Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture | Scientific Reports - Nature.com

Nearly 97% of drugs developed for oncology interventions neglect during objective trials owed to the reliance connected inadequate cause models, specifically successful vitro 2-dimensional (2D) compartment civilization models¹. Though successful vitro 2D compartment civilization models are convenient to use, they bash not decently correspond the analyzable tumor microenvironment arsenic cellular behaviour is impacted by the level morphology of the stiff, planar integrative typically used^2,3. To code the concerns with 2D compartment civilization models, determination has been a displacement towards three-dimensional (3D) crab compartment civilization models arsenic much physiologically applicable mimics of the tumor microenvironment⁴. Overall, 3D tumor models tin instrumentality galore forms, specified arsenic spheroids, organoids, oregon matrix-based cultures and co-cultures to sanction a few^5,6. To adhd complexity and heighten physiological relevance, 3D tumor models tin beryllium combined with microfluidic devices to let perfusion oregon mimic vascularization⁷. Microfluidic systems that harvester cells, an extracellular matrix, and perfusion are bully mimics of the successful vivo tumor microenvironment and are debased outgo and reproducible, alteration the usage of tiny civilization volumes, and person a controllable plan that tin beryllium optimized for a desired application^8,9. Perfusion-based compartment civilization systems supply nutrients to cells and region metabolic discarded successful a mode meant to mimic successful vivo wide transport and support soluble factors adjacent biologic concentrations¹⁰. In addition, temporal and spatial gradients tin beryllium generated wrong microfluidic systems, further expanding their worth arsenic cause screening platforms⁸. However, designing and operating a robust microfluidic perfusion strategy for 3D civilization of adherent mammalian cells is challenging³. Challenges could beryllium related to instrumentality design, specified arsenic choosing the due civilization configuration, materials, oregon microfluidic web fabrication, oregon purely technical, specified arsenic sterilization, compartment seeding successful the device, optimizing the wide transport and travel shear stresses to maximize compartment viability, and avoiding aerial bubbles successful the perfusion channels.

Gradient mixers person been developed for microfluidic systems that tin beryllium utilized to supply on-chip mixing to proviso discrete concentrations of soluble factors to compartment civilization systems that tin beryllium utilized to support cells, differentiate stem cells oregon reply assorted biologic questions¹¹. Many of these microfluidic mixers utilize tree-like structures with aggregate stages that necessitate implicit mixing anterior to the extremity of each stage^8,12,13, akin to the plan utilized herein. On-chip gradient enactment eliminates the anticipation for pipetting errors, decreases chances for contamination, and allows for less microfluidic inlets that simplify setup compared to microfluidic devices with externally generated gradients^14,15.

Recognizing the enhanced physiological relevance of perfusion 3D compartment civilization systems, a increasing assemblage of probe has focused connected microfluidic compartment civilization devices for cause screening applications with accent connected liver and crab insubstantial models^16,17. For example, tumor spheroid models were grown successful compartment civilization microwells connected with a attraction gradient generator successful microfluidic chips and utilized successful cause screening applications^18,19. These 3D systems did not incorporated an extracellular matrix mimic and relied connected compartment seizure to capable the compartment civilization chambers. Utilizing a clever design, a 3D microfluidic compartment array incorporated some crab cells and endothelial cells to emulate the tumor environment⁷. The bottommost furniture had microchambers with hydrogel-embedded crab cells, separated from an precocious microchannel furniture of endothelial cells done a permeable membrane with clustered pores, and the instrumentality did not see a attraction gradient generator. The authors past compared cause responses successful their strategy to cause responses successful a static 3D civilization and noted a delayed effect perchance owed to the endothelium formed successful the apical microchannels, serving arsenic a obstruction for cause penetration. Another capillary-force based microfluidic instrumentality for 2D and 3D compartment civilization incorporating endothelial and crab compartment layers was developed for cause screening applications, wherever a gradient could beryllium generated without the request for specialized equipment²⁰. Toh et al. developed a 3D microfluidic compartment array spot to trial cause hepatotoxicity, wherever hepatocytes were cultured and exposed to gradient cause doses²¹. The 3D microenvironment was generated via collagen and terpolymer coacervation and maintained with a micropillar array. Overall, microfluidic compartment civilization devices tin harvester aggregate utile features specified arsenic an extracellular matrix mimic, cells and compartment co-cultures, physiologically applicable perfusion, multiplexed compartment civilization chambers, and attraction gradient oregon dilution generators, which marque them charismatic for applications specified arsenic cause screening²².

Here, we picture a microfluidic instrumentality designed for hydrogel-based 3D compartment cultures and showcase its inferior with 2 antithetic hydrogel matrices and gathering dose–response curves with 2 chemotherapeutics. The 2D array of compartment civilization chambers are perfused by a tree-like MCGG that creates a serial fractional dilution that proviso dilution ratios of 1, ½, ¼, and 0. The cells and gel precursor solution are easy loaded into the instrumentality and the hydrogels signifier aft photoinitiation oregon Michael-type summation without occlusion of the perfusion channels oregon unit buildup that could impact compartment viability. Using this microfluidic system, we cultured glioblastoma cells successful 3D polyethylene glycol (PEG)-based hydrogels, and measured cause transportation via perfusion and compartment viability aft attraction with temozolomide (TMZ) and carmustine (BCNU).

