Rat Adipose Tissue-Derived Stem Cells Transplantation Attenuates Cardiac Dysfunction Post Infarction and Biopolymers Enhance Cell Retention

Background

Cardiac cell transplantation is compromised by low cell retention and poor graft viability. Here, the effects of co-injecting adipose tissue-derived stem cells (ASCs) with biopolymers on cell cardiac retention, ventricular morphometry and performance were evaluated in a rat model of myocardial infarction (MI).

Methodology/Principal Findings

^99mTc-labeled ASCs (1×10⁶ cells) isolated from isogenic Lewis rats were injected 24 hours post-MI using fibrin a, collagen (ASC/C), or culture medium (ASC/M) as vehicle, and cell body distribution was assessed 24 hours later by γ-emission counting of harvested organs. ASC/F and ASC/C groups retained significantly more cells in the myocardium than ASC/M (13.8±2.0 and 26.8±2.4% vs. 4.8±0.7%, respectively). Then, morphometric and direct cardiac functional parameters were evaluated 4 weeks post-MI cell injection. Left ventricle (LV) perimeter and percentage of interstitial collagen in the spare myocardium were significantly attenuated in all ASC-treated groups compared to the non-treated (NT) and control groups (culture medium, fibrin, or collagen alone). Direct hemodynamic assessment under pharmacological stress showed that stroke volume (SV) and left ventricle end-diastolic pressure were preserved in ASC-treated groups regardless of the vehicle used to deliver ASCs. Stroke work (SW), a global index of cardiac function, improved in ASC/M while it normalized when biopolymers were co-injected with ASCs. A positive correlation was observed between cardiac ASCs retention and preservation of SV and improvement in SW post-MI under hemodynamic stress.

Conclusions

We provided direct evidence that intramyocardial injection of ASCs mitigates the negative cardiac remodeling and preserves ventricular function post-MI in rats and these beneficial effects can be further enhanced by administrating co-injection of ASCs with biopolymers.     

Proinflammatory interleukins' production by adipose tissue‐derived mesenchymal stromal cells: the impact of cell culture conditions and cell‐to‐cell interaction

The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL‐6 and IL‐8 production by adipose‐derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O₂ concentrations, some highly up‐regulated at hypoxia. IL‐6 and IL‐8 production was inversely dependent on cell culture density. In early (first–third) passages, IL‐6 and IL‐8 concentration was higher at 20% O₂ and in late (8th‐12th) passages under 5% O₂. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL‐6 and did not result in the elevation of IL‐8 concentration. Thereby, the production of proinflammatory interleukins (IL‐6 and IL‐8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood‐borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE PARAGRAPH Ex vivo expansion is widely used for increasing the number of adipose‐derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.     

Biophysical cues enhance myogenesis of human adipose derived stem/stromal cells

Adipose-derived stem/stromal cell (ASC)-based tissue engineered muscle grafts could provide an effective alternative therapy to autografts – which are limited by their availability – for the regeneration of damaged muscle. However, the current myogenic potential of ASCs is limited by their low differentiation efficiency into myoblasts. The aim of this study was to enhance the myogenic response of human ASCs to biochemical cues by providing biophysical stimuli (11% cyclic uniaxial strain, 0.5 Hz, 1 h/day) to mimic the cues present in the native muscle microenvironment. ASCs elongated and fused upon induction with myogenic induction medium alone. Yet, their myogenic characteristics were significantly enhanced with the addition of biophysical stimulation; the nuclei per cell increased approximately 4.5-fold by day 21 in dynamic compared to static conditions (23.3 ± 7.3 vs. 5.2 ± 1.6, respectively), they aligned at almost 45° to the direction of strain, and exhibited significantly higher expression of myogenic proteins (desmin, myoD and myosin heavy chain). These results demonstrate that mimicking the biophysical cues inherent to the native muscle microenvironment in monolayer ASC cultures significantly improves their differentiation along the myogenic lineage.     

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells

BACKGROUNDAdipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.AIMTo test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).METHODSASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco''s Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).RESULTSDepending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSIONThe presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.     

