Micropatterning of Nanoengineered Surfaces to Study Neuronal Cell Attachment in Vitro
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a recent study by Lee et al., in which the release of VEGF from alginate hydrogels was similarly increased by enhanced fluid pressure during mechanically-induced hydrogel collapse. 15 Therefore, we speculate that the CaM conformational change promoted collapse of PEG-CaM-PEG-containing hydrogels, which in turn increased the internal fluid pressure in the hydrogels and led to an increased VEGF release rate (Fig. 6). Future applications of these dynamic, protein-based materials may ultimately optimize network properties to more efficiently limit protein release in the absence of TFP, with the goal of creating a material from which protein release can be switched on or off dependent on the presence and concentration of a specific biochemical ligand. Specific methods for decreasing the release rate in the absence of TFP could involve tailoring the initial hydrogel mesh size by, for example, decreasing molecular weight between crosslinks or increasing the crosslink density. However, it is important to note that ultimate application of protein-based, dynamic materials for growth factor delivery in vivo will also require a more extensive analysis of the immunogenicity of these materials in future studies. The 4 of PEG-CaM-PEG3400 hydrogels also had a substantial effect on the total amount of VEGF released. Both 10% and 12.5% PEG-CaM-PEG3400 hydrogels released $100% of their VEGF in 48 h in the presence of TFP. Alternatively, 15% hydrogels absorbed the most VEGF but only released $58% of their VEGF over 48 h in the presence of TFP (Fig. 5A). To explain this phenomenon we hypothesize that two distinct mechanisms control the total amount of VEGF released from PEG-CaM-PEG3400 hydrogels: (1) The 4 of PEG-CaM- PEG3400, which modulates the amount of absorbed VEGF via electrostatic interactions (Fig. 3A–B); and (2) the magnitude of hydrogel collapse (Fig. 2C), which modulates the internal fluid pressure and release of VEGF. Therefore, we hypothesize that although 15% PEG-CaM-PEG3400 hydrogels absorb the most VEGF through electrostatic interactions (Fig. 3A–B), they also exhibited the smallest volume collapse (Fig. 2C), which results in the least amount of total VEGF released (Fig. 5A). As in the case of pure PEG-CaM-PEG3400 hydrogels, PEG-CaM-PEG3400/ PEGDA3400 hydrogels that could undergo greater dynamic changes released more total VEGF when exposed to TFP. However, 9/1% or 7/3% hydrogels were capable of releasing >85% of their loaded VEGF even in the absence of TFP (Fig. 5B). The ability of these hydrogels to release nearly 100% their absorbed VEGF may be indicative of decreased charge-charge interactions between VEGF and CaM in the hydrogels due to the inclusion of more synthetic, neutral PEGDA molecules. In summary, we have developed an adaptable scheme to synthesize, purify, and assess the activity of polymer–protein conjugates for construction of dynamic hydrogels. The dynamic response of these materials is based on a specific biochemical interaction between a dynamic protein and a soluble ligand, which provides enhanced specificity when compared to mecha- nisms commonly used to generate pH-, temperature-, or light- responsive materials. These hydrogels can be used to modulate the release of an important, biologically active protein, VEGF. The approach described here may have broad applicability in biomedical applications that require controlled protein delivery, including tissue engineering, bio-microfluidics, and in vivo drug delivery. In addition, this general mechanism for controlling network properties could be useful in applications that extend beyond protein delivery, including biosensing and soft robotics. There are hundreds of proteins that undergo well-characterized, dynamic changes in conformation, 35 and these proteins represent a large library of building blocks for synthesis of dynamic materials. Fig. 6 Schematic representation of a swollen PEG-CaM-PEG hydrogel network loaded with a therapeutic drug (top) and releasing the drug at a slower rate (bottom left) in its swollen state and faster (bottom right) rates in response to stimulation with a specific ligand. 2404 | Soft Matter, 2009, 5, 2399–2406 This journal is ª The Royal Society of Chemistry 2009 Conclusion The conformational change of the protein calmodulin upon ligand binding can be translated into dynamic volume shifts in a hydrogel network. Specifically, dynamic hydrogels were cre- ated by synthesizing and purifying PEG-CaM-PEG conju- gates, and the CaM protein component remained active through this process. Increasing the 4 of PEG-CaM-PEG3400 networks or increasing the 4 of PEGDA3400 within PEG-CaM- PEG3400/PEGDA3400 networks resulted in less dynamic hydrogels. Importantly, the physiochemical and dynamic properties of these dynamic, protein-based hydrogel networks can be used to modulate absorption and dynamic release of a therapeutic protein, VEGF. The 4 of PEG-CaM-PEG3400 in hydrogels was linearly related to the amount of VEGF absor- bed. Release of VEGF from PEG-CaM-PEG3400 hydrogels was significantly influenced by both the initial concentration of PEG-CaM-PEG3400 during hydrogel formation and the dynamic properties of the hydrogels. Importantly, an increased rate of VEGF release from hydrogels could be triggered at multiple time points by inducing the CaM conformational change. This study provides a first demonstration that protein conformational changes in a hydrogel network can be used as a mechanism to trigger changes in the release of a therapeutic protein. The physical parameters found to be important for modulating VEGF release here may be extended to future applications of dynamic, protein-based materials in controlled drug delivery. Experimental Synthesis of PEG-CaM-PEG PEGDA 575 was purchased from Sigma-Aldrich (St. Louis, MO, USA). PEG 3400 was purchased from Fisher Scientific (Fair- lawn, NJ, USA) and hydroxyl end-groups were derivatized with acrylate groups using a previously described method. 8,23
Trifluoperazine (TFP) and all other reagents (Ellman’s Reagent, Dithiothreitol (DTT), NaCl, Tris, MES, EDTA, EGTA, and CaCl 2
CaM (T34C, T110C) was recombinantly expressed and puri- fied as previously described. 21–23 After CaM was purified using a Phenyl Sepharose column (GE Healthcare, Chalfont St. Giles, United Kingdom), it was placed in 10,000 MWCO dialysis tubing (Pierce, Rockford, IL, USA) and dialyzed at 4
C for 24 h against a solution containing 10 mM Tris and 5 mM CaCl 2 (pH 8.0). CaM was then reacted with 10 Â molar excess of PEGDA for 2.5 h at 37
