Infection courses, virological features and IFN-α responses of HBV genotypes in cell culture and animal models.

BACKGROUND & AIMS: HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment. METHODS: Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model. RESULTS: HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. CONCLUSIONS: Herein, we generated stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models. LAY SUMMARY: Stable cell lines producing high-titer cell culture-generated hepatitis B virus (HBV) of various genotypes were established. HBV genotypes showed stable infectivity in both in vitro and in vivo models, which are valuable tools for antiviral development.

Cell culture techniques in gastrointestinal research: Methods, possibilities and challenges.

Cell culture is one of the most valuable tools which is being applied in both fundamental and applied gastrointestinal research. The cells are isolated from their natural location (in vivo) and further propagated in vitro or artificial environment and studied. Over the years, several methods have been devised to isolate animal cells derived from the gut and culture them in vitro to study the functions and biology in the context of complex gastrointestinal diseases. This mini-review briefly describes the types and methods of cell culture covering the simplest monoculture models to more recent 3D organoid models, highlighting its importance in personalized precession medicine and other aspects of translational research. It also throws light upon the major challenges and outlines the future directions for using cell culture as a model system.

Culture and Real-time Polymerase Chain reaction sensitivity in the diagnosis of invasive meningococcal disease: Does culture miss less severe cases?

BACKGROUND: Invasive meningococcal disease (IMD) is a highly lethal disease. Diagnosis is commonly performed by culture or Realtime-PCR (qPCR). AIMS: Our aim was to evaluate, retrospectively, whether culture positivity correlates with higher bacterial load and fatal outcome. Our secondary aim was to compare culture and qPCR sensitivity. METHODS: The National Register for Molecular Surveillance was used as data source. Cycle threshold (CT), known to be inversely correlated with bacterial load, was used to compare bacterial load in different samples. RESULTS: Three-hundred-thirteen patients were found positive for Neisseria meningitidis by qPCR, or culture, or both; 41 died (case fatality rate 13.1%); 128/143 (89.5%) blood samples and 138/144 (95.8%) CSF were positive by qPCR, 37/143 (25.9%) blood samples and 45/144 (31.2%) CSF were also positive in culture. qPCR was 3.5 times (blood) or 3.1 times (CSF) more sensitive than culture in achieving a laboratory diagnosis of IMD (OR 24.4; 95% CI 12.2-49.8; p < .10-4; Cohen's κ 0.08 for blood and OR 49.0; 95% CI 19.1-133.4; p<10-4; Cohen's κ 0.02; for CSF). Positivity of culture did not correlate with higher bacterial loads in blood (mean CT 27.7±5.71, and CT 28.1±6.03, p = 0.739 respectively in culture positive or negative samples) or in CSF (mean CT 23.1±4.9 and 24.7±5.4 respectively in positive or negative CSF samples, p = 0.11).CT values in blood from patients who died were significantly lower than in patients who survived (respectively mean 18.0, range 14-23 and mean 29.6, range 16-39; p<10-17). No deaths occurred in patients with CT in blood over 23. Positive blood cultures were found in 10/25 (40%) patients who died and in 32/163 (19.6%) patients who survived, p = 0.036, OR 2.73; 95% CL 1.025-7.215), however 60% of deaths would have remained undiagnosed with the use of culture only. CONCLUSIONS: In conclusion our study demonstrated that qPCR is significantly (at least 3 times) more sensitive than culture in the laboratory confirmation of IMD. The study also demonstrated that culture negativity is not associated with lower bacterial loads and with less severe cases. On the other side, in patients with sepsis, qPCR can predict fatal outcome since higher bacterial load, evaluated by qPCR, appears strictly associated with most severe cases and fatal outcome. The study also showed that molecular techniques such as qPCR can provide a valuable addition to the proportion of diagnosed and serotyped cases of IMD.

Software tools for cell culture-related 3D printed structures.

Three-dimensional (3D) printing technology allowed fast and cheap prototype fabrication in numerous segments of industry and it also became an increasingly versatile experimental platform in life sciences. Yet, general purpose software tools to control printer hardware are often suboptimal for bioprinting applications. Here we report a package of open source software tools that we developed specifically to meet bioprinting requirements: Machine movements can be (i) precisely specified using high level programming languages, and (ii) easily distributed across a batch of tissue culture dishes. To demonstrate the utility of the reported technique, we present custom fabricated, biocompatible 3D-printed plastic structures that can control cell spreading area or medium volume, and exhibit excellent optical properties even at 50 ul sample volumes. We expect our software tools to be helpful not only to manufacture customized in vitro experimental chambers, but for applications involving printing cells and extracellular matrices as well.

