Supplementary Information

Large‐scalePatterning of Living Colloids for Dynamic Studies of Neutrophil‐Microbe Interactions

Jae Jung Kima†, Eduardo Reátegui b,c,d †, Alex Hopkeb, Fatemeh Jalalib, Maedeh Roushanb, Patrick S. Doylea*, and Daniel Irimiab*,aDepartment of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.bBioMEMS Resource Center, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, MA, 02129, USA.cMassachusetts General Hospital Cancer Center, Harvard Medical School, MA, 02129, USA.d current address: William G. Lowrie Department of Chemical and Biomolecular Engineering and Comprehensive Cancer Center. The Ohio State University, Columbus, OH, 43210, USA.

Supplementary Figures

Fig. S1 Detail configuration of platforms in the assembly (a) and imaging processes (b).

Fig. S2 Washing step with and without confinement.

Fig. S3 3D image of C. albicans array.

Fig. S4 Patterning of heterogeneous microorganisms.

Fig. S5 Large-scale patterns of various bacteria species generated by porous microwells.

Fig. S6 Host-pathogen interactions between patterned S. aureus and human neutrophils.

Supplementary Movies

Movie S1 Time-lapse of patterned C. albicans.

Movie S2 Time-lapse of radiation of C. albicans hyphae.

Movie S3 Time-lapse of neutrophil migration toward patterned C. albicans.

Movie S4 Time-lapse of neutrophil migration toward patterned S. aureus.

Electronic Supplementary Material (ESI) for Lab on a Chip.This journal is © The Royal Society of Chemistry 2018

Fabrication of micro-wells

Porous micro-well arrays are fabricated using a previously reported process[23]. Briefly, PDMS

(Sylgard 184, Dow Corning) is mixed in a 10:1 ratio and poured on top of SU-8 master

(Microchem) fabricated by a standard photolithography procedure, to make a PDMS mold for

porous micro-wells. After the PDMS is fully cured (65 °C, overnight), the mold is punched to

make an inlet. Prepared PDMS mold and flat PDMS are placed on the top and bottom of porous

PET membrane (Millipore, MCSP06H48, diameter of pore = 3 µm). NOA 81 drop is placed on

top of the inlet, and the whole ensamble is placed in a vacuum desiccator. After the vacuum is

applied, all the air trapped by the mold escapes through the inlet. As vacuum is removed, NOA 81

is injected into the mold through the inlet and it spreads between the molded and flat PDMS pieces

due to the pressure difference. Injected NOA 81 is cured by exposure to ultraviolet light for 6 min.

The PDMS parts are then removed to leave the porous micro-wells. A PDMS wall is bonded to

micro-well arrays by using biocompatible adhesive (Sigma-Aldrich, GBL654008). Note that the

PDMS mold can be deformed due to the pressure difference while the PDMS mold is vacuumed.

Such deformation does not cause the problem when the PDMS mold is in contactwith the

membrane. However, severe deformation can remove the contact. Such severe deformation can be

minimized by using molds with high modulus, low aspect ratio, short inter-well distance, and low-

pressure difference.

Patterning of microorganisms

Microorganism patterning is aided by vacuum suction. To apply a negative pressure to the micro-

wells, we use a sterile filter unit (Nalgene) and a PDMS block with a hole. The filter unit is

connected to a vacuum pump after its filter membrane is removed. To ensure conformal contact,

we sequentially place the punched PDMS block and micro-well platform on top of the filter unit.

The microorganism solution is dispensed on top of porous micro-wells, inside the PDMS wall

(height of PDMS wall ~ 3 mm, Fig. S1a). Vacuum (150-400 mmHg) is then applied to the whole

system in order to guide and arrange microorganisms inside the wells. Excess microorganisms are

washed away using a squeeze bottle. We found that the washing step can be improved by adding

the confinement (Fig. S2). Arrangement and washing steps are repeated as necessary to efficiently

fill all the wells of the arrays.

