Cell Isolation and Culture
Human umbilical vein endothelial cells (HUVECs) were derived from umbilical cords according to established protocols.15 Umbilical cords were kindly provided by the Department of Gynecology and Obstetrics (RWTH Aachen University Hospital) following approval by the ethics committee at the medical faculty of the RWTH Aachen University (EK 019) and informed consent provided by the patients. Briefly, the vein was washed with phosphate-buffered saline (PBS, Thermo Fisher Scientific) and HUVECs were isolated by enzymatic dissociation in 1 mg mL−1 collagenase (Sigma-Aldrich). These endothelial cells (ECs) were cultured in flasks coated with 2% gelatin and incubated with EC growth medium 2 (EGM-2, PromoCell) in a humidified incubator at 37 °C and 5% CO2. Cell passages 3–4 sourced from three different donors were used for all experiments.
Microfluidic Flow Chamber with RGD-Conjugated Films
Thin polydimethylsiloxane (PDMS) films (100 µm, Wacker Chemie) were thoroughly cleaned using soap and absolute ethanol (Merck), rinsed with ultra-pure water (Sartorius) and air dried. The films were mounted on commercially available microfluidic flow chambers (sticky µ-slides I0.1, Ibidi) with a chamber height of 150 µm. The assembled devices were placed in an oven at 60 °C for 8 h and allowed to cool to room temperature to prevent membrane detachment. To enable cell attachment, RGD peptides were conjugated to the cell growth area of the PDMS films as previously described,12 to generate the RGD-PDMS membranes. The microfluidic channels were disinfected twice with 70% ethanol for 10 min, rinsed with sterile PBS and stored at 4 °C.
Model System
For long-term dynamic culture, separate model systems were used for each of the three donors. In each model system, up to four microfluidic flow chambers were connected in series. Each model system comprised a 2-L medium reservoir (Schott), a peristaltic pump (ISMATEC IPC-N 8 with PHARMED ISMAPRENE pump tubes, both Cole-Parmer) as well as disposable tubing (Extension Line Type Heidelberger, B. Braun Melsungen or Fresenius Kabi). The microfluidic flow chambers were placed in PDMS-cast mountings and secured with a metal tenter frame (Figs. 1a and 1b). The mounting also formed the gas pathway for the gas transfer measurements (Fig. 1c).
Before connecting the microfluidic flow chambers, the model system was flushed with ~ 200 mL EGM–2 supplemented with 1% antibiotic-antimycotic solution (ABM, Thermo Fisher Scientific) and equilibrated to incubator conditions (37 °C, 5% CO2). During experiments, equilibration to incubator conditions and sufficient gas exchange was guaranteed by the reservoir, which was open to the incubator atmosphere in a sterile manner.
Static and Dynamic Culture
For the endothelialization of RGD-PDMS films, HUVECs were suspended in EGM-2 with 1% ABM and transferred to the microfluidic channel at a concentration of 8 × 106 cells mL−1 (equivalent to 1.2 × 105 cells cm−2). Cell-seeded microfluidic flow chambers were incubated in a humidified incubator at 37 °C and 5% CO2, and the medium was exchanged every 30 min. After 2 h of static culture, the cell layer was reviewed by bright-field microscopy. As soon as the cells showed sufficient adhesion to the membrane (~ 2 to 2.5 h), the endothelialized flow chambers were connected to the model system in a sterile manner. Dynamic culture was initiated by applying a laminar flow of 0.77 mL min−1 to the cells with a corresponding physiological WSS of 0.5 Pa (equivalent to 5 dyn cm−2).29 The culture medium was exchanged twice weekly.
In advance of the gas transfer tests, three endothelialized membranes with ECs from different donors were each dynamically cultured for 3, 19 or 33 days. As a reference, the same number of endothelialized membranes was dynamically cultured for immunocytochemical staining.
Venous Blood for Gas Transfer Testing
To test gas transfer rates, we used fresh, pooled and heparinized porcine blood from an abattoir. Initially, 15,000 IU L−1 heparin-sodium (B. Braun Melsungen), 1.8 mL L−1 50% glucose solution (B. Braun Melsungen), 1.2 mL L−1 antibiotics solution (10 mg mL−1 gentamycin, Biochrom) and 6 mL L−1 0.9% sodium chloride (B. Braun Melsungen) were added to the porcine whole blood.
All tests were performed with blood inlet conditions according to DIN EN ISO 7199.6 To meet the specifications, the heparinized blood was transferred to a simplified heart–lung machine setup comprising an open reservoir, a roller pump and an oxygenator with a heat exchanger to adjust the blood parameters according to Table 1. The hemoglobin concentration and the base excess were adjusted by adding 0.9% sodium chloride and 8.4% sodium bicarbonate (Fresenius Kabi), respectively. The oxygenator was supplied with an adjustable gas mixture of oxygen, nitrogen and carbon dioxide. Throughout the experiments, the blood parameters were checked regularly by blood gas analysis (ABL 800 Flex, Radiometer) and the settings were adjusted accordingly.
