Literatura científica selecionada sobre o tema "Peau perfusée ex-Vivo"

We compared the perfluorochemical emulsion Fluosol-43 and an erythrocyte-based solution as support media for ex vivo working rabbit hearts functioning with a physiological workload. Both groups of hearts (n = 5/group) exhibited stable function (left ventricular peak systolic pressure, peak rates of left ventricular pressure rise and relaxation, aortic flow, peak aortic flow rate, stroke work, and peak power) for the first 6 h of perfusion. Coronary flow, coronary venous O2 content, and O2 supply-to-demand ratio declined similarly in both groups during the first 6 h. Both groups of hearts preferentially utilized pyruvate to glucose. The Fluosol-43-perfused hearts had higher heart rate, left ventricular peak systolic pressure, peak rate of left ventricular pressure rise, aortic flow, coronary flow, and myocardial O2 consumption compared with the erythrocyte-perfused hearts. The Fluosol-43 hearts produced more lactate and released more creatine phosphokinase than did the erythrocyte-perfused hearts, but the rates were low and constant throughout perfusion, indicating that the hearts were not progressively ischemic. After the first 6 h, function of the Fluosol-43 hearts declined, resulting in their earlier failure compared with the erythrocyte-perfused hearts. The data indicate that Fluosol-43 had sufficient O2- carrying capacity to support stable function of a rabbit heart at a physiological workload for 6 h, and differences in function and ex vivo longevity of the two groups of hearts suggested that a component or contaminant of Fluosol-43 altered sarcolemmal function and/or that a component needed for membrane integrity was lacking in the Fluosol-43 perfusate.

Objectives.To determine maternal-fetal transplacental passage of meropenem in the ex vivo human perfusion model.Study design.Term placentae (n= 6) were collected immediately after delivery. A single cotyledon was localized, perfused and stabilized with physiologic Eagles minimal essential medium containing 3% bovine albumin and heparin as described by Chalier (Chalier JC. Criteria for evaluating perfusion experiments and presentation results. Contrib Gynecol Obstet 1985; 13:32–39). Meropenem was added to the maternal medium in concentrations similar to maternal serum peak and trough levels, then perfused through the maternal circulation of the cotyledon. To assess transfer and accumulation, fluid aliquots from both the maternal and fetal compartments were collected over an hour at defined intervals in an open and closed system. AntipyrineC14was added to the medium in order to calculate the transport fraction and clearance indexes. Meropenem and antipyrineC14concentrations were determined by High-pressure Liquid Chromatography and liquid scintillation, respectively.Results.Mean antipyrine transport fraction was 2.33 + 0.25. Maternal and fetal mean meropenem peak concentrations were 54.3 + 3.3μg/ml and 2.2 + 0.18μg/ml, respectively. Whereas, maternal and fetal mean trough concentrations were 12.7 + 1.3μg/ml and 0.41 + 0.10μg/ml, respectively. Mean peak clearance index was 0.077 + 0.007 and the mean trough was 0.052 + 0.015. Mean accumulation for the peak and trough concentrations of meropenem were 0.9 and 2.95μg/ml, respectively.Conclusions.Transplacental passage of meropenem was incomplete in the ex vivo human placental perfusion model. Accumulation was also noted in the fetal compartment

Background When perfused neonatal brain slices are studied ex vivo with nuclear magnetic resonance (NMR) spectroscopy, it is possible to use 31P detection to monitor levels of intracellular adenosine triphosphate (ATP), cytosolic pH, and other high-energy phosphates and 1H detection to monitor lactate and glutamate. Adult brain slices of high metabolic integrity are more difficult to obtain for such studies, because the adult cranium is thicker, and postdecapitation revival time is shorter. A common clinical anesthesia phenomenon--loss of temperature regulation during anesthesia, with surface cooling and deep hypothermia, was used to obtain high-quality adult rat cerebrocortical slices for NMR studies. Methods Spontaneously breathing adult rats (350 g), anesthetized with isoflurane in a chamber, were packed in ice and cooled until rectal temperatures decreased to approximately 