Perfusion Medium Research Articles

Perfusable vascular tree like construction in 3D cell-dense tissues using artificial vascular bed.

Perfusable vascular structures in cell-dense tissues are essential for fabricating functional three-dimensional (3D) tissues in vitro. However, it is challenging to introduce functional vascular networks observable as vascular tree, finely spaced at intervals of tens of micrometers as in living tissues, into a 3D cell-dense tissue. Herein, we propose a method for introducing numerous vascular networks that can be perfused with blood into 3D tissues constructed by cell sheet engineering. We devise an artificial vascular bed using a hydrogel that is barely deformed by cells, enabling perfusion of the culture medium directly beneath the cell sheets. Triple-layered cell sheets with an endothelial cell network prepared by fibroblast co-culture are transplanted onto the vascular bed and subjected to perfusion culture. We demonstrate that numerous vascular networks are formed with luminal structures in the cell sheets and can be perfused with India ink or blood after a five-day perfusion culture. Histological analysis also demonstrates that perfusable vascular structures are constructed at least 100μm intervals uniformly and densely within the tissues. The results suggest that our perfusion culture method enhances vascularization within the 3D cell-dense tissues and enables the introduction of functional vasculature macroscopically observable as vascular tree in vitro. In conclusion, this technology can be used to fabricate functional tissues and organs for regenerative therapies and in vitro experimental models.

A Novel Approach for Non-Invasive Continuous In-Line Control of Perfusion Cell Cultivations by Raman Spectroscopy.

Continuous manufacturing is becoming more important in the biopharmaceutical industry. This processing strategy is favorable, as it is more efficient, flexible, and has the potential to produce higher and more consistent product quality. At the same time, it faces some challenges, especially in cell culture. As a steady state has to be maintained over a prolonged time, it is unavoidable to implement advanced process analytical technologies to control the relevant process parameters in a fast and precise manner. One such analytical technology is Raman spectroscopy, which has proven its advantages for process monitoring and control mostly in (fed-) batch cultivations. In this study, an in-line flow cell for Raman spectroscopy is included in the cell-free harvest stream of a perfusion process. Quantitative models for glucose and lactate were generated based on five cultivations originating from varying bioreactor scales. After successfully validating the glucose model (Root Mean Square Error of Prediction (RMSEP) of ∼0.2 g/L), it was employed for control of an external glucose feed in cultivation with a glucose-free perfusion medium. The generated model was successfully applied to perform process control at 4 g/L and 1.5 g/L glucose over several days, respectively, with variability of ±0.4 g/L. The results demonstrate the high potential of Raman spectroscopy for advanced process monitoring and control of a perfusion process with a bioreactor and scale-independent measurement method.

Quenching Epigenetic Drug Resistance Using Antihypoxic Microparticles in Glioblastoma Patient‐Derived Chips (Adv. Healthcare Mater. 8/2022)

Epigenetic Alterations In article number 2102226, Young Min Shin, Hak-Joon Sung, and co-workers demonstrate that loading anti-hypoxic microparticles onto glioblastoma (GBM) masses reduces epigenetic drug resistance and suppresses subsequent cell invasiveness. The results are verified using a 3D GBM chip with cell spheres in vitro under media perfusion and in vivo through chip implantation into mouse ischemic hind limbs.

Accumulation of 3-aminopropylphosphonate in the ex vivo brain observed by phosphorus-31 nuclear magnetic resonance.

3‐aminopropylphosphonate (3‐APP) is known for its use as an exogenous indicator of extracellular volume and pH in phosphorus‐31 nuclear magnetic resonance (31P NMR) studies. We used 3‐APP for estimating the extracellular volume in NMR studies of several ex vivo preparations including retrograde perfused mouse heart (n = 4), mouse liver slices (n = 2), xenograft breast cancer tumors (n = 7, MCF7), and rat brain slices (n = 4). In the former three preparations, the 3‐APP signal was stable in lineshape and intensity for hours and the chemical shift of the signal in the presence of the biological sample was the same as in the perfusion medium without the biological sample. However, in studies of brain slices, the 3‐APP signal appeared split into two, with an upfield component (0.7 ± 0.1 ppm to the left) increasing with time and showing a wider linewidth (66.7 ± 12.6 vs. 39.1 ± 7.6 Hz, the latter is of the perfusion medium signal). This finding suggests that 3‐APP inadvertently accumulated in brain slices, most likely as a membrane bound form. This observation limits the use of 3‐APP as an inert biochemical indicator in brain preparations and should be taken into account when using 3‐APP in vivo.

