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An advanced 3D lymphatic system for assaying human cutaneous lymphangiogenesis in a microfluidic platform – NPG Asia Materials

Fabrication of the microfluidic chip

The microfluidic devices were fabricated by soft lithography. The SU-8-100 photoresist spin-coated 16-inch silicon wafer was baked at 95 °C for 1 h, and then, microfluidic channels were patterned by selectively exposing ultraviolet (UV) light through a mask. A polydimethylsiloxane (PDMS; Dow Chemical, Sylgard 184) solution, which is a representative biocompatible substance23, was poured on the wafer and cured at 80 °C for over 1 hour in a dry oven. The wafer was coated with polytetrafluoroethylene, and the PDMS curing could be repeated more than 80 times. After the cured PDMS was detached from the wafer, reservoirs were punched using 3 mm and 1 mm biopsy punches. The punched PDMS and cover glass were sterilized at 120 °C for 30 minutes and then dried at 80 °C overnight. The dried cover glass was bonded onto the PDMS with an oxygen plasma treatment (FEMTO Science). Then, the hydrophilic microchannels were coated with 2 mg/mL biocompatible PDA solution24 and incubated at 37 °C for 1 hour 30 minutes. After the channels were washed with deionized distilled water (DDW) twice, the microfluidic chip was dried at 80 °C overnight to allow the microchannels to be hydrophobic. Collagen type 1 (COL1) solution (Corning, 354236) at 2 mg/ml, 3 mg/ml, and 5 mg/ml was prepared by mixing with 10X phosphate buffered saline (PBS) and DDW. In addition, sodium hydroxide (NaOH, 1 N) was added to adjust the pH to 7.4. The mixed COL1 solutions were injected inside of each of the 3 ECM gel channels in series and incubated at 37 °C for 40 minutes for gelation. After that, the medium channels were filled with cell culture medium.

Cell preparation

For lymphatic endothelial cell experiments, human dermal lymphatic endothelial cells and juvenile foreskin (HDLEC; Promocell, C-12216) were purchased and cultured in an EGM2-MV bullet kit (Lonza, CC3202) at 37 °C in a humidified 5% CO2 incubator. For keratinocyte experiments, Human Epidermal Keratinocyte, Adult, Single Donor (NHEK-Ad; Lonza, 00192627) were purchased and cultured with the KGM Gold Keratinocyte Growth Medium BulletKit (Lonza, 00192060). HDLECs and NHEKs were grown to ~ 70% and ~ 80% confluence in 75 T flasks, respectively.

Cell culture in a microfluidic chip

For lymphatic endothelial barrier formation, HDLECs were detached using Accutase (Innovative Cell Technologies, AT104) at 37 °C for 10 minutes, and the cell suspension was prepared at a density of 2 × 106 cells/ml. All medium in the microfluidic chip was removed, and 40 µl of the cell suspension was injected into each lymphatic seeding channel. Then, the devices were tilted by 30° at 37 °C for 1 hour so that HDLECs could be attached mainly to the COL1 hydrogel wall. Subsequently, unattached HDLECs were eliminated, and then, all medium channels were filled with fresh medium. After the lymphatic single layer was established for 2 days, lymphangiogenesis was induced for the next 6 days.

Since KGM Gold medium contains 5% more fetal bovine serum (FBS) than EGM-2 MV medium, KGM Gold with 5% FBS was used from this stage. This medium was supplemented with 100 nM sphingosine-1-phosphate (S1P; Sigma, 73914) for all four conditions, 100 ng/ml of vascular endothelial growth factor-A (VEGF-165; PeproTech, 100-20) and VEGF-C (PeproTech, 100-20CD) except for the negative control. For combined treatment, 50 ng/ml and 250 ng/ml Lymphanax were added to the medium of the V100 + L50 and V100 + L250 conditions, respectively. In addition, considering that Lymphanax was dissolved in 10% DMSO solution, the DMSO ratio of all culture media was adjusted to be the same.

For the skin and lymphatic endothelial barrier coculture system, NHEKs were detached using Accutase at 37 °C for 10 minutes, and the cell suspension was prepared at a density of 2.4 × 106 cells/ml. All medium in the microfluidic chip was removed, and 40 µl of the cell suspension was injected into each skin seeding channel. NHEKs were cultured for 3 days to form a skin barrier, and HDLECs were introduced to the lymphatic seeding channel. After we confirmed that the HDLECs were well attached, an air-liquid interface was established by removing KGM Gold from the skin seeding channel and tilting the device above 45°. Moreover, ascorbic acid (Sigma, AA4403), which can enhance the differentiation of keratinocytes, was added. Induction of lymphangiogenesis began on D5, which was 2 days after HDLECs were seeded.