30 degrees C. An intraaortic injection of heparinized saline at 4 degrees C further cooled the brain to approximately 18 degrees C. Slices were obtained and then recovered at 37 degrees C in oxygenated medium. Interleaved 31P/1H NMR spectra were acquired continually before, during, and after 20 min of no-flow hypoxia (PO2 approximately 0 mmHg). Histologic (Nissl stain) measurements were made from random slices removed at different times in the protocol. Three types of pretreatment were compared in no-flow hypoxia studies. The treatments were: (1) hyperoxia; (2) hypercapnia (50% CO2); and (3) hypoxia, which was accomplished by washing the slices with perfusate equilibrated with 100% N2 and maintaining a 100% N2 gas flow in the air space above the perfusate. Results During hyperoxia, 31P NMR metabolite ratios were identical to those seen in vivo in adult brains, except that, in vitro, the Pi peak was slightly larger than in vivo. A lactate peak was seen in in vitro 1H spectra of slices after metabolic recovery from decapitation, although lactate is barely detectable in vivo in healthy brains. The in vitro lactate peak was attributed to a small population of metabolically impaired cells in an injury layer at the cut edge. NMR spectral resolution from the solenoidal coil exceeded that obtained in vivo in surface coil experiments. Phosphocreatine and ATP became undetectable during oxygen deprivation, which also caused a three- to sixfold increase in the ratio of lactate to N-acetyl-aspartate. Within experimental error, all metabolite concentrations except pHi recovered to control values within 2 h after oxygen restoration. Nissl-stained sections suggested that pretreatment with hypercapnia protected neurons from cell swelling during the brief period of no-flow oxygen deprivation. Conclusions Perfused, respiring adult brain slices having intact metabolic function can be obtained for NMR spectroscopy studies. Such studies have higher spectral resolution than can be obtained in vivo. During such NMR experiments, one can deliver drugs or molecular probes to brain cells and obtain brain tissue specimens for histologic and immunochemical measures of injury. Important ex vivo NMR spectroscopy studies that are difficult or impossible to perform in vivo are feasible in this model.

Acute liver failure (ALF) is a rare but devastating disease associated with substantial morbidity and a mortality rate of almost 45%. Medical treatments, apart from supportive care, are limited and liver transplantation may be the only rescue option. Large animal models, which most closely represent human disease, can be logistically and technically cumbersome, expensive and pose ethical challenges. The development of isolated organ perfusion technologies, originally intended for preservation before transplantation, offers a new platform for experimental models of liver disease, such as ALF. In this study, female domestic swine underwent hepatectomy, followed by perfusion of the isolated liver on a normothermic machine perfusion device. Five control livers were perfused for 24 h at 37 °C, while receiving supplemental oxygen and nutrition. Six livers received toxic doses of acetaminophen given over 12 h, titrated to methemoglobin levels. Perfusate was sampled every 4 h for measurement of biochemical markers of injury (e.g., aspartate aminotransferase [AST], alanine aminotransferase [ALT]). Liver biopsies were taken at the beginning, middle, and end of perfusion for histological assessment. Acetaminophen-treated livers received a median dose of 8.93 g (8.21–9.75 g) of acetaminophen, achieving a peak acetaminophen level of 3780 µmol/L (3189–3913 µmol/L). Peak values of ALT (76 vs. 105 U/L; p = 0.429) and AST (3576 vs. 4712 U/L; p = 0.429) were not significantly different between groups. However, by the end of perfusion, histology scores were significantly worse in the acetaminophen treated group (p = 0.016). All acetaminophen treated livers developed significant methemoglobinemia, with a peak methemoglobin level of 19.3%, compared to 2.0% for control livers (p = 0.004). The development of a model of ALF in the ex vivo setting was confounded by the development of toxic methemoglobinemia. Further attempts using alternative agents or dosing strategies may be warranted to explore this setting as a model of liver disease.

