Perfusion Mode Research Articles

Process Intensification Using a One‐Step Inoculum Production and High‐Seeded Fed‐Batch Processes

AbstractVarious approaches to process intensification are currently being investigated to ensure time and cost savings when producing biopharmaceuticals. In the present study, a one‐step inoculum production was established based on an ultra‐high cell density working cell bank with immunoglobulin G producing Chinese hamster ovary cells. Cryovials were used for direct inoculation of 1‐L wave‐mixed perfusion bioreactors. When reaching around 180 · 106 cells mL−1 in N−1 perfusion mode, low‐seed and high‐seed fed‐batch experiments in shake flasks were inoculated, as was the case for 250‐mL stirred single‐use bioreactors. Additionally, proof‐of‐concept runs at 50‐L and 200‐L scale were successfully performed. The intensification approach presented allows manufacturing capacity to be increased by up to 50 %.

Effect of dental tissue thickness on the measurement of oxygen saturation by two different pulse oximeters.

This study aimed to evaluate the influence of different dental tissue thickness on the measurement of oxygen saturation (SpO2) levels in high (HP) and low (LP) blood perfusion by comparing the values obtained from two different pulse oximeters (POs) - BCI and Sense 10. Thirty freshly extracted human teeth had their crowns interposed between the POs and an optical simulator, which emulated the SpO2 and heart beats per minute (bpm) at HP (100% SpO2/75 bpm) and LP (86% SpO2/75 bpm) modes. Afterwards, the palatine/lingual surfaces of the dental crowns were worn with diamond drills. The reading of SpO2 was performed again using the POs alternately through the buccal surface of each dental crown. Data were analyzed by the Wilcoxon, Mann-Whitney and Kendall Tau-b tests (α=5%). The results showed significant difference at the HP and LP modes in the SpO2 readouts through the different dental thicknesses with the use of BCI, and at the LP mode with the use of Sense 10, which had a significant linear correlation (p<0.0001) and lower SpO2 readout values in relation to the increase of the dental thickness. Irrespective of tooth thickness, Sense 10 had significantly higher readout values (p<0.0001) than BCI at both perfusion modes. The interposition of different thicknesses of enamel and dentin influenced the POs measurement of SpO2, specially at the low perfusion mode. The POs were more accurate in SpO2 measurement when simulated perfusion levels were higher.

Laser speckle contrast imaging compared with indocyanine green in renal perfusion of a porcine model.

When viewed under near-infrared light, indocyanine green (ICG) signal for kidney perfusion can be utilized in partial nephrectomy. Laser speckle contrast imaging (LSCI) uses coherent light to detect perfusion during real-time laparoscopic surgery. Laser speckle contrast imaging or ActivSight, an imaging sensor adapter, was used during laparoscopy of an anesthetized porcine kidney model. ActivSight's "perfusion mode" and "quantification mode" displayed the blood flow as a heatmap and numerical signal intensity, respectively. After the upper segmental renal artery was clamped, ICG was seen in the lower pole, and LSCI showed low unit (dark color) quantification and perfusion in the upper pole. Indocyanine green was retained in the lower pole after the upper segmental artery was unclamped, and LSCI perfusion was demonstrated in the entire kidney. Laser speckle contrast imaging is a dye-free, repeatable, real-time adjunct for renal parenchymal perfusion assessment applicable to minimally invasive renal surgery to complement the technology of ICG near-infrared fluorescence and advance digital surgery.

Production of Lentiviral Vectors Using a HEK-293 Producer Cell Line and Advanced Perfusion Processing.

The field of lentiviral vector (LV) production continues to face challenges in large-scale manufacturing, specifically regarding producing enough vectors to meet the demand for treating patients as well as producing high and consistent quality of vectors for efficient dosing. Two areas of interest are the use of stable producer cell lines, which facilitates the scalability of LV production processes as well as making the process more reproducible and robust for clinical applications, and the search of a cell retention device scalable to industrial-size bioreactors. This manuscript investigates a stable producer cell line for producing LVs with GFP as the transgene at shake flask scale and demonstrates LV production at 3L bioreactor scale using the Tangential Flow Depth Filtration (TFDF) as a cell retention device in perfusion mode. Cumulative functional yields of 3.3 x 1011 and 3.9 x 1011 transducing units were achieved; the former over 6 days of LV production with 16.3 L of perfused media and the latter over 4 days with 16 L. In comparing to a previously published value that was achieved using the same stable producer cell line and the acoustic filter as the perfusion device at the same bioreactor scale, the TFDF perfusion run produced 1.5-fold higher cumulative functional yield. Given its scale-up potential, the TFDF is an excellent candidate to be further evaluated to determine optimized conditions that can ultimately support continuous manufacturing of LVs at large scale.