C in a solution containing 10 mM Tris and 5 mM CaCl 2 (pH 8.0). The reaction mixture was then cooled to 4 C and purified using identical conditions to the initial CaM (T34C T110C) purification, but in the absence of DTT. The collected fractions of purified PEG-CaM-PEG were pooled and placed in 10,000 MWCO dialysis tubing (Pierce, Rockford, IL, USA) and dialyzed at 4
C for 24 h against ultrapure H 2 O (18 MU resis- tivity). Raffinose (1.0 mg/ml) was added to the PEG-CaM-PEG solution in all cases (except when evaluating the effect of lyophilization on CaM activity without Raffinose), flash frozen in liquid N 2 , and lyophilized. Characterization of PEG-CaM-PEG synthesis CaM and PEG-CaM-PEG was desalted for analysis using a C18 ZipTip (Millipore, Billerica, MA, USA). The molecular weights of CaM and PEG-CaM-PEG were evaluated by using a REFLEX II MALDI-TOF mass spectrometer (Bruker, Billerica, MA, USA). The purity of CaM and PEG-CaM-PEG conjugates was evaluated using a HPLC with a C18 analytical column (Shi- madzu, Kyoto, Japan) with a gradient of 0–90% H 2 O + 0.1%
trifluoroacetic acid (TFA) (Fisher Scientific, Fairlawn, NJ, USA)/acetonitrile (Sigma Aldrich, St. Louis, MO, USA) and a flow rate of 0.9 ml/minute. Characterization of PEG-CaM-PEG activity The activity of PEG-CaM-PEG was assessed by adapting a cal- cineurin activity kit (Biomol International, Plymouth Meeting, PA, USA). Briefly, the concentration of samples of CaM from different purification and processing steps were normalized by first measuring the protein concentration using the MicroBCA Assay (Pierce, Rockford, IL, USA), then diluting CaM samples to equal concentrations. The diluted CaM solutions were then added to a solution containing the enzyme calcineurin and the RII phosphopeptide a calcineurin substrate. Calmodulin increases the phosphatase activity of calcineurin which removes the phosphate group from the RII phosphopeptide. The liber- ated phosphate in turn complexes with malachite green and molybdate and forms particles in solution which can be quanti- fied by measuring absorption of light at 620 nm. The reaction was stopped by adding the Biomol Green Reagent and the absorption of light of the samples was compared to a standard curve of free phosphate. The activity of CaM was calculated by normalizing the concentration of phosphate liberated by calci- neurin in the presence of calmodulin from the assay kit to 100% and the concentration of phosphate of calcineurin in the absence of CaM was set to 0%. Hydrogel formation The ratio of PEG-CaM-PEG to total weighed mass of lyophi- lized PEG-CaM-PEG samples was determined by dividing the measured PEG-CaM-PEG from the MicroBCA assay by the weight of the lyophilized solid. To form hydrogels entirely of PEG-CaM-PEG, 10–20% (w/v) PEG-CaM-PEG was mixed with 0.05% Irgacure 2959 (Ciba, Basel, Switzerland) photoinitiator in aqueous buffer (20 mM HEPES, 150 mM NaCl, 10 mM CaCl 2 (pH 7.4)). Hydrogel precursor solutions were cast between two Sigma Cote treated glass slides with a 1 mm spacer and exposed to 4 mW/cm 2 , 365 nm UV light for 8 min. In some cases, this procedure was used to create 10% (w/v) hydrogels using combi- nations of PEG-CaM-PEG conjugates and PEGDA molecules, as indicated in the text. After crosslinking, hydrogels were swollen in aqueous buffer (20 mM MES, 150 mM NaCl, 10 mM CaCl 2
Characterizing dynamic properties of PEG-CaM-PEG hydrogels The dynamic properties of PEG-CaM-PEG hydrogels were examined by varying (1) the ratio of PEG-CaM-PEG3400/ This journal is ª The Royal Society of Chemistry 2009 Soft Matter, 2009, 5, 2399–2406 | 2405
PEGDA3400 and (2) 4 of PEG-CaM-PEG3400. PEG-CaM- PEG hydrogels were swollen for 24 h and bright field micro- graphs were obtained using an inverted microscope (Olympus, Center Valley, PA, USA) controlled by SimplePCI software (Hamamatsu, Sewickley, PA, USA). Then, hydrogels were incubated in a solution of 10 mM TFP (20 mM MES, 150 mM NaCl, 10 mM CaCl 2 (pH 6.3)) for 24 h and micrographs were acquired again. To remove TFP from the hydrogels they were first washed in EGTA buffer for 12 h to remove Ca 2+ dependent ligand binding then incubated in Ca 2+ -containing buffer for 12 h (20 mM MES, 150 mM NaCl, 10 mM CaCl 2 (pH 6.3)). Images were analyzed using ImageJ software (Freeware, Bethesda, MD, USA). The change in volume was calculated by assuming isotropic expansion and contraction as outlined previously by Sui et al. 23 The swelling ratio (Q m ) was calculated by dividing swollen hydrogel mass by hydrogel mass, measured after lyophilization. Absorption and release of VEGF 5 ml PEG-CaM-PEG hydrogels were formed and swelled for 24 h in Ca 2+
2 (pH
6.3)). The hydrogels were then incubated for 24 h in a solution of Ca 2+ buffer with 444 ng/ml VEGF (Invitrogen, Carlsbad, CA, USA), with 9 ng/ml 125 I-labeled VEGF included as a tracer (Perkin Elmer, Fremont, CA, USA). VEGF-loaded hydrogels were then placed in scintillation tubes and their radioactivity was measured using a Packard Cobra II Gamma Counter (Meriden, CT, USA). The coefficient of determination R 2 value of VEGF release was calculated using Microsoft Excel 2007. Hydrogels were then placed back into 1 ml Ca 2+ buffer that either contained 10 mM TFP (+TFP) or did not contain TFP ( ÀTFP). The buffer was removed and refreshed after 1, 2, 4, 12, 24, and 48 h. The radioactivity of the removed buffer was analyzed using the gamma counter. In one set of experiments, hydrogels were maintained in TFP-free, Ca 2+ buffer for 24 h and then were switched to TFP-containing buffer. The buffer in these hydro- gels, as well as the hydrogels that had never been exposed to TFP, was refreshed after 1, 2, 4, and 12 h. Acknowledgements The authors are grateful to the support by the National Science Foundation for a Graduate Fellowship to WJK and for a CAREER award (0745563) to WLM. References 1 J. L. West and J. A. Hubbell, Reactive Polymers, 1995, 25, 139–147. 2 S. Young, M. Wong, Y. Tabata and A. G. Mikos, Journal of Controlled Release, 2005, 109, 256–274. 3 H. H. Tonnesen and J. Karlsen, Drug Development and Industrial Pharmacy, 2002, 28, 621–630. 4 K. S. Anseth, A. T. Metters, S. J. Bryant, P. J. Martens, J. H. Elisseeff and C. N. Bowman, Journal of Controlled Release, 2002, 78, 199–209. 5 G. M. Eichenbaum, P. F. Kiser, S. A. Simon and D. Needham, Macromolecules, 1998, 31, 5084–5093. 6 H. Kawaguchi, M. Kawahara, N. Yaguchi, F. Hoshino and Y. Ohtsuka, Polymer Journal, 1988, 20, 903–909. 7 N. A. Peppas, Y. Huang, M. Torres-Lugo, J. H. Ward and J. Zhang, Annu. Rev. Biomed. Eng., 2000, 2, 9–29. 8 G. M. Cruise, D. S. Scharp and J. A. Hubbell, Biomaterials, 1998, 19, 1287–1294. 9 F. Gu, B. Amsden and R. Neufeld, J. Control Release, 2004, 96, 463– 472.