Abstract Purpose To evaluate dynamic contrast-enhanced ultrasound (DCEUS) as a tool for measuring blood flow in the macro- and microcirculation of an ex-vivo machine-perfused pig liver and to confirm the ability of DCEUS to accurately detect induced flow rate changes so that it could then be used clinically for monitoring flow changes in liver tumors. Materials and Methods Bolus injections of contrast agents in the hepatic artery (HA) and portal vein (PV) were administered to 3 machine-perfused pig livers. Flow changes were induced by the pump of the machine perfusion system. The induced flow rates were of clinical relevance (150 – 400 ml/min for HA and 400 – 1400 ml/min for PV). Quantification parameters from time-intensity curves [rise time (RT), mean transit time (MTT), area under the curve (AUC) and peak intensity (PI)] were extracted in order to evaluate whether the induced flow changes were reflected in these parameters. Results A linear relationship between the image intensity and the microbubble concentration was confirmed first, while time parameters (RT and MMT) were found to be independent of concentration. The induced flow changes which propagated from the larger vessels to the parenchyma were reflected in the quantification parameters. Specifically, RT, MTT and AUC correlated with flow rate changes. Conclusion Machine-perfused pig liver is an excellent test bed for DCEUS quantification approaches for the study of the hepatic vascular networks. DCEUS quantification parameters (RT, MTT, and AUC) can measure relative flow changes of about 20 % and above in the liver vasculature. DCEUS quantification is a promising tool for real-time monitoring of the vascular network of tumors.

Non-invasive therapeutic-focused ultrasound (US) can be used for the mechanical dissociation of tissue and is described as histotripsy. We have performed US histotripsy in viable perfused ex vivo porcine livers as a step in the development of a novel approach to hepatocyte cell transplantation. The histotripsy nidus was created with a 2 MHz single-element focused US transducer, producing 50 pulses of 10 ms duration, with peak positive and negative pressure values of P+ = 77.7 MPa and P− = –13.7 MPaat focus, respectively, and a duty cycle of 1%. Here, we present the histological analysis, including 3D reconstruction of histotripsy sites. Five whole porcine livers were retrieved fresh from the abattoir using human transplant retrieval and cold static preservation techniques and were then perfused using an organ preservation circuit. Whilst under perfusion, histotripsy was performed to randomly selected sites on the live. Fifteen lesional sites were formalin-fixed and paraffin-embedded. Sections were stained with Haematoxylin and Eosin and picro-Sirius red, and they were also stained for reticulin. Additionally, two lesion sites were used for 3D reconstruction. The core of the typical lesion consisted of eosinophilic material associated with reticulin loss, collagen damage including loss of birefringence to fibrous septa, and perilesional portal tracts, including large portal vein branches, but intact peri-lesional hepatic plates. The 3D reconstruction of two histotripsy sites was successful and confirmed the feasibility of this approach to investigate the effects of histotripsy on tissue in detail.

Despite a similar mechanism of action underlying their glucose-lowering effects in type 2 diabetes, dipeptidyl peptidase-4 (DPP-4) inhibitors have diverse molecular structures, raising the prospect of agent-specific, glucose-independent actions. To explore the issue of possible DPP-4 inhibitor cardiac heterogeneity, we perfused different DPP-4 inhibitors to beating mouse hearts ex vivo, at concentrations equivalent to peak plasma levels achieved in humans with standard dosing. We studied male and female mice, young non-diabetic mice, and aged diabetic high fat diet-fed mice and observed that linagliptin enhanced recovery after ischemia-reperfusion, whereas sitagliptin, alogliptin, and saxagliptin did not. DPP-4 transcripts were not detected in adult mouse cardiomyocytes by RNA sequencing and the addition of linagliptin caused ≤0.2% of cardiomyocyte genes to be differentially expressed. In contrast, incubation of C166 endothelial cells with linagliptin induced cell signaling characterized by phosphorylation of Akt and endothelial nitric oxide synthase, whereas the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine increased serine 16 phosphorylation of the calcium regulatory protein, phospholamban in cardiomyocytes. Furthermore, linagliptin increased cardiomyocyte cGMP when cells were co-cultured with C166 endothelial cells, but not when cardiomyocytes were cultured alone. Thus, at a concentration comparable to that achieved in patients, linagliptin has direct effects on mouse hearts. The effects of linagliptin on cardiomyocytes are likely to be either off-target or indirect, mediated through NO generation by the adjacent cardiac endothelium.