3D In Vitro Platform for Cell and Explant Culture in Liquid-like Solids.

Existing 3D cell models and technologies have offered tools to elevate cell culture to a more physiologically relevant dimension. One mechanism to maintain cells cultured in 3D is by means of perfusion. However, existing perfusion technologies for cell culture require complex electronic components, intricate tubing networks, or specific laboratory protocols for each application. We have developed a cell culture platform that simply employs a pump-free suction device to enable controlled perfusion of cell culture media through a bed of granular microgels and removal of cell-secreted metabolic waste. We demonstrated the versatile application of the platform by culturing single cells and keeping tissue microexplants viable for an extended period. The human cardiomyocyte AC16 cell line cultured in our platform revealed rapid cellular spheroid formation after 48 h and ~90% viability by day 7. Notably, we were able to culture gut microexplants for more than 2 weeks as demonstrated by immunofluorescent viability assay and prolonged contractility.

Transparent and Highly Flexible Hierarchically Structured Polydimethylsiloxane Surfaces Suppress Bacterial Attachment and Thrombosis Under Static and Dynamic Conditions.

The surface fouling of biomedical devices has been an ongoing issue in healthcare. Bacterial and blood adhesion in particular, severely impede the performance of such tools, leading to poor patient outcomes. Various structural and chemical modifications have been shown to reduce fouling, but all existing strategies lack the combination of physical, chemical, and economic traits necessary for widespread use. Herein, a lubricant infused, hierarchically micro- and nanostructured polydimethylsiloxane surface is presented. The surface is easy to produce and exhibits the high flexibility and optical transparency necessary for incorporation into various biomedical tools. Tests involving two clinically relevant, priority pathogens show up to a 98.5% reduction in the biofilm formation of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. With blood, the surface reduces staining by 95% and suppresses thrombin generation to background levels. Furthermore, the surface shows applicability within applications such as catheters, extracorporeal circuits, and microfluidic devices, through its effectiveness in dynamic conditions. The perfusion of bacterial media shows up to 96.5% reduction in bacterial adhesion. Similarly, a 95.8% reduction in fibrin networks is observed following whole blood perfusion. This substrate stands to hold high applicability within biomedical systems as a means to prevent fouling, thus improving performance.

3D Microvascularized Tissue Models by Laser‐Based Cavitation Molding of Collagen

Abstract3D tissue models recapitulating human physiology are important for fundamental biomedical research, and they hold promise to become a new tool in drug development. An integrated and defined microvasculature in 3D tissue models is necessary for optimal cell functions. However, conventional bioprinting only allows the fabrication of hydrogel scaffolds containing vessel‐like structures with large diameters (>100 µm) and simple geometries. Recent developments in laser photoablation enable the generation of this type of structure with higher resolution and complexity, but the photo‐thermal process can compromise cell viability and hydrogel integrity. To address these limitations, the present work reports in situ 3D patterning of collagen hydrogels by femtosecond laser irradiation to create channels and cavities with diameters ranging from 20 to 60 µm. In this process, laser irradiation of the hydrogel generates cavitation gas bubbles that rearrange the collagen fibers, thereby creating stable microchannels. Such 3D channels can be formed in cell‐ and organoid‐laden hydrogel without affecting the viability outside the lumen and can enable the formation of artificial microvasculature by the culture of endothelial cells and cell media perfusion. Thus, this method enables organs‐on‐a‐chip and 3D tissue models featuring complex microvasculature.