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Microfluidic device for multimodal characterization of pancreatic islets Javeed Shaikh Mohammed, a Yong Wang, b Tricia A. Harvat, b Jose Oberholzer ab and David T. Eddington* ac Received 6th June 2008, Accepted 22nd September 2008 First published as an Advance Article on the web 21st October 2008 DOI: 10.1039/b809590f A microfluidic device to perfuse pancreatic islets while simultaneously characterizing their functionality through fluorescence imaging of the mitochondrial membrane potential and intracellular calcium ([Ca 2+
i ) in addition to enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin was developed and characterized. This multimodal characterization of islet function will facilitate rapid assessment of tissue quality immediately following isolation from donor pancreas and allow more informed transplantation decisions to be made which may improve transplantation outcomes. The microfluidic perfusion chamber allows flow rates of up to 1 mL min À1 , without any noticeable perturbation or shear of islets. This multimodal quantification was done on both mouse and human islets. The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay. Introduction Diabetes Mellitus (DM) is a group of metabolic disorders in which the body does not regulate blood glucose levels properly, resulting in elevated levels of blood glucose. There are two major types of DM: Type I and II. Type I DM is an autoimmune disease which involves the destruction of b-cells (insulin secreting cells in islets of Langerhans) leading to insulin deficiency. 1 In Type II DM, insulin (a key regulator of carbohydrate metabo- lism) is produced but there is insulin resistance in peripheral tissues (such as adipose and muscle), and at later stages of Type II DM, insulin secretion is damaged because of glucotoxicity and lipotoxicity. For Type I DM, maintaining blood glucose levels under tight control with exogenous insulin injection represents the most effective way to prevent the onset or to reduce progression of the chronic complications. Currently, intensive insulin therapy has shown to reduce most complications associ- ated with Type I DM patients, 2,3 however, it can never approx- imate pulsatile insulin secretory patterns 4–7
of the normal b-cell and comes with risks of major hypoglycemic episodes. Whole pancreas transplantation is the only therapeutic modality that can stop the progression of diabetic complications without increasing the incidence of hypoglycemic events. Unfortunately, the procedure has a high morbidity and significant mortality rate. 8
patients reaching insulin-independence after islet transplantation from multiple donors using steroid-free, sirolimus based immu- nosuppression. 51 With the new protocol, islet transplantation is progressively becoming a promising treatment for Type I DM with benefits of minimal surgery, less mortality and morbidity. 9–13
In brief, the donated pancreas from a cadaver is digested by collagenase and then further purified by continuous Biocoll gradients. After a short culture period, islets are trans- planted into the liver via the portal vein system. Currently, prior to transplantation, the islets are put through a rigorous quality control to assess their purity, morphology, sterility, pyrogenicity, viability, and potency (static glucose-stimulation studies). 9,14
However, the standard assays for evaluating islet quality prior to transplantation such as static glucose incubation and viability assay provide us with little information about b-cell function and does not address defects in b-cell morphology, metabolism, and signaling levels. Transplantation of isolated islets to diabetic nude mice models has been used as an in vivo model to assess the islet potency (ability of the transplanted islets to reverse DM). The time to reverse DM and the proportion of mice in which DM reverses provides potency of that batch of islets. This potency information along with glucose tolerance tests allows the effi- ciency of transplanted islets in the recipient to be predicted, but in a retrospect manner as the mouse model takes as long as 45 days. Therefore, it is essential to develop a standardized, accurate, real-time assessment of b-cell function based on b-cell physiology that can be used as a gold standard for assessing the functionality of islet preparations post isolation prior to trans- plantation. 14,15
In this paper we present the design, fabrication, and testing of a microfluidic device for multiplexing several well- accepted islet functional assays. Temporally resolved analysis of the kinetics of insulin secre- tion, mitochondria potential, and [Ca 2+ ] i will provide in depth understanding of the functionality of the islets. While all of these assays can currently be multiplexed in a 96-well plate, these experiments rely on static incubation of the islets. The static analysis of islets can not provide the details on temporal dynamics of insulin secretion of islets and therefore necessitates a Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA b Department of Surgery, University of Illinois at Chicago, Chicago, IL, USA c Department of Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, USA This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 97–106 | 97 PAPER www.rsc.org/loc | Lab on a Chip dynamic perfusion of islets to obtain comprehensive information on the islets. Glucose-dependent islet signaling begins with uptake of glucose that is rapidly phosphorylated by glucokinase and converted to pyruvate by the glycolytic pathway, and then oxidized within the mitochondria by the tricarboxylic acid cycle and oxidative phosphorylation. Glucose catabolism generates ATP that consequently closes ATP-sensitive K + (K ATP ) chan-
nels, initiating plasma membrane depolarization, and increases [Ca
2+ ] i through the voltage-dependent calcium channel. As a result, the glucose-stimulated rise in [Ca 2+ ]