Use of real-time cellular analysis and Plackett-Burman design to develop the serum-free media for PC-3 prostate cancer cells.

In this study, we developed a rapid strategy to screen a serum-free medium for culturing the anchorage-dependent PC-3 prostate cancer cells, which was going to be prepared in large scale to generate GM-CSF/TNFα-surface-modified whole cell prostate cancer vaccine. Automated real-time cellular analysis as a rapid and non-invasive technology was used to monitor the growth of PC-3 cells in 16-well plates. At the same time, Plackett-Burman design was employed to identify the most influential formulation by integrating relevant information statistically. The effects of the 16 selected factors were evaluated during exponential cell growth and three medium constituents (EGF, FGF and linoleic acid) were identified to have significant effects on the cell growth. Subsequently, the response surface methodology with central composite design was applied to determine the interactions among the three factors so that these factors were optimized to improve cell growth. Finally, the prediction of the best combination was made under the maximal response to optimize cell growth by Design-Expert software 7.0. A total of 20 experiments were conducted to construct a quadratic model and a second-order polynomial equation. With the optimized combination validated by the stability test of serial passaging PC-3 cells, the serum-free medium had similar cell density and cell viability to the original serum medium. In summary, this high-throughput scheme minimized the screening time and may thus provide a new platform to efficiently develop the serum-free media for adherent cells.

Drug Discovery Approaches Utilizing Three-Dimensional Cell Culture.

Historically, two-dimensional (2D) cell culture has been the preferred method of producing disease models in vitro. Recently, there has been a move away from 2D culture in favor of generating three-dimensional (3D) multicellular structures, which are thought to be more representative of the in vivo environment. This transition has brought with it an influx of technologies capable of producing these structures in various ways. However, it is becoming evident that many of these technologies do not perform well in automated in vitro drug discovery units. We believe that this is a result of their incompatibility with high-throughput screening (HTS). In this study, we review a number of technologies, which are currently available for producing in vitro 3D disease models. We assess their amenability with high-content screening and HTS and highlight our own work in attempting to address many of the practical problems that are hampering the successful deployment of 3D cell systems in mainstream research.

Burden of tuberculosis in intensive care units in Cape Town, South Africa, and assessment of the accuracy and effect on patient outcomes of the Xpert MTB/RIF test on tracheal aspirate samples for diagnosis of pulmonary tuberculosis: a prospective burden of disease study with a nested randomised controlled trial.