Studies of skinned fibers suggest that the rate of ATP turnover in skeletal muscle is depressed by acidosis. To examine whether this occurs in intact muscles, the ATP cost of isometric contractions was measured in ex vivo, arterially perfused cat biceps (predominantly fast-twitch) and soleus (slow-twitch) muscles under normocapnic (5% CO2) and hypercapnic (70% CO2) conditions. Hypercapnia decreased extracellular pH from 7.4 to 6.7 and intracellular pH from 7.1 to 6.5 (soleus) or 6.6 (biceps) but had no significant effect on the phosphocreatine (PCr)-to-ATP ratio in muscles at rest. The ATP cost of contraction was estimated from PCr changes, measured by gating the acquisition of 31P-nuclear magnetic resonance spectra to times before and after brief tetani (1 s at 100 Hz and 2 s at 25 Hz for biceps and soleus, respectively) or 10-s trains of twitches (2 and 1 Hz, respectively). Peak isometric force and the ATP cost of tetanic contraction (PCr/force x time integral) were not significantly different under hypercapnic compared with normocapnic conditions in either muscle (mean: 7.97 and 2.44 micromol x kg(-1) x s(-1) for biceps and soleus, respectively). Twitch force and the ATP cost per twitch decreased by nearly 50% during hypercapnic perfusion in both muscle types. The results indicate that hypercapnic acidosis has no significant effect on the ATPase rate per active myosin head in intact mammalian skeletal muscle.

Consistent with a critical role in respiratory and autonomic stress responses, the carotid bodies are strongly excited by pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide implicated in stress responses throughout the sympathetic nervous system. PACAP excites isolated carotid body glomus cells via activation of PAC1 receptors, with one study suggesting PAC1-induced excitation is due entirely to protein kinase A (PKA)-mediated inhibition of TASK channels. However, in other systems, PAC1 is known to be coupled to multiple intracellular signaling pathways, including PKA, phospholipase C (PLC), phospholipase D (PLD), and protein kinase C (PKC), that trigger multiple downstream effectors including increased Ca2+ mobilization, inhibition of various K+ channels, and activation of nonselective cation channels. This study tests if non-PKA/TASK channel signaling helps mediate the stimulatory effects of PACAP on the carotid body. Using an ex vivo arterially perfused rat carotid body preparation, we show that PACAP-38 stimulates carotid sinus nerve activity in a biphasic manner (peak response, falling to plateau). PKA blocker H-89 only reduced the plateau response (∼41%), whereas the TASK-1-like K+ channel blocker/transient receptor potential vanilloid 1 channel agonist anandamide only inhibited the peak response (∼48%), suggesting involvement of additional pathways. The PLD blocker CAY10594 significantly inhibited both peak and plateau responses. The PLC blocker U73122 decimated both peak and plateau responses. Brefeldin A, a blocker of Epac (cAMP-activated guanine exchange factor, reported to link Gs-coupled receptors with PLC/PLD), also reduced both phases of the response, as did blocking signaling downstream of PLC/PLD with the PKC inhibitors chelerythrine chloride and GF109203X. Suggesting the involvement of non-TASK ion channels in the effects of PACAP, the A-type K+ channel blocker 4-aminopyridine, and the putative transient receptor potential channel (TRPC)/T-type calcium channel blocker SKF96365 each significantly inhibited the peak and steady-state responses. These data suggest the stimulatory effect of PACAP-38 on carotid body sensory activity is mediated through multiple signaling pathways: the PLC-PKC pathways predominates, with TRPC and/or T-type channel activation and Kv channel inactivation; only partial involvement is attributable to PKA and PLD activation.