and insulin secre- tion are tightly coupled. 16 Previous studies 17 in conjunction with our laboratory observations indicate that mitochondrial damage, insulin degranulation, and cell surface membrane defects play key roles in determining human b-cell function and viability. The glucose-stimulated insulin secretion exhibits a biphasic response, an initial burst of insulin followed by a sustained basal level of insulin release. The first phase is characterized by a transient increase in insulin release that occurs 1–3 minutes after glucose stimulation and is 3–5 minutes in duration and then the second phase where insulin secretion decreases to a suprabasal level and gradually increases as long as the stimulating concentration of glucose is present. Loss of glucose-stimulated biphasic insulin secretion of islets has been observed in some diabetic mice models. Static incubation only provides the information of bulk insulin secretion (secretory capacity) 9 during the glucose-exposed period and will not reveal any information on the biphasic response. As the static glucose assay is currently the gold stan- dard of islet characterization prior to transplantation, an incomplete assessment of islet functionality may result in poor transplantation outcomes. 18 In summary, current standard assays for evaluating human islet functionality prior to trans- plantation do not provide adequate information about human islet function and defects caused by organ procurement, cold ischemia, and islet isolation process. Therefore, a simple, quick, and predictable method to evaluate human islets prior to trans- plantation would prove immensely useful to address these shortcomings. Microfluidics offers a practical solution to this unmet clinical need to rapidly assess islet function. 19 Parallel microchannels enable multiple experiments to be performed simultaneously and imaging of cells within the microchannels, which is currently not possible with commercially available dynamic perfusion platforms. Additionally, microfluidic delivery of stimulatory solutions reduces the mechanical perturbation that will reduce stress to the islets. Microfluidic assessment of islet function has been previously demonstrated including continuous perfusion and online electrophoresis immunoassay to study single 20,21 and
multiple (four) 19 mouse islets, for automated sampling, 22 fluo-
rogenic labeling, 18 capillary electrophoresis analysis of islet proteins, 21,23
and for partially stimulating mouse islets allowing observation of the NAD(P)H and individual b-cell [Ca 2+ ]
responses. 7 However, none have aimed to provide a simple device to characterize the kinetics of insulin secretion through imaging and collecting the perfusate for off-chip analysis in a platform designed to plug into the already complex transplantation arena. While these devices have demonstrated new biological insight and offer unique advantages not possible with current tech- niques, they also share complex designs and require expert users to operate. To successfully deploy a device at multiple transplantation centers, the device must be simple, robust, user- friendly, and provide quick, reproducible results that are predictive of transplant outcome, 15 as a device failure during a characterization process may lead to discarding potentially lifesaving donor tissue. We have developed a new device to simultaneously quantify the mitochondrial potential, [Ca 2+ ] i and
biphasic response of islets in response to dynamic glucose stim- ulation to provide a complete assessment of islet function. 9,18 Multimodal islet characterization experiments can be rapidly preformed within the islet isolation arena to directly assess tissue function without sacrificing valuable tissue or time. The micro- fluidic device consists of three layers (one with tiny circular wells, another with a large circular well, and a layer with rectangular microchannel) of polydimethylsiloxane (PDMS), and contains six identical perfusion chambers for assay multiplexing. Fig. 1(a) contains the cross-sectional and isometric view of a single perfusion chamber. In each chamber, the bottom-most layer consists of an array of tiny circular wells that help to gently immobilize the islets without shielding them from the perfusion fluids to maximize the amount of islet surface area exposed to flow. The diameter and depth of the wells are 500 mm and 150 m m, respectively. Each well is separated from another well by 60 m m. It has been confirmed that the well design can successfully immobilize the islets with a size range from 50–500 mm at flow rate as large as 1 mL min À1 . Use of high flow rates prevents build up of high concentrations of insulin within the perfusion chamber before being washed out. 19 The next layer is a 7 mm diameter by 3 mm deep circular well that encompasses the array of the tiny wells. The top-most layer is a rectangular micro- channel that feeds into the larger well. The channel introduces the perfusate at one end of the large well and collects it from the opposite end of the well. The dimensions of the channel are 19 mm
 2 mm  500 mm. The height of the channels was chosen to be 500 mm in order to accommodate the expected largest size of human islets. The simple design of the perfusion chamber allows the use of the microfluidic device for multiple experiments after a simple cleaning procedure with alcohol and DI water; also, the soft lithography processes used to fabricate the device facilitate easy replication of devices. The device design leverages previously proven microfluidic techniques and is in itself not novel, however, multimodal characterization assays of islet tissue is a new and exciting application that will have a real and immediate impact in the transplantation clinic. Materials and method Fabrication of the microfluidic device Fig. 2 depicts the fabrication flow used for the microfluidic perfusion chamber. Standard SU8 lithography was used to create the masters for the array of small wells and the rectangular channels as previously described. 24 Briefly, silicon wafers (3 inch) were cleaned in acetone, methanol, isopropanol and dried in a stream of N 2 . The wafer was then exposed to oxygen plasma for 30 s at a power of 425 W (Terra Universal) to oxidize any remaining organics. Next, SU8 was spun on the cleaned silicon wafer for 30 s at 2000 rpm (SU8-100) or 1000 rpm (SU8-2150) to achieve thicknesses of 150 mm and 500 mm, respectively. The wafer was then exposed and developed to achieve a mold master. 98 | Lab Chip, 2009, 9, 97–106 This journal is ª The Royal Society of Chemistry 2009 Device characterization Fluorescence intensity of perfused solutions was measured over time across different regions in the perfusion chamber to assess the ability of the device to switch perfused solutions in the chamber. DI water was perfused for 30 s, followed by fluorescein isothiocyanate (2 mM FITC, Sigma, MO) for 60 s, and finally flushing out the fluorescein with DI water at a flow rate of 1 mL min
