment epithelial cells (RPE, D407) in this system to determine if 3D structures can be generated from non-transformed progenitors. Our second goal was to determine if the formation of 3D structures correlates with the upregulation of neurotrophins, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGFa), ciliary neurotrophic factor (CNTF), and brain-delivered neurotrophic factor (BDNF). These factors have been implicated in progenitor cell proliferation, commitment, differentiation, and survival. We also investigated the expression of the following retinal specific proteins in this system: neuron specific enolase (NSE); tyrosine hydroxylase (TH); D2D3, D-4 receptors; protein kinase-C alpha (PKC alpha), and calbindin. The 3D structures generated were characterized by phase and scanning transmission electron microscopy. Retinal progenitors, cultured alone or as a co-culture in the rotating bioreactor, formed 3D structures with some degree of differentiation, accompanied by the upregulation of bFGF, CNTF, and TGFa. Brain-derived neurotrophic factor, which is expressed ill vivo in RPE (D407), was not expressed in monolayer cultures of RPE but expressed in the rotating bioreactor-cultured RPE and retinal progenitors (Ret 1-4). Upregulation of neurotrophins was noted in all rotating bioreactor-cultured cells. Also, upregulation of D-4 receptor, calbindin, and PKC alpha was noted in the rotating bioreactor-cultured cells. We conclude that non-transformed retinal progenitors can be grown in the rotating bioreactor to form 3D structures with some degree of differentiation. We relied on molecular and biochemical analysis to characterize differentiation in cells grown in the rotating bioreactor.

Related URLs:
<Go to ISI>://WOS:000235844900015

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon (TM)) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.

Related URLs:
<Go to ISI>://WOS:000283899600001

Scale-Up of Mouse Embryonic Stem Cell Expansion in Stirred Bioreactors

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The aim of this study was to develop a robust, quality controlled and reproducible large-scale culture system using serum-free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 +/- 0.4) and (5.6 +/- 0.8) x 10(6) cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 +/- 7 and 108 +/- 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale-up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 +/- 15-fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct-4, Nanog, and SSEA-1 and their differentiation potential into cells of the three embryonic germ layers. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1421-1432, 2011

Related URLs:
<Go to ISI>://WOS:000296061900026

Limiting Cell Aggregation During Mesenchymal Stem Cell Expansion on Microcarriers

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T-flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex-1 (R), by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50 x 10(6) cell g(-1) Cytodex-1 (R)). Then, the MSC aggregates were put into contact with additional available surfaces (T-flask, fresh and used Cytodex-1 (R)) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T-flasks or fresh Cytodex-1 (R) carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex-1 (R) carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex-1 (R) microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number. (C) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 780-787, 2012

Related URLs:
<Go to ISI>://WOS:000312675200001

Attachment to Cytodex Beads Enhances Differentiation of Human Retinal Progenitors in 3-D Bioreactor Culture

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Retinal degenerations are the leading cause of genetically inherited blindness. One of the strategies currently being tested for the treatment is cell/tissue transplantation. As such stem cells and tissue engineered constructs are of great importance. This report describes the growth of multipotential human retinal progenitors (cell line) in a 3-D bioreactor culture vessel with (adhesive substrate) laminin coated collagen 1/cytodex beads and without adhesive substrate (beadless culture). The study demonstrates that progenitors are capable of growth and differentiation in the bioreactor with or without beads. The presence of adhesive substrate accelerates and enhances photoreceptor differentiation in the bioreactor, reflected by significantly higher level expressions of several photoreceptor specific proteins; N acetyl transferase (AaNat), rhodopsin and cone transducin GNB3. Both monomeric and dimeric forms of rhodopsin are expressed in cells attached to beads, whereas, only the monomeric form is expressed in beadless culture. Similarly, a different isomeric form of tyrosine hydroxylase (a doublet) is expressed in cell bead attached cultures. Co-culturing retinal progenitors with retinal pigment epithelium (RPE) in cell bead cultures further stabilizes the photoreceptor phenotype and rhodopsin expression. Most of the retinal neuronal phenotypes are confirmed by an expression of specific proteins. The adhesive substrate in the form of collagen 1, laminin coated cytodex beads, could be just an effector for stabilization or a positive signal, modulating extracellular matrix (ECM) molecules and/or neurotrophins. In the future, the bioreactor culture system could be utilized to grow retina-like structures from ciliary epithelium by incorporating biodegradable substrates.