Cerebral Perfusion Patterns of Anxiety State in Patients With Pulmonary Nodules: A Study of Cerebral Blood Flow Based on Arterial Spin Labeling.

Background and PurposeThe proportion of patients with somatic diseases associated with anxiety is increasing each year, and pulmonary nodules have become a non-negligible cause of anxiety, the mechanism of which is unclear. The study focus on the cerebral blood flow (CBF) of anxiety in patients with pulmonary nodules to explore the cerebral perfusion pattern of anxiety associated with pulmonary nodules, blood perfusion status and mode of pulmonary nodule induced anxiety state.Materials and MethodsPatients with unconfirmed pulmonary nodules were evaluated by Hamilton Anxiety Scale (HAMA). The total score > 14 was defined as anxiety group, and the total score ≤ 14 points was defined as non-anxiety group. A total of 38 patients were enrolled, of which 19 patients were the anxiety group and 19 were the non-anxiety group. All subjects underwent arterial spin labeling imaging using a 3.0 T MRI. A two-sample t-test was performed to compare the CBF between the two groups. The CBF was extracted in brain regions with difference, and Spearman correlation was used to analyze the correlation between CBF and HAMA scores; ROC was used to analyze the performance of CBF to distinguish between the anxiety group and the non-anxiety group.ResultsThe CBF in the right insula/Heschl’s cortex of the anxiety group decreased (cluster = 109, peak t = 4.124, and P < 0.001), and the CBF in the right postcentral gyrus increased (cluster = 53, peak t = −3.912, and P < 0.001) in the anxiety group. But there was no correlation between CBF and HAMA score. The ROC analysis of the CBF of the right insula/Heschl’s cortex showed that the AUC was 0.856 (95%CI, 0.729, 0.983; P < 0.001), the optimal cutoff value of the CBF was 50.899, with the sensitivity of 0.895, and specificity of 0.789. The ROC analysis of CBF in the right postcentral gyrus showed that the AUC was 0.845 (95%CI, 0.718, 0.972; P < 0.001), the optimal cutoff value of CBF was 43.595, with the sensitivity of 0.737, and specificity of 0.842.ConclusionThe CBF of the right insula/Heschl’s cortex decreased and the CBF of the right postcentral gyrus increased in patients with pulmonary nodules under anxiety state, and the CBF of the aforementioned brain regions can accurately distinguish the anxiety group from the non-anxiety group.

Seed Train Intensification Using an Ultra-High Cell Density Cell Banking Process

A current focus of biopharmaceutical research and production is seed train process intensification. This allows for intermediate cultivation steps to be avoided or even for the direct inoculation of a production bioreactor with cells from cryovials or cryobags. Based on preliminary investigations regarding the suitability of high cell densities for cryopreservation and the suitability of cells from perfusion cultivations as inoculum for further cultivations, an ultra-high cell density working cell bank (UHCD-WCB) was established for an immunoglobulin G (IgG)-producing Chinese hamster ovary (CHO) cell line. The cells were previously expanded in a wave-mixed bioreactor with internal filter-based perfusion and a 1 L working volume. This procedure allows for cryovial freezing at 260 × 106 cells mL−1 for the first time. The cryovials are suitable for the direct inoculation of N−1 bioreactors in the perfusion mode. These in turn can be used to inoculate subsequent IgG productions in the fed-batch mode (low-seed fed-batch or high-seed fed-batch) or the continuous mode. A comparison with the standard approach shows that cell growth and antibody production are comparable, but time savings of greater than 35% are possible for inoculum production.

Productivity and quality improvement for a symmetric bispecific antibody through the application of intensified perfusion cell culture.