The effect of interleukin-1 alpha (IL-1) on the synthesis of glomerular basement membrane heparan sulfate-proteoglycan (HS-PG) was investigated. An ex vivo recirculating organ perfusion system was used. Kidneys were perfused with a medium containing (35S) sulfate and IL-1 (0.625 to 6.25 ng/mL). After radiolabeling was performed, a small cortical piece was saved for tissue autoradiography; the remaining kidney and perfusion medium were used for biochemical studies. Renal cortices were dissected out, and glomeruli were isolated; the PG were extracted and characterized. With exposure to IL-1 (5 ng/mL), an approximately twofold increase of the radioactivity in the glomerular fraction was noted. The increase was unaffected by indomethacin treatment. By Sepharose CL-6B chromatography, a single peak of radioactivity with Kav = 0.25 (macromolecular form of PG) was observed in the control group. The IL-treated group had two peaks of radioactivity with Kav = 0.25 and 0.45: the first peak contained PG, and the second peak consisted of free glycosaminoglycan (GAG) chains. Elution profiles of hydrolyzed tissue GAG chains were similar. No change in the ratio of chondroitin to heparan sulfate was observed. By DEAE-Sephacel chromatography, the glomerular PG/GAG of the IL-treated group eluted at a relatively lower salt concentration, suggesting a change in the charge density characteristics. No differences in the elution profiles of media PG/GAG were observed. (35S)methionine labeling of proteins showed no significant increase of the total radioincorporation in the IL-treated group. Immunoprecipitation studies revealed an approximately 88% increase in the de novo synthesis of PG. Tissue autoradiography revealed an approximately twofold increase of (35S) sulfate radioactivity over the glomerular mesangium, epithelium, and basement membranes. These results indicate that IL-1 enhances the synthesis of the macromolecular form of PG and the generation of free chains. Conceivably, such alterations may lead to defective macromolecular interactions among various components of the glomerular basement membrane and compromise the integrity of the ultrafiltration unit of the glomerulus.

Le tissu adipeux de l’Homme est un réel organe dynamique dont l’intérêt en chirurgie est grandissant. Sa composante sous cutanée, dense en adipocytes, se compose d’un stroma riche en cellules souches mésenchymateuses. Les ADSC (cellules souches mésenchymateuses dérivées du tissus adipeux ou Adipose-derived Stem cells) peuvent en être isolées au laboratoire après digestion enzymatique, ou mécaniquement (émulsification mécanique et nanofat), les rendant disponibles immédiatement au bloc opératoire. La pluripotence des ADSC en fait un atout majeur pour leur utilisation en médecine régénératrice.La chirurgie plastique est amenée à réparer des défects pouvant, entre autres, affecter les tissus nerveux périphérique, osseux et cutané. Ainsi, la lipoaspiration, geste chirurgical effectué quotidiennement au bloc opératoire, offre la possibilité de disposer de lipoaspirat issu du tissu adipeux sous cutané, potentiellement riche en ADSC. Ainsi, après avoir réalisé une revue de la littérature recensant 51 marqueurs phénotypiques depuis 2006, 5 marqueurs différenciants se démarquaient depuis les dernières recommandations de 2019 : CD34-, CD45-, CD73+, CD90+ et CD105+. Alors, nous avons appliqué cette nouvelle stratégie à une cohorte de 21 patients en analysant la cellularité en ADSC de la Fraction Vasculaire Stromale (SVF) selon la technique, l’âge, le sexe, la localisation et l’indice de masse corporelle. Ensuite, la culture cellulaire avait pour but de confirmer la fonctionnalité des ADSC en comparant la production d’IL-6 et de TNF- dans 4 sous populations issus de lipoaspirats. Les variations phénotypiques secondaires à l’amplification cellulaire étaient observées. L’objectif suivant était d’user de la pluripotence des ADSC dans 3 indications : différenciation neurogène, ostéogénique et cutanée.Ainsi, le potentiel neurogénique des ADSC contenus dans la SVF isolée mécaniquement (nanofat) était étudié dans la repousse nerveuse. Pour cela, un défect de nerf sciatique chez le rat était reconstruit à l’aide d’un neurotube de chitosan, associé ou non à l’adjonction d’ADSC. L’environnement favorable à une repousse nerveuse médiée par l’ajout de nanofat était objectivé, entre autres, par analyses histologiques et immunohistochimiques (PGP9.5 et protéine S100).Ensuite, la différenciation ostéoblastique était étudiée après culture cellulaire 3D des ADSC sur une matrice osseuse commerciale. La production d’hydroxyapatite et d’ostéocalcine supportaient la preuve d’une différenciation ostéogénique des ADSC.Enfin, nous avons développé un modèle d’étude de peau humaine isolée et perfusée viable pendant 5 jours, support d’études futures entre ADSC et matrice dermique équivalente. Ainsi, une revue de la littérature étudiait les caractérisations biomécaniques de ce type de biomatériaux (electrospinnés) comme substitut cutané
