This study is financially supported by ZonMw as part of the LSH 2Treat program (436001003) and the Dutch Kidney Foundation (14a2d507). N.A.K. acknowledges support from the European Research Council (851960). We gratefully acknowledge the Gravitation Program &#8220;Materials Driven Regeneration&#8221;, funded by the Netherlands Organization for Scientific Research (024.003.013).

The authors have nothing to disclose.

The bioreactor described herein allows for the systematic evaluation of the contributions of the individual and combined effects of shear stress and cyclic stretch on inflammation and tissue regeneration in tubular resorbable scaffolds. This approach also enables a large variety of analyses to be performed on vascular constructs, as exemplified in the representative results section. These results show the distinctive impact of the different hemodynamic loading regimes (i.e., different combinations of shear and stretch) on both growth and remodeling of the TEVG construct. These insights, collected via this in vitro platform, aid in the optimization of scaffold design parameters for newly developed in situ TEVGs. To ensure a proper experimental workflow, an understanding of the critical steps and the limitations of this protocol is important.
The most critical steps in the protocol are related to the application of stretch to the samples. For stretch application, it is essential that the setup is leak-free. There are two weak points in the system: the knots mounting the electrospun grafts to the pressure conduits and the connection between the bioreactor base and the flow culture chambers. As described in steps 2.5.2 and 2.5.4, multiple, tight knots have to be positioned exactly at the engraved groove. If the knot is not tight enough or is placed slightly above or below the groove, a slight leakage of hydraulic fluid into the flow culture chamber may occur. This leakage may be detected as a pressure drop in the hydraulic reservoir and a steadily increasing voltage on the strain pump to reach the set pressure value. Besides, this leads to a disturbed flow in the flow culture chamber, an increased risk of contamination of the cell culture, and a dilution of the medium. Once this happens, the flow culture chamber has to be taken out of the experiment, and the screw thread on the hydraulic reservoir should be closed with a white Luer cap. This major measure of taking out a complete sample, which is needed to ensure proper continuation of the experiment for the other seven flow culture chambers, emphasizes the importance of accurately placing tight knots, exactly in the engraved grooves. Only then can proper separation be ensured between the water in the hydraulic reservoir and the medium in the flow culture chambers. To improve robustness of the mounting of the scaffolds, the grooves in the pressure conduits will be made slightly deeper in a revised version of the bioreactor, allowing for easier and better knot placement and thus, secure separation of the hydraulic fluid from the medium.
Another potential source of leakage is between the screw threads of the bioreactor base and the white Luer connectors of the flow culture chamber. Because of possible wear and tear of the Teflon material, an extra silicone O-ring can be added to prevent leakage (step 5.2.3). Moreover, the Teflon bellow of the strain pump should be allowed to slightly expand O/N one day before the experiment (step 1.4). If leakage occurs, the lost hydraulic fluid should be compensated by adding a small amount of ultrapure water via one of the eight screw threads (use a syringe with needle and flexible wire). Place the flow culture chamber back with a small piece of parafilm between the screw thread and the white Luer connector on the bottom compartment of the flow culture chamber. To overcome this leakage issue in future experiments, the current Teflon screw threads on the bioreactor base will be replaced by stainless steel threads in the next-generation version of the bioreactor to prevent wear-out of the system.
The variation in stretch (Figure 3C) is larger than the variation in WSS (Figure 3D), as stretch is more difficult to control. Nevertheless, besides the measures to prevent leakage, there are other measures that limit the variation in stretch: (i) avoiding air bubbles in the flute (step 1.4 and 5.2.1), (ii) ensuring consistent pre-stretch among the different samples (step 2.5.3), and (iii) ensuring consistent scaffold properties among different samples (step 1.3).
Finally, extra caution is needed when mounting the electrospun scaffold onto the silicone tubing (step 2.3). Specifically, when the fragile electrospun scaffold needs to be pulled over the stretched silicone tubing, it is important not to apply too much force to prevent the electrospun graft from being damaged, especially on the inside of the electrospun graft. If damage occurs on the inside of the electrospun graft, either from forceful pulling or from too weak stretching of the silicone tube, the extent of the damage to the electrospun fibers only becomes visible after the construct is harvested and analyzed. In the current setup, the success of the seeding can only be confirmed upon immunofluorescence&#160;or immunohistochemical analysis, after sacrificing the sample. However, the seeding procedure with fibrin as the cell carrier is a well-established method67 that typically leads to a homogenous cell distribution for scaffolds with a sufficiently large pore size. One of the most important aspects with respect to cell seeding is to ensure that the scaffold is as dry as possible prior to seeding (step 4.4), to prevent the fibrin gel with the cells from passing through the wet scaffold, resulting in inhomogeneous seeding. Finally, although the decontamination method of the electrospun graft by UV radiation and dipping in 30% ethanol is not as stringent as the sterilization of electrospun grafts prepared for in vivo studies, which are often sterilized by ethylene oxide or gamma irradiation, it is sufficient for in vitro culture experiments that can last up to 20 days without any signs of contamination (for examples, see Figure 3, Figure 4, Figure 5, Figure 6, and Van Haaften and Wissing (2020)44). Moreover, the PCL-BU material that is used here, does not allow for long exposure to high ethanol concentrations. The most suitable sterilization method can be chosen depending on the material used.
In addition to the results of previously performed co-culture studies, a broader variety of studies can be performed with the same system. The system was previously employed to perform dynamic monocultures of myofibroblasts (Supplementary Figure 1A&#8211;B) and macrophages (Supplementary Figure 1C&#8211;D) to investigate the effects of hemodynamic loading on individual cell types and their paracrine signaling19,43. Different hemodynamic loading regimes resulted in clear actin fiber orientation in myofibroblasts (Supplementary Figure 1A) and distinctly different collagen deposition (Supplementary Figure 1B) after 10 days. The cytokine production by THP1-derived macrophages was drastically different between the different hemodynamic loads (Supplementary Figure 1C) and showed a more pro-inflammatory profile when loaded (Supplementary Figure 1D). Other validated possibilities include the application of oscillatory flow, by using an extra pump and fluidic unit. The viscosity of the medium can be increased towards the range of blood viscosity (e.g., by adding xanthan gum)69. Modulating the medium viscosity represents an additional variable to broaden the range of applicable shear stresses. Lastly, although the described protocol employs the &#39;Ibidi&#39; flow-conditioning setup, setups from other manufacturers can be used as well, as long as similar flow regimes can be applied.
One of the major advantages of using this bioreactor system is the relatively large construct (approximately 15 mm x 10.5 mm) that can be hemodynamically loaded, allowing for a wide variety of possible readout parameters to be extracted from a single sample. At the same time, the construct size may be viewed as a limitation as well, as this setup requires a relatively large amount of (sometimes costly) material, especially if primary cells are used, or if the culture medium requires costly additives. Moreover, the throughput of the setup is relatively low. Consequently, the current setup is particularly suited for hypothesis-driven research in which a limited number of variables is comprehensively tested, rather than the screening of a large number of variables with limited readout. For future experiments, small improvements on the current setup are being made to enable the option for mounting smaller scaffolds and downscaling the size of the medium reservoirs. With respect to the latter, the current volume of the medium reservoirs is required to enable sufficient volumetric flow rates to achieve the desired shear stresses. The required flow rates&#8212;and with that, the volume of the medium reservoirs&#8212;can be reduced by increasing the viscosity of the medium (e.g., by adding xanthan gum, as previously established69).
To conclude, this bioreactor allows for the quantification of the individual and combined effects of shear stress and cyclic stretch on tissue growth and remodeling on a wide variety of elastomeric 3D biomaterial scaffolds. The bioreactor can culture up to eight vascular constructs under various loading conditions. Owing to its design, the bioreactor is especially suited to study the interplay between hemodynamics and in situ vascular TE processes.