Precision radiotherapy by SPECT lung functional imaging in NSCLC

Background: Single Photon Emission Computed Tomography (SPECT) could be used to avoid the non-affected perfusion areas in patients with non-small-cell lung cancer (NSCLC) and to potentially reduce lung toxicity. The aim of this study is to compare dosimetric differences between two different 3D-conformal treatment plans, with and without CT/SPECT contribution. Methods: Simulation Computed tomography (CT) scans were accurately co-registered with SPECT scans and three different areas, based on SPECT intensity perfusion, were contoured: low perfusion (LP), medium perfusion (MP) and high perfusion (HP). Two different 3D-conformal plans, with co-planar and nonco-planar fields, were generated; one without SPECT information (anatomic plan), and one using the perfusion area identified with functional imaging (functional plan). Results: 9 patients were planned and a total of 18 plans were available for analysis. Anatomical and functional plans resulted in comparable planning target volume (PTV) coverage. In the functional plans, a significant reduction of dose in high perfusion areas was reported. The reduction of HP-V20 Gy values ranged from 15% to 8% (p = 0.046), the ipsiHP-V20 Gy from 38% to 22% (p = 0.028) and ipsiHP-Dmean reduction from 16 Gy to 12 Gy (p = 0.039). No significant differences in other organs at risk (OARs) metrics were reported between anatomical and functional plans. Conclusions: Despite the few cases reported, the strength of our study lies in the reported benefit of functional lung information in 3D conformal radiation planning, without compromising target coverage or worsening dose distribution to the OARs. There is an urgent need for prospective clinical and randomized trials in order to define the role of lung functional imaging in reducing toxicity in clinical practice.

Myocardial Perfusion Imaging Failed to Improve Patient Risk Classification Compared to the Revised Cardiac Risk Index for Early Cardiac Complications After Major Non-Cardiac Surgery

Cardiac evaluation is desirable before major non-cardiac surgery with myocardial perfusion imaging (MPI) frequently utilised for risk assessment. However, the prognostic utility of MPI above a simple clinical risk calculator, the Revised Cardiac Risk Index (RCRI), is unknown. We conducted a retrospective cohort study of patients who underwent MPI before major non-cardiac surgery, incorporating 635 surgical procedures in 629 patients over six years. Major adverse cardiac events (MACE) within 30 days of surgery, including any myocardial infarction, acute pulmonary oedema, ventricular arrhythmia, or cardiac death, occurred in 47 (7.4%) cases. We analysed predictive value of MPI for MACE using multivariable logistic regression and categorical net reclassification index. MPI-identified medium or large-sized reversible perfusion defects (p=0.02, odds ratio 2.9 [95% CI 1.1-7.1]) and RCRI score two or more (p=0.03, odds ratio 2.3 [(95% CI 1.1-4.8]) were significantly associated with MACE after adjusting for age, coronary revascularisation, surgical acuity, need for general anaesthesia, left ventricular ejection fraction (LVEF) and fixed perfusion defects. Net reclassification index comparing models with and without MPI risk factors (LVEF, reversible perfusion and fixed perfusion defects) did not significantly improve risk classification. MPI risk factors are weak predictors for early cardiac complications after major non-cardiac surgery and failed to improve patient risk classification beyond a simple risk assessment using age, RCRI and surgical priority. Clinicians should consider alternative risk assessment strategies because of MPI's poor prognostic utility and its associated time and financial costs.

Effect of experimental hyperglycemia on intestinal elimination and biliary excretion of ibuprofen enantiomers in hyperglycemic rats

Diabetic complications are mostly due to hyperglycemia. Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the activities of drug-metabolizing enzymes and membrane-integrated transporters, which can modify the fate of drugs and other xenobiotics. An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion of ibuprofen enantiomers in the rat. Experimental diabetes was induced by intravenous (i.v.) administration of streptozotocin. The intestinal perfusion medium contained 250 µM racemic ibuprofen. A validated isocratic chiral HPLC method with UV detection was developed to determine the amount of the two enantiomers in the intestinal perfusate and the bile. The results indicated that experimental diabetes doesn’t cause a statistically significant difference in the disappearance of ibuprofen enantiomers from the small intestine. Analysis of the bile samples detected only the (S)-IBP enantiomer. Excretion of the ibuprofen enantiomer to the bile decreased in experimental diabetes. The observed changes can affect the pharmacokinetics of drugs administered in hyperglycemic individuals.