À1 . Images were collected with a high-speed, high-resolution charge-coupled device (CCD) (Retiga-SRV, Fast 1394, QImag- ing) and analyzed using image processing software (SlideBook 4.2, Olympus). Islet isolation and culture Mouse pancreatic islets were isolated and cultured as previously described. 25 In brief, the pancreata of 8–10 week-old C57/B6 or BALB/c (Jackson Laboratory) were perfused with 0.22 mg mL À1 Collagenase (Sigma, MO) and then digested for 11 min at 37 C. The digested pancreata were shaken vigorously for 15 s and further purified through discontinuous Ficoll gradient. The purified islets were cultured in 95% air and 5% CO 2 at 37
C with
RPMI-1640 medium supplemented with 10% fetal bovine serum. All procedures were approved by the Animal Care and Use Committee at the University of Illinois at Chicago. Human islets were isolated and purified using the standard Edmonton protocol at the University of Illinois at Chicago (UIC) following informed consent by donor relatives. The pan- creata were digested using a modified automated method as described previously 14,26 and further purified by continuous gradients on a cell separator (COBE 2991 Ô, COBE Laboratories Inc, Lakewood, CO) at 4
C and then cultured at 37 C. Experimental setup for perfusion and imaging Two multi-syringe infusion pumps (Harvard Apparatus) were used to supply different solutions at the inlet of each microfluidic perfusion chamber. One pump was used to supply basal glucose solution to the perfusion chambers and the other pump was used to supply either stimulatory glucose, potassium chloride (KCl), tolbutamide (TB), or alpha-ketoisocaproate (KIC) solutions. All the input solutions were pre-warmed to 37
C until used. A flow rate of 1 mL min À1 was set for both the pumps to achieve adequate Fig. 1 (a) Design of the microfluidic device and image of the actual device. The schematic depicts the cross-section view and experiment setup (left part) and the isometric view of a single-chamber of PDMS microfluidic device is shown in the insert image (right part). The device contains three layers. The bottom layer consists of an array of small circular (150 mm deep, 500 mm diameter) wells that help immobilize the islets exposed to flow. The next layer is a large circular well (3 mm deep, 7 mm diameter) that encompasses the array of the tiny wells and the top-most layer is a rectangular micro- channel (500 mm deep, 2 mm wide) that provides access to these wells. (b) Plots of fluorescence intensity versus time across different regions in the perfusion chamber during the perfusion (flow rate of 1 mL min À1 ) of DI water for 30 s followed by fluorescein isothiocyanate (2 mM FITC) for 60 s, and finally flushing out the fluorescein with DI water (intensity values are area averages of n ¼ 3 scans at a region of interest). This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 97–106 | 99
volume for later analysis. Bovine serum albumin is first injected through the device to coat the channel walls to prevent non- specific insulin adsorption. All experiments were performed at 37 C. Fig. 1(a) shows the experimental setup used for this work. Islet loading into the device The islets are introduced into the access ports in the microfluidic chamber using a micropipette. For mouse islet experiments 25 islets were introduced into each perfusion chamber and for human islet experiments 100 islets were introduced. The islets are introduced from one of the access ports of the perfusion chamber. Care was taken while introducing the islets into the perfusion chamber to prevent rupturing of islets. When the microfluidic device is tilted, the islets roll down along the microchannel and fall into the large circular well and eventually the islets (represented by the spheres in Fig. 1(a)) settle down into the smaller circular wells at the bottom of the large circular well. After the islets have been introduced into the perfusion chambers, the inlet and outlet tubing were integrated with the microfluidic device using connectors. To eliminate any effects of manual handling, the islets were again perfused with 2 mM basal glucose solutions for 10–15 minutes before starting any experiments. Dynamic perfusion of islets Dynamic glucose stimulation of islets was achieved by exposing islets to steps in glucose concentrations while collecting the perfusate for quantifying the amount of secreted insulin using a standard ELISA kit. The microfluidic perfusion chambers were loaded with islets and each perfusion chamber was perfused with 2 mM basal glucose solution for 10–15 min prior to the start of collecting data as described above. After this initial phase 2 mM basal glucose solution was perfused for an additional 5 min and stimulatory glucose solution (8 mM, 12 mM, 16.7 mM glucose solutions in separate channels) for 20 min, followed by 2 mM basal glucose solution for another 5 min. The perfusate from each chamber was collected in centrifuge tubes and ELISA assay was performed. As islets range from 50–500 mm in diameter, they easily settle in the 500 mm diameter wells. It was observed that with this device design, even at a flow rate of 1 mL min À1 there was no noticeable perturbation or shear of islets. The islets were stationary for long periods of time even during switching of fluids at the inlet port of the perfusion chamber facilitating time-lapse imaging of multiple islets.