Effects of In Vitro Low Oxygen Tension Preconditioning of Adipose Stromal Cells on Their In Vivo Chondrogenic Potential: Application in Cartilage Tissue Repair

Purpose

Multipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair.

Methods

MSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O₂. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O₂ was finally evaluated using real-time PCR.

Results/Conclusions

5% O₂ increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O₂. These data together indicate that although 5% O₂ enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.     

Soluble factors from ASCs effectively direct control of chondrogenic fate

Background and objectives: Adipose tissue‐derived stem cells (ASCs) have great potential for regenerative medicine. For molecular understanding of specific functional molecules present in ASCs, we analysed 756 proteins including specific chondrogenic functional factors, using high‐throughput nano reverse‐phase liquid chromatography–electrospray ionization–tandem mass spectrometry. Materials, methods and results: Of these proteins, 33 were identified as chondrogenic factors or proteins including type 2 collagen, biglycan, insulin‐like growth factor‐binding protein and transforming growth factor‐beta 1 (TGF‐β1). ASCs are a possible cell source for cartilage regeneration as they are able to secrete a number of functional cytokines including chondrogenesis‐inducing molecules such as TGF‐β1 and bone morphogenetic protein 4 (BMP4). The chondrogenic phenotype of cultured ASCs was effectively induced by ASC‐culture media (CM) containing BMP4 and TGF‐β1, and maintained after pre‐treatment for 14 days in vitro and subcutaneous implantation in vivo. Chondrogenic differentiation efficiency of cultured ASCs and cultured mouse skin‐derived progenitor cells (SPCs) depended absolutely on ASC CM‐fold concentration. Cell density was also a very important factor for chondrogenic behaviour development during differentiation of ASCs and SPCs. Conclusion: ASC CM‐derived TGF‐β1‐induced chondrogenic differentiation of ASCs resulted in significant reduction in chondrogenic activity after inhibition of the p38 pathway, revealing involvement of this MAPK pathway in TGF‐β1 signalling. On the other hand, TGF‐β1 signalling also led to SMAD activation that could directly increase chondrogenic activity of ASCs.     

In Situ Normoxia Enhances Survival and Proliferation Rate of Human Adipose Tissue-Derived Stromal Cells without Increasing the Risk of Tumourigenesis

Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O₂ concentration (21% O₂) or in situ normoxia (2% O₂). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O₂ concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O₂ as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.     

Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation

BackgroundAdipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.MethodsNormal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated.ResultsASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia.ConclusionASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.     

Hierarchical Fabric Decorated with Carbon Nanowire/Metal Oxide Nanocomposites for 1.6 V Wearable Aqueous Supercapacitors

Aqueous asymmetric supercapacitors (ASCs) may offer comparable or higher energy density than electric double‐layer capacitors (EDLCs) based on organic electrolytes. As such, ASCs may be more suitable for integration into smart textiles, where the use of flammable organic solvents is not acceptable. However, reported ASC devices typically suffer from poor rate capability and low areal loadings. This study demonstrates the development of nitrogen‐doped carbon (N‐C) nanowire/metal oxide (Fe₂O₃ and MnO₂) nanocomposite electrodes directly produced on the internal surface of a conductive fabric for use as high‐rate electrodes for solid‐state ASCs. The N‐C nanowires provide fast and efficient pathways for electrons, while short diffusion paths within nanosized metal oxides enable fast ion transport, leading to greatly enhanced performance at high rates. The porous structure of the fabric enables high areal capacitance loading in each electrode (≈150 mF cm^?2). Both electrodes show high specific capacitance of ≈180 F g^?1 (Fe₂O₃) and ≈250 F g^?1 (MnO₂) and excellent rate capability. Solid‐state ASCs assembled by using an aqueous gel electrolyte operate at 1.6 V and deliver over 60 mF cm^?2 during ≈50 s charging/discharging time and over 30 mF cm^?2 for ≈5 s discharge.     

