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Research Containing: Bioreactors

Stem cell cultivation in bioreactors

by on 9 June 2015in Biology & Biotechnology No comment

Cell-based therapies have generated great interest in the scientific and medical communities, and stem cells in particular are very appealing for regenerative medicine, drug screening and other biomedical applications. These unspecialized cells have unlimited self-renewal capacity and the remarkable ability to produce mature cells with specialized functions, such as blood cells, nerve cells or cardiac muscle. However, the actual number of cells that can be obtained from available donors is very low. One possible solution for the generation of relevant numbers of cells for several applications is to scale-up the culture of these cells in vitro. This review describes recent developments in the cultivation of stem cells in bioreactors, particularly considerations regarding critical culture parameters, possible bioreactor configurations, and integration of novel technologies in the bioprocess development stage. We expect that this review will provide updated and detailed information focusing on the systematic production of stem cell products in compliance with regulatory guidelines, while using robust and cost-effective approaches. (C) 2011 Elsevier Inc. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000296821900023

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor

by on 9 June 2015in Biology & Biotechnology No comment

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 x 10(5) cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

Related URLs:
<Go to ISI>://WOS:000293752000009

Plant molecular biology in the space station era: utilization of KSC fixation tubes with RNAlater

by on 9 June 2015in Biology & Biotechnology No comment

Spaceflight experiments involving biological specimens face unique challenges with regard to the on orbit harvest and preservation of material for later ground-based analyses. Preserving plant material for gene expression analyses requires that the tissue be prepared and stored in a manner that maintains the integrity of RNA. The liquid preservative RNAlater (Ambion) provides an effective alternative to conventional freezing strategies, which are limited or unavailable in current spaceflight experiment scenarios. The spaceflight use of RNAlater is enabled by the Kennedy space center fixation tube (KFT), hardware designed to provide the necessary containment of fixatives during the harvest and stowage of biological samples in space. Pairing RNAlater with the KFT system provides a safe and effective strategy for preserving plant material for subsequent molecular analyses, a strategy that has proven effective in several spaceflight experiments. Possible spaceflight scenarios for the use of RNAlater and KFTs are explored and discussed.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/15736319

Microbial responses to microgravity and other low-shear environments

by on 9 June 2015in Biology & Biotechnology No comment

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/15187188

Novel quantitative biosystem for modeling physiological fluid shear stress on cells

by on 9 June 2015in Biology & Biotechnology No comment

The response of microbes to changes in the mechanical force of fluid shear has important implications for pathogens, which experience wide fluctuations in fluid shear in vivo during infection. However, the majority of studies have not cultured microbes under physiological fluid shear conditions within a range commonly encountered by microbes during host-pathogen interactions. Here we describe a convenient batch culture biosystem in which (i) the levels of fluid shear force can be varied within physiologically relevant ranges and quantified via mathematical models and (ii) large numbers of cells can be planktonically grown and harvested to examine the effect of fluid shear levels on microbial genomic and phenotypic responses. A quantitative model based on numerical simulations and in situ imaging analysis was developed to calculate the fluid shear imparted by spherical beads of different sizes on bacterial cell cultures grown in a rotating wall vessel (RWV) bioreactor. To demonstrate the application of this model, we subjected cultures of the bacterial pathogen Salmonella enterica serovar Typhimurium to three physiologically-relevant fluid shear ranges during growth in the RVW and demonstrated a progressive relationship between the applied fluid shear and the bacterial genetic and phenotypic responses. By applying this model to different cell types, including other bacterial pathogens, entire classes of genes and proteins involved in cellular interactions may be discovered that have not previously been identified during growth under conventional culture conditions, leading to new targets for vaccine and therapeutic development.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/17142365

Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel

by on 9 June 2015in Biology & Biotechnology No comment

Expansion of umbilical cord blood mononuclear cells (UCB MNCs) was carried out in a rotating wall vessel (RWV) bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 3.33 ng/ml G-CSF, 2.13 ng/ml GM-CSF, 7.47 ng/ml FL and 7.47 ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24 It. Flow cytometric assay for CD34(+) cells was carried out at 0, 144 and 197 h and methylcellulose colony assays were performed at 0, 72, 144 and 197 It. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for hematopoietic stem cells (HSCs) and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs. At the end of 200 h culture, the total cell number was multiplied by 435.5 +/- 87.6 times, and CD34(+) cells 32.7 +/- 15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7 +/- 4.9 times. While in T-flasks, however, total cells density changed mildly, CD34(+) cells and CFU-GM decreased in number. It is demonstrated that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flask. (c) 2006 Elsevier B.V. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000239156700012