27 Calcium imaging Dual-wavelength excitation microfluorometry was used to measure [Ca 2+ ]
as described previously. 25 Islets were loaded with 5 mM Fura-2/AM (Molecular Probes Inc., Eugene, OR) through a 25 min incubation at 37
C in Krebs–Ringer buffer (KRB) solution with 2 mM glucose (KRB2), then loaded into the microfluidic perfusion device and mounted on an inverted epifluorescence microscope (IX71, Olympus) equipped with Fig. 2
Fabrication process flow for the microfluidic perfusion device. Standard SU8 lithography was used to create the masters for the array of small wells and the rectangular channels. The microfluidic device contains three layers of PDMS. 100 | Lab Chip, 2009, 9, 97–106 This journal is ª The Royal Society of Chemistry 2009
a heating stage and perfused by a continuous flow (1 mL min À1 ) of KRB2 at 37
C (pH 7.4). KRB containing different glucose concentrations was administered to the islets after rinsing in KRB2 for 10–15 min. Multiple islets were simultaneously imaged with 10 –20 objectives. Fura-2 dual-wavelength excitation at 340 and 380 nm (shift in excitation wavelength occurs upon binding Ca 2+ ),
and detection of fluorescence emission at 510 nm was accomplished using an image acquisition and analysis soft- ware (SlideBook 4.2, Olympus); images were collected with a high-speed, high-resolution charge-coupled device (Retiga- SRV, Fast 1394, QImaging). The images were acquired near the equatorial plane of the islets. As the handpicked islets were of similar size, the equatorial plane of all the islets being imaged was approximately the same. All experiments were performed at 37
tation imaging, the islets were perfused with KRB2 for 5 min, 14 mM glucose solution for 15 min, KRB2 for another 10 min, 30 mM KCl solution for 10 min and finally with KRB2 for 5 min. The perfusate was collected for Insulin ELISA. Mitochondria potential imaging Rhodamine 123 (Rh123) was used as an indicator of mitochon- drial membrane potential. Rh123 is a lipophilic cation that partitions selectively into the negatively-charged mitochondrial membrane. Hyper-polarization of the mitochondrial membrane causes uptake of Rh123 into mitochondria and a decrease in fluorescence due to intermolecular crowding and quenching. 25 Islets were incubated in KRB2 supplemented with 10 mg mL À1 Rh123 for 20 min at 37
C, then loaded into the microfluidic perfusion device and mounted on an inverted epifluorescence microscope equipped with a heating stage and perfused by a continuous flow (rate 1 mL min À1 ) of KRB2 at 37 C (pH 7.4). KRB containing different glucose concentrations was adminis- tered to the islets after rinsing in KRB2 for 10–15 min. Rh123 fluorescence was excited at 540 nm and emission measured at 590 nm. Images were collected with a CCD as described above. All experiments were performed at 37
C. Once the software settings were set for Rhodamine 123 imaging, the islets were perfused with KRB2 for 5 min, next with 14 mM glucose solution for 15 min, followed by KRB2 for 5 min. The perfusate was collected for Insulin ELISA. Insulin ELISA method The perfusate samples were frozen at À80
with the ELISA kit. Insulin was measured using Mammalian or Mice Insulin ELISA (Mercodia AB, Uppsala, Sweden) kits. The protocol provided by the manufacturer was followed. Results and discussion Fig. 1(b) shows the fluorescence intensity of perfused solutions measured over time across different regions in the perfusion chamber during the perfusion of DI water and fluorescein solu- tions. The intensity values are area averages of three different scans. The intensity changes in the plots clearly illustrate the ability of the large circular well to completely exchange its stimulatory solution in approximately two minutes. The fluid flow within the perfusion chamber is an important factor in switching fluids with a step change and to provide uniform concentrations of fluids around the islets. 19 The loading of islets and the relative position of the islets being imaged within the perfusion chamber need to be standardized to obtain comparable results and can be improved in future iterations. After the characterization of the microfluidic device was done, it was applied to test different assays with mouse islets. The glucose-stimulated insulin secretion in islets is coupled to the metabolic state of the b-cells and involves both glycolytic and Krebs cycle metabolism. 29 The stimulatory glucose is phos- phorylated after it enters the cells and undergoes glycolysis leading to increased metabolic flux and closure of the plasma membrane-associated ATP-sensitive K + (K ATP ) channels. 29,30 This channel closure in turn depolarizes the membrane leading to the activation of the voltage-sensitive Ca 2+ channels and a concomitant rise in [Ca 2+ ] i , and increased insulin secre- tion. 6,7,28,29,31–42 Mouse islets were dynamically perfused with basal and stimulatory glucose solutions and the secreted insulin profiles are shown in Fig. 3(a). From the plots it is clear that the microfluidic perfusion device successfully characterizes the biphasic insulin release profile of mouse islets; an initial sharp rise followed by a moderate elevation. 16,18,22,28,31,43,44 The mouse islets show a clear dose-response illustrated with a marked increase in the insulin secretion with increasing stimulatory glucose concentrations, and is consistent with previous reports. 25 There is slight delay in the onset of the peak after the islets are exposed to the stimulatory glucose consistent with previous results.
18,44 After successfully applying the microfluidic device to perform dynamic glucose stimulation of mouse islets, the device was applied to perform simultaneous perfusion and imaging experi- ments. [Ca 2+ ] i imaging and insulin secretion analysis of mouse islets was performed as this correlates to the functional capacity of the islets. 45 We tracked the whole-islet [Ca 2+ ] i changes in multiple islets under basal and stimulatory glucose concentra- tions, and eventually under KCl stimulation. An absorption shift in excitation wavelength of Fura-2 loaded in the islets occurs upon binding of Ca 2+ . 28 High extracellular KCl will collapse and depolarize the cell membrane leading to opening of voltage- dependent calcium channels, calcium influx, and eventually insulin secretion. 46 Fig. 3(b) contains the plot of temporal insulin secretion of the C57/B6 mice islets superimposed on the plot of temporal changes in the ratio of fluorescence intensities (340/380 nm) of the islets imaged. Fig. 3(c) contains the plot of temporal insulin secretion of the BALB/c mice islets superimposed on the plot of temporal changes in the ratio of fluorescence intensities (340/380 nm) of the islets imaged. The secondary Ca 2+ oscilla-
tions can be vividly seen in the Fura-2 plot, indicating that the device can successfully capture finer details of islet functionality. It is evident from the plots that the 14 mM stimulatory glucose and 30 mM KCl stimulation correlates to increased insulin secretion and [Ca 2+ ] i as expected. 25,45 There is a time lag between the insulin release and Ca 2+ release and can be ascribed to the limitations of the current system that will be addressed in future iterations (random location of islets in the perfusion chamber, diffusion of insulin, and flow within the chamber). 16 It is noteworthy that the time lag could partly be attributed to the time required for the [Ca 2+ ]