Human adipose tissue is a truly dynamic organ with an increasing interest in surgery. Its subcutaneous component, dense in adipocytes, is composed of a stroma rich in mesenchymal stem cells. Adipose-derived stem cells (ADSCs) can be isolated from this stroma after enzymatic digestion in the laboratory, or mechanically (mechanical emulsification and nanofat), making them immediately available at the operating room. Moreover, t ADSCs pluripotency makes them a major asset for use in regenerative medicine.Plastic and reconstructive surgery is required to repair defects that can affect peripheral nerve, bone and skin tissue. Liposuction, a surgical procedure performed daily at the operating room, offers the possibility of using liposuction from subcutaneous adipose tissue, which is potentially rich in ADSCs. So, after carrying out a literature review listing 51 phenotypic markers since 2006, 5 different markers stood out since the latest 2019 recommendations: CD34-, CD45-, CD73+, CD90+and CD105+. We therefore took up this new strategy in a cohort of 21 patients by analyzing the cellularity of Stromal Vascular Fraction (SVF) in ADSC according to technique, age, sex, location and body mass index. Cell culture was then used to confirm ADSC functionality by comparing IL-6 and TNF-a production in 4 sub-populations derived from lipoaspirates. Phenotypic variations secondary to cell amplification were observed. The next objective was to use the pluripotency of ADSCs in 3 indications: neurogenic, osteogenic and cutaneous differentiation.The neurogenic potential of ADSCs contained in mechanically isolated SVF (nanofat) was studied in nerve regrowth. A rat sciatic nerve defect was reconstructed using a chitosan neurotube, with or without the addition of ADSCs. The favorable environment for nerve regrowth mediated by the addition of nanofat was assessed by histological and immunohistochemical analyses (PGP9.5 and S100 protein).Osteoblastic differentiation was then studied after 3D cell culture of ADSCs on commercially available bone matrix. Production of hydroxyapatite and osteocalcin supported evidence of osteogenic differentiation.Finally, we developed an ex vivo model of isolated and perfused human skin viable for 5 days, to support future studies between ADSCs and dermal matrix equivalent. A review of the literature looked at the biomechanical characterizations of such electrospun biomaterials as skin substitutes

Les modèles organotypiques tels que les explants de peau humaine sont les modèles les plus complexes et parmi les plus représentatifs de la peau in vivo existants à ce jour pour tester l’efficacité ou l’innocuité de molécules d’intérêts thérapeutiques au stade des études pré-cliniques. Cependant, l’absence de circulation sanguine et lymphatique dans ces modèles reste une limite importante dans l’homéostasie du tissu, notamment pour prédire les réponses de la peau à un traitement. De plus, les échanges en nutriments et en oxygène n’étant possibles que par diffusion, la durée de vie de ces modèles reste limitée. Différentes stratégies ont été mises en place afin de contrôler les mécanismes de transports moléculaires au sein de tissus biologiques. La microfluidique offre un fort potentiel pour contrôler la convection et la diffusion permettant l’échange de composés dans ces modèles de peau.L’objectif de ce projet est de développer, caractériser et valider un modèle de peau humaine ex vivo perfusé. Le but de cette perfusion intra-tissulaire est de favoriser les échanges de nutriments, d’oxygène ou de médicaments, mais également d’améliorer l’élimination des déchets métaboliques.Le premier objectif de mes travaux a consisté à mettre en place un flux intra-tissulaire dans un explant de peau humaine, et à développer un procédé permettant de maintenir l’explant perfusé en culture pendant plusieurs jours. Pour cela, un dispositif poreux a été implanté dans le derme du modèle ex vivo de peau humaine NativeSkin, développé par la société Genoskin, puis relié à un système microfluidique permettant l’infusion de composés au sein du tissu.Le deuxième objectif a consisté à développer des méthodes d’analyse de la diffusion de composés dans des explants de peau. Quatre méthodes ont été développées : l’évaluation macroscopique et qualitative de la diffusion à l’aide d’un colorant, l’étude de la diffusion en temps réel par radiographie à rayons X, l’étude de la diffusion en trois dimensions par tomographie à rayons X, et enfin l’analyse de la diffusion de dextrans fluorescents de différents poids moléculaires, sur coupes histologiques. Un modèle numérique permettant de simuler la diffusion dans le modèle de peau a également été développé sur le logiciel COMSOL, permettant de prédire le profil de diffusion d’un composé.Le troisième et dernier objectif a consisté à déterminer les paramètres de perfusion permettant une bonne diffusion des composés dans l’explant de peau, sans toutefois endommager le