Materials

Sylgard™ 184 silicone elastomer kit (referred to arsenic polydimethyl siloxane oregon PDMS) was obtained from Dow Silicones Corporation (Midland, MI). Polyester (PETE) membrane filters (transparent, 0.2 µm pore size, 12 µm thickness, 2e⁶ pores/cm²) were from Sterlitech Corporation (Kent, WA). Pre-cleaned 75 mm × 50 mm × 1 mm plain solid slides were from Corning Incorporated (Corning, NY). INTRAMEDIC polyethylene (PE) tubing (I.D. 1.40 mm, O.D. 1.90 mm; and I.D. 1.14 mm, O.D. 1.57 mm) were from Clay Adams. Silicon (SI) wafers were from University Wafer Inc (Boston, MA). SU-8 2025 photoresist and SU-8 developer (98–100% 1-Methoxy-2-propyl acetate) were from Kayaku Advanced Materials Inc (Westborough, MA). MIL PRF 131k Class 1 foil, 150 µm heavy (MarvelSeal^® 470) was from Berry Global (Evansville, IN). A1 frame, a bladed polymer movie (A4 inkjet waterproof film) was from CisInks (South El Monte, CA). Polyethylene glycol diacrylate (PEGDA; 5 kDA), 4-arm poly(ethylene glycol)-acrylate (4-arm PEG-Ac; 10 kDa), and poly(ethylene glycol)-dithiol (PEG-diSH; 3.4 kDa) were from Laysan Bio Inc. (Arab, AL). Phosphate buffered saline (PBS, 10 X, pH 7.4), Trypan Blue (0.4%), fluorescent polystyrene beads (Fluoro-Max™ Fluorescent Red, 542/612 nm, d = 2 µm), and Temozolomide (TMZ) were from Thermo Fisher Scientific (Waltham, MA). Irgacure 2959 was from BASF corp (Florham Park, NJ). Fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were from Hyclone (Logan, UT). Roswell Park Memorial Institute (RPMI)-1640 mean and 0.05% trypsin/0.02% ethylenedinitrilotetraacetic acerb (EDTA) were from Coring (Coring, NY). Acridine orangish (AO) and carmustine (BCNU) were from Millipore Sigma (St Louis, MO). Propidium iodide (PI) was from MP Biomedical LLC (Solon, OH). Cell stain 3,3’-dioctafecyloxacarbocyanine perchlorate (DiOC) was from Life Technologies (Carlsbad, CA). Brilliant Blue FCF (CAS Number: 3844-45-9) was purchased arsenic bluish nutrient coloring from a section market store. Homo sapiens encephalon glioblastoma U87 cells were from ATCC (Manassas, VA). Glycine–Arginine–Cysteine–Aspartic Acid–Arginine–Glycine–Aspartic Acid–Serine (GRCD-RGDS), Glycine–Arginine–Cysteine–Aspartic Acid–Arginine–Glycine–Aspartic Acid–Serine–FITC (GRCD-RGDS-FITC), and Aspartic Acid–Arginine–Cysteine–Glycine–Valine–Proline–Methionine–Serine–Methionine–Arginine–Glycine–Cysteine–Arginine–Aspartic Acid (DRCG-VPMSMR-GCRD) peptides were from Genic Bio (Shanghai, China).

Microfluidic instrumentality preparation

The microfluidic instrumentality was fabricated by assembling a enactment solid microscope descent (75 mm × 50 mm × 1 mm), 2 layers of patterned PDMS (top perfusion furniture and a bottommost compartment civilization chambers furniture with compartment loading channels), sixteen PETE membranes (capping the sixteen compartment civilization microwells), and 3 tubes—two inlet tubes (PE tubing; I.D. 1.14 mm, O.D. 1.57 mm) and 1 outlet conduit (PE tubing; I.D. 1.40 mm, O.D. 1.90 mm) (Fig. 1, Supplemental Fig. S1). The apical perfusion furniture was 3 mm heavy and the bottommost compartment civilization microwells furniture was 250 µm thick. The tallness of the perfusion and compartment loading channels was 50 µm. A good extent of 250 µm was chosen to beryllium larger than an idiosyncratic compartment portion inactive allowing imaging done the full sample. Most perfusion channels were 150 µm wide with the cardinal exceptions of 50 µm and 75 µm, which were incorporated into the plan to execute fractional dilution series. These fractional dilution channels were utilized to nutrient a attraction gradient of 100%, 50%, 25%, and 0% of archetypal solute concentration. The compartment loading channels connected the bottommost furniture were besides 150 µm wide.

Figure 1

Microfluidic instrumentality design. (A) Photo of the microfluidic instrumentality with the inlet (left-hand broadside of image) and outlet (right-hand broadside of image) tubing. Black standard barroom represents 1 cm. (B) Channel widths of the microchannels and the attraction dilutions generated with these transmission widths. (C) Side presumption of the fabricated microfluidic devices displaying the inlet tubes (left), mixing chambers (red), compartment civilization chambers (green), and outlet conduit (right).

Both apical and bottommost PDMS layers were fabricated via curing PDMS precursor connected patterned Si masters (fabricated by modular photolithography SU-8 2025 procedure: https://kayakuam.com/wp-content/uploads/2019/09/SU-82000DataSheet2025thru2075Ver4-3.pdf) astatine 75 °C overnight. Briefly, a 50 µm heavy SU-8 2025 photoresist was spin-coated connected a Si wafer astatine 1700 rpm (SP-100 spinner, BidTec) and brushed baked connected a hotplate astatine 65 °C for 3 min and past astatine 95 °C for 9 min. The patterns connected the pre-printed masks were transferred to the photoresist furniture done UV radiation (160 mJ/cm² with Flood Exposure Model 60, ABM-USA, Inc., San Josa, California, USA), post-exposure baked (on a hotplate astatine 65 °C for 6 min and past astatine 95 °C for 7 min) and developed by a modular process shown successful Supplemental Fig. S2. UV radiation crosslinked the exposed SU-8, and SU-8 developer dissolved the uncrosslinked SU-8, leaving lone the crosslinked SU-8 patterns.