Cardiovascular tissue engineering (TE) is being pursued as an alternative treatment option to the currently used permanent cardiovascular prostheses (e.g., vascular grafts, heart valve replacements), which are suboptimal for large cohorts of patients1,2,3,4. Much sought-after applications include tissue-engineered vascular grafts (TEVGs)5,6 and heart valves (TEHVs)7,8. Most often, cardiovascular TE methodologies make use of resorbable biomaterials (either natural or synthetic) that serve as an instructive scaffold for the new tissue to be formed. The formation of new tissue can either be engineered completely in vitro, by seeding the scaffold with cells and culturing in a bioreactor prior to implantation (in vitro TE)9,10,11, or directly in situ, in which the synthetic scaffold is implanted without pre-culturing in order to induce the formation of new tissue directly in the body (in situ TE)12,13,14. For both in vitro and in situ cardiovascular TE approaches, successful functional regeneration is dominantly dependent on both the host immune response to the implanted construct and appropriate biomechanical loading.
The importance of biomechanical loading for cardiovascular TE is well-acknowledged15. In the case of cardiovascular implants, the cells that populate the scaffold are exposed to cyclic stretch and shear stresses that arise as a result of the hemodynamic environment. Numerous studies have reported the stimulatory effect of (cyclic) stretch on the formation of matrix components, such as collagen16,17,18,19, glycosaminoglycans (GAGs)20, and elastin21,22, by various cell types. For example, Huang et al. demonstrated that biaxial stretch elevated the deposition and organization of collagen and elastin in in vitro TEVGs by using a vascular bioreactor23. While the emphasis typically lies on stretch as the dominant load, these studies often make use of flow-driven bioreactors in which the sample is also exposed to shear flow. Although relatively little is known about the isolated influence of shear stresses on tissue formation and inflammation in 3D, some data are available. For example, Hinderer et al. and Eoh et al. demonstrated that shear flow, in addition to a 3D scaffold microstructure, was important for the formation of mature elastin by human vascular smooth muscle cells in an in vitro model system24,25. Altogether, these findings illustrate the relevance of both cyclic stretch and shear stress for cardiovascular TE.
Another important determinant for the success or failure of TE implants is the host&#8217;s immune response to the implanted graft26. This is particularly important for material-driven in situ TE strategies, which actually rely on the acute inflammatory response to the scaffold to kickstart the subsequent processes of cellular influx and endogenous tissue formation and remodeling27. The macrophage is a critical initiator of functional tissue regeneration, which has been shown by multiple studies28,29,30. Analogous to wound healing, the regeneration of tissue is governed by paracrine signaling between macrophages and tissue-producing cells such as fibroblasts and myofibroblasts31,32,33. In addition to coordinating new tissue deposition, macrophages are involved in the active resorption of foreign scaffold material34,35. As such, the in vitro macrophage response to a biomaterial has been identified as a predictive parameter for the in vivo success of implants36,37,38.
The macrophage response to an implanted scaffold is dependent on scaffold design features such as material composition and microstructure35,39,40. In addition to scaffold properties, the macrophage response to a scaffold and their crosstalk with myofibroblasts is also impacted by hemodynamic loads. For example, cyclic stretch was shown to be an important modulator of macrophage phenotype41,42,43,44 and the secretion of cytokines43,44,45,46 in 3D electrospun scaffolds. Using a co-culture system of macrophages and vascular smooth muscle cells, Battiston et al. demonstrated that the presence of macrophages led to increased levels of elastin and GAGs and that moderate levels of cyclic stretch (1.07&#8211;1.10) stimulated the deposition of collagen I and elastin47. In previous works, we have&#160;demonstrated that shear stress is an important determinant for monocyte recruitment into 3D electrospun scaffolds48,49, and that both shear stress and cyclic stretch impact the paracrine signaling between human monocytes and mesenchymal stromal cells50. Fahy et al. demonstrated that shear flow increased the secretion of pro-inflammatory cytokines by human monocytes51.
