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Selective biofunctionalization of 3D cell-imprinted PDMS with collagen immobilization for targeted cell attachment

cell culture

The ethics committee approved all the experiments of the Pasteur Institute of Iran. Human umbilical vein endothelial cells (HUVECs, Cell Bank of Pasteur institute of Iran) were used to prepare the aligned cell pattern. After obtaining informed consent, the adipose-derived stem cells (ADSCs) were isolated from adipose tissue withdrawn from healthy 20 years old human bodies and were used for culture on modified PDMS. First, adipose tissue was washed three times in phosphate-buffered solution (PBS) with 3% Penicillin/Streptomycin (Sigma, USA), then they were cut into 1–2 mm pieces and digested in 0.02 mg/ml collagenase type I (Sigma , United States) at 37 °C for 2 h. The solution was passed through a 75 μm filter to remove undigested tissue, followed by neutralization of the enzyme with Gibco Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco). Finally, they were centrifuged at 1300 rpm for 5 min to separate the cellular pellets. The obtained solution consisting of ADSCs was transferred to the culture medium consisting of DMEM/Ham’s F12 supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, and 10% Fetal bovine serum (FBS) and was incubated at 37 °C in a 5% COtwo incubator18. After 24 h, the non-adherent cells and debris were discarded, and a new culture medium was added. ADSCs in the third passage were used for cell seeding.

Preparing the aligned cell-imprinted PDMS substrate using a microfluidic chip

The aligned cell-imprinted substrate was prepared better to display the protein immobilization into the desired regions. The aligned cell-imprinted substrate by microfluidic chip was created based on the previous study19. In brief, a set of 128 micro-channels with a length of 20 mm and a depth of 50 μm was considered. The micro-channels have a 40 μm width and the ability to accommodate about 3 × 106 HUVEC cells in aligned lines. Microfluidic chip after washing with ethanol and sterilization in the autoclave was placed on a cell culture plate with channels side facing down. HUVEC cell solution with the concentration of 6 × 106 in 150 μl of culture medium DMEM/Ham’s F12 was injected into the chip at a flow rate of 50 μl/min by a syringe pump. After filling up the microchannel with the cells, the cell culture plate and microfluidic chip were incubated for 7 h for complete cell adhesion to the cell culture plate. At this point, we took out the microfluidic chip and washed the cells in PBS before fixing them in a 4% glutaraldehyde solution for 1 h. To transfer the ordered arrangement of the fixed cells to the PDMS, a layer of PDMS 10:1 was poured over them. After peeling the cured PDMS away from the fixed cells, a 1 M NaOH solution wash was used to remove any leftover cells or residues (Fig. 1A-i,ii).

Figure 1

Schematic illustration of (A-i, ii) creating aligned cell-imprinted PDMS substrate by micro-channels, (A-iii, iv) modification of aligned cell imprinted PDMS surface by collagen immobilization, (B.) functionalization of the glass