The bottommost compartment civilization PDMS furniture was prepared likewise with a modification to power thickness (Supplemental Fig. S3). To execute a thickness of 250 µm for the bottommost PDMS layer, a 150 µm Al foil framework spacer was used. After pouring PDMS precursor connected the Si maestro and the Al frame, a bladed polymer movie and a rigid solid descent were enactment connected top. The flexible polymer movie was added to debar the interaction of PDMS precursor and the solid slide, different the rigid solid descent could not beryllium removed aft PDMS was cured. A 5 lb. value was added connected the apical to guarantee a azygous thickness of the PDMS layer, and the PDMS was cured astatine 80 °C overnight. The thickness was measured by an Alpha-Step IQ profilometer (KLA-TENCOR Alpha D500, KLA Corp., Milpitas, California, USA). Note that the PDMS furniture was thicker than the spacer arsenic not each excess PDMS was extruded done the edges anterior to polymerization owed to the precocious viscosity of the PDMS precursor.

After the PDMS layers were cured, 16 compartment chambers (2 mm I.D.), 2 inlets (1.5 mm I.D.) and 1 outlet (2 mm I.D.) were chopped with biopsy punches. Sixteen circular PETE membranes (pore size 0.2 µm), chopped with 3 mm diameter biopsy punch, were individually placed implicit each of the 2 mm ID compartment civilization microwells to abstracted the 2 PDMS layers and forestall cells and worldly successful the civilization chambers from escaping into the perfusion channels. Individual membrane pieces were chosen arsenic opposed to 1 portion to alteration amended adhesion betwixt the 2 PDMS layers (top and bottommost layers of the device), to debar leakage, and to guarantee stableness implicit time. Individual pieces besides forestall the crosstalk betwixt wells, which could hap with 1 portion membrane layer. The 3 mm diameter membranes are somewhat larger than the 2 mm ID wells, truthful the edges of the membranes interaction the creaseless surfaces of the PDMS, adhere to it spontaneously, and enactment dependable during the pursuing oxygen plasma and alignment. A laboratory corona attraction instrumentality (BD-20AC model, Electro-Technic Products, Chicago, IL, USA) was past utilized to dainty the 2 PDMS surfaces with plasma for 1 min to oxidize them. The 2 PDMS layers were aligned with 4 registration marks connected the corners by eye. After the 2 PDMS layers were pressed unneurotic with PETE membranes in-between, the solid descent surfaces and PDMS assembly were further oxidized with plasma for 1 min to amended attachment. Inlets and outlet tubing were inserted into the inlets and outlet locations and sealed with uncured PDMS. The devices were heated astatine 75 °C for astatine slightest 2 h to amended sealing and cure the PDMS. A elaborate instrumentality assembly schematic is shown successful Supplemental Fig. S4.

Mixer show and dilution analysis

For perfusion, perfusion fluids were placed successful 3 mL syringes successful a dual syringe pump (New Era Pump Systems Inc, Farmingdale, NY) truthful the inputs into some sides of the mixers had the aforesaid travel rate. The syringes were connected to the inlet ports of the instrumentality with needles (21 G) and tubing (PE tubing I.D. 1.14 mm, O.D. 1.57 mm). Volumetric travel rates tested were 1, 1.5, 2, 5, and 10 µL/min. To survey mixing efficiency, a solution of Brilliant Blue FCF (to assistance successful visualization) was dissolved successful PBS for a last attraction of 25 μM (measured via Beer-Lambert Law)²³ and utilized successful operation with plain PBS to find the show of the micromixers. Using an inverted microscope (Zeiss, Axiovert 200 M, Oberkochen Germany) astatine 5X magnification, images were captured astatine 3 regions—entrance, middle, and exit—of the micromixers during perfusion (Fig. 2B). ImageJ software²⁴ was past utilized to divided retired RGB channels and measurement the strength of the bluish RGB transmission wrong the micromixers (as a relation of micromixer width) utilizing the ‘Plot Profile’ function. Values were normalized from 0–1 for each acceptable of images, with a grey worth of 1 being assigned to the bluish color, and a grey worth of 0 assigned to the inheritance colour of the microfluidic chip. The implicit mixing scale (AMI) was calculated as:

where I_i is the section pixel intensity, < I > is the mean pixel strength successful the transverse section, and N I the full fig of pixels²⁵.

Figure 2

Loading and perfusion set-up. (A) Schematic of loading the hydrogel precursor solution into the membrane-capped microwells of the bottommost loading furniture of the instrumentality via the loading ports utilizing an insulin needle. (B) Schematic of perfusion set-up successful which tubing connects the 3 mL syringes containing the perfusion fluids to the inlet ports of the microfluidic device. Red circles bespeak wherever images were taken wrong the micromixer for mixing investigation (entrance, middle, and exit). Blue circles bespeak wherever images were taken astatine the extremity of the micromixer for attraction gradient analysis.

In addition, to find the attraction gradient generated by the microfluidic instrumentality astatine the acceptable volumetric travel rates, images were besides captured astatine the extremity of each micromixer and ImageJ bundle was past utilized to comparison the mean strength of the dye produced by the micromixers.

Calculation of diffusion coefficient, hydrodynamic radius, and diffusion clip of molecules done the width of the mixing channel

Modeling molecules arsenic hard spheres, we utilized the Stokes–Einstein equation to cipher the diffusion coefficient for molecules:

where D is the diffusion coefficient, \(k\) is the Boltzmann constant, \(\mu\) is the dynamic viscosity of h2o astatine 37 °C, and \(r\) is the hydrodynamic radius of the particle. Assuming that the molecular measurement of the particle corresponds to a spherical shape, the hydrodynamic radius of each molecule was calculated as:

where MW is the molecular value of the particle (provided by the Royal Society of Chemistry), N_A is Avogadro’s Number, and \(\rho\) is the density of the particle (provided by the Royal Society of Chemistry). The clip required for a molecule to diffuse crossed the width of the transmission was past calculated as:

where \(t\) is the clip required for diffusion to instrumentality spot and \(x\) is the diffusion distance.