After microorganism patterning, the whole platform is transferred and adhered on top of a glass

plate by using a biocompatible double sided adhesive tape (Sigma-Aldrich, GBL654008). Using

gentle pipetting, we then exchange the remaining solution inside the PDMS wall with the

appropriate media. It is important that the platform does not dry out during experiments. Drying

is avoided by filling the area surrounding the platform with the same media (Fig. S1b, the levels

of media inside/outside the PDMS wall are ~ 3 mm). A cover glass is placed on top of the PDMS

wall of the platform to avoid evaporation of media during time-lapse experiments.

Fig. S1 Detail configuration of platforms in the assembly (a) and imaging processes (b). During microbe patterning, a suspension of microorganisms is added on top of the microwells. Hydrodynamic force guides microorganisms to microwells. After arrangement, redundant microorganisms are washed away. For imaging, the device with microwells is covered with a coverslip and submerged in media inside a glass-bottom plate.

Fig. S2 Washing step without and with confinement. Washing redundant microorganisms can be more readily achieved in the presence of confinement (b, d). Confinement is generated by placing flat PDMS block on top of arrays with 120 µm spacing. Without confinement (a, c), we rely on the turbulent flow for washing away any redundant microorganism. However, with the confinement, we are able to generate streamlines parallel to the platform, and accomplish a better washing. Figure S2c, d are taken while focusing on the top surface of microwell arrays. Brightness and contrast are adjusted to visualize individual, residual C. albicans. In this project, all experiment were accomplished without confinement. In the future, it would be beneficial to add the microfluidic channel on top this array to more precisely control the washing step.

Fig. S3 3D image of C. albicans array. The image is generated using a confocal microscope (LSM710, AxioObserver, Zeiss, New York, USA). Different colors represent different heights in the sample. Blue dots (H = 35 µm) represent the redundant C. albicans on top of the 35 µm deep microwells. The image has the poor resolution in z-direction, likely because of the porous membrane at the bottom of the platform. The volume of the filled well was roughly estimated based on the color in this image, and the relative distance between three focal points: porous membrane, the top surface of C. albicans cluster, and the top surface of microwells.

Neutrophil Isolation and Staining

Fresh human blood samples from healthy volunteers were purchased from Research Blood

Components (Allston, MA, USA). Human neutrophils were isolated within 2 h of blood draw

using an EasySep Human Neutrophil Enrichment Kit (STEMCELL Technologies, Vancouver,

Canada). After isolation, neutrophils were counted and stained with Hoechst 33342

trihydrochloride (Life Technologies, Woburn, MA, USA). Stained neutrophils were then

resuspended at 1.5 x 106 cells/mL in cell culture media (IMDM media with 20 % FBS).

Live-cell Imaging and Tracking

Patterned microorganisms and neutrophils were imaged using time-lapse imaging at 10x and 20x

magnification using a fully automatic Nikon TiE microscope (Microdevices Instruments, Avon,

MA, USA) with an incubator stage set at 37 °C and 5 % CO2. Successive frames were acquired at

either 15 or 5 sec, depending on the type of analysis. Neutrophils were automatically tracked using

Imaris spot detection and tracking (Bitplane). Tracks with durations of at least 60 s were considered

for the anlysis. The chemotactic index of each neutrophil was calculated as the cosine of the angle

between the radial velocity towards the target and the velocity.

Chemotactic index calculation

The chemotactic index (CI) of individual neutrophils at time t was calculated as . 𝐶𝐼(𝑡)=

‒ 𝑅'(𝑡)𝑋'(𝑡)

indicates the rate of change of the distance between the neutrophil’s position (x) 𝑅'(𝑡) =

∂∂𝑡‖𝑥 ‒ 𝑧‖

and the target, C. albicans pattern, position (z). We choose the centroid of this object for z.

Neutrophil migration speed is estimated as .𝑋'(𝑡) = ‖∂𝑥∂𝑡‖

Confocal Microscopy

Patterned microorganisms on microporous wells were mounted on a 1”x3” cover slip for direct

imaging. Micrographs were captured with a confocal microscope (LSM710, AxioObserver, Zeiss,

New York, USA) equipped with a 20x Zeiss Plan-APOCHROMAT DIC air objective, and an

incubator stage set at 37 °C and 5 % CO2. HeNe laser and Argon lasers were utilized to image with

following excitation/emission wavelength: 633/699 nm; 514/614 nm; and 488/526 nm.