Neuroprotective role of human neuritin 1 on axotomized human eyes pressurized using the Translaminar Autonomous System

Purpose: Primary open-angle glaucoma is a leading cause of vision loss worldwide characterized by retinal ganglion cell death (RGC) and visual field loss. Glaucoma is most commonly associated with an increase in intraocular pressure (IOP), and current clinical management is limited to lowering intraocular pressure. We have previously shown that the secreted human protein neuritin 1 (NRN1) exhibits neuroprotection, regeneration, and preservation of RGC function after RGC axonal injury. We propose to overcome RGC degeneration by over-expressing NRN1 in our novel ex-vivo perfused pressurized Translaminar Autonomous System (TAS). 
 Methods: Human donor eyes were obtained from eye banks. To model glaucomatous conditions, dissected human posterior eye cups were cultured in the TAS model with IOP:ICP conditions of 16:12 mmHg over 6 days. Flow rate of perfusion medium to the interior and exterior (around optic nerve) of the eye cup (IOP and ICP, respectively) was controlled by automated pumps to induce the translaminar pressure gradient.We assessed survival of RGCs with and without hNRN1 treatment by TaqMan array analysis of apoptosis/inflammatory markers (retina). Expression of FN and COLIV was examined from conditioned medium of both groups by Western analysis. 
 Results: We successfully maintained IOP: ICP differentials over 6 days. The RGCs degenerated faster in non-treated conditions compared to NRN1 treated eyes. In contrast to control, NRN1-treated group displayed differential expression of FN and COLIV. We observed dysregulated expression of GFAP, BAX and TLR4 expression between control and experimental conditions. 
 Conclusion: We utilized our unique TAS model to recreate the human optic nerve head environment of elevated translaminar pressure for testing a unique neuroprotective and regenerative gene therapy. Using this approach, we hope to identify human Neuritin 1 as a therapeutic gene to save the visual neurons and develop a new treatment for glaucoma.

Injectable, Pore-Forming, Perfusable Double-Network Hydrogels Resilient to Extreme Biomechanical Stimulations.

Biological tissues hinge on blood perfusion and mechanical toughness to function. Injectable hydrogels that possess both high permeability and toughness have profound impacts on regenerative medicine but remain a long‐standing challenge. To address this issue, injectable, pore‐forming double‐network hydrogels are fabricated by orchestrating stepwise gelation and phase separation processes. The interconnected pores of the resulting hydrogels enable direct medium perfusion through organ‐sized matrices. The hydrogels are amenable to cell encapsulation and delivery while promoting cell proliferation and spreading. They are also pore insensitive, tough, and fatigue resistant. When tested in biomimetic perfusion bioreactors, the hydrogels maintain physical integrity under prolonged, high‐frequency biomechanical stimulations (>6000 000 cycles at 120 Hz). The excellent biomechanical performance suggests the great potential of the new injectable hydrogel technology for repairing mechanically dynamic tissues, such as vocal folds, and other applications, such as tissue engineering, biofabrication, organs‐on‐chips, drug delivery, and disease modeling.

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.

Engineering a 3D human intracranial aneurysm model using liquid-assisted injection molding and tuned hydrogels