Table 1 Blood conditions for in vitro testing of oxygen and carbon dioxide transfer rates specified by DIN EN ISO 7199:20176. Gas Transfer Tests
Gas transfer tests were carried out on three endothelialized microfluidic flow chambers (n = 3) for each culture period (3, 19 and 33 days). Three blank RGD-PDMS flow chambers served as references. The RGD-PDMS film of the flow chamber acts as a gas exchange membrane between the microfluidic channel (blood pathway) and the gas compartment (gas pathway). For gas transfer testing, the device was perfused on the principle of counter flow (Fig. 1c).
In more detail, the blood pathway consisted of a syringe pump (LA-120, Landgraf Laborsysteme HLL) placed on a platform shaker, a 50-mL syringe (PERFUSOR, B. Braun Melsungen), extension lines (Type Heidelberger, 30 cm, Fresenius Kabi) with three-way valves for sampling (DISCOFIX, B. Braun Melsungen), the flow chamber to be tested, and a waste container. The gas pathway provided pure oxygen and was equipped with a thermal mass flow meter (TSI 4100 series, TSI) to ensure a constant gas flow rate. The blood flow rate was set by the syringe pump.
All gas transfer experiments were conducted in a climatic chamber at 37 °C. Initially, blood meeting the standards of ISO 7199 was drawn into a 50-mL syringe and connected to the blood pathway, as was the microfluidic flow chamber. Blood and gas flow were started and slowly increased to the target values of 0.77 and 500 mL min−1, respectively, to prevent any damage to the cell layer. Both flows were stopped immediately after sampling, which corresponds to an exposure time of approximately 20 min. The blood flow rate corresponds to a hemodynamic WSS of 2.5 Pa (assuming a blood viscosity of 3.6 mPa s).11 Based on our unpublished results showing sufficient stability of the cell layer after short-term blood exposure, this supraphysiological WSS was chosen in favor of higher flow rates and more accurate sampling.
With 1-mL syringes (B. Braun Melsungen), samples of at least 0.5 mL volume were taken in triplicate beginning at the outlet (arterial samples) and then at the inlet (venous samples) of the microfluidic flow chamber. Venous samples were drawn after arterial samples to avoid any impact on the blood residence time within the channel. Sampling was time-controlled to avoid accidentally affecting the flow conditions inside the microfluidic chamber. Prior to sampling, the microfluidic flow chamber was perfused with at least three times its volume to ensure an equilibrium state. Moreover, the dead volume in the sampling port was discarded. Possible air bubbles were removed and syringes were hermetically sealed. Samples were stored on ice until analysis in a blood gas analyzer, but no longer than 20 min.
Evaluation of Gas Transfer Measurements
The blood gas analysis determines the concentration of total oxygen and total carbon dioxide in whole blood (tO2 and tCO2; the underlying calculations were previously published3,25) taking into account the respective fractions of physically dissolved and chemically bound gas. The oxygen and carbon dioxide transfer were then computed as the difference in mean gas concentrations between the inlet and outlet samples. Relating the transfer to the blood flow rate yielded the transfer rates for oxygen and carbon dioxide, as shown below:
$$ {\text{OTR = }}\dot{V} ( {\text{tO}}_{{ 2 , {\text{arterial}}}} - {\text{tO}}_{{ 2 , {\text{venous}}}} ) $$
(1)
$$ {\text{CTR = }}\;\dot{V} ( {\text{tCO}}_{{ 2 , {\text{arterial}}}} - {\text{tCO}}_{{ 2 , {\text{venous}}}} ) $$
(2)
Immunocytochemistry and Fluorescence Microscopy
All endothelialized RGD-PDMS samples were fixed for immunocytochemical staining with antibodies for CD31 and von Willebrand factor (vWf) as described elsewhere.15 Prior to immunocytochemical staining, endothelialized membranes exposed to blood were carefully rinsed with PBS containing 2% ABM to remove blood residues.
Briefly, the ECs were fixed in ice-cold methanol and nonspecific binding sites were blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. The cells were incubated sequentially with primary antibodies against CD31 (monoclonal, mouse, P8590, Sigma-Aldrich) and vWf (monoclonal, rabbit, A0082, Dako) as well as their corresponding secondary antibodies (Alexa Fluor 594, goat anti-mouse, A11005, and Alexa Fluor 488, goat anti-rabbit, A11008, both Thermo Fisher Scientific) for 1 h each at 37 °C. Cells were counterstained with DAPI (Carl Roth). Samples were viewed by fluorescence microscopy (AxioObserver Z1; Carl Zeiss) and images were acquired using a high-resolution CCD camera (AxioCam MRm, Carl Zeiss) and AxioVision software.
Statistical Analysis
Results are presented as means ± standard deviations (SD). Both outcome variables OTR and CTR were separately analyzed by one-way ANOVA to test for differences in the mean outcome between the four test conditions (three different culture periods and the blank reference). Data were analyzed using Excel 2016 (Microsoft Corp.) and SAS v9.4 software (SAS Institute). The statistical analysis was conducted in an explorative manner, and a p value below 0.05 was considered to be statistically significant (labeled *).