BackgroundAggregation, fragmentation, and low yield are issues frequently found during the cell culture process of bispecific antibodies (bsAbs), whose inherent complexity likely plays a role in causing these issues.MethodsIn this study, we made a head-to-head comparison between fed-batch cell culture and intensified perfusion cell culture with a symmetric bsAb case.ResultsIn comparison with the fed-batch culture, a 6.6-fold improvement in integrated viable cell density and a 10.9-fold improvement in volumetric productivity were achieved with the intensified perfusion mode. In addition, a significant decrease in aggregation and fragmentation was observed with the intensified perfusion cell culture. Furthermore, product homogeneity was improved, which was reflected by the increased percentage of capillary isoelectric focusing main group. The quality improvement with intensified perfusion cell culture can be attributed to the shortened product retention in the bioreactor.ConclusionsThese findings suggest that intensified perfusion cell culture could be a better choice than traditional fed-batch especially for complex molecules like bsAbs. As this is a single case report, future studies on other cases are needed to further confirm the general applicability of this strategy.

Carotid artery vulnerable plaque model for cerebrovascular events by conventional ultrasound &amp; contrast-enhanced ultrasound: A preliminary study

Conventional ultrasound and contrast-enhanced ultrasound play an important role in the application of carotid plaque. To establish carotid artery vulnerable plaques model by conventional ultrasound combined with contrast-enhanced ultrasound, identify high-risk plaques that may lead to cerebrovascular events, and provide clinical risk warning of high-risk plaques of stroke. 205 cases of patients selected in 5053 patients with symptoms from 2018 to 2019 who were verified carotid plaques by conventional ultrasound and contrast-enhanced ultrasound image characteristics, 147 cases as a training set, establishing the carotid artery plaque model, analyzing the characteristic of the plaques and the relationship between cerebrovascular event, with 58 cases as a test set, verify the model. Routine carotid ultrasound and contrast-enhanced carotid ultrasound were performed in all enrolled patients. The gray-level characteristics of conventional ultrasound in the training concentration showed statistical differences in plaque morphology, fibrous cap morphology, uniformity and calcification degree in cerebrovascular events. The contrast enhanced ultrasound characteristics of plaques showed statistical differences in neovascularization and perfusion mode in cerebrovascular events. In the test set, there were statistical differences in the above conventional gray scale features and CEUS features. The vulnerable plaque model established by conventional ultrasound combined with contrast-enhanced ultrasound has good diagnostic value for the characteristic plaque of carotid artery with cerebrovascular events.

Experience with Transcranial Electrical Stimulation in the Assessment of the Microvascular Bed by Contrast-Free Magnetic Resonance Perfusion Imaging in Patients with Chronic Cerebral Ischemia

Objective: to define the role of transcranial electrical stimulation in the assessment of the microvascular bed by contrast-free magnetic resonance (MR) perfusion imaging in patients with chronic cerebral ischemia. Material and methods. Examinations were made in 37 people aged 65-70 years who were diagnosed with chronic cerebral ischemia. The patients were divided into two groups: with and without cognitive impairment (CI). The investigation algorithm involved brain magnetic resonance imaging (MRI) (Toshiba Vantage Titan, 1.5 T), transcranial electrical stimulation (TES) using a TETOS computer hardware diagnostics (Research and Production Firm “BIOSS”) and repeated brain MRI in the arterial spin labelling (ASL) perfusion mode. ASL perfusion data were used to assess cerebral blood flow in the regions of interest before and after TES. Results. Contrast-free MR perfusion imaging revealed relative hyperperfusion within the cortex of the frontal and parietal lobes concurrent with hypoperfusion of the subcortical nuclei and cerebral white matter in patients with CI, which suggests that enhanced cortical blood flow is ineffective and shunting blood flow forms. TES caused a statistically significant change in cerebral perfusion in the non-CI group of patients with chronic cerebral ischemia. After TES by ASL perfusion imaging, the patients without CI showed a diffuse increase in cerebral blood flow (p = 0.002), whereas the group of apparently healthy volunteers exhibited this increase in blood flow. Conclusion. Cerebral perfusion indices before and after TES in patients of all the examined groups define its role as a complementary tool to assess the compensatory capabilities of the microvascular bed by contrast-free MR perfusion imaging in chronic cerebral ischemia.