Physiologically relevant intracranial aneurysm (IA) models are crucially required to facilitate testing treatment options for IA. Herein, we report the development of a new in vitro tissue-engineered platform, which recapitulates the microenvironment, structure, and cellular complexity of native human IA. A new modified liquid-assisted injection molding technique was developed to fabricate a three-dimensional hollow IA model with clinically relevant IA dimensions within a mechanically tuned Gelatin Methacryloyl (GelMA) hydrogel. An endothelium lining was created inside the IA model by culturing human umbilical vein endothelial cells over pre-cultured human brain vascular smooth muscle cells. These cellularized IA models were subjected to medium perfusion at flow rates between 6.3 and 15.75 mL/min for inducing biomimetic vessel wall shear stress (10–25 dyn/cm2) to the cells for ten days. Both cell types maintained their secretome profiles and showed more than 96% viability, demonstrating the biocompatibility of the hydrogel during perfusion cell culture at such flow rates. Based on the characterized viscoelastic properties of the GelMA hydrogel and with the aid of a fluid-structure interaction model, the capability of the IA model in predicting the response of the IA to different fluid flow profiles was mathematically shown. With physiologically relevant behavior, our developed in vitro human IA model could allow researchers to better understand the pathophysiology and treatment of IA. Statement of significanceA three-dimensional intracranial aneurysm (IA) tissue model recapitulating the microenvironment, structure, and cellular complexity of native human IA was developed. • An endothelium lining was created inside the IA model over pre-cultured human brain vascular smooth muscle cells over at least 10-day successful culture. • The cells maintained their secretome profiles, demonstrating the biocompatibility of hydrogel during a long-term perfusion cell culture. • The IA model showed its capability in predicting the response of IA to different fluid flow profiles. • The cells in the vessel region behaved differently from cells in the aneurysm region due to alteration in hemodynamic shear stress. • The IA model could allow researchers to better understand the pathophysiology and treatment options of IA.

Modeling of the Human Bone Environment: Mechanical Stimuli Guide Mesenchymal Stem Cell-Extracellular Matrix Interactions.

In bone tissue engineering, the design of in vitro models able to recreate both the chemical composition, the structural architecture, and the overall mechanical environment of the native tissue is still often neglected. In this study, we apply a bioreactor system where human bone-marrow hMSCs are seeded in human femoral head-derived decellularized bone scaffolds and subjected to dynamic culture, i.e., shear stress induced by continuous cell culture medium perfusion at 1.7 mL/min flow rate and compressive stress by 10% uniaxial load at 1 Hz for 1 h per day. In silico modeling revealed that continuous medium flow generates a mean shear stress of 8.5 mPa sensed by hMSCs seeded on 3D bone scaffolds. Experimentally, both dynamic conditions improved cell repopulation within the scaffold and boosted ECM production compared with static controls. Early response of hMSCs to mechanical stimuli comprises evident cell shape changes and stronger integrin-mediated adhesion to the matrix. Stress-induced Col6 and SPP1 gene expression suggests an early hMSC commitment towards osteogenic lineage independent of Runx2 signaling. This study provides a foundation for exploring the early effects of external mechanical stimuli on hMSC behavior in a biologically meaningful in vitro environment, opening new opportunities to study bone development, remodeling, and pathologies.

P–459 Ex vivo perfusion of whole ewe ovaries with follicular maturation for up to seven days: towards the development of an alternative fertility preservation method