to reach the threshold to trigger exocytosis. 6,28 Post-stimulation basal levels are very close to the This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 97–106 | 101
pre-stimulation basal levels (as expected from the device char- acterization results), indicating that the stimulatory perfusion solutions exchanged properly inside the chamber. In another set of simultaneous imaging and perfusion experiments, mitochondrial integrity and insulin secretion of mouse islets was evaluated as this correlates with functional capacity of the islets. 17 The mitochondrial membrane potential of b-cells is controlled by the ATP/ADP ratio through the K ATP channel. Under stimulatory glucose, the ratio increases causing the depolarization of the b-cells and leading to insulin secretion. Rh123 (a lipophilic cation) partitions selectively into the negatively-charged mitochondrial membrane and hyper- polarization of the mitochondrial membrane causes uptake of Rh123 into mitochondria and a decrease in fluorescence due to intermolecular crowding and quenching. 25 Fig. 3(d) contains the plot of temporal insulin secretion of the mouse islets superimposed on the plot of temporal changes in the fluores- cence intensity (corresponding to the mitochondrial membrane potential) of the mouse islets imaged. It is evident from the plots that the 14 mM stimulatory glucose correlates to increased insulin secretion and decreased mitochondrial membrane potential as expected. 25 Here again, the post-stimulation basal levels are very close to the pre-stimulation basal levels, indi- cating that the stimulatory perfusion solutions exchanged properly inside the chamber. After successful application of the microfluidic device to perform different assays on mouse islets, the device was applied to perform assays on human islets. Perfusion experiments were performed with 100 human islets as opposed to 25 mouse islets due to the relatively lower secreted insulin levels in human islets and higher variability of human islet function. Human islets were dynamically perfused with basal and stimulatory glucose solu- tions, and the representative secreted insulin profiles for a human pancreatic isolation batch is shown in Fig. 4(a). It is evident that the microfluidic perfusion device successfully characterizes the insulin release profile of human islets in response to step changes in glucose concentration; similar to the results observed with mouse islets. The human islets also show a clear dose-response illustrated with a marked increase in the insulin secretion with increasing stimulatory glucose concentrations, and is consistent with the results seen with mouse islets. Here again, a slight delay in the onset of the peak after the islets are exposed to the stim- ulatory glucose is seen. Islets from different human pancreatic isolation batches have highly variable responses as opposed to more repeatable Fig. 3
(a) Temporal insulin secretion profiles of mouse islets perfused with basal and different stimulatory glucose solutions, (b) temporal insulin secretion and [Ca 2+ ] i [indicated by Fura-2 ratio of the fluorescence intensities (340/380 nm)] profiles of C57/B6 mice islets perfused with basal and stimulatory glucose, KCl solutions, (c) temporal insulin secretion and [Ca 2+
i [indicated by Fura-2 ratio of the fluorescence intensities (340/ 380 nm)] profiles of BALB/c mice islets perfused with basal and stimu- latory glucose, KCl solutions, (d) temporal insulin secretion and mito- chondrial membrane potential profiles (indicated by fluorescence intensity of Rh123) of the mouse islets perfused with basal and different stimulatory glucose solutions. In all the experiments, 25 mouse islets were loaded per perfusion chamber. These are results from single representa- tive experiments with n ¼ 3 replicates at each point for Fura-2 ratios and Rh123 intensities. 102 | Lab Chip, 2009, 9, 97–106 This journal is ª The Royal Society of Chemistry 2009 responses to islets from genetically similar experimental mice. Therefore, the results for human islets (in response to step changes in glucose concentration) from different human pancreatic isolation batches have been summarized in Table 1. Area under the curve (AUC) for insulin levels under 2 mM basal glucose (KRB2) followed by stimulation with 8, 12, 16.7 mM stimulatory glucose, and again under KRB2 in human islets have been presented separately for different human pancreatic isolation batches. It is clear from the data in Table 1 that although there is a distinct difference between basal and stimu- latory insulin levels in all cases, there is a large batch-to-batch variability in responses to the different stimulatory glucose solutions. Also, the dose-response to increasing glucose concentrations is not the same in all the batches. Another factor leading to the variability in the response could be due to multiple sizes of cells or inherent variability in cells. 28 In another set of experiments, response of human islets to different commonly employed secretagogues (30 mM KCl, 150 mM TB, 10 mM KIC 34 in separate channels) was analyzed using the microfluidic device. Although these stimuli induce insulin secretion in islets, the mechanisms are different from the glucose induced insulin secretion. High extracellular KCl will collapse and depolarize cell membranes leading to the opening of voltage-dependent calcium channels, calcium influx, and even- tually insulin secretion. 46 TB is known as K + -ATP channel blocker 16,25,30,35 that causes the cells to depolarize and raise Ca 2+ levels, 6,36 thereby making the cells secrete insulin. 38 TB is a sul- phonylurea drug used to treat non-insulin-dependent DM patients. 6 KIC (mitochondrial fuel) 45 serves as a substrate for mitochondrial metabolism generating ATP and other signaling molecules independent of the glycolytic pathway. 5 Human islets were dynamically perfused with basal and secretagogue solutions and the representative secreted insulin profiles for a human pancreatic isolation batch is shown in Fig. 4(b). Our results clearly demonstrate that the microfluidic device can successfully characterize different islet physiological functions and produces repeatable and consistent data with mouse islets. 25,30,34,45 The
results for human islets (in response to step changes in different secretagogue solutions) from different human pancreatic isola- tion batches have been summarized in Table 2. AUC for insulin levels under KRB2 followed by stimulation with 30 mM KCl, 150 mM TB, 10 mM KIC, and again under KRB2 in human islets have been presented separately for different human pancreatic isolation batches. It is clear from the data in Table 2 that although there is a distinct difference between basal and stimu- latory insulin levels in all cases, there is a large batch-to-batch variability in responses to the different secretagogue solutions; similar to the results for human islet responses to step changes in glucose solutions (Table 1). The microfluidic device was next applied to perform simul- taneous perfusion and imaging experiments with human islets. Representative results for a human pancreatic isolation batch is shown in Fig. 4(c) that contains the plot of temporal insulin secretion of the human islets superimposed on the plot of temporal changes in the ratio of fluorescence intensities (340/ 380 nm) of the human islets imaged. It is evident from the plots that the 14 mM stimulatory glucose and 30 mM KCl stimulation correlates to increased insulin secretion and [Ca 2+ ] i as expected and consistent with mouse islets. The peaks are not as distinct and not as intense as observed in the case of mouse islets as it is known that the human islets have a wider range of mitochon- drial membrane potential and [Ca 2+ ]