Taken together, the above evidence shows that an adequate understanding of and control over hemodynamic loads is crucial for cardiovascular TE, and that it is important to consider the inflammatory response to achieve this. Numerous bioreactors have been described previously for the in vitro52,53,54,55,56,57,58 or ex vivo59,60,61 culture of cardiovascular tissues. However, all these systems are designed to mimic the physiological hemodynamic loading conditions as much as possible. While this is highly valuable for the purpose of creating cardiovascular tissues in vitro or maintaining ex vivo cultures, such systems do not allow for systematic studies into the individual effects of individual cues. This is because the application of both cyclic stretch and shear stress in these bioreactors is driven by the same pressurized flow, which intrinsically links them. While microsystems that allow for accurate multi-cue mechanical manipulation have been described for 2D substrates62 or 3D hydrogel setups63,64, such setups do not allow for the incorporation of elastomeric 3D biomaterial scaffolds.
Here, we present the application of a tubular bioreactor system that uniquely enables the decoupling of shear stress and cyclic stretch and helps to mechanistically investigate their individual and combined effects. This system allows for testing of a broad variety of tissue engineered vascular grafts (e.g., synthetic or natural origin, different micro-architecture, various porosities). To effectively decouple the application of shear stress and stretch, the key concepts that the bioreactor uses are (1) separation of the control of shear stress and stretch using distinct pump systems and (2) stimulation of the scaffolds in an &#8216;inside-out&#8217; manner with computationally driven dimensions. Flow is applied on the outside surface of the tubular scaffold through the use of a flow pump, whereas circumferential stretch of the scaffold is induced by expanding a silicone tube on which the scaffold is mounted through the use of a separate strain pump. The dimensions of the silicone tube and the glass tube that contains the construct are carefully chosen and validated using computational fluid dynamics simulations, to ensure that the shear stress on the scaffold (due to flow) and the circumferential stretch (due to tube expansion) do not significantly affect each other. This inside-out design has several practical rationales. If stretch is applied by the luminal fluid pressure (similar to physiological loading), it&#160;inherently requires the sample design to be leak-free. In addition, the pressure required to stretch the sample would be completely determined by the sample stiffness, which may vary between samples and within a sample over time, making it difficult to control the stretch. This bioreactor mounts the tissue engineered graft around a silicone tube and allows for wall shear stress (WSS) application on the outer wall of the graft and pressurizes the graft from the inside. This way, equal loading conditions between samples and within samples over time can be ensured, and moreover, the samples are allowed to be leaky, as is common for porous vascular scaffolds19. This inside-out bioreactor is specifically intended for systematic studies on the effects of shear and/or stretch, rather than the engineering of a native-like blood vessel in vitro, for which traditional vascular bioreactor setups are more suitable. See Figure 1A&#8211;B for the bioreactor design drawings, and its corresponding Table 1 for a functional description and rationale behind the main components of the bioreactor.
The use of the bioreactor is demonstrated on the basis&#160;of a series of recent studies by our group in which we investigated the individual and combined influences of shear stress and cyclic stretch on inflammation and tissue formation in resorbable electrospun scaffolds for in situ cardiovascular tissue19,43,44. To that end, we used human macrophages and myofibroblasts either in mono- or in co-culture to simulate the various phases of the in situ regenerative cascade. We have&#160;demonstrated that cytokine secretion by human macrophages is distinctly impacted by both cyclic stretch and shear stress, affecting the matrix deposition and organization by human myofibroblasts in these scaffolds, both via paracrine signaling and direct contact19,43,44. Notably, these studies revealed that in the case of combined application of shear stress and stretch, the effects on tissue formation and inflammation are either dominated by one of the two loads, or there are synergistic effects of both loads. These findings illustrate the relevance of decoupling both loads to gain a better understanding of the contribution of the mechanical environment on TE processes. This understanding can be applied to systematically optimize scaffold design parameters in relevant hemodynamic loading regimes. In addition, the mechanistic data from such well-controlled environments may serve as input for numerical models that are being developed to predict the course of in situ tissue remodeling, as recently reported for TEVGs65 or TEHVs66, to further improve predictive capacity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >advanced Dulbecco&#8217;s modified EagleMedium (aDMEM)</td>