tissu. Une première série d’expériences (8 donneurs) a été réalisée sur des modèles perfusés à flux constant (2,5µL/min) avec du milieu de culture, pendant 10 jours. Les résultats ont montré qu’à l’issue de la culture, les modèles de peau ne présentent pas d’altération de la viabilité cellulaire ni de l’intégrité du tissu, avec un maintien de la prolifération et du métabolisme cellulaire. Cependant, la caractérisation de la diffusion dans le modèle a démontré un manque de reproductibilité dans les expériences, avec d’importantes variabilités inter et intra-donneurs. De plus, la perfusion de dextrans de différents poids moléculaires a démontré que la diffusion de composés de hauts poids moléculaires était limitée. Afin de pallier ces limites, nous avons proposé une nouvelle méthode de perfusion basée sur une modulation de la pression au sein du dispositif. L’application d’une légère surpression au sein du dispositif poreux permet d’améliorer la reproductibilité et l’efficacité des échanges moléculaires au sein de l’explant.Les résultats obtenus positionnent le modèle FlowSkin ainsi développé comme un nouvel outil pertinent pour évaluer l’efficacité ou la toxicité de molécules administrées par voie intraveineuse, directement sur de la peau humaine. De plus, la perfusion de transporteurs d’oxygène via ce système pourrait permettre de prolonger la durée de vie et donc d’améliorer encore la pertinence du modèle de peau ex vivo
Organotypic models as human skin explants are the most complex and among the most representative of in vivo skin existing today to test the efficacy or the safety of molecules of therapeutic interest during preclinical studies. However, the loss of vascularization and lymphatic system in these models remains a major limitation in tissue homeostasis that impedes the prediction of skin responses to a treatment. In addition, exchanges of nutrients and oxygen being limited to diffusion, models lifetime is limited. Different strategies have been implemented to study and improve mass transport mechanism in such models. Microfluidics offers a great potential to control diffusion and convection mechanisms that permit molecular exchanges in skin models.The objective of this project is to develop, characterize and validate an ex vivo perfused human skin model. The purpose of this intra-tissue infusion is to promote the exchanges of nutrients, oxygen or drugs, but also to improve metabolic waste elimination.The first objective of my work consisted in implementing an intra-tissue flow in a human skin explant, and in setting up a process to maintain the perfused model in culture for several days. To this end, a porous device was implanted in the dermis of the ex vivo human skin model NativeSkin, developed by the company Genoskin. The implantable device is then connected to a microfluidic system allowing the infusion of compounds within the tissue.The second objective was to develop analysis methods of the diffusion of compounds in skin explants. Four methods have been developed: macroscopic and qualitative evaluation of the diffusion using a dye, the study of the diffusion in real time by X-ray radiography, the study of the diffusion in three dimensions by X-ray tomography, and finally the analysis of the diffusion of fluorescent dextrans of different molecular weights, on histological sections. A numerical model allowing to simulate the diffusion in the skin model has also been developed using COMSOL software, allowing to predict the diffusion profile of a compound.The third and last objective of my work was to determine perfusion parameters allowing efficient molecular exchanges of compounds in the skin explant, but without damaging the tissue. A first series of experiments (8 donors) was carried out on models perfused with a constant flow-rate (2.5 µL/min) with culture medium, for 10 days. The results showed that at the end of the culture, skin models did not show any alteration in cell viability or tissue integrity, with maintenance of cell proliferation and metabolism. However, diffusion characterization in the model demonstrated a lack of reproducibility in the experiments, with significant inter and intra-donor variability. In addition, the infusion of different molecular weights dextrans has demonstrated that the mass transport of high molecular weight compounds was limited through the implantable device. We demonstrated that the control of the fluid pressure is critical and that imposing a pulsatile injection with slight overpressures improves the efficiency and reproducibility of the molecular species delivery and collection in the explant.These results have shown the potential of the developed FlowSkin model as a new tool to study the efficacy or toxicity of intravenously administered drugs directly onto human skin. In addition, the combination of FlowSkin with perfusion of oxygen carriers offers unique opportunities to extend the lifetime and further improve the relevance of such ex vivo skin model