response to glucose as compared to mouse islets. 6 There is a time lag between the insulin release and Ca 2+ release and can be ascribed to the limitations of the current system as previously mentioned. Post- stimulation basal levels are very close to the pre-stimulation Fig. 4 (a) Temporal insulin secretion profiles of human islets perfused with basal and different stimulatory glucose solutions, (b) temporal insulin secretion profiles of human islets perfused with basal and different secretagogue (KCl, TB, KIC) solutions, (c) temporal insulin secretion and [Ca
2+ ] i [indicated by Fura-2 ratio of the fluorescence intensities (340/ 380 nm)] profiles of human islets perfused with basal and stimulatory glucose, KCl solutions. In all the experiments, 100 human islets were loaded per perfusion chamber. These are results from single representa- tive experiments with n ¼ 3 replicates at each point for Fura-2 ratios. This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 97–106 | 103
basal levels as expected, indicating that the stimulatory perfu- sion solutions exchanged properly inside the chamber. The insulin secretion and [Ca 2+ ] i results for human islets from different human pancreatic isolation batches have been summarized in Table 3. AUC for FURA-2 ratio (representing [Ca 2+
i ) and insulin levels under KRB2, next by stimulation with 14 mM stimulatory glucose, under KRB2, followed by stimu- lation with 30 mM KCl, and finally in KRB2 in human islets have been presented separately for different human pancreatic isolation batches. It is evident from the data in Table 3 that although there is a distinct difference between basal and stim- ulatory insulin levels in all cases, there is a large batch-to-batch variability in responses; similar to the previous human islet results (Table 1 and Table 2). From the results it is evident that the microfluidic device will prove very useful in investigating islet physiology and drug action mechanisms on islets. 22,38
The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay. The authors understand that further experiments on multiple human islet tissue are required to statistically correlate these functional assays with transplantation outcomes; however the device pre- sented here provides a simple route to collect this data. All the biochemical reactions involved in the functional assays utilize oxygen,
47–50 and the oxygen changes can be measured as oxygen consumption rate 15 can be used to improve the predictive power of islet functionality in future iterations. Islet function is currently being assessed by transplantation of isolated human islets to diabetic nude mice models. The long term in vivo transplantation results will be used as a benchmark to correlate the in vitro functional assay results. Table 1
Area under the curve (AUC) for insulin levels under 2 mM basal glucose (KRB2) followed by stimulation with 8, 12, 16.7 mM stimulatory glucose, and again under KRB2 in human islets (100 islets) from different human pancreatic isolation batches. These are results from single repre- sentative experiments for each isolation batch Batch no. AUC-8 mM AUC-12 mM AUC-16.7 mM KRB2
8 mM KRB2
KRB2 12 mM
KRB2 KRB2
16.7 mM KRB2
1 0.79
104.10 15.52
38.43 197.20
17.95 1.45
165.20 21.19
2 0.21
188.40 17.07
0.71 201.40
4.71 0.24
193.90 12.65
3 0.13
293.40 1.61
11.42 480.40
35.88 26.70
802.10 24.41
4 0.00
105.00 4.11
1.87 94.17
4.88 0.39
171.60 3.10
5 2.48
101.20 8.83
1.41 360.00
8.07 5.33
420.80 38.14
6 0.17
105.90 7.80
4.70 196.20
14.32 0.32
160.10 23.50
Table 2 Area under the curve (AUC) for insulin levels under 2 mM basal glucose (KRB2) followed by stimulation with 30 mM KCl, 150 mM TB, 10 mM KIC, and again under KRB2 in human islets (100 islets) from different human pancreatic isolation batches. These are results from single representative experiments for each isolation batch Batch no. AUC-30 mM KCl AUC-150 mM TB AUC-10 mM KIC KRB2 KCl
KRB2 KRB2
TB KRB2
KRB2 KIC
KRB2 2 23.96 370.20 18.38
17.86 42.93
5.38 ND ND ND 3 12.33 312.60 13.47
29.03 168.50
9.57 ND ND ND 4 31.22 108.00 11.81
0.00 16.07
28.37 7.06
53.37 2.53
5 1.03
440.10 40.36
0.14 167.50
5.58 1.11
145.90 18.70
6 0.85
30.01 0.76
0.17 73.61
7.51 ND ND ND Table 3
Area under the curve (AUC) for FURA-2 ratio (representing [Ca 2+ ] i ) and insulin levels under 2 mM basal glucose (KRB2), next by stimulation with 14 mM stimulatory glucose, under KRB2, followed by stimulation with 30 mM KCl, and finally in KRB2 in human islets (100 islets). These are results from single representative experiments for each isolation batch with n ¼ 3 replicates at each point for Fura-2 ratios Batch no. AUC-FURA2 AUC-Insulin ELISA KRB2 14 mM
KRB2 KCl
KRB2 KRB2
14 mM KRB2
KCl KRB2
1 0.13
1.80 0.42
1.56 0.14
2.26 82.40
5.28 140.60
29.03 2 0.21 0.65 0.12
1.43 0.08
0.05 40.56
2.54 154.50
2.31 5 0.00 0.17 0.03
0.51 0.05
0.71 89.40
9.41 210.00
0.10 6 0.01 0.35 0.11
0.82 0.27
0.88 375.50
58.06 263.20
3.33 7 0.01 0.16 0.05
0.20 0.03
1.43 125.00
2.18 94.36
7.61 104 | Lab Chip, 2009, 9, 97–106 This journal is ª The Royal Society of Chemistry 2009 Conclusions In conclusion, we have developed a simple microfluidic device that can provide a comprehensive analysis of the functionality of islets prior to transplantation. We have successfully demon- strated that the microfluidic device can be used to perform multimodal islet functional assays. The different types of anal- yses that can be performed using this microfluidic device are not limited to the demonstrations presented in this paper. We intend to use this microfluidic device as a new gold standard to obtain an empirical relation between the different results obtained using multimodal functional assays to predict the islet transplantation efficiency in a time frame acceptable for the clinic. Acknowledgements Support for JO was provided through the National Institutes of Health Center Grant, ‘‘Chicago Islet Consortium ICR at UIC’’ and by ‘‘The Chicago Project’’. Additional support to DTE was provided by DARPA through the US Army Research Office Award (Agreement # W911NF-07-1-0360) and by the Alfred P. Sloan foundation. References 1 Y. Okubo, A. Shimada, Y. Kanazawa, T. Shigihara, Y. Oikawa, T. Imai, J. Miyazaki and H. Itoh, Hyperplastic islets observed in ‘‘reversed’’ NOD mice treated without hematopoietic cells, Diabetes Res. Clin. Pract., 2008, 79(1), 18–23. 2 Diabetes Control and Complications Trial/Epidemiology of, D., Interventions and Complications Research, G., Retinopathy and nephropathy in patients with type 1 diabetes four years after a trial of intensive therapy, New Engl. J. Med., 2000, 342(6), 381–389. 3 B. Keymeulen, E. Vandemeulebroucke, A. G. Ziegler, C. Mathieu, L. Kaufman, G. Hale, F. Gorus, M. Goldman, M. Walter, S. Candon,
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