Neural precursor cells form rudimentary tissue-like structures in a rotating-wall vessel bioreactor

by on 9 June 2015in Biology & Biotechnology No comment

We have analyzed the biology of embryonic, epidermal growth factor-responsive murine neural precursor cells cultured in the high-aspect ratio vessel (HARV). Within 2-3 d of rotary-cell culture, such cells formed multiple, macroscopic, three-dimensional structures that were orders of magnitude larger than the cellular clusters ("neurospheres") formed by these cells in conventional stationary-flask cultures. Each HARV structure was composed of a multilayered cellular shell surrounding one or more central cavities that were bordered by pyknotic cell nuclei. Although the cells in the HARV structures were more pleomorphic than those in neurospheres, the structures did not appear to represent primitive neural tumors: the formation of HARV structures by precursor cells was not an irreversible phenotypic change, and the structures dill not originate front the clonal expansion of single-progenitor cells; the growth rate and invasiveness of the cells in HARVs were less than those in flasks; and HARV-cultured cells did not form tumors after subcutaneous inoculation into the Hanks of NOD-scid/scid mice. Immunohistochemical analysis suggested that HARV structures might be novel "proto-tissues" characterized by a crude. but organized, architecture, with a surface laver of immature proliferating cells (nestin- and proliferating cell nuclear antigen-positive) that enclosed strata of more differentiated cells (beta -tubulin III- and glial fibrillary acidic protein-positive) within. Rotary-cell culture may have significant implications for the eventual utility. of neural precursors for clinical neurotransplantation.

Related URLs:
<Go to ISI>://WOS:000168690900004

Reconstruction of Functional Cortical-like Tissues from Neural Stem and Progenitor Cells

by on 9 June 2015in Biology & Biotechnology No comment

Neural stem and progenitor cells isolated from embryonic day 13 rat cerebral cortex were immobilized in three-dimensional type I collagen gels, and then the cell-collagen constructs were transferred to rotary wall vessel bioreactors and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor for up to 10 weeks. Remarkably, the collagen-entrapped cells formed a complex two-layered structure that emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The surface layer (layer I) composed primarily of proliferating neural progenitor cells (nestin(+), vimentin(+), and PCNA(+)) predominantly expressed functional neurotransmitter receptors for cholinergic and purinergic agonists while differentiating cells (TuJ1(+) and GFAP(+)) in the deeper layer (layer II) contained differentiated neurons and astrocytes and mainly responded to GABAergic and glutamatergic agonists and to veratridine, which activates voltage-dependent Na(+) channels. An active synaptic vesicle recycling was demonstrated by neuronal networks in the deeper layer using the endocytotic marker FM1-43. Cell polarization forming the characteristic two-layered structure was found to associate with the bFGF and FGF receptor signaling. These engineered functional tissue constructs have a potential use as tissue surrogates for drug screening and detection of environmental toxins, and in neural cell replacement therapy.

Related URLs:
<Go to ISI>://WOS:000259998000009

Neural stem cell differentiation in a cell-collagen-bioreactor culture system

by on 9 June 2015in Biology & Biotechnology No comment

Neural stem cells and neural progenitors (NSCs/NPs) are capable of self-renewal and can give rise to both neurons and glia. Such cells have been isolated from the embryonic brain and immobilized in three dimensional collagen gels. The collagen-entrapped NSCs/NPs recapitulate CNS stem cell development and form functional synapses and neuronal circuits. However, the cell-collagen constructs from static conditions contain hypoxic, necrotic cores and the cells are short-lived. In the present study, NSCs/NPs isolated from embryonic day 13 rat cortical neuroepithelium are immobilized in type 1 collagen gels and cultured in NASA-designed rotating wall vessel (RWV) bioreactors for up to 9 weeks. Initially, during the first 2 weeks of culture, a lag phase of cellular growth and differentiation is observed in the RWV bioreactors. Accelerated growth and differentiation, with the cells beginning to form large aggregates (similar to1 mm in diameter) without death cores, begins during the third week. The collagen-entrapped NSCs/NPs cultured in RWV show active neuronal generation followed by astrocyte production. After 6 weeks in rotary culture, the cell-collagen constructs contain over 10 fold greater nestin(+) and GFAP(+) cells and two-fold more TuJ1 gene expression than those found in static cultures. In addition, TuJ1(+) neurons in RWV culture give rise to extensive neurite outgrowth and considerably more synapsin 1(+) pre-synaptic puncta surrounding MAP2(+) cell bodies and dendrites. These results strongly suggest that the cell-collagen-bioreactor culture system supports long-term NSC/NP growth and differentiation, and RWV bioreactors can be useful in generating neural tissue like constructs, which may have the potential for cell replacement therapy. (C) 2004 Elsevier B.V. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000225471700002

NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure

by on 9 June 2015in Biology & Biotechnology No comment

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a "stimulatory'' environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Related URLs:
<Go to ISI>://WOS:000297350800010