Synthesis, double stranded DNA-binding and photocleavage studies of a functionalized ruthenium(II) complex with 7,7′-methylenedioxyphenyldipyrido[3,2-a:2′,3′-c]-phenazine

A new Ru(II) complex [Ru(phen)₂(mdpz)]²⁺ (phen = 1,10-phenanthroline, mdpz = 7,7′-methylenedioxyphenyl-dipyrido-[3,2-a:2′,3′-c]phenazine) has been synthesized and characterized in detail by elemental analysis, mass spectrometry and ¹H NMR spectroscopy. The interaction of the complex with calf thymus DNA was investigated by spectroscopic and viscosity measurements. The results suggest that the complex binds to DNA via an intercalative mode and serves as a molecular “light switch” for DNA. Moreover, the complex has been found to promote the photocleavage of plasmid DNA pBR322 under irradiation at 365 nm. The mechanism studies reveal that singlet oxygen (¹O₂) plays a significant role in the photocleavage.     

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic,Induced Pluripotent and Adipose Stem Cells

Background

The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.

Methodology/Principal Findings

Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.

Conclusion/Significance

Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.     

Syntheses and characterizations of two lanthanide(III)-copper(II) coordination polymers constructed by pyridine-2,6-dicarboxylic acid

The hydrothermal reaction of La₂O₃ and Pr₂O₃ with pyridine-2,6-dicarboxylic acid (H₂pydc), CuO, and H₂O with a mole ratio of 1:2:4:300 resulted in the formation of two polymeric Cu(II)-Ln(III) complexes, [{Ln₄Cu₂(pydc)₈(H₂O)₈} · 18H₂O]_n (Ln = La (1); Pr (2)). 1 and 2 are isomorphous and crystallize in monoclinic space group C2/c. Complexes 1 and 2 have one-dimensional infinite chains with “∞” shape. The 1D chains are linked by the hydrogen bonds and π?π stacking interactions to form layer structures which are further linked by the hydrogen bonds and π?π stacking interactions to form the three-dimensional (3D) structures with nanoscale porosities. Temperature-dependent magnetic susceptibilities and the thermal stabilities of complexes 1 and 2 were studied.     

Advancements in adipose-derived stem cell therapy for skin fibrosis

Pathological scarring and scleroderma, which are the most common conditions of skin fibrosis, pathologically manifest as fibroblast proliferation and extracellular matrix (ECM) hyperplasia. Fibroblast proliferation and ECM hyperplasia lead to fibrotic tissue remodeling, causing an exaggerated and prolonged wound-healing response. The pathogenesis of these diseases has not been fully clarified and is unfortunately accompanied by exceptionally high medical needs and poor treatment effects. Currently, a promising and relatively low-cost treatment has emerged-adipose-derived stem cell (ASC) therapy as a branch of stem cell therapy, including ASCs and their derivatives-purified ASC, stromal vascular fraction, ASC-conditioned medium, ASC exosomes, etc., which are rich in sources and easy to obtain. ASCs have been widely used in therapeutic settings for patients, primarily for the defection of soft tissues, such as breast enhancement and facial contouring. In the field of skin regeneration, ASC therapy has become a hot research topic because it is beneficial for reversing skin fibrosis. The ability of ASCs to control profibrotic factors as well as anti-inflammatory and immunomodulatory actions will be discussed in this review, as well as their new applications in the treatment of skin fibrosis. Although the long-term effect of ASC therapy is still unclear, ASCs have emerged as one of the most promising systemic antifibrotic therapies under development.     

Human Adipose-Tissue Derived Stromal Cells in Combination with Hypoxia Effectively Support Ex Vivo Expansion of Cord Blood Haematopoietic Progenitors

