Neuroregenerative Potential of Prenyl- and Pyranochalcones: A Structure-Activity Study.

Loss of neuronal tissue is a hallmark of age-related neurodegenerative diseases. Since adult neurogenesis has been confirmed in the human brain, great interest has arisen in substances stimulating the endogenous neuronal regeneration mechanism based on adult neural stem cells. Medicinal plants are a valuable source of neuroactive small molecules. In the structure-activity study presented here, the activities of prenyl- and pyranochalcones were compared to each other, using a differentiation assay based on the doublecortin promoter sequences. The latter revealed that the pyrano ring is a crucial structural element for the induction of neuronal differentiation of adult neural stem cells, while compounds with a prenyl group show significantly lower activities. Furthermore, a decrease of pro-differentiation activity was observed following structural modifications, such as substitutions on the pyrano ring and on the B-ring of the chalcone. We also initiated the elucidation of the structural characteristics of the newly discovered lead substance xanthohumol C, which correlated with the activation of the doublecortin promoter during neuronal differentiation.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

Cre mRNA Is Not Transferred by EVs from Endothelial and Adipose-Derived Stromal/Stem Cells during Vascular Network Formation.

Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs.

Aging restricts the ability of mesenchymal stem cells to promote the generation of oligodendrocytes during remyelination.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that leads to severe neurological deficits. Due to their immunomodulatory and neuroprotective activities and their ability to promote the generation of oligodendrocytes, mesenchymal stem cells (MSCs) are currently being developed for autologous cell therapy in MS. As aging reduces the regenerative capacity of all tissues, it is of relevance to investigate whether MSCs retain their pro-oligodendrogenic activity with increasing age. We demonstrate that MSCs derived from aged rats have a reduced capacity to induce oligodendrocyte differentiation of adult CNS stem/progenitor cells. Aging also abolished the ability of MSCs to enhance the generation of myelin-like sheaths in demyelinated cerebellar slice cultures. Finally, in a rat model for CNS demyelination, aging suppressed the capability of systemically transplanted MSCs to boost oligodendrocyte progenitor cell (OPC) differentiation during remyelination. Thus, aging restricts the ability of MSCs to support the generation of oligodendrocytes and consequently inhibits their capacity to enhance the generation of myelin-like sheaths. These findings may impact on the design of therapies using autologous MSCs in older MS patients.

Improvement of adipose tissue-derived cells by low-energy extracorporeal shock wave therapy.

BACKGROUND: Cell-based therapies with autologous adipose tissue-derived cells have shown great potential in several clinical studies in the last decades. The majority of these studies have been using the stromal vascular fraction (SVF), a heterogeneous mixture of fibroblasts, lymphocytes, monocytes/macrophages, endothelial cells, endothelial progenitor cells, pericytes and adipose-derived stromal/stem cells (ASC) among others. Although possible clinical applications of autologous adipose tissue-derived cells are manifold, they are limited by insufficient uniformity in cell identity and regenerative potency. METHODS: In our experimental set-up, low-energy extracorporeal shock wave therapy (ESWT) was performed on freshly obtained human adipose tissue and isolated adipose tissue SVF cells aiming to equalize and enhance stem cell properties and functionality. RESULTS: After ESWT on adipose tissue we could achieve higher cellular adenosine triphosphate (ATP) levels compared with ESWT on the isolated SVF as well as the control. ESWT on adipose tissue resulted in a significantly higher expression of single mesenchymal and vascular marker compared with untreated control. Analysis of SVF protein secretome revealed a significant enhancement in insulin-like growth factor (IGF)-1 and placental growth factor (PLGF) after ESWT on adipose tissue. DISCUSSION: Summarizing we could show that ESWT on adipose tissue enhanced the cellular ATP content and modified the expression of single mesenchymal and vascular marker, and thus potentially provides a more regenerative cell population. Because the effectiveness of autologous cell therapy is dependent on the therapeutic potency of the patient's cells, this technology might raise the number of patients eligible for autologous cell transplantation.

The adipose tissue-derived stromal vascular fraction cells from lipedema patients: Are they different?

BACKGROUND AIMS: Lipedema is a hormone-related disease of women characterized by enlargement of the extremities caused by subcutaneous deposition of adipose tissue. In healthy patients application of autologous adipose tissue-derived cells has shown great potential in several clinical studies for engrafting of soft tissue reconstruction in recent decades. The majority of these studies have used the stromal vascular fraction (SVF), a heterogeneous cell population containing adipose-derived stromal/stem cells (ASC), among others. Because cell identity and regenerative properties might be affected by the health condition of patients, we characterized the SVF cells of 30 lipedema patients in comparison to 22 healthy patients. METHODS: SVF cells were analyzed regarding cell yield, viability, adenosine triphosphate content, colony forming units and proliferative capacity, as well as surface marker profile and differentiation potential in vitro. RESULTS: Our results demonstrated a significantly enhanced SVF cell yield isolated from lipedema compared with healthy patients. In contrast, the adipogenic differentiation potential of SVF cells isolated from lipedema patients was significantly reduced compared with healthy patients. Interestingly, expression of the mesenchymal marker CD90 and the endothelial/pericytic marker CD146 was significantly enhanced when isolated from lipedema patients. DISCUSSION: The enhanced number of CD90+ and CD146+ cells could explain the increased cell yield because the other tested surface marker were not reduced in lipedema patients. Because the cellular mechanism and composition in lipedema is largely unknown, our findings might contribute to a better understanding of its etiology.