Related URLs:
<Go to ISI>://WOS:000301640300005

Dynamic Cultivation of Human Mesenchymal Stem Cells in a Rotating Bed Bioreactor System Based on the Z (R) RP Platform

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three-dimensional functional tissue-like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three-dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stein cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram (R). Furthermore, hydroxyapatite coated Sponceram (R) was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP-expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor-system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram (R) compared to hydroxyapatite coated Sponceram (R). With this study, we Present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1762-1771 2009

Related URLs:
<Go to ISI>://WOS:000273152000025

Rhythmicity of engraftment and altered cell cycle kinetics of cytokine-cultured murine marrow in simulated microgravity compared with static cultures

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Space flight with associated microgravity is complicated by "astronaut's anemia" and other hematologic abnormalities. Altered erythroid differentiation, red cell survival, plasma volume, and progenitor numbers have been reported. We studied the impact of microgravity on engraftable stem cells, culturing marrow cells in rotary wall vessel (RWV) culture chambers mimicking microgravity and in normal gravity nonadherent Teflon bottles. A quantitative competitive engraftment technique was assessed under both conditions in lethally irradiated hosts. We assessed 8-wk engraftable stem cells over a period spanning at least one cell cycle for cytokine (FLT-3 ligand, thrombopoietin [TPO], steel factor)-activated marrow stem cells. Engraftable stem cells were supported out to 56 h under microgravity conditions, and this support was superior to that seen in normal-gravity Teflon bottle cultures out to 40 h, with Teflon bottle culture support superior to RWV from 40 to 56 h. A nadir of stem cell number was seen at 40 h in Teflon and 48 h in RWV, suggesting altered marrow stem cell cycle kinetics under microgravity. This is the first study of engraftable stem cells under microgravity conditions, and the differences between microgravity and normal gravity cultures may present opportunities for unique future stem cell expansion strategies.

Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system

by cfynanon 9 June 2015in Biology & Biotechnology No comment

In parallel to the active search for therapeutic and industrial applications of human embryonic stem cells (hESCs), designing automated means of producing those cells is a timely goal. Slow-turning lateral vessels (STLVs) with low shear stress have shown promise for expanding the cells at the embryoid body stage. We have improved this technology by developing two complementary systems, allowing continuous optimization of the culture conditions. First, perfused STLV bioreactors were set up, to provide continuous delivery of culture medium to the cells growing in the rotating chamber. This allowed the external control of the culture medium, and consequently optimized oxygenation, pH, nutrient supply, and waste elimination. Second, a dialysis chamber was adapted. This led to a further enhanced controlled environment and a decrease in the quantity of adjunct products (e.g., growth factors) necessary to the cells inside the bioreactor chamber. hESC aggregation and initial differentiation-taking neural induction as an example-were compared between the perfused and dialyzed STLV system and static cultures. Perfused and dialyzed STLV bioreactors promoted formation of embryoid bodies that were differentiated more rapidly and were homogeneously synchronized in a statistically significant manner.

Computational modeling for the optimization of a cardiogenic 3D bioprocess of encapsulated embryonic stem cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

We present a computational fluid dynamics (CFD)-based model aimed at the identification of optimized culture conditions promoting efficient cardiogenesis of hydrogel-bead-encapsulated embryonic stem cells (ESCs) within a rotating bioreactor. The numerical approach, integrating diffusion, convection, and multiphase fluid dynamics calculations, allowed to evaluate (i) the microgravity motion of the floating beads, (ii) the O-2 delivery to the cells, also (iii) taking into account the cellularO(2) consumption, as a function of different rotation speeds of the breeding chamber. According to our results, a 25rpm rotation (i) enhances an adequate mixing of the cell carriers, avoiding sedimentation and excessive packing, also maintaining a quite homogeneous distribution of the suspended beads and (ii) imparts a proper cellular O-2 supply, providing cells close to a normoxia condition. The bioreactor working conditions derived from the numerical analysis allowed the attainment of in vitro long-term cell viability maintenance, supporting efficient large-scale generation of ESC-derived cardiomyocytes (ESC-DCs) through a chemical-based conditioning bioprocess. In conclusion, we demonstrated the feasibility of using CFD-based tools, as a reliable and cost-effective strategy to assist the design of a 3D cardiogenic bioprocess.

Related URLs:
<Go to ISI>://WOS:000300230600020

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Research Containing: Bioreactors

Enhanced neurotrophin synthesis and molecular differentiation in non-transformed human retinal progenitor cells cultured in a rotating bioreactor

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

Scale-Up of Mouse Embryonic Stem Cell Expansion in Stirred Bioreactors

Limiting Cell Aggregation During Mesenchymal Stem Cell Expansion on Microcarriers

Attachment to Cytodex Beads Enhances Differentiation of Human Retinal Progenitors in 3-D Bioreactor Culture

Dynamic Cultivation of Human Mesenchymal Stem Cells in a Rotating Bed Bioreactor System Based on the Z (R) RP Platform

Rhythmicity of engraftment and altered cell cycle kinetics of cytokine-cultured murine marrow in simulated microgravity compared with static cultures

Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system

Computational modeling for the optimization of a cardiogenic 3D bioprocess of encapsulated embryonic stem cells