Abstract Study question To develop an alternative fertility preservation method for young female cancer patients based on an ex vivo perfusion of whole ovaries serving as a platform for future ovarian stimulation studies. Summary answer It is possible to maintain viable follicles and to retrieve oocytes after ex vivo perfusion of ewe ovaries for up to 7 days. What is known already Some progress has been made in terms of follicular growth and the isolation of mature oocytes in vitro. However, full development, from early follicular stages to a viable offspring, has only been described in rodent models. The complex events controlling follicular expansion and the long time required for folliculogenesis and oocyte maturity in large mammalian species creating challenges and limitations for in vitro studies. Ex vivo perfusion of a whole ovary could potentially be a solution by exploiting the intact ovarian architecture to support folliculogenesis and oocyte maturation. Study design, size, duration Thirty-one ewe ovaries were divided into 4 groups and ex vivo perfused in a bioreactor. Group 1 (n = 14) perfusion for 48 hours with no hormone supplementation; Group 2 (n = 4) perfusion 96–101 hours with follicle stimulating hormone (FSH); Group 3 (n = 3) perfusion 120–168 hours with human menopausal gonadotropin (hMG); Group 4 (n = 10) perfusion 72–144 hours with hMG. Participants/materials, setting, methods Ewe ovaries from sexually mature ewes were ex vivo perfused in a bioreactor under normothermic conditions for up to 7 days (max total 168 hours). Histomorphological, immunohistochemical, hormonal and biochemical analyses were performed to assess ovarian structure and viability after cold ischemia and after perfusion which was subsequently compared to control ovaries. Main results and the role of chance The perfused ovaries in group 2 and 3 showed no significant differences in follicular density, viability and oocyte quality after ischemia and perfusion compared to control ovaries. Estradiol and progesterone levels did not increase during the perfusion. The perfused ovaries in group 1 and 4 showed a significant decrease in the ovarian reserve and oocyte quality. In total, 16 GV-MI oocytes were retrieved from groups 3 and 4. Limitations, reasons for caution 1. Ovaries were retrieved from ewes of unknown cycle and reproductive history. 2. The perfusion medium was changed after 24 hours from perfusion start to remove detrimental metabolites and this could affect the measured concentrations of hormones and metabolites in the perfusion medium. Wider implications of the findings: These results pave the way to propose ex vivo perfusion as a good platform for fertility preservation studies on whole mammalian and human ovaries to retrieve fully mature oocytes. Trial registration number Not applicable

QS2: DIY Bioreactors for Tissue Engineering - An Ultra-low-cost Platform for Perfusion Cell Culture and Live Imaging

Purpose:While tissue engineering offers the promise of revolutionary innovation, scalable three-dimensional tissue culture is limited by the diffusion of nutrients and oxygen making media perfusion obligatory. Unfortunately, the cost of bioreactors for large construct tissue culture can be prohibitive, with a typical perfusion chamber costing several thousand dollars, and even small petri-dish-sized devices costing hundreds of dollars each. We have developed a low-cost perfusion setup that seals collagen-based perfusable cellular constructs within a sterile PDMS well between coverslips, allowing for repeated live-imaging of perfused 3D engineered tissues. Herein we describe fabrication of this novel system and validate its utility.Methods:Molds and frames were designed on 3D-modeling software (Fusion 360) and printed on a Prusa i3 MK3S 3D printer in poly(lactic acid) (PLA). Molds were filled with poly(dimethyl siloxane) (PDMS), which was cured to form chambers, bubble traps, mason jar lid chambers, and media reservoir lid adapters. In total, the tissue culture chamber device, mason jar lid inset, media reservoir lid, and bubble trap require 4, 1, 2, and 4 unique printed components, respectively.Results:Each perfusion chamber can be assembled for under 8 USD per device and reused repeatedly. The current model has a tissue chamber custom-built with 18x10x4 (LxWxH)mm3 dimensions, but this chamber can be readily customized to experiment-specific dimensions. These devices allow cellular hydrogel constructs to be maintained in a sterile environment after assembly, perfused at varying rates to expose cells to different levels of shear stress, and the cells can be intermittently imaged with light, fluorescent or confocal microscopy - an unparalleled benefit for monitoring of experiments and collection of timepoint imaging data. The perfusion circuit consists of autoclavable glass and PDMS components, including a bubble trap, a crucial component of the circuit for preventing air bubbles that can damage cells and block microchannels, and a lid adapter, which allows 50 mL conical tubes to serve as self-oxygenating media reservoirs. Media changes can be performed via peristaltic pump perfusion or with syringe-based cell culture techniques for static culture. Constructs have been perfused within standard incubators for up to 14 days demonstrating normal cell viability without contamination or evidence of infection, with longer perfusion culture intervals (>1 month) currently being tested.Conclusion:The increasing accessibility of 3D-modeling and 3D-printing has enabled rapid prototyping of devices to address the problems that we face as surgeon-scientists. We have developed a low-cost tissue-engineering perfusion circuit that facilitates 3D-tissue-culture while allowing for repeated live-imaging as the cultured tissue develops. The instructions for our setup can be utilized to replicate our devices in other labs, and these designs can be readily customized to meet the needs of specific experimental aims.