Cell maintenance

Human glioblastoma U87 cells were cultured successful RPMI-1640 mean supplemented with 10% FBS and 1% P/S and incubated successful a humidified incubator astatine 37 °C and 5% CO₂. Cells were passaged by a 5 min vulnerability to Trypsin/EDTA erstwhile an ~ 80% confluency was achieved, and media was changed each 2 days. Cell passages 10–20 were utilized for experiments. Cells were cultured with DiOC (20 μM) for 24 h to stain each cells anterior to each further experimentation.

Loading of PEG hydrogels successful the compartment civilization chambers of the microfluidic device

Non-degradable, non-adhesive PEGDA hydrogels were prepared by UV photo-polymerization. Briefly, to hole a banal solution of the photoinitiator, 1% w/v Irgacure 2959 was dissolved successful de-ionized h2o (DI water), sonicated (Branson, Model #2800, 40 kHz) for 90 min and stored astatine country temperature, protected from the airy for up to 2 weeks. PEGDA hydrogel precursor solution (250 µL) of 20% w/v and 0.1% w/v successful Irgacure were prepared successful PBS and vortexed for 30 s to guarantee implicit mixing. The hydrogel precursor solution was loaded into the compartment civilization microwells of the microfluidic instrumentality done the loading ports by utilizing an insulin needle (Fig. 2A). Each microwell contained astir 0.2 µL of the hydrogel precursor solution; hence 3.14 µL of solution were needed to capable each 16 of the microwells. The excess magnitude of precursor solution was indispensable to guarantee that the solution reached the microwells alternatively of remaining wrong the loading channels. Relief channels were incorporated into the plan of the loading furniture of the instrumentality to alteration the excess precursor solution to exit the instrumentality alternatively of pushing against the membrane capping the microwells. The hydrogels were past polymerized nether UV lamp (4.81 mW cm², 1 W, 365 nm; Blak-Ray^® XX-15L UV seat lamp, UVP, Upland, CA) for 10 min²⁶. These gels are referred to arsenic PEGDA throughout.

Degradable, adhesive 4-arm PEG-Ac hydrogels were prepared by Michael-type addition. First, 4-arm PEG-RGDS was prepared pursuing a antecedently developed protocol²⁷. We modified connected mean 1 of the acrylate groups of each 4-arm PEG-Ac with RGDS (80% modification efficiency) and stored the lyophilized merchandise nether argon successful a desiccated instrumentality at − 20 °C for up to 6 months. Hydrogels were past formed utilizing a substance of 4-arm PEG-Ac and 4-arm PEG-RGDS (0.8 mM successful RGDS) and a 50:50 crosslinker substance of PEG-diSH and an enzymatically degradable peptide crosslinker DRCG-VPMS↑MR-GCRD. For each gels, 20% w/v banal solutions of 4-arm PEG-Ac, PEG-diSH, and 4-arm PEG-RGDS were prepared successful TEA buffer pH 8 instantly anterior to use. Stock solutions were mixed and the peptide crosslinker was added arsenic pulverization to springiness a hydrogel precursor solution with a 1:1 acrylate to thiol molar ratio and a last polymer attraction of 10% w/v and a last peptide crosslinker attraction of 7.8 mM. The hydrogel precursor solution was mixed via pipetting for 30 s, injected successful the microfluidic instrumentality arsenic described above, and allowed to gel for 20 min successful a humidified incubator astatine 37 °C and 5% CO₂. These gels are referred to arsenic 4-arm PEG-Ac throughout.

To measure the reproducibility of hydrogel and compartment loading into the compartment civilization microwells of the microfluidic device, DiOC-stained U87 cells were added to the PEGDA hydrogel precursor solution astatine 10⁶ cells/mL. The precursor solution was past loaded into the microwells utilizing an insulin needle and gelled nether UV arsenic described above. Images of each good were taken utilizing an inverted fluorescence microscope and ImageJ was utilized to number the fig of ells wrong each well. The organisation of the fluorescently labelled cells wrong the hydrogel was imaged utilizing a confocal microscope (Leica Confocal SP8, Leica Microsystems Inc, Buffalo Grove, IL) astatine × 10 magnification and processed utilizing Fiji bundle (free download http://fiji.sc). For confocal imaging the hydrogel was besides rendered fluorescent by covalently incorporating a fluorescent GRCD-RGDS-FITC peptide arsenic described by america previously²⁷.

Drug screening

A hydrogel precursor solution containing 20% w/v PEGDA, 0.1% v/v Irgacure, and 10⁶ cells/mL (DiOC-stained U87 cells) was injected and polymerized successful the microfluidic instrumentality compartment civilization microwells arsenic described supra to hole the non-degradable, non-adhesive PEGDA hydrogels. Immediately aft gelation, RPMI media supplemented with 10% FBS and 1% P/S was perfused done the instrumentality for 48 h. At 48 h the perfusion media was replaced with supplemented media containing 0.2 μg/mL PI (staining nuclei of dormant cells) successful some inlets and 2 mM TMZ oregon 10 µM BCNU successful 1 inlet and perfused done the instrumentality for 48 h. Similarly, degradable, adhesive 4-arm PEG-Ac hydrogels containing 10⁶ cells/mL of DiOC-stained U87 cells, were prepared arsenic described above, cultured for 48 h and exposed to TMZ for 48 h. Encapsulated cells were imaged utilizing Axiovert 200 M inverted fluorescence microscope at × 10 magnification. Cell viability was calculated as:

where N_L represents the fig of unrecorded cells and N_D represents the fig of dormant cells.

Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS; Zeiss LSM 510 Fluorescence Microscope, Zeiss, Germany) was utilized to measurement successful situ diffusivity of a exemplary fluorophore (Atto 655) successful a 20% w/v PEGDA hydrogel slab. Hydrogels were formed arsenic described above, with the modification that Atto 655 (0.5 nM) was added to the hydrogel precursor solutions. For FCS measurements, hydrogels (150 µL) were prepared successful an 8 enclosure coverglass with #1 German borosilicate bottom. Hydrogels were past soaked overnight successful DMEM mean without phenol reddish and containing 0.5 nM Atto 655 to debar concentration-driven diffusion of Atto 655 from the hydrogel into the surrounding media. Atto 655 (0.5 nM) successful deionized h2o was besides utilized to calibrate the confocal measurement of the FCS instrument. A 633 nm ps pulsed laser was utilized for six measurements of 300 s for each illustration location. An autocorrelation relation G(τ) was obtained for each measurement:

where N is the fig of fluorescent particles, p = r_o/z_o is an instrumental constant, r_o is the radius and z_o is the axial magnitude of the focused laser beam spot, τ_d is the solute diffusion time, T is triplet authorities amplitude, and τ_T is the triplet lifetime. The autocorrelation relation was acceptable utilizing a Triplet exemplary to relationship for the imaginable excitation of molecular triplet states astatine higher laser intensities. Lastly, the autocorrelation relation was normalized arsenic follows:

where G(τ_D) is the worth of Eq. (6) astatine each clip constituent and G(τ₀) is the worth of Eq. (6) astatine the archetypal clip point. The effectual tracer diffusion coefficient for each macromolecule successful solution was calculated from τ_D as:

Assuming the diffusivities of TMZ and Atto 655 were akin owed to their comparatively akin molecular weights (194 g/mol and 887 g/mol, respectively), we past modeled the anticipated TMZ attraction successful the gels arsenic a relation of gel extent and time. TMZ attraction was calculated via Fick’s 2nd instrumentality of diffusion for 1-D geometry (a bladed slab) with the pursuing bound conditions: c(x = 0) = 2 mM TMZ and c(x = ∞) = 0 mM TMZ astatine t = 0 h, wherever c is the TMZ concentration, x is the gel determination and t is time:

Statistical analysis

All information are presented arsenic mean values (± SD) determined from 3 to 4 autarkic experiments. Statistical investigation for attraction gradient investigation and loading reproducibility was performed utilizing one-way ANOVA with Holm–Sidak oregon Dunn’s aggregate examination tests utilizing GraphPad Prism 6^®. Statistical investigation for the slope of the strength illustration vs volumetric travel complaint was performed utilizing linear-regression investigation utilizing GraphPad Prism 6^®. A p < 0.05 was considered statistically significant.

Device design, advantages and limitations

Here we designed a microfluidic instrumentality which allowed america to load hydrogel-encapsulated cells into membrane-capped compartment civilization microwells to beryllium perfused with fluids containing desired biomolecules oregon drugs of varying concentrations (Fig. 1, Supplemental Fig. S1). The instrumentality has a operation of respective features that marque it a bully prime for 3D hydrogel-based perfusion compartment culture: (i) it is an enclosed operation with compartment loading channels; (ii) determination is simply a 2D array of compartment civilization chambers capped by a membrane to forestall contamination oregon compartment flight into the perfusion layers; (iii) cells are loaded wrong a hydrogel for biomimetic 3D compartment culture; (iv) the inverted plan allows for imaging from the bottom; and (v) the MCGG uses transmission width arsenic opposed to magnitude to alteration mixing ratios.

The microfluidic instrumentality was constructed with PDMS, which is commonly utilized successful microfluidic applications arsenic it is elemental to fabricate, inert, non-cytotoxic, state permeable, and suitable for fluorescence imaging^3,28,29. Importantly for our design, PDMS is ailment and enabled the usage of an insulin needle for loading cells and gels successful the microwells. The microfluidic strategy consisted of 2 microfluidic transmission layers, separated by membranes to seal the bottommost cell-loading channels. The apical perfusion furniture (Figs. 1, 2B) contains the serpentine micromixers starring to the perfusion channels that supply travel implicit the membrane to provender the cells successful the civilization chambers below. The two-stage micromixers person provender transmission widths of 150/75/150/150 μm upstream of the archetypal signifier and 150/50/150/50/150 μm upstream of the 2nd signifier to nutrient the fractional attraction dilution bid of 1:½:¼:0 (Fig. 1B). The perfusion channels fluidically code the compartment civilization chambers successful parallel to debar crosstalk. The instrumentality provides unchangeable cognition implicit respective days, consuming < 1.5 mL/day and perfuses the cells with caller media to debar cytotoxicity. At the volumetric flowrate of 1 μL/min, the linear velocity is ~ 1.1 mm/s wrong the provender transmission to the perfusion furniture supra the compartment civilization chamber, which is akin to the successful vivo flowrate done capillaries³⁰. For example, Giulitti et al. demonstrated that if perfusion is excessively dilatory (e.g. 0.6 µL/h), downstream cells tin acquisition cytotoxicity from the heterogeneous organisation of nutrients (higher attraction upstream) and discarded (higher attraction downstream)³¹. Higher flowrates (e.g. 30–60 μL/h, akin to the 1 μL/min utilized successful this study) effect successful much homogenous compartment civilization wrong the full microfluidic device. Based connected instrumentality design, travel rates that are excessively precocious (e.g. 40 μL/min) could besides effect successful incomplete mixing oregon shear stress-induced cytotoxicity³².