Immunofluorescence staining

Cells in the devices were fixed with 4% paraformaldehyde (PFA; Biosesang, PC2031-100-00) for 30 minutes and permeabilized with 0.2% Triton X-100 (Sigma, T8787) for 5 minutes at room temperature. Then, the cells were blocked using 5% bovine serum albumin (BSA, Sigma, A9418) for 90 minutes at room temperature. Forty microliters of primary antibody solution diluted in PBS containing anti-LYVE1 (1:100, Thermo Fisher Scientific, MA5-32512), anti-RELN (1:100, Thermo Fisher Scientific, PA5-78413), anti-PROX1 (1:100, Invitrogen, PA5-85552), anti-VE cadherin (1:100, Abcam, ab33168), anti-VEGFR3 (1:100, Thermo Fisher Scientific, MA5-15651), anti-CD31 (1:200, Abcam, ab24590), anti-Ki67 (1:500, Abcam, ab15580), anti-K10 (1:100, Abcam, ab76318), anti-K14 (1:100, Abcam, ab7800), and anti-Loricrin (1:200, Novus Biologicals, NBP1-33610) in PBS was added to each channel and stored at 4 °C overnight. After that, appropriate secondary antibodies at 1:200 dilutions were added to the samples for 2 hours at room temperature. For staining of F-actin and DNA, 1:500 diluted phalloidin and DAPI in PBS were added. All procedures were performed with devices rocking gently on a shaker, and three sets of PBS washes were included in each step. Images were obtained using a confocal microscope (Carl Zeiss, LSM-700).

Quantification of lymphangiogenesis

All images used for analysis were immunostained with phalloidin and captured using Celena® X (Logos Biosystems). Due to their tree-like form, vascular networks can be structurally categorized as branches and nodes. Since the innermost layer of a vessel consists of endothelial cells that combine to form a tube, branches and nodes consist of multiple endothelial cells and have a 3D structure25,26,27. Based on F-actin-stained images (1600 × 1200 pixels), 3D lymphangiogenic morphogenesis was quantified by 12 indicators. The algorithm recognized the sprouting region between nodes as a branch. Curvature refers to the curvature of a branch; Bra. Area refers to the area of a branch; Nod.Rad refers to the radius of a node; Bra.Euc.Leng refers to the Euclidian distance between the endpoints of each branch; Bra.Width refers to the average width of a branch; N.C.B. refers to the number of branches connected to a node; Bra.Length refers to the total distance between the endpoints of each branch; Bra.Num indicates the number of branches; Area refers to the area of the entire vasculature; Nod.Num refers to the number of nodes; Bra.∆.Width refers to the mean value of width differentiation at each branch point. For manual image analysis, values of whole vessels containing several branches were measured using ImageJ software. Ves.Num refers to the number of vessels; Area refers to the area of the entire vessel; Ves.Leng indicates the total distance from the starting point to the endpoint of each vessel in a straight line; Separation refers to the number of vessels separated from the vessel divided by the total Ves.Num. The code for this algorithm was released on GitHub28. [https://github.com/moonchildz/angio_analysis].

Preparation of lymphanax

For Lymphanax, fresh ginseng was washed 3 times and dried for 30 minutes. The ginseng was then cut to 1 cm long, cultured in anaerobic conditions for 3 weeks, and dried under hot air at 60 °C. Subsequently, Lymphanax was immersed in 50%-80% EtOH at 50–80 °C for 5 hours. After this process was repeated 3 times, the extracted material was vacuum filtered, concentrated and purified with a rotary evaporator. The obtained Lymphanax was dissolved in DMSO and ready for cell treatment.

Permeability analysis

For measurement of the permeability of the skin layer in the microfluidic chip, 10 µM 0.4 kDa fluorescein sodium salt (Sigma, F6377), 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran; Sigma, FD4), and 40 kDa fluorescein isothiocyanate-dextran (FITC-dextran; Sigma, FD40S) solutions were added to the medium channel of the skin layer. Fluorescence images of the gel channel immediately next to the skin layer were captured after 0 min, 60 min, and 120 min of incubation at 37 °C. The intensity profile of diffusion was measured by ImageJ software, and then, the permeability value P was calculated using Fick’s first law as follows:

$$J=-Dfrac{partial C}{partial x}$$

$${Flux}=-PDelta C$$

where J is the flux, D is the diffusion coefficient, C is the concentration, x is the position, and P is the permeability of the skin layer.

Statistical analysis

Data are presented as the mean ± standard error of the mean (SEM), and statistical significance was determined using GraphPad Prism software (version 5.01). Comparisons among the groups were determined by one-way analysis of variance (ANOVA) followed by post hoc pairwise comparison testing using Tukey’s method. Significance was defined as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.