Unlocking Potential: Low Bovine Serum Albumin Enhances the Chondrogenicity of Human Adipose-Derived Stromal Cells in Pellet Cultures.

Bovine serum albumin (BSA) plays a crucial role in cell culture media, influencing cellular processes such as proliferation and differentiation. Although it is commonly included in chondrogenic differentiation media, its specific function remains unclear. This study explores the effect of different BSA concentrations on the chondrogenic differentiation of human adipose-derived stromal/stem cells (hASCs). hASC pellets from six donors were cultured under chondrogenic conditions with three BSA concentrations. Surprisingly, a lower BSA concentration led to enhanced chondrogenesis. The degree of this effect was donor-dependent, classifying them into two groups: (1) high responders, forming at least 35% larger, differentiated pellets with low BSA in comparison to high BSA; (2) low responders, which benefitted only slightly from low BSA doses with a decrease in pellet size and marginal differentiation, indicative of low intrinsic differentiation potential. In all cases, increased chondrogenesis was accompanied by hypertrophy under low BSA concentrations. To the best of our knowledge, this is the first study showing improved chondrogenicity and the tendency for hypertrophy with low BSA concentration compared to standard levels. Once the tendency for hypertrophy is understood, the determination of BSA concentration might be used to tune hASC chondrogenic or osteogenic differentiation.

Single molecule studies of dynamic platelet interactions with endothelial cells.

A biotechnological platform consisting of two-color 3D super-resolution readout and a microfluidic system was developed to investigate platelet interaction with a layer of perfused endothelial cells under flow conditions. Platelet activation has been confirmed via CD62P clustering on the membrane and mitochondrial morphology of ECs at the single cell level were examined using 3D two-color single-molecule localization microscopy and classified applying machine learning. To compare binding of activated platelets to intact or stressed ECs, a femtosecond laser was used to induced damage to single ECs within the perfused endothelial layer. We observed that activated platelets bound to the perfused ECs layer preferentially in the proximity to single stressed ECs. Platelets activated under flow were ∼6 times larger compared to activated ones under static conditions. The CD62P expression indicated more CD62P proteins on membrane of dynamically activated platelets, with a tendency to higher densities at the platelet/EC interface. Platelets activated under static conditions showed a less pronounced CD62P top/bottom asymmetry. The clustering of CD62P in the platelet membrane differs depending on the activation conditions. Our results confirm that nanoscopic analysis using two-color 3D super-resolution technology can be used to assess platelet interaction with a stressed endothelium under dynamic conditions.

Intra-Articular Application of Autologous, Fat-Derived Orthobiologics in the Treatment of Knee Osteoarthritis: A Systematic Review.

The aim of this study was to review the current literature regarding the effects of intra-articularly applied, fat-derived orthobiologics (FDO) in the treatment of primary knee osteoarthritis over a mid-term follow-up period. A systematic literature search was conducted on the online databases of Scopus, PubMed, Ovid MEDLINE, and Cochrane Library. Studies investigating intra-articularly applied FDO with a minimum number of 10 knee osteoarthritis patients, a follow-up period of at least 2 years, and at least 1 reported functional parameter (pain level or Patient-Reported Outcome Measures) were included. Exclusion criteria encompassed focal chondral defects and techniques including additional arthroscopic bone marrow stimulation. In 28 of 29 studies, FDO showed a subjective improvement in symptoms (pain and Patient-Reported Outcome Measures) up to a maximum follow-up of 7.2 years. Radiographic cartilage regeneration up to 3 years postoperatively, as well as macroscopic cartilage regeneration investigated via second-look arthroscopy, may corroborate the favorable clinical findings in patients with knee osteoarthritis. The methodological heterogeneity in FDO treatments leads to variations in cell composition and represents a limitation in the current state of knowledge. However, this systematic review suggests that FDO injection leads to beneficial mid-term results including symptom reduction and preservation of the affected joint in knee osteoarthritis patients.

Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue.