			<td >Gibco</td>
			<td >12491-015</td>
			<td >cell culture medium for fibroblasts</td>
		</tr>
		<tr >
			<td >Aqua Stabil</td>
			<td >Julabo</td>
			<td >8940012</td>
			<td >prevent microorganism growth in bioreactor-hydraulic reservoir</td>
		</tr>
		<tr >
			<td >Bovine fibrinogen</td>
			<td >Sigma</td>
			<td >F8630</td>
			<td >to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Bovine thrombin</td>
			<td >Sigma</td>
			<td >T4648</td>
			<td >to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Eppendorf</td>
			<td >5804</td>
			<td >to spin down cells and conditioned medium</td>
		</tr>
		<tr >
			<td >Clamp scissor - &#34;kelly forceps&#34;</td>
			<td >Almedic</td>
			<td >P-422</td>
			<td >clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >CO2 cell culture incubators</td>
			<td >Sanyo</td>
			<td >MCO-170AIC-PE</td>
			<td >for cell culturing</td>
		</tr>
		<tr >
			<td >Compressed air reservoir</td>
			<td >Festo</td>
			<td >CRVZS-5</td>
			<td >smoothing air pressure fluctuations and create time delays for pressure build-up</td>
		</tr>
		<tr >
			<td >Custom Matlab script to calculate the maximum stretches</td>
			<td >Matlab</td>
			<td >R2017. The Mathworks, Natick, MA</td>
			<td >calculate the minimum and maximum outer diameter of the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Data acquisition board</td>
			<td >National Instruments</td>
			<td >BNC-2090</td>
			<td >data processing in between amplifier system and computer</td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >VWR</td>
			<td >VWRK4096-9005</td>
			<td >to keep sterile working conditions</td>
		</tr>
		<tr >
			<td >Fetal bovine calf serum (FBS)</td>
			<td >Greiner</td>
			<td >758087</td>
			<td >cell culture medium supplement; serum-supplement</td>
		</tr>
		<tr >
			<td >Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushing</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >flow culture chamber compartments (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Glass Pasteur pipet</td>
			<td >Assistant</td>
			<td >HE40567002</td>
			<td >apply vacuum on electrospun scaffold (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Glass tubes of the flow culture chamber</td>
			<td >Custon made, Equipment &#38; Prototype Center, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td>
		</tr>
		<tr >
			<td >GlutaMax</td>
			<td >Gibco</td>
			<td >35050061</td>
			<td >cell culture medium amino acid supplement, minimizes ammonia build-up</td>
		</tr>
		<tr >
			<td >High speed camera</td>
			<td >MotionScope</td>
			<td >M-5</td>
			<td >to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td>
		</tr>
		<tr >
			<td >High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td>
			<td >Nikon</td>
			<td >JAA616AB</td>
			<td >to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td>
		</tr>
		<tr >
			<td >Hose clip</td>
			<td >ibidi GmbH</td>
			<td >10821</td>
			<td >block medium flow (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td>
			<td >ibidi GmbH</td>
			<td >10902</td>
			<td >set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.</td>
		</tr>
		<tr >
			<td >Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td>
			<td >Swann Morton</td>
			<td >0301; 0933</td>
			<td >to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td>
		</tr>
		<tr >
			<td >LabVIEW Software</td>
			<td >National Instruments</td>
			<td >version 2018</td>
			<td >to control the stretch applied to the scaffolds</td>
		</tr>
		<tr >
			<td >Laminar flow biosafety cabinet with UV light</td>
			<td >Labconco</td>
			<td >302310001</td>
			<td >to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td>
		</tr>
		<tr >
			<td >Large and small petri dishes</td>
			<td >Greiner</td>
			<td >664-160</td>
			<td >for sterile working conditions</td>
		</tr>
		<tr >
			<td >L-ascorbic acid 2-phosphate (vitamin C)</td>
			<td >Sigma</td>
			<td >A8960</td>
			<td >cell culture medium supplement, important for collagen production</td>
		</tr>
		<tr >
			<td >LED light cold source KL2500</td>
			<td >Zeiss</td>
			<td >Schott AG</td>
			<td >to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td>
		</tr>
		<tr >
			<td >Luer (female and male) locks and connectors, white luer caps</td>
			<td >ibidi GmbH</td>
			<td >various, see (https://ibidi.com/26-flow-accessories)</td>
			<td >to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Measuring amplifier (PICAS)</td>
			<td >PEEKEL instruments B.V.</td>
			<td >n.a.</td>
			<td >to amplify the signal from the pressure sensor and feedback to LabView</td>
		</tr>
		<tr >
			<td >Medium reservoir (large syringes 60 mL) and reservoir holders</td>