An optimized 3D-printed perfusion bioreactor for homogeneous cell seeding in bone substitute scaffolds for future chairside applications

A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailor-made bioreactor design. A perfusion system for efficient, homogeneous, and rapid seeding with human adipogenic stem cells in bone substitute scaffolds was designed. Variants concerning medium inlet and outlet port geometry, i.e. cylindrical or conical diffuser, cell concentration, perfusion mode and perfusion rates were simulated in silico. Cell distribution during perfusion was monitored by dynamic [18F]FDG micro-PET/CT and validated by laser scanning microscopy with three-dimensional image reconstruction. By iterative feedback of the in silico and in vitro experiments, the homogeneity of cell distribution throughout the scaffold was optimized with adjustment of flow rates, cell density and perfusion properties. Finally, a bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode. During this short period of time, the cells initially adhere within the entire scaffold and stay viable. After two weeks, the formation of several cell layers was observed, which was associated with an osteogenic differentiation process. This newly designed bioreactor may be considered as a prototype for chairside application.

Multi-objective optimization of monoclonal antibody production in bioreactor

The increasing demand for monoclonal antibodies (mAbs) based therapeutics stipulates their efficient production. mAbs are predominantly produced in a fed-batch bioreactor with temperature and media composition as key control variables. In this paper, we have developed a mathematical model incorporating the effect of temperature, biomass, glucose, protein, and lactate concentration on mAb productivity. The model parameter values are estimated by minimizing the normalized error function using the particle swarm optimization (PSO) algorithm. The model is further used to find the optimum temperature and reactor input-output flow rate strategy for maximum mAb production in minimum reactor run time for a traditional sequential combination of the batch, fed-batch, and perfusion modes of operation. The non-dominated sorting genetic algorithm (NSGA-II) has been successfully implemented for bi-objective optimization, which estimated a 5% increase in mAb production with respect to the experimentally produced mAb following combined mode of operation, and the same reactor run time and nutrients’ consumption using the optimized operating conditions. Continuous production is becoming increasingly popular for the intensification of biopharmaceutical processes. Therefore, the simulation is further extended to continuous mAb production, and multiple flow strategies are compared to maximize daily mAb production while minimizing nutrients’ requirement, wastage, and reactor volume.

Contrast-Enhanced Ultrasound in the Bladder: Critical Features to Differentiate Occupied Lesions.

Objective To study the clinical diagnostic value of contrast-enhanced ultrasound (CEUS) in bladder occupied lesions. Methods 38 cases of conventional-ultrasound-found bladder occupied lesions did color Doppler flow imaging (CDFI) and CEUS checks. By comparing the difference between two types of blood flow imaging technologies in displaying the flow of bladder occupied lesions and observing the perfusion modes of contrast agents to enter lesions, the perfusion characteristics of CEUS were analyzed. Finally, they were contrasted with the surgical pathology results. Results Of all the 38 cases, there were 51 bladder occupied lesions, including 43 bladder malignant tumors, 2 bladder inverted papillomas, and 6 glandular cystitis lesions. The blood flow display rate of bladder occupied lesions was 100% using CEUS. Apparently, it was higher than that of CDFI (62.7%), and the result of these showed a statistically significant difference (P < 0.05). Using CEUS, 46 malignant lesions and 5 glandular cystitis lesions were indicated, and the diagnostic accuracy rate was 86.3%. Conclusion CEUS can improve the blood flow display rate of bladder occupied lesions, and it can also observe the real-time blood flow of these lesions. It can help judge their nature and has a higher clinical value in differentiating the benign from the malignant.

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis.

By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have beenimproved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 1011 TCID50 /Lbioreactor /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.

Cell culture\u2013based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act “universally” against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key points• An automated perfusion process for production of IAV DIPs was established.• DIP titers of 7.40E + 9 plaque forming units per mL were reached.• A novel tubular cell retention device enabled continuous virus harvesting.

A new perfusion mode of culture for WJ-MSCs expansion in a stirred and online monitored bioreactor.