Acting as the largest energy reservoir in the body, adipose tissue is involved in longevity and progression of age-related metabolic dysfunction. Here, cellular senescence plays a central role in the generation of a pro-inflammatory environment and in the evolution of chronic diseases. Within the complexity of a tissue, identification and targeting of senescent cells is hampered by their heterogeneity. In this study, we generated stress-induced premature senescence 2D and 3D in vitro models for the stromal vascular niche of human adipose tissue. We established treatment conditions for senescence induction using Doxorubicin (Dox), starting from adipose-derived stromal/stem cells (ASCs), which we adapted to freshly isolated microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. Senescence hallmarks for the established in vitro models were verified on different cellular levels, including morphology, cell cycle arrest, senescence-associated ß-galactosidase activity (SA-ßgal) and gene expression. Two subsequent exposures with 200 nM Dox for six days were suitable to induce senescence in our in vitro models. We demonstrated induction of senescence in the 2D in vitro models through SA-ßgal activity, at the mRNA level (LMNB1, CDK1, p21) and additionally by G2/M phase cell cycle arrest in ASCs. Significant differences in Lamin B1 and p21 protein expression confirmed senescence in our MT-SVF 3D model. MT-SVF 3D cultures were composed of multiple cell types, including CD31, CD34 and CD68 positive cells, while cell death remained unaltered upon senescence induction. As heterogeneity and complexity of adipose tissue senescence is given by multiple cell types, our established senescence models that represent the perivascular niche embedded within its native extracellular matrix are highly relevant for future clinical studies.

Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema.

Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease.

Occurrence of Lymphangiogenesis in Peripheral Nerve Autografts Contrasts Schwann Cell-Induced Apoptosis of Lymphatic Endothelial Cells In Vitro.

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types' migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC-LEC interaction.

Label-free characterization of an extracellular vesicle-based therapeutic.

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers. Furthermore, analysis of QCM dissipation versus frequency allowed us to quantitatively determine the ratio of marker-specific EVs versus non-vesicular particles (NVPs) - a parameter that cannot be obtained by any other technique so far. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and NVPs in our EV preparations. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV therapeutics and opens the door to improved quality control.

Dual Channel Microfluidics for Mimicking the Blood-Brain Barrier.

High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in in vitro models of the endothelium. In this study, we demonstrate a microfluidic system consisting of two channels horizontally separated by a cell-growth-promoting membrane. Its design allows for high-resolution (down to single-molecule level) imaging using a high numerical aperture objective with a short working distance. To reduce optical aberrations and enable single-molecule-sensitive imaging, an observation window was constructed in the membrane via laser cutting with subsequent structuring using 3D multiphoton lithography for improved cell growth. The upper channel was loaded with endothelial cells under flow conditions, which showed polarization and junction formation. A coculture of human vascular endothelial cells with pericytes was developed that mimics the blood-brain barrier. Finally, this dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles.

SVF-derived extracellular vesicles carry characteristic miRNAs in lipedema.

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs (miRNAs) from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular miRNAs in concentrated conditioned medium (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEVmiRNA profiles - but not cCMmiRNAs - were impacted by lipedema. Seven sEVmiRNAs (miR-16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR-144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy individuals, whereas only one cCMmiRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

Microvascular Networks From Endothelial Cells and Mesenchymal Stromal Cells From Adipose Tissue and Bone Marrow: A Comparison.

A promising approach to overcome hypoxic conditions in tissue engineered constructs is to use the potential of endothelial cells (EC) to form networks in vitro when co-cultured with a supporting cell type in a 3D environment. Adipose tissue-derived stromal cells (ASC) as well as bone marrow-derived stromal cells (BMSC) have been shown to support vessel formation of EC in vitro, but only very few studies compared the angiogenic potential of both cell types using the same model. Here, we aimed at investigating the ability of ASC and BMSC to induce network formation of EC in a co-culture model in fibrin. While vascular structures of BMSC and EC remained stable over the course of 3 weeks, ASC-EC co-cultures developed more junctions and higher network density within the same time frame. Both co-cultures showed positive staining for neural glial antigen 2 (NG2) and basal lamina proteins. This indicates that vessels matured and were surrounded by perivascular cells as well as matrix molecules involved in stabilization. Gene expression analysis revealed a significant increase of vascular endothelial growth factor (VEGF) expression in ASC-EC co-culture compared to BMSC-EC co-culture. These observations were donor-independent and highlight the importance of organotypic cell sources for vascular tissue engineering.

Every Breath You Take: Non-invasive Real-Time Oxygen Biosensing in Two- and Three-Dimensional Microfluidic Cell Models.