			<td >ibidi GmbH</td>
			<td >10974</td>
			<td >medium reservoir (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td>
			<td >Rubber BV</td>
			<td >1805</td>
			<td >to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing</td>
		</tr>
		<tr >
			<td >Motion Studio Software</td>
			<td >Idtvision</td>
			<td >2.15.00</td>
			<td >to make the high speed time lapse images for stretch monitoring</td>
		</tr>
		<tr >
			<td >Needle (19G)</td>
			<td >BD Microlance</td>
			<td >301700</td>
			<td >together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td>
		</tr>
		<tr >
			<td >Needle driver</td>
			<td >Adson</td>
			<td >2429218</td>
			<td >to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Paper tissues</td>
			<td >Kleenex</td>
			<td >38044001</td>
			<td >for cleaning of the equipment with 70% ethanol</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td >Sigma</td>
			<td >P7793-1EA</td>
			<td >quick fix if leakage occurs</td>
		</tr>
		<tr >
			<td >Penicillin/streptomycin (P/S)</td>
			<td >Lonza</td>
			<td >DE17-602E</td>
			<td >cell culture medium supplement; prevent bacterial contamination</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS)</td>
			<td >Sigma</td>
			<td >P4417-100TAB</td>
			<td >for storage and washing steps (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Plastic containers (60 mL) with red screw caps</td>
			<td >Greiner</td>
			<td >206202</td>
			<td >to prepare the fibrinogen solution</td>
		</tr>
		<tr >
			<td >Pneumatic cylinder</td>
			<td >Festo</td>
			<td >AEVC-20-10-I-P</td>
			<td >to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &#181;m wall thickness, 20 mm length)</td>
			<td >SyMO-Chem, Eindhoven, The Netherlands</td>
			<td >n.a.</td>
			<td >produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details</td>
		</tr>
		<tr >
			<td >Pressure conduit without holes (for static control)</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Pressure sensor and transducer</td>
			<td >BD</td>
			<td >TC-XX and P 10 EZ</td>
			<td >the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td>
		</tr>
		<tr >
			<td >Proportional air pressure control valve and pressure sensor</td>
			<td >Festo</td>
			<td >MPPES-3-1/8-2-010, 159596</td>
			<td >provides compressed air to the pneumatic actuated pump</td>
		</tr>
		<tr >
			<td >Roswell Park Memorial Institute 1640 (RPMI-1640)</td>
			<td >Gibco</td>
			<td >A1049101</td>
			<td >cell culture medium for monocyte/macrophage</td>
		</tr>
		<tr >
			<td >Safe lock Eppendorf tubes (1.5 mL)</td>
			<td >Eppendorf</td>
			<td >30120086</td>
			<td >multiple applications (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Sodium dodecyl sulfate solution 20%</td>
			<td >Sigma</td>
			<td >5030</td>
			<td >Used to clean materials, at a concentration of 0.1%.&#160;&#160;</td>
		</tr>
		<tr >
			<td >Silicone O-rings</td>
			<td >Technirub</td>
			<td >1250S</td>
			<td >to prevent leakage (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td>
			<td >Rubber BV</td>
			<td >1805</td>
			<td >to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Sterile tube (15 mL)</td>
			<td >Falcon</td>
			<td >352095</td>
			<td >multiple applications</td>
		</tr>
		<tr >
			<td >Suture, 5-0 prolene with pre-attached taper point needle</td>
			<td >Ethicon, Johnson&#38;Johnson</td>
			<td >EH7404H</td>
			<td >Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td>
		</tr>
		<tr >
			<td >Syringe (24 mL)</td>
			<td >B. Braun Melsungen AG</td>
			<td >2057932</td>
			<td >to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td>
		</tr>
		<tr >
			<td >Syringe filter (0.2 &#181;m)</td>
			<td >Satorius</td>
			<td >17597-K</td>
			<td >to filter the fibrinogen solution</td>
		</tr>
		<tr >
			<td >T150 cell culture flask with filter cap</td>
			<td >Nunc</td>
			<td >178983</td>
			<td >to degas culture medium</td>
		</tr>
		<tr >
			<td >T75 Cell culture flask with filter cap</td>
			<td >Nunc</td>
			<td >156499</td>
			<td >to culture static control samples</td>
		</tr>
		<tr >
			<td >Teflon bellow</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Tray (stainless steel)</td>
			<td >PolarWare</td>
			<td >15-248</td>
			<td >for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)</td>
		</tr>
		<tr >
			<td >Tweezers</td>
			<td >Wironit</td>
			<td >4910</td>
			<td >sterile handling of individual parts (autoclave at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Ultrapure water</td>
			<td >Stakpure</td>
			<td >Omniapure UV 18200002</td>
			<td >to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td>
		</tr>
		<tr >
			<td >UV light</td>
			<td >Philips</td>
			<td >TUV 30W/G30 T8</td>
			<td >for decontamination of grafts and bioreactor parts before seeding</td>
		</tr>
	</tbody>
</table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