Asa clinical dose requires a minimum of 106 cells per kilogram of patients, it is, therefore, crucial to develop a scalable method of production of Wharton Jelly mesenchymal stem cells (WJ-MSCs) with maintained inner characteristics. Scalable expansion of WJ-MSCs on microcarriers usually found in cell culture, involvesspecific cell detachment using trypsin and could have harmful effects on cells. In this study,the performance of batch, fed-batch, and perfused-continuous mode of culture were compared. The batch and fed-batch modes resulted in expansion factors of 5 and 43, respectively. The perfused-continuous mode strategy consisted of the implementation of a settling tube inside the bioreactor. The diameter of the tube was calculated to maintain microcarriers colonized by cells in the bioreactor whereas empty microcarriers (responsible for potentially damaging collisions) were removed, using a continuous flow rate based on MSCs physiological requirements. Thanks to this strategy, a maximal number of 800 million cells was obtained in a 1.5 L bioreactor in 10 days. Lastly, online dielectric spectroscopy was implemented in the bioreactor and indicated that cell growth could be monitored during the culture.

Effect of hirudin on arterialized venous flap survival in rabbits

Arterialized venous flap (AVF) is limited in clinical application because its survival remains inconsistent and its exact survival mechanism is still unclear. Hirudin is an effective thrombin specific inhibitor, which is isolated from the salivary gland secretions of the leech. Our study evaluated the impact of hirudin on the viability of AVFs in rabbits. Thirty-six rabbits were randomly divided into three groups: sham group (physiological perfusion), control group (AVF), and hirudin group (AVF + hirudin). In hirudin group, 20 antithrombin units (ATU) hirudin (2.5 ml) were injected into each flap. In sham group and control group, the same amount of normal saline was injected into each flap. Status of flap survival, water content, vascular perfusion, histopathology, expression of CD34, VEGF, eNOS and HIF-1α were analyzed in each group. Analysis of oxidative stress was performed by measuring the activity of superoxide dismutase (SOD) and malondialdehyde (MDA). Compared with flaps in sham group with physiological perfusion mode, results of survival rate, perfusion status, SOD activity, expression of CD34, VEGF, and eNOS of AVFs in control group were significantly lower, while water content, MDA level and expression of HIF-1α were higher. The flap condition of AVFs injected with hirudin in hirudin group was improved significantly, and the results were similar to sham group. Our findings revealed that hirudin can effectively improve survival of AVF.

The Pulsatile Modification Improves Hemodynamics and Attenuates Inflammatory Responses in Extracorporeal Membrane Oxygenation.

BackgroundCOVID-19 is still a worldwide pandemic and extracorporeal membrane oxygenation (ECMO) is vital for extremely critical COVID-19 patients. Pulsatile flow impacts greatly on organ function and microcirculation, however, the effects of pulsatile flow on hemodynamics and inflammatory responses during ECMO are unknown. An in vivo study was launched aiming at comparing the two perfusion modes in ECMO.MethodsFourteen beagles were randomly allocated into two groups: the pulsatile group (n=7) and the non-pulsatile group (n=7). ECMO was conducted using the i-Cor system for 24 hours. Hemodynamic parameters including surplus hemodynamic energy (SHE), energy equivalent pressure (EEP), oxygenator pressure drop (OPD), and circuit pressure drop (CPD) were monitored. To assess inflammatory responses during ECMO, levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, and transforming growth factor-β1 (TGF-β1) were measured.ResultsEEP and SHE were markedly higher in pulsatile circuits when compared with the conventional circuits. Between-group differences in both OPD and CPD reached statistical significance. Significant decreases in TNF-α were seen in animals treated with pulsatile flows at 2 hours, 12 hours, and 24 hours as well as a decrease in IL-1β at 24 hours during ECMO. The TGF-β1 levels were significantly higher in pulsatile circuits from 2 hours to 24 hours. The changes in IL-6 and IL-8 levels were insignificant.ConclusionThe modification of pulsatility in ECMO generates more hemodynamic energies and attenuates inflammatory responses as compared to the conventional non-pulsatile ECMO.

Application of metabolic modeling for targeted optimization of high seeding density processes.