BACKGROUND: There are few prospective data about the incidence and mortality associated with pulmonary tuberculosis in intensive care units (ICUs), and none on the accuracy and clinical effect of the Xpert-MTB/RIF assay in this setting. We aimed to measure the frequency of culture-positive tuberculosis in ICUs in Cape Town, South Africa and to assess the performance and effect on patient outcomes of Xpert MTB/RIF versus smear microscopy for diagnosis of tuberculosis. METHODS: We did a prospective burden of disease study with a randomised controlled substudy at the ICUs of four hospitals in Cape Town. Mechanically ventilated adults (≥18 years) with suspected pulmonary tuberculosis admitted between Aug 1, 2010, and July 31, 2013 (irrespective of the reason for admission), were prospectively investigated by culture, and by Xpert-MTB/RIF testing or smear microscopy, of tracheal aspirate samples. In the substudy, patients were randomly assigned (1:1), via a computer-generated allocation list, to smear microscopy or Xpert MTB/RIF. Participants, caregivers, and outcome assessors were not masked to group assignment. Only the laboratory staff were blinded to the clinical details of the participants. In November, 2012, Xpert MTB/RIF was adopted as the initial diagnostic test for respiratory samples in Western Cape province. Thereafter, patients received Xpert MTB/MIF and culture as standard of care. For the whole study cohort, the primary outcome was the frequency of bacteriologically confirmed tuberculosis. The primary endpoint of the randomised substudy was the proportion of culture-positive patients on treatment at 48 h after enrolment. The randomised substudy is registered with ClinicalTrials.gov, number NCT01530568. FINDINGS: We investigated 341 patients for suspected pulmonary tuberculosis out of a total of 2309 ICU admissions. 46 (15%) of 317 patients included in the final analysis had a positive test for tuberculosis (Xpert MTB/RIF or culture). Culture-positive patients who failed to initiate treatment (adjusted HR 4·49, 95% CI 1·45-13·89) or who received inotropes (4·33, 1·49-12·60) were more likely to die. However, tuberculosis status was not associated with 28-day or 90-day mortality. In the substudy, we randomly assigned 115 patients to smear microscopy and 111 to Xpert MTB/RIF. Smear microscopy detected six (43%) of 14 culture-positive patients, and Xpert MTB/RIF detected 11 (100%) of 11 culture-positive patients (p=0·002). The proportion of culture-positive patients on treatment at 48 h was higher in the Xpert MTB/RIF group than in the smear microscopy group (11 [92%] of 12 vs nine [53%] of 17; p=0·043), although use of Xpert MTB/RIF had no effect on mortality or other patient outcomes. INTERPRETATION: Tuberculosis is fairly common in ICUs in high-burden settings, and clinicians should screen and test patients for tuberculosis with Xpert MTB/RIF where available. This test improves diagnostic yield and rates of treatment initiation, and reduces unnecessary treatment, but might not increase the total number of patients on treatment when empirical treatment is widely used. A suspected diagnosis of pulmonary tuberculosis should not exclude patients from ICU care in resource-limited settings because mortality is unaffected by the presence of this disease. FUNDING: European and Developing Countries Clinical Trials Partnership, South African Medical Research Council, and the Discovery Foundation.

Determinants of culture success in an airway epithelium sampling program of young children with cystic fibrosis.

AIM OF THE STUDY: The bronchial brushing technique presents an opportunity to establish a gold standard in vitro model of Cystic Fibrosis (CF) airway disease. However, unique obstacles exist when establishing CF airway epithelial cells (pAECCF). We aimed to identify determinants of culture success through retrospective analysis of a program of routinely brushing children with CF. MATERIALS AND METHODS: Anaesthetised children (CF and non-CF) had airway samples taken which were immediately processed for cell culture. Airway data for the CF cohort was obtained from clinical records and the AREST CF database. RESULTS: Of 260 brushings processed for culture, 114 (43.8%) pAECCF successfully cultured to passage one (P1) and 63 (24.2% of total) progressed to passage two (P2). However, >80% of non-CF specimens (pAECnon-CF) cultured to P2 from similar cell numbers. Within the CF cohort, specimens successfully cultured to P2 had a higher initial cell count and lower proportion of severe CF mutation phenotype than those that did not proliferate beyond initial seeding. Elevated airway IL-8 concentration was also negatively associated with culture establishment. Contamination by opportunistic pathogens was observed in 81 (31.2% of total) cultures and brushings from children with lower respiratory tract infections were more likely to co-culture contaminating flora. CONCLUSIONS: Lower passage rates of pAECCF cultures uniquely contrasts with pAECnon-CF despite similar cell numbers. An equivalent establishment rate of CF nasal epithelium reported elsewhere, significant associations to CFTR mutation phenotype, elevated airway IL-8 and opportunistic pathogens all suggest this is likely related to the CF disease milieu.

Mammalian cell cultures for biologics manufacturing.

Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations.

Feeder-free and serum-free production of hepatocytes, cholangiocytes, and their proliferating progenitors from human pluripotent stem cells: application to liver-specific functional and cytotoxic assays.

We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.

[Biological effects and potential applications of mesenchymal stem cell culture under low oxygen pressure]. / Effet et applications potentielles de la culture des cellules souches mésenchymateuses de moelle osseuse en condition d'hypoxie.

As for hemopoietic stem cells, it is thought although not formally demonstrated that bone marrow mesenchymal stem cells (BMMSC) reside in a specific microenvironment or niche characterized by a low O(2) tension. In support of this hypothesis is the observation that MSC can be amplified in vitro under 1-8% O(2) while retaining multipotent capacities. Culture in hypoxic condition may therefore be useful in therapy as the low number of MSC is a major limitation to their use in regenerative medicine and to a lesser extent in the treatment of some autoimmune and overt inflammatory diseases. However hypoxia may modify MSC with significant effects on their metabolism and gene expression hence modifications in their differentiation abilities to mature in specialized cells. This review discusses the various effects of hypoxia on the fate and behavior of MSC and potential clinical applications of culture under hypoxic conditions in regenerative medicine and immune/inflammatory disorders.