a-multi-cue-bioreactor-to-evaluate-inflammatory-regenerative-capacity

Nanyang Technological University

Research

JoVE Journal

Engineering

5.4K Views.  Eindhoven University of Technology. The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

Video: A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Experimental Protocol to Investigate Particle Aerosolization of a Product Under Abrasion and Under Environmental Weathering

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering, which is a fundamental element to the approach of nanosafety-by-design of nanostructured products for their durable development. This approach is basically a preemptive one in which the focus is put on minimizing the emission of engineered nanomaterials' aerosols during the usage phase of the product's life cycle. This can be attained by altering its material properties during its design phase without compromising with any of its added benefits. In this article, an experimental protocol is presented to investigate the nanosafety-by-design of three commercial nanostructured products with respect to their mechanical solicitation and environmental weathering. The means chosen for applying the mechanical solicitation is an abrasion process and for the environmental weathering, it is an accelerated UV exposure in the presence of humidity and heat. The eventual emission of engineered nanomaterials is studied in terms of their number concentration, size distribution, morphology and chemical composition. The purpose of the protocol is to study the emission for test samples and experimental conditions which are corresponding to real life situations. It was found that the application of the mechanical stresses alone emits the engineered nanomaterials' aerosols in which the engineered nanomaterial is always embedded inside the product matrix, thus, a representative product element. In such a case, the emitted aerosols comprise of both nanoparticles as well as microparticles. But if the mechanical stresses are coupled with the environmental weathering, the experimental protocol reveals then the eventual deterioration of the product, after a certain weathering duration, may lead to the emission of the free engineered nanomaterial aerosols too.

In this article, an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering is presented. Results on the emission of engineered nanomaterials, in the form of aerosols are presented. The specific experimental set-up is described in detail.

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental ...

Design and Use of a Full Flow Sampling System (FFS) for the Quantification of Methane Emissions