Process intensification by application of perfusion mode in pre‐stage bioreactors and subsequent inoculation of cell cultures at high seeding densities (HSD) has the potential to meet the increasing requirements of future manufacturing demands. However, process development is currently restrained by a limited understanding of the cell's requirements under these process conditions. The goal of this study was to use extended metabolite analysis and metabolic modeling for targeted optimization of HSD cultivations. The metabolite analysis of HSD N‐stage cultures revealed accumulation of inhibiting metabolites early in the process and flux balance analysis led to the assumption that reactive oxygen species (ROS) were contributing to the fast decrease in cell viability. Based on the metabolic analysis an optimized feeding strategy with lactate and cysteine supplementation was applied, resulting in an increase in antibody titer of up to 47%. Flux balance analysis was further used to elucidate the surprisingly strong synergistic effect of lactate and cysteine, indicating that increased lactate uptake led to reduced ROS formation under these conditions whilst additional cysteine actively reduced ROS via the glutathione pathway. The presented results finally demonstrate the benefit of modeling approaches for process intensification as well as the potential of HSD cultivations for biopharmaceutical manufacturing.

Interaction with biliary and pancreatic fluids drives supersaturation and drug absorption from lipid-based formulations of low (saquinavir) and high (fenofibrate) permeability poorly soluble drugs

Drug absorption from lipid-based formulations (LBFs) in the gastrointestinal (GI) tract is the result of a series of processes, including formulation dispersion, interaction with biliary and pancreatic secretions, drug solubilisation and supersaturation, and finally intestinal permeability. Optimal formulation design is dependent on a good understanding of the limitations to, and drivers of, absorption, but for LBFs the complexity of these processes makes data interpretation complex. The current study has re-examined a previous in vitro digestion–in situ perfusion model to increase physiological relevance and has used this model to examine drug absorption from LBFs. The composition of rat bile and jejunal fluid was also characterised to identify in vivo-relevant conditions. Digestion was initiated using rat bile/pancreatic fluid and the formulation and digestive enzymes mixed immediately prior to entry into the jejunum (allowing dilution/digestion to occur at the absorptive site). These conditions were employed to study drug absorption from LBFs of high (fenofibrate, FFB) and low (saquinavir, SQV) permeability compounds. The impact of polymeric precipitation inhibitors (PPIs) was also evaluated. For FFB, supersaturation, initiated by formulation interaction with biliary/pancreatic fluids, appeared to drive absorption and the addition of the PPIs poly(glycidyl methacrylate) (PPGAE) and hydroxypropylmethyl cellulose (HPMC), reduced drug precipitation, increased FFB supersaturation and increased absorption from a Type IV LBF of FFB. For a Type IIIB LBF however, PPIs were ineffective at increasing absorption. The impact of PPIs on the absorption of a less permeable drug, SQV, was similarly evaluated and again drug absorption appeared to be related to the extent of supersaturation, although in this case PPI were unable to promote absorption. For both FFB and SQV, drug absorption patterns obtained with the in vitro digestion–in situ perfusion mode, correlated well with in vitro supersaturation data and in vivo drug exposure data from oral bioavailability studies. The data are consistent with a mode of drug absorption where rapid dilution of LBFs with biliary and pancreatic secretions at the absorptive site in the upper small intestine drives transient supersaturation, that supersaturation is a significant driver of drug absorption for both low and high permeability drugs, and that PPIs delay drug precipitation, enhance supersaturation and promote drug absorption in a drug and formulation specific manner.

Improvement of multiterritory perforator flap survival supported by a hybrid perfusion mode: A novel strategy and literature review

Perforator flaps have been widely used in clinical Settings, however, unexpected necrosis was still commonly encountered in the distal portions of multiterritory flaps known as Choke zone III. In this study, we introduced a novel hybrid perfusion technique which is different from the established one of arterial supercharging or venous superdrainage to improve multiterritory flap survival with success. In order to ensure the entire flap survival of multiterritory flaps extending to choke zone III, a “hybrid perfusion” mode by anastomosing a distal vein of the flap with a recipient artery was carried out in two cases based on our previous basic study. In addition, a systematic literature review regarding the established microsurgical assistant techniques of arterial supercharging and venous superdrainage techniques were performed. Both flaps survived uneventfully. At a minimal follow-up of six months, both patients were satisfied with the results. This novel hybrid perfusion technique provides a simple new concept in solving partial necrosis of multiterritory flaps. Further practice is guaranteed for better understanding this unconventional attempt.