ImagCell: a computer tool for cell culture image processing applications in bioimpedance measurements.

This paper presents a computer tool for automatic analysis of cell culture images. The program allows the extraction of relevant information from biological images for pre- and postsystem analysis. In particular, this tool is being used for electrical characterization of electrode-solution-cell systems in which bioimpedance is the main parameter to be known. The correct modeling of this kind of systems enables both electronic system characterization for circuit design specifications and data decoding from measurements. The developed program allows cell culture image processing for geographic information extraction and generates cell count and equivalent circuit descriptions useful for system simulations.

Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach.

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.

Opportunities to replace the use of animals in sepsis research. The report and recommendations of a Focus on Alternatives workshop.

Sepsis and multiple organ failure are common causes of death in patients admitted to intensive care units. The incidence of sepsis and associated mortalities has been steadily increasing over the past 20 years. Sepsis is a complex inflammatory condition, the precise causes of which are still poorly understood. Animal models of sepsis have the potential to cause substantial suffering, and many of them have been poorly representative of the human syndrome. However, a number of non-animal approaches, including in vitro, in silico and clinical studies, show promise for addressing this situation. This report is based on discussions held at an expert workshop convened by Focus on Alternatives and held in 2004 at the Wellcome Trust, London. It provides an overview of some non-animal approaches to sepsis research, including their strengths and weaknesses, and argues that they should be prioritised for further development.

From Poisson distribution to interleukins.

Abstract In the mid-1970s, Ivan Lefkovits and co-workers introduced limiting dilution analysis of T and B cells as a method to quantify antigen-specific T and B cells. In this study, I describe how my co-operation with Ivan on the subject led not only to heated discussions on the validity of the approach but also to the identification of interleukin-2 as T-cell-derived factor, capable of replacing T-cell help required for the activation of B cells.

Stochastic model of T cell proliferation: a calculus revealing IL-2 regulation of precursor frequencies, cell cycle time, and survival.

The outcome of Ag exposure is dictated by complex regulation of T cell proliferation. The rates of proliferation and survival are altered by numerous signals that the cell receives and integrates to achieve a net response. We have illustrated previously how small changes in kinetic parameters can lead to large differences, even under conditions of saturating IL-2. In this study, we examine the effect of varying IL-2 concentration on T cell response and develop a model incorporating additional parameters of proliferation and survival. Strikingly, the proportion of cells that enter the first division, but not the time at which they enter, is dramatically altered by IL-2. Furthermore, the survival and average division time of cells in later divisions are also altered by IL-2 concentration. Together, the small simultaneous effects on these parameters result in large differences in total cell number. These results reveal how in vitro systems may exaggerate the contribution of IL-2, and thus how costimuli or additional helper cells that alter IL-2 concentration, even by relatively small amounts, will generate large in vitro differences in cell number and therefore appear obligatory. Furthermore, they illustrate how a quantitative model of T cell activation can clarify how complex signal integration is handled by T cells in situ, and therefore more appropriately aid development of a theory of behavior.

Integration of cell culture and microfabrication technology.

Recent progress in cell culture and microfabrication technologies has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. The cell-based biosensors are composed of two transducers, where the primary transducer is cellular and the secondary transducer is typically electrical. Advances in gene manipulation and cell culture techniques have contributed to the development of the cell as a transducer, while microfabrication techniques have been applied to the development of integrating the cell with the second transducer. Cellular patterning using microfabrication techniques is essential for cell-based biosensors, cell culture analogues, tissue engineering, and fundamental studies of cell biology. The photolithographic technique is highly developed and has been widely used for patterning cells. Recently, a set of alternative techniques, largely based on soft lithoghraphy, has been developed for biological applications. Those techniques include microcontact printing, microfluidic patterning using microchannels, and laminar flow patterning. A classical metallic stencil patterning method has been improved by employing a rubber-like stencil. These cellular micropatterning techniques have been usefully employed to understand questions in fundamental cell biology, especially cellular interactions with various materials and other cells. Using these micropatterning tecchniques and insights into the interaction of cellular biology with surfaces, a wide array of biosensors have been developed. In this manuscript examples of cell-based biosensors are described. Neurons have a great potential for use in a cell-based biosensor because they are electrically excitable cells, from which electrical signals are generated with the binding of detecting molecules. Consequently, the electrical signals generated in the cell can be determined in a noninvasive manner. A microphysiometer is a device to detect functional responses from cells by measuring the change of extracellular pH. The main application of the microphysiometer is the analysis of functional responses of cells upon receptor stimulation. Development of a microscale cell culture analogue system, an in vitro animal or human surrogate, is another promising area using cell culture and microfabrication technologies. Such devices are potentially very useful in the fields of toxicology and drug testing because they may increase the accuracy of in vitro predictions, simplify testing procedures, and reduce the cost of such tests, allowing many more tests to be done with a limited set of resources.