The use of natural gas continues to grow with increased discovery and production of unconventional shale resources. At the same time, the natural gas industry faces continued scrutiny for methane emissions from across the supply chain, due to methane&#39;s relatively high global warming potential (25-84x that of carbon dioxide, according to the Energy Information Administration). Currently, a variety of techniques of varied uncertainties exists to measure or estimate methane emissions from components or facilities. Currently, only one commercial system is available for quantification of component level emissions and recent reports have highlighted its weaknesses.
In order to improve accuracy and increase measurement flexibility, we have designed, developed, and implemented a novel full flow sampling system (FFS) for quantification of methane emissions and greenhouse gases based on transportation emissions measurement principles. The FFS is a modular system that consists of an explosive-proof blower(s), mass airflow sensor(s) (MAF), thermocouple, sample probe, constant volume sampling pump, laser based greenhouse gas sensor, data acquisition device, and analysis software. Dependent upon the blower and hose configuration employed, the current FFS is able to achieve a flow rate ranging from 40 to 1,500 standard cubic feet per minute (SCFM). Utilization of laser-based sensors mitigates interference from higher hydrocarbons (C2+). Co-measurement of water vapor allows for humidity correction. The system is portable, with multiple configurations for a variety of applications ranging from being carried by a person to being mounted in a hand drawn cart, on-road vehicle bed, or from the bed of utility terrain vehicles (UTVs). The FFS is able to quantify methane emission rates with a relative uncertainty of &#177; 4.4%. The FFS has proven, real world operation for the quantification of methane emissions occurring in conventional and remote facilities.

Design and Use of a Full Flow Sampling System FFS for the Quantification of Methane Emissions

We have designed, developed, and implemented a novel full flow sampling system (FFS) for quantification of methane emissions and greenhouse gases from across the natural gas supply chain.

The use of natural gas continues to grow with increased discovery and production of unconventional shale resources. At the same time, the natural gas ...

A Robotic Platform to Study the Foreflipper of the California Sea Lion

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most of their thrust with their large foreflippers. This protocol describes a robotic platform designed to study the hydrodynamic performance of the swimming California sea lion (Zalophus californianus). The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;). The kinematics of the sea lion&#39;s propulsive stroke are extracted from video data of unmarked, non-research sea lions at the Smithsonian Zoological Park (SNZ). Those data form the basis of the actuation motion of the robotic flipper presented here. The geometry of the robotic flipper is based a on high-resolution laser scan of a foreflipper of an adult female sea lion, scaled to about 60% of the full-scale flipper. The articulated model has three joints, mimicking the elbow, wrist and knuckle joint of the sea lion foreflipper. The robotic platform matches dynamics properties&#8212;Reynolds number and tip speed&#8212;of the animal when accelerating from rest. The robotic flipper can be used to determine the performance (forces and torques) and resulting flowfields.

A robotic platform is described that will be used to study the hydrodynamic performance&#8212;forces and flowfields&#8212;of the swimming California sea lion. The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;).

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most ...

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each thermoplastic of interest. Solvent bonding is a simple and versatile method that can be used to fabricate devices from a variety of plastics. An appropriate solvent is added between two device layers to be bonded, and heat and pressure are applied to the device to facilitate the bonding. By using an appropriate combination of solvent, plastic, heat, and pressure, the device can be sealed with a high quality bond, characterized as having high bond coverage, bond strength, optical clarity, durability over time, and low deformation or damage to microfeature geometry. We describe the procedure for bonding devices made from two popular thermoplastics, poly(methyl-methacrylate) (PMMA), and cyclo-olefin polymer (COP), as well as a variety of methods to characterize the quality of the resulting bonds, and strategies to troubleshoot low quality bonds. These methods can be used to develop new solvent bonding protocols for other plastic-solvent systems.

Solvent bonding is a simple and versatile method for fabricating thermoplastic microfluidic devices with high quality bonds. We describe a protocol to achieve strong, optically clear bonds in PMMA and COP microfluidic devices that preserve microfeature details, by a judicious combination of pressure, temperature, an appropriate solvent, and device geometry.

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each ...

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions

To help address production issues in refineries caused by the fouling of process units and lines, we have developed a setup as well as a method to visualize the behavior of petroleum samples under process conditions. The experimental setup relies on a custom-built micro-reactor fitted with a sapphire window at the bottom, which is placed over the objective of an inverted microscope equipped with a cross-polarizer module. Using reflection microscopy enables the visualization of opaque samples, such as petroleum vacuum residues, or asphaltenes. The combination of the sapphire window from the micro-reactor with the cross-polarizer module of the microscope on the light path allows high-contrast imaging of isotropic and anisotropic media. While observations are carried out, the micro-reactor can be heated to the temperature range of cracking reactions (up to 450 &#176;C), can be subjected to H2 pressure relevant to hydroconversion reactions (up to 16 MPa), and can stir the sample by magnetic coupling. 
Observations are typically carried out by taking snapshots of the sample under cross-polarized light at regular time intervals. Image analyses may not only provide information on the temperature, pressure, and reactive conditions yielding phase separation, but may also give an estimate of the evolution of the chemical (absorption/reflection spectra) and physical (refractive index) properties of the sample before the onset of phase separation.

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions

This article describes a setup and method for the in situ visualization of oil samples under a variety of temperature and pressure conditions that aim to emulate refining and upgrading processes. It is primarily used for studying isotropic and anisotropic media involved in the fouling behavior of petroleum feeds.