Microchannels for loading cells into civilization chambers were incorporated into the bottommost layer. Each enactment of compartment civilization chambers is loaded from a azygous inlet. If the aforesaid compartment containing solution is loaded into each inlet, 4 biologic replicates are created arsenic performed successful this study. Loading is astir accordant erstwhile each compartment civilization enclosure has the aforesaid magnitude of the microchannel walk done it (the sum of the transmission magnitude from the inlet to the microwell and from the microwell to the exit) to let microwells to beryllium filled successful parallel simultaneously (Fig. 2A). The channels from each compartment civilization microwell to the exit acted arsenic a illustration alleviation transmission to easiness the loading process and trim unit required portion loading the hydrogel precursor solution into the microwells. The alleviation channels forestall the hydrogel-encapsulated cells from escaping into the perfusion furniture and blocking perfusion upon gelation nether excess loading pressure. Each compartment civilization good was capped with a PETE membrane (pore size of 0.2 µm), which enabled the speech of nutrients and discarded betwixt the cells and the perfusing fluids but was restrictive for ample particles, bacteria, and cells, assuring that nary contamination could hap owed to perfusion. Lastly, the cells contained wrong the gel matrix were shielded from hydrodynamic shear accent of the circulating perfusion fluids for improved recapitulation of the successful vivo system. The determination of the civilization chambers successful narration to the perfusion watercourse is important arsenic cells located straight successful the flow-path are subjected to hydrodynamic shear stress, which tin impact the compartment morphology, cytoskeleton organization, proliferation, cellular signaling pathways, and cistron and macromolecule expression³.

Tree-like microfluidic attraction gradient generator

Here, successful opposition to a emblematic MCGG, a fractional serial dilution is achieved by varying the widths of the provender channels. Diffusive-based mixers person a maximum velocity astatine which implicit mixing is achieved, hence, varying the provender channels width arsenic opposed to magnitude allows for a much compact mixing structure. Previously, a non-dimensional parameter has been created to find the required mixer magnitude for assorted geometries³³. The maximum velocity for gradient mixing is experimentally measured utilizing a microscope successful our squared country 18.5 mm agelong serpentine channels arsenic shown successful Fig. 2B. Images that measurement mixing astatine the entrance, middle, and extremity of the channel astatine flowrates of 1, 5, and 10 μL/min are shown (Fig. 3A). In total, 5 volumetric travel rates (1, 1.5, 2, 5, and 10 µL/min) were utilized to find the grade of mixing astatine the exit of the mixing channel. Complete mixing is indicated by a homogenous solution attraction crossed the microchannel width, which yields a slope of zero for a crippled of strength vs. distance. In Fig. 3B an AMI of 0 indicates implicit mixing. At a travel complaint of 1 μL/min, the AMI worth was calculated to beryllium 0.012 astatine a flowrate of 1 μL/min. At greater flowrates, the AMI increased, indicating incomplete mixing, wherever the grade of mixing decreases arsenic the flowrate increases. Overall, mixing is linearly babelike connected the volumetric travel complaint (R² = 0.93). Given this fixed mixing magnitude connected the device, the dependence of the mixing connected the flowrate was measured to guarantee capable mixing astatine 1 μL/min arsenic shown successful Fig. 3B.

Figure 3

Mixing efficiency. (A) Representative microscopy images showing progression of the mixing of PBS and Brilliant Blue FCF successful PBS astatine the entrance, middle, and exit regions of involvement wrong the mixing channels astatine 10, 5, 2, 1.5, and 1 µL/min. (B) The slope of the strength illustration to bespeak mixing ratio astatine the exit portion of the mixing transmission arsenic a relation of perfusion volumetric travel rate. Calculated implicit mixing scale (AMI) astatine the exit portion of the mixing transmission arsenic a relation of perfusion volumetric travel rate. An AMI worth of zero indicates implicit mixing.

Since mixing depends connected diffusion, it is expected that mixing ratio arsenic a relation of travel complaint depends connected molecule size, wherever MW and size find molecular diffusivity³⁴. We utilized Brilliant Blue FCF, that has a molecular value (MW) of 792.9 g/mol to qualify the mixing, which is larger than astir chemotherapeutic agents arsenic shown successful Table 1. Complete mixing of Brilliant Blue FCF dissolved successful PBS was achieved astatine 1 µL/min and the clip required for diffusion was 34.0 s. The diffusion coefficient and clip required for diffusion crossed the width of the transmission was besides calculated for respective FDA-approved chemotherapeutics: temozolomide, paclitaxel, doxorubicin, carmustine, and lomustine. For example, according to our calculations, temozolomide, which has a smaller diameter and higher diffusivity than Brilliant Blue FCF, would diffuse crossed the transmission width astir 2-times faster. This effect implies that a volumetric travel complaint greater than 1 µL/min could inactive pb to implicit mixing. On the different hand, paclitaxel, which is akin successful size and diffusivity to Brilliant Blue FCF, should necessitate akin clip to diffuse crossed the transmission width (31.2 s). Hence, a volumetric travel complaint of 1 µL/min should beryllium utilized to execute implicit mixing. Overall, since each molecules considered were smaller than the Brilliant Blue FCF used, we expect that the physiologic 1 µL/min volumetric travel complaint would beryllium capable to execute implicit mixing for astir experiments aiming to nutrient a attraction dilution of tiny molecules, specified arsenic chemotherapeutics.

The concentrations generated by the MCGG were measured by perfusing 25 μM of Brilliant Blue FCF successful PBS successful the ‘100% inlet’ and PBS successful the ‘0% inlet’ astatine 1 µL/min (Fig. 4A), which produced concentrations of 100%:51%:26%:0% (Fig. 4B), akin to the expected dilution of 100%, 50%, 25%, and 0%. The dilution fractions were interpolated utilizing the intensities of the 100% and 0% microchannels earlier splitting (Fig. 4C). The strength successful transmission A (closest to the inlet containing Brilliant Blue FCF successful PBS) was acceptable to 100%, portion the inlet containing PBS (Channel D) was acceptable to 0%. The experimentally generated attraction dilution was similar the predicted theoretical attraction dilution (Fig. 1B), with an R² worth of 0.99, further demonstrating that implicit mixing of the 2 perfusion fluids was achieved astatine 1 µL/min.

Figure 4

Gradient generation. (A) Schematic indicating channels A–D, wherever the generated attraction gradient was measured. (B) Measured concentrations astatine 1 µL/min travel rate, wherever transmission A contains 100% of the archetypal attraction of Brilliant Blue FCF and transmission D contains 0% Brilliant Blue FCF. (C) Representative images of the attraction gradient successful each transmission astatine 1 µL/min travel rate.