Supernatant Analysis

Isotated neutrophils were suspened in IMDM media supplemented with 4 % human serum albumin

(HSA). Neutrophils were added to the platform and allowed to interact with the patterned

microorganisms for 1h. After that, the supernatant was extracted from the platform and filtered

using a Spin-X centrifuge tube filter (0.45 m cellulose acetate, Costar) for 5 min at 800 RPM.

This step assures that cells are removed before protein analysis. A human cytokine array detection

kit was used (ARY005B, R&D Systems, Minneapolis, MN, USA) to identify proteins. The

concentrations of 36 inflammatory cytokines were measured. Results were compared with

measurements from control samples. Pure media and media with only neutrophils (no microbes)

were used for the two control conditions.

Microorganism Culture

C. albicans was streaked out from a glycerol stock onto YPD agar plates and grown at 37°C

overnight. A single colony was picked and transferred to 15mL YPD liquid and grown at 37°C

overnight with shaking. The strain SC5314-FarRed670 was primarily used for experiments. Two

additional strains, SC5314-GFP and Caf2-dTom, were also used during three color Candida

patterning[28, 38] (Fig. S3). The concentration of C. albicans used in this study was ~ 7 × 107/ml.

The SH1000-GFP Staphylococcus aureus (S. aureus) strain, expressing green florescent protein

(GFP), was provided from the laboratory of Mary Mullins at the University of Sheffield (Sheffield,

UK) as a generous gift. S. aureus were cultured in brain heart infusion (BHI) agar (Remel, Lenexa,

KS, USA). Single colonies were transferred and suspended in 7 ml of BHI broth medium. After

overnight incubation with shaking at 37 °C in an aerobic condition, 1ml of bacterial suspension

was added to 49 ml of BHI broth and sub-cultured for 4 hours. The Actinomyces graevenitzii (A.

graevenitzii) and Corynebacterium matruchotii (C. matruchotii) were cultured in Chacollate II

agar (GC II Agar with hemoglobin and IsoVltalexTM, BD, USA). Single colonies from agar plates

were picked and suspended separately in 10 mL of BHI broth medium (Remel, Lenexa, KS, USA)

and then incubated at 37°C in anaerobic incubator. After overnight incubation, bacterial

suspensions were used for the experiment. A. graevenitzii and C. matruchotii were stained by

BacLight Red and Green bacterial stains (Molecular Probes, OR, USA) by following the protocol

provided by vendor, respectively.

The concentration of S. aureus, A. graevenitzii, and C. matruchotii used in this study was about 5

× 108, 4 × 107, and 1 × 106/ml, respectively. As a rule of thumb, concentration of microorganism

can be adjusted by considering following points. At higher concentrations, a large number of

microorganisms will grow outside the microwells. At lower concentrations, microorganisms

would not pack efficiently, and could be easily removed during the washing steps of the loading

protocol. These details were added in the supplementary information.

Fig. S4 Patterning of heterogeneous microorganisms. C. albicans strains expressing three different fluorophores were patterned simultaneously. Representative images are taken with an epifluorescent microscope. Inset shows the image taken by confocal microscopy. Individual yeast cells can be defined within micro-wells.

Fig. S5 Large-scale patterns of various bacteria species generated by porous microwells. a) S. aureus. b) Corynebacterium matruchotii. c) Actinomyces graevenitzii.

Fig. S6 Host-pathogen interactions between patterned S. aureus and human neutrophils. Neutrophils migrates toward the patterned S. aureus aggregations. In this demonstration, before adding neutrophils on top of arrays, a drop of 4 % agarose solution is added between glass plate and platform (Fig. S1) and cured at 4 °C, to minimize any S. aureus leaking through the pore during the experiment.

Movie S1 Time-lapse of patterned C. albicans. Patterned C. albicans were incubated in YPD

liquid at 37 °C.

Movie S2 Time-lapse of radiation of C. albicans hyphae. Patterned C. albicans were incubated

in YPD liquid containing 10 ng/ml caspofungin at 37 °C.

Movie S3 Time-lapse of neutrophil migration toward patterned C. albicans.

Movie S4 Time-lapse of neutrophil migration toward patterned S. aureus.