Broth recycling to reduce process noise resulting from concentrated substrate addition in fed-batch cultivation of Escherichia coli.

In this work feed hardware for fed-batch cultivation is presented (broth recycle feed injection system or BRFIS). BRFIS proved superior to conventional submerged or dripped feed systems in reducing dissolved oxygen (DO) oscillations during Escherichia coli fed-batch cultivation (5 min coefficient of variation of 0.7% for BRFIS as compared to 26% or greater for conventional feeding hardware in a 2 L test reactor). Hence, BRFIS is useful for fed-batch cultivation systems where the DO signal is used in measurement or control.

Erythropoietin fosters both intrinsic and extrinsic neuronal protection through modulation of microglia, Akt1, Bad, and caspase-mediated pathways.

1. Erythropoietin (EPO) plays a significant role in the hematopoietic system, but the function of EPO as a neuroprotectant and anti-inflammatory mediator requires further definition. We therefore examined the cellular mechanisms that mediate protection by EPO during free radical injury in primary neurons and cerebral microglia. 2. Neuronal injury was evaluated by trypan blue, DNA fragmentation, phosphatidylserine (PS) exposure, Akt1 phosphorylation, Bad phosphorylation, mitochondrial membrane potential, and cysteine protease activity. Microglial activation was assessed through proliferating cell nuclear antigen and PS receptor expression. 3. EPO provides intrinsic neuronal protection that is both necessary and sufficient to prevent acute genomic DNA destruction and subsequent membrane PS exposure, since protection by EPO is completely abolished by cotreatment with an anti-EPO neutralizing antibody. 4. Extrinsic protection by EPO is offered through the inhibition of cerebral microglial activation and the suppression of microglial PS receptor expression for the prevention of neuronal phagocytosis. In regards to microglial chemotaxis, EPO modulates neuronal poptotic membrane PS exposure necessary for microglial activation primarily through the regulation of caspase 1. 5. EPO increases Akt1 activity, phosphorylates Bad, and maintains neuronal nuclear DNA integrity through the downstream modulation of mitochrondrial membrane potential, cytochrome c release, and caspase 1, 3, and 8-like activities. 6. Elucidating the intrinsic and extrinsic protective pathways of EPO that mediate both neuronal integrity and inflammatory microglial activation may enhance the development of future therapies directed against acute neuronal injury.

Improved methods for the isolation and purification of porcine islets.

Recent progress in human islet transplantation demonstrates the feasibility of using purified human islets for treatment of type 1 diabetes mellitus; however, a shortage of human pancreata remains a major obstacle. This report describes methods to isolate porcine islets using a modification of the automated chamber method. The pancreata from 2-year-old sows were trimmed and injected intraductally with Sevac, Sigma, or Liberase PI collagenase. The pancreata was placed in the chamber, shaken, and recirculated at 70 ml/min until an adequate number of islets were liberated. The digest was centrifuged and the pellets pooled with University of Wisconsin Solution + 10% horse serum and incubated at 4 degrees C for 1 h. The islets were purified using a continuous gradient of Hypaque Euroficoll on a refrigerated COBE 2991. The islets were collected in fractions, assessed for purity, sized, and then suspended in Medium 199. Collagenase preparations obtained from Sevac (2919 islet equivalents [IE]/g), Sigma (2543 IE/g), and Liberase PI (2901 IE/g) gave similar results with 94%-95% purity. In summary, we report a successful method for efficient isolation and purification of porcine islets, yielding nearly 3000 IE/gm, with different collagenase products.