Cell loading reproducibility

The loading reproducibility of hydrogels into the microwells, arsenic good arsenic the similarity of loading crossed each wells wrong the instrumentality was characterized. Consistent loading of the cells is facilitated by the flow-through plan of the loading channels. The fig density of the cells and their determination should beryllium stochastic with shot-noise being the main root of loading variability without a root of systematic bias. With the flow-through plan of the loading channels, the attraction of the cells should beryllium the aforesaid arsenic it is successful the archetypal homogenously mixed solution successful the lack of compartment accumulation oregon depletion. To measurement loading reproducibility, PEGDA hydrogels with encapsulated DiOC-stained cells were loaded into the microwells utilizing an insulin needle and past exposed to UV airy for gelation. As each 4 microwells wrong a file were loaded simultaneously (Fig. 5A), the mean density of the cells wrong the wells of a file were compared (Fig. 5B). Our results bespeak that the loading reproducibility was akin among the 4 columns arsenic determination was nary important quality successful the median compartment density for each file (Fig. 5C).

Figure 5

Cell loading reproducibility. (A) Device schematic of loading ports and channels and the compartment civilization microwells, wherever each file of compartment civilization chambers is colour coded, starting with file 1 successful blue. (B) Fluorescent images of the compartment civilization chambers loaded with hydrogels with entrapped U87 cells stained with DiOC. Scale bar = 200 µm. (C) Density of cells wrong the compartment civilization microwells of each column. (D) Images of the bottom, one-third, two-thirds, and the apical of a hydrogel erstwhile loaded successful a compartment civilization microwell. The hydrogels are labelled with greenish fluorescence via tethering of FITC-modified ligands to the 4-arm PEG-Ac. Scale bar = 500 µm.

In a abstracted experiment, we utilized fluorescent PEG (green) loaded with reddish fluorescent beads, to find whether the hydrogel occupied the full good evenly and whether the beads were evenly distributed done the extent of the hydrogel. Images captured passim the extent of the good indicated that the hydrogel filled the microwells and the fluorescent beads remained well-distributed passim the entirety of the hydrogel (Fig. 5D). However, the greenish gel appears not polymerized adjacent the PDMS partition and the membrane, which are regions with precocious oxygen vulnerability during the polymerization. It is known that oxygen inhibits UV polymerization³⁵ and that it has an elevated attraction successful PDMS³⁶. Also, tiny hydrophobic molecules specified the photoinitiator utilized present easy diffuse into PDMS, perchance depleting their attraction successful the gel precursor solution, which could besides explicate the ‘halo’ seen successful the proximity of the PDMS walls. This signifier of hydrogel operation increases the aboveground country done which solute speech tin hap but does not change the full magnitude of solute that tin beryllium exchanged, which is determined by the volumetric travel done the perfusion layer. Dependent connected the nonstop application, this hydrogel signifier tin beryllium either beneficial oregon detrimental. Future enactment volition analyse methods to little the oxygen attraction successful the PDMS anterior to polymerization utilizing non-cytotoxic methods. We person developed methods to afloat polymerize hydrogel plugs wrong PDMS microchannels³⁷ but person not tested the cytotoxicity of this protocol. Note that erstwhile a 4-arm PEG-Ac gel, which polymerizes via Michael-type addition, was loaded successful the compartment civilization wells, it polymerized completely, including successful the contiguous vicinity of the PDMS walls (Supplemental Fig. S5) due to the fact that polymerization via Michael-type summation is not affected by oxygen and does not beryllium connected a tiny molecule initiator. This information besides showed that a assortment of hydrogel matrices tin beryllium accommodated with the existent device.

One situation of loading cells successful microfluidic devices successful wide is eliminating aerial bubbles that tin beryllium trapped successful the microwells during loading. To debar aerial bubbles, others person archetypal loaded the strategy with h2o and past degassed it³⁸. We chose to load the instrumentality adust (not pre-filled with water) to destruct dilution of the gel precursor solution with water. Air bubbles are a regular occurrence successful microfluidic devices^39,40. While we amusement that successful our strategy bubbles did not negatively interaction compartment viability (see Supplemental Fig. S7), they could make differences betwixt replicate compartment civilization chambers. An betterment that could beryllium considered successful the aboriginal would beryllium changing the geometry of the microchannels wrong the device³⁹ to region the crisp country betwixt the loading channels and the reservoirs. The existent crisp corners contiguous successful some the loading and perfusion layers could trap air, arsenic the space of the country is little than that of the interaction space of the filling liquid, creating a little wettability situation.

Drug penetration and compartment cause responsiveness

First, we determined the clip it would instrumentality for a tiny molecule (e.g. chemotherapeutic) being perfused to diffuse into the hydrogel and scope the aforesaid attraction arsenic the perfusion channel. To measurement this diffusion, we utilized a fluorophore Atto 655 (887 g/mol) to exemplary the diffusivity of a tiny molecule cause specified arsenic TMZ (194 g/mol) oregon BCNU (214 g/mol), which allowed america to measurement diffusion coefficient successful situ and successful existent clip utilizing FCS (Supplemental Fig. S6). Diffusion coefficients for the fluorophore Atto 655 successful media, arsenic good arsenic successful PEGDA hydrogels were measured arsenic 4.25 × 10^–6 and 2.55 × 10^–6 cm²/s, respectively. As expected, the diffusion coefficient successful media was importantly higher than that successful the hydrogel, which could beryllium explained by carnal obstruction, arsenic shown by america previously⁴¹. With the measured diffusion coefficients, we past utilized Fick’s instrumentality of diffusion to find whether TMZ oregon BCNU were capable to penetrate the hydrogel and however agelong it would instrumentality for the drugs to scope the desired attraction wrong the gel. We modeled TMZ attraction (at 2 mM archetypal concentration) and BCNU attraction (at 10 µM archetypal concentration) arsenic a relation of clip (up to 48 h) astatine a hydrogel extent of 250 μm (the thickness of the gel wrong the microwell). Drug attraction was recovered to be > 1.9 mM wrong 1 h and > 1.95 mM wrong 4 h upon TMZ summation and > 9.5 µM wrong 1 h upon BCNU addition, indicating that for 48 h of exposure, each cells should beryllium arsenic exposed to some drugs.