The optimisation of haematopoietic stem and progenitor cell expansion is on demand in modern cell therapy. In this work, haematopoietic stem/progenitor cells (HSPCs) have been selected from unmanipulated cord blood mononuclear cells (cbMNCs) due to adhesion to human adipose-tissue derived stromal cells (ASCs) under standard (20%) and tissue-related (5%) oxygen. ASCs efficiently maintained viability and supported further HSPC expansion at 20% and 5% O₂. During co-culture with ASCs, a new floating population of differently committed HSPCs (HSPCs-1) grew. This suspension was enriched with СD34⁺ cells up to 6 (20% O₂) and 8 (5% O₂) times. Functional analysis of HSPCs-1 revealed cobble-stone area forming cells (CAFCs) and lineage-restricted colony-forming cells (CFCs). The number of CFCs was 1.6 times higher at tissue-related O₂, than in standard cultivation (20% O₂). This increase was related to a rise in the number of multipotent precursors - BFU-E, CFU-GEMM and CFU-GM. These changes were at least partly ensured by the increased concentration of MCP-1 and IL-8 at 5% O₂. In summary, our data demonstrated that human ASCs enables the selection of functionally active HSPCs from unfractionated cbMNCs, the further expansion of which without exogenous cytokines provides enrichment with CD34⁺ cells. ASCs efficiently support the viability and proliferation of cord blood haematopoietic progenitors of different commitment at standard and tissue-related O₂ levels at the expense of direct and paracrine cell-to-cell interactions.     

Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate

 This paper presents a mathematical model which enables the semi-quantification of ozone (O₃) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments – based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O₃ reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O₃ taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O₃ in the cell wall. Received: 13 May 1999 / Accepted: 5 August 1999     

Quantitation of Hydroxyl Radicals Produced by Radiation and Copper-Linked Oxidation of Ascorbate by 2-Deoxy-d-Ribose Method

We have established controlled conditions for studying the reaction of chemically and radiolytically produced hydroxyl radical (OH) with 2-deoxy-D-ribose (2-DR). Ascorbate (ASC) or dithiothreitol (DTT) and cuprous or cupric ions were used to generate the OH-radical. The OH-radical was detected using the classical method of measuring the amount of thiobarbituric acid reactive products (TBARP) formed by OH-mediated 2-DR degradation, but using sensitive fluorescent detection of the TBARP production to quantify the OH-radical. All experiments were performed with adequate O₂ concentrations. The copper reaction with ASC consumes O₂ in a manner that is strongly dependent on copper concentration, and less dependent on ascorbate concentration. For an independent check of the Cu²⁺ catalyzed ASC oxidation kinetics, the decay of ASC absorbency at 265 nm, as well as the increase of H₂O₂ absorbency at 240 nm, were also monitored. These spectral changes agree well with the O₂ consumption data. TBARP production from 2-DR incubated with a Cu²⁺–ASC mixture or γ-irradiated were also compared. γ-Irradiation of 2-DR solutions shows a dose and 2-DR concentration dependent increase of TBARP generation. Other electron donors, such as DTT, are more complicated in their mechanism of OH-radical production. Incubation of 2-DR with Cu²⁺-DTT mixtures shows a delay (50 min) before OH-radical generation is detected. Our results suggest that the Cu²⁺-ASC reaction can be used to mimic the effects of ionizing radiation with respect to OH-radical generation. The good reproducibility and relative simplicity of the 2-DR method with fluorescence detection indicates its usefulness for the quantitation of the OH-radical generated radiolytically or chemically in carefully controlled model systems. © 1997 Elsevier Science Inc.     

The effect of headspace renewal in a Temporary Immersion Bioreactor on plantain (Musa AAB) shoot proliferation and quality

The positive effect of ventilation of the culture container on in vitro shoot proliferation and quality was already proven for different species. Hereafter we report on the evolution of the headspace during in vitro culture of plantain in a Temporary Immersion Bioreactor (TIB) on the one hand, and culture on semi-solid medium on the other hand. The CO₂ and C₂H₄ concentration reached a maximum of 12% and 0.45 μl l⁻¹, respectively in the control treatment on semi-solid medium, compared to 5.7% CO₂ and 0.06 μl l⁻¹ C₂H₄ in TIB. The minimal O₂-concentration on semi-solid medium was 15.1%, compared to 19.3% in TIB. The multiplication rate was best in TIB, 6.4 compared to 4.3 in semi-solid conditions, and this was also the case for shoot height (4.3 cm compared to 3.3 cm), and leaf number (2.6 compared to 1.6). Moreover shoots produced on semi-solid medium showed distorted leaves. A typical day-night pattern in CO₂ and O₂ concentration was observed in TIB, as well as on semi-solid medium; this is illustrative for the photosynthetic capacity of the plant material produced in both systems.