Perfusable System Using Porous Collagen Gel Scaffold Actively Provides Fresh Culture Media to a Cultured 3D Tissue.

Culturing three-dimensional (3D) tissues with an appropriate microenvironment is a critical and fundamental technology in broad areas of cutting-edge bioengineering research. In addition, many technologies have engineered tissue functions. However, an effective system for transporting nutrients, waste, or oxygen to affect the functions of cell tissues has not been reported. In this study, we introduce a novel system that employs diffusion and convection to enhance transportation. To demonstrate the concept of the proposed system, three layers of normal human dermal fibroblast cell sheets are used as a model tissue, which is cultured on a general dish or porous collagen scaffold with perfusable channels for three days with and without the perfusion of culture media in the scaffold. The results show that the viability of the cell tissue was improved by the developed system. Furthermore, glucose consumption, lactate production, and oxygen transport to the tissues were increased, which might improve the viability of tissues. However, mechanical stress in the proposed system did not cause damage or unintentional functional changes in the cultured tissue. We believe that the introduced culturing system potentially suggests a novel standard for 3D cell cultures.

Highly parallelized human embryonic stem cell differentiation to cardiac mesoderm in nanoliter chambers on a microfluidic chip

Human stem cell-derived cells and tissues hold considerable potential for applications in regenerative medicine, disease modeling and drug discovery. The generation, culture and differentiation of stem cells in low-volume, automated and parallelized microfluidic chips hold great promise to accelerate the research in this domain. Here, we show that we can differentiate human embryonic stem cells (hESCs) to early cardiac mesodermal cells in microfluidic chambers that have a volume of only 30 nanoliters, using discontinuous medium perfusion. 64 of these chambers were parallelized on a chip which contained integrated valves to spatiotemporally isolate the chambers and automate cell culture medium exchanges. To confirm cell pluripotency, we tracked hESC proliferation and immunostained the cells for pluripotency markers SOX2 and OCT3/4. During differentiation, we investigated the effect of different medium perfusion frequencies on cell reorganization and the expression of the early cardiac mesoderm reporter MESP1mCherry by live-cell imaging. Our study demonstrates that microfluidic technology can be used to automatically culture, differentiate and study hESC in very low-volume culture chambers even without continuous medium perfusion. This result is an important step towards further automation and parallelization in stem cell technology.

Hydrogen sulfide (H2S) reduces oxygen and ATP consumption in the isolated perfused pig kidney

Abstract Objective Organ donation after circulatory death [DCD] has the potential to reduce the shortage of kidneys available for transplantation. However, many DCD grafts are discarded because of long warm ischemia times. Strategies reducing oxygen demand may minimize damages caused by ischemia/reperfusion injury. Ex-vivo, Hydrogen sulfide (H2S) reduces oxygen and ATP consumption of the isolated perfused kidney, reduces inflammation and improves renal function following ischemia reperfusion injury in rodents. However, the benefits and applicability of H2S in clinically relevant model remain unknown. Methods To mimic DCD, pig kidneys underwent 0 or 60 min of warm ischemia, before oxygenated hypothermic machine perfusion (HMP). NaHS (100µM), an H2S donor, was added to the perfusion media or injected as an intra-arterial bolus before warm ischemia. After 2 hours of HMP, kidneys were transplanted and reperfused for 1 hour before harvest. Kidney function was assesses before, after and during ex vivo perfusion by measuring energy metabolites, Gadolinium elimination by pMRI and histopathological scoring. Results Warm ischemia (60 min) induced significant histological damages, delayed cortical and medullary Gadolinium elimination (perfusion), and reduced ATP levels, but not its precursors (AMP). As expected, ATP levels and kidney perfusion both inversely correlated with the severity of kidney histological injury. NaHS reduced metabolism during warm ischemia, and seemed to increase kidney ATP levels and viability after reperfusion. Conclusion Our preliminary data suggest that the H2S donor NaHS reduces kidney metabolism and protects from warm ischemia. Further experiments will identify the best administration protocol and the clinical relevance of H2S supplementation in the context of organ preservation.