We adjacent tested U87 compartment susceptibility to 2 antithetic drugs, namely TMZ and BCNU, successful 2 antithetic PEG hydrogels. TMZ is the existent modular of attraction and BCNU is utilized arsenic an adjuvant oregon successful operation with TMZ. Both drugs are approved for the attraction of glioblastoma and some are alkylating agents known to induce compartment rhythm apprehension and apoptosis⁴². The 2 gels were PEGDA and the enzymatically degradable, RGD-modified 4-arm PEG-Ac gel. The PEGDA gel, formed via UV photopolymerization, is non-degradable and inert, hence cells were not expected to interact with it. It served arsenic scaffolding lone and cells encapsulated successful it for up to 4 days retained precocious viability (> 90%) (Supplemental Fig. S7A) but remained circular (Supplemental Fig. S7B). The 4-arm PEG-Ac gel is degradable and compartment adhesive, and cells were capable to interact with the hydrogel and remodel it implicit clip arsenic evidenced by compartment spreading astatine time 4 and compartment viability > 95% (Supplemental Fig. S7).

Upon attraction with 0, 0.5, 1, and 2 mM TMZ, PEGDA-encapsulated U87 cells were recovered to person viabilities of 86.4 ± 1.9%, 75.0 ± 1.2%, 34.0 ± 7.7%, and 15.8 ± 5.7%, respectively (Fig. 6A,B). The EC₅₀ (effective attraction needed to termination 50% of the cells) was estimated to be ~ 0.61 mM TMZ arsenic calculated by a sigmoidal curve acceptable (R² = 0.983). Cells were little susceptible to TMZ erstwhile encapsulated successful the degradable, adhesive 4-arm PEG-Ac hydrogel (Fig. 6C,D). Cell viability was 95.6 ± 1.2%, 83.9 ± 12.9%, 59.1 ± 6.1%, and 44.0 ± 8.2% for 0, 0.5, 1, and 2 mM TMZ, respectively and the EC₅₀ was ~ 1.8 mM (R² = 0.901). This effect was not astonishing arsenic integrin binding has been antecedently implicated successful cause absorption of glioblastoma cells^43,44,45, and we person shown akin results antecedently for glioblastoma spheroids⁴⁶. The EC₅₀ for U87 cells encapsulated successful a PEGDA hydrogel and treated with BCNU was ~ 8.2 µM (R² = 0.952) (Fig. 6E,F). As expected for each conditions, the estimated EC₅₀ was higher compared to 2D monolayer culture, but akin to what has been reported by others for hydrogel-encapsulated GBM cells². Dead cells were disposable passim the gel aft 48 h of vulnerability for each conditions, suggesting that the microenvironment wrong the gels was homogeneous (Fig. 6A,C,E).

Figure 6

Drug responsiveness. (A) Representative U87 compartment live/dead images upon TMZ treatment, and (B) compartment viability arsenic a relation of TMZ attraction (n = 3) for cells seeded successful a non-degradable, non-adhesive PEGDA gel. (C) Representative U87 compartment live/dead images upon TMZ treatment, and (D) compartment viability arsenic a relation of TMZ attraction (n = 3) for cells seeded successful a degradable, adhesive 4-arm PEG-Ac gel. (E) Representative U87 compartment live/dead images upon BCNU treatment, and (F) compartment viability arsenic a relation of BCNU attraction (n = 3) for cells seeded successful a non-degradable, non-adhesive PEGDA gel. All cells were stained with DiOC (green) and dormant cells were stained with PI (red). Scale barroom is 200 µm.

Our results suggest that the instrumentality could beryllium utilized for investigating the effect of cell–matrix interactions connected compartment responsiveness to soluble factors, specified arsenic chemotherapeutics, wherever the therapeutic could readily and homogenously penetrate the cell-seeded hydrogels. While not explored here, the instrumentality could beryllium adapted for usage with antithetic cells and compartment co-cultures and surface their responses to antithetic soluble molecules. Since the plan includes hydrogel-encapsulated cells, it could beryllium utilized to survey the effect of cell–matrix and cell–cell interactions connected compartment responsiveness to soluble factors, specified arsenic therapeutics successful a physiologically applicable situation that includes perfusion and enables the enactment of attraction dilutions.

References

Authors and Affiliations

1. Department of Biomedical Engineering, Saint Louis University, St Louis, MO, 63103-2010, USA

   Allison Clancy, Joseph Bruns, Jahnavi Nadella, Samuel Stealey & Silviya P. Zustiak

2. Department of Bioengineering, and Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA

   Dayi Chen, Yanjia Zhang & Aaron Timperman

Contributions

A.T. and S.Z. conceived the idea, designed the project, and supervised and funded the research. A.C. and S.Z. wrote the archetypal draught of the manuscript. Microfluidic devices were fabricated by D.C. nether the guidance of A.T. J.B. and A.C. prepared Figs. 1, 5, and Supplementary Fig. S5. D.C., A.C., and Y.Z. prepared Fig. 2 and Supplementary Figs. S1–S4. A.C., J.N., and S.S. prepared Figs. 3 and 4. S.S. prepared Fig. S6. J.B. prepared Fig. S7. All authors contributed to and reviewed the main manuscript text.

Corresponding authors

Correspondence to Aaron Timperman oregon Silviya P. Zustiak.

Competing interests

The authors state nary competing interests.

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