Knowledge on the availability of dissolved oxygen inside microfluidic cell culture systems is vital for recreating physiological-relevant microenvironments and for providing reliable and reproducible measurement conditions. It is important to highlight that in vivo cells experience a diverse range of oxygen tensions depending on the resident tissue type, which can also be recreated in vitro using specialized cell culture instruments that regulate external oxygen concentrations. While cell-culture conditions can be readily adjusted using state-of-the-art incubators, the control of physiological-relevant microenvironments within the microfluidic chip, however, requires the integration of oxygen sensors. Although several sensing approaches have been reported to monitor oxygen levels in the presence of cell monolayers, oxygen demands of microfluidic three-dimensional (3D)-cell cultures and spatio-temporal variations of oxygen concentrations inside two-dimensional (2D) and 3D cell culture systems are still largely unknown. To gain a better understanding on available oxygen levels inside organ-on-a-chip systems, we have therefore developed two different microfluidic devices containing embedded sensor arrays to monitor local oxygen levels to investigate (i) oxygen consumption rates of 2D and 3D hydrogel-based cell cultures, (ii) the establishment of oxygen gradients within cell culture chambers, and (iii) influence of microfluidic material (e.g., gas tight vs. gas permeable), surface coatings, cell densities, and medium flow rate on the respiratory activities of four different cell types. We demonstrate how dynamic control of cyclic normoxic-hypoxic cell microenvironments can be readily accomplished using programmable flow profiles employing both gas-impermeable and gas-permeable microfluidic biochips.

Correction to: The role of fibrinolysis inhibition in engineered vascular networks derived from endothelial cells and adipose-derived stem cells.

The original article [1] contains numerous value errors in the graphs in Fig. 2b regarding the markers describing the values for total tubule length and mean tubule length without aprotinin at 2.5 mg/ml concentration of fibrinogen. The corrected version of this figure can be viewed ahead.

The role of fibrinolysis inhibition in engineered vascular networks derived from endothelial cells and adipose-derived stem cells.

BACKGROUND: Co-cultures of endothelial cells with mesenchymal stem cells currently represent one of the most promising approaches in providing oxygen and nutrient supply for microvascular tissue engineering. Still, to translate this model into clinics several in vitro parameters including growth medium and scaffold degradation need to be fine-tuned. METHODS: We recently described the co-culture of adipose-derived stem cells with endothelial cells in fibrin, resulting in capillary formation in vitro as well as their perfusion in vivo. Here, we aimed to further characterise microvascular tube formation in fibrin by determining the role of scaffold degradation, thrombin concentration and culture conditions on vascularisation. RESULTS: We observed that inhibition of cell-mediated fibrin degradation by the commonly used inhibitor aprotinin resulted in impaired vascular network formation. Aprotinin had no effect on laminin and collagen type IV deposition or formation of tube-like structures in scaffold-free co-culture, indicating that poor vascularisation of fibrin clots is primarily caused by inhibition of plasminogen-driven fibrinolysis. Co-culture in plasminogen- and factor XIII-depleted fibrin did not result in different vascular network density compared to controls. Furthermore, we demonstrate that thrombin negatively affects vascular network density at high concentrations. However, only transient activation of incorporated endothelial cells by thrombin could be observed, thus excluding a long-term inflammatory response in tissue-engineered micro-capillaries. Finally, we show that vascularisation of fibrin scaffolds in basal medium is undermined because of increased fibrinolytic activity leading to scaffold destabilisation without aprotinin. CONCLUSIONS: Taken together, our data reveal a critical role of fibrinolysis inhibition in in vitro cell-mediated vascularisation of fibrin scaffolds.

Engineering of three-dimensional pre-vascular networks within fibrin hydrogel constructs by microfluidic control over reciprocal cell signaling.

Reengineering functional vascular networks in vitro remains an integral part in tissue engineering, since the incorporation of non-perfused tissues results in restricted nutrient supply and limited waste removal. Microfluidic devices are routinely used to mimic both physiological and pathological vascular microenvironments. Current procedures either involve the investigation of growth factor gradients and interstitial flow on endothelial cell sprouting alone or on the heterotypic cell-cell interactions between endothelial and mural cells. However, limited research has been conducted on the influence of flow on co-cultures of these cells. Here, we exploited the ability of microfluidics to create and monitor spatiotemporal gradients to investigate the influence of growth factor supply and elution on vascularization using static as well as indirect and direct flow setups. Co-cultures of human adipose-derived stem/stromal cells and human umbilical vein endothelial cells embedded in fibrin hydrogels were found to be severely affected by diffusion limited growth factor gradients as well as by elution of reciprocal signaling molecules during both static and flow conditions. Static cultures formed pre-vascular networks up to a depth of 4 mm into the construct with subsequent decline due to diffusion limitation. In contrast, indirect flow conditions enhanced endothelial cell sprouting but failed to form vascular networks. Additionally, complete inhibition of pre-vascular network formation was observable for direct application of flow through the hydrogel with decline of endothelial cell viability after seven days. Using finite volume CFD simulations of different sized molecules vital for pre-vascular network formation into and out of the hydrogel constructs, we found that interstitial flow enhances growth factor supply to the cells in the bulk of the chamber but elutes cellular secretome, resulting in truncated, premature vascularization.