To help address production issues in refineries caused by the fouling of process units and lines, we have developed a setup as well as a method to ...

A Novel Biaxial Testing Apparatus for the Determination of Forming Limit under Hot Stamping Conditions

The hot stamping and cold die quenching process is increasingly used to form complex shaped structural components of sheet metals. Conventional experimental approaches, such as out-of-plane and in-plane tests, are not applicable to the determination of forming limits when heating and rapid cooling processes are introduced prior to forming for tests conducted under hot stamping conditions. A novel in-plane biaxial testing system was designed and used for the determination of forming limits of sheet metals at various strain paths, temperatures, and strain rates after heating and cooling processes in a resistance heating uniaxial testing machine. The core part of the biaxial testing system is a biaxial apparatus, which transfers a uniaxial force provided by the uniaxial testing machine to a biaxial force. One type of cruciform specimen was designed and verified for the formability test of aluminum alloy 6082 using the proposed biaxial testing system. The digital image correlation (DIC) system with a high-speed camera was used for taking strain measurements of a specimen during a deformation. The aim of proposing this biaxial testing system is to enable the forming limits of an alloy to be determined at various temperatures and strain rates under hot stamping conditions.

This protocol proposes a novel biaxial testing system used on a resistance heating uniaxial tensile test machine in order to determine the forming limit diagram (FLD) of sheet metals under hot stamping conditions.

The hot stamping and cold die quenching process is increasingly used to form complex shaped structural components of sheet metals. Conventional ...

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

Flow-assisted Dielectrophoresis: A Low Cost Method for the Fabrication of High Performance Solution-processable Nanowire Devices

Flow-assisted dielectrophoresis (DEP) is an efficient self-assembly method for the controllable and reproducible positioning, alignment, and selection of nanowires. DEP is used for nanowire analysis, characterization, and for solution-based fabrication of semiconducting devices. The method works by applying an alternating electric field between metallic electrodes. The nanowire formulation is then dropped onto the electrodes which are on an inclined surface to create a flow of the formulation using gravity. The nanowires then align along the gradient of the electric field and in the direction of the liquid flow. The frequency of the field can be adjusted to select nanowires with superior conductivity and lower trap density. 
In this work, flow-assisted DEP is used to create nanowire field effect transistors. Flow-assisted DEP has several advantages: it allows selection of nanowire electrical properties; control of nanowire length; placement of nanowires in specific areas; control of orientation of nanowires; and control of nanowire density in the device. 
The technique can be expanded to many other applications such as gas sensors and microwave switches. The technique is efficient, quick, reproducible, and it uses a minimal amount of dilute solution making it ideal for the testing of novel nanomaterials. Wafer scale assembly of nanowire devices can also be achieved using this technique, allowing large numbers of samples for testing and large-area electronic applications.

In this paper, flow assisted dielectrophoresis is demonstrated for the self-assembly of nanowire devices. The fabrication of a silicon nanowire field effect transistor is shown as an example.

Flow-assisted dielectrophoresis (DEP) is an efficient self-assembly method for the controllable and reproducible positioning, alignment, and selection of ...

Measurements of Waves in a Wind-wave Tank Under Steady and Time-varying Wind Forcing

This manuscript describes an experimental procedure that allows obtaining diverse quantitative information on temporal and spatial evolution of water waves excited by time-dependent and steady wind forcing. Capacitance-type wave gauge and Laser Slope Gauge (LSG) are used to measure instantaneous water surface elevation and two components of the instantaneous surface slope at a number of locations along the test section of a wind-wave facility. The computer-controlled blower provides airflow over the water in the tank whose rate can vary in time. In the present experiments, the wind speed in the test section initially increases quickly from rest to the set value. It is then kept constant for the prescribed duration; finally, the airflow is shut down. At the beginning of each experimental run, the water surface is calm and there is no wind. Operation of the blower is initiated simultaneously with the acquisition of data provided by all sensors by a computer; data acquisition continues until the waves in the tank fully decay. Multiple independent runs performed under identical forcing conditions allow determining statistically reliable ensemble-averaged characteristic parameters that quantitatively describe wind-waves&#39; variation in time for the initial development stage as a function of fetch. The procedure also allows characterizing the spatial evolution of the wave field under steady wind forcing, as well as decay of waves in time, once the wind is shut down, as a function of fetch.

This manuscript describes a fully computer-controlled procedure that allows obtaining reliable statistical parameters from experiments of water waves excited by steady and unsteady wind forcing in a small-scale facility.

This manuscript describes an experimental procedure that allows obtaining diverse quantitative information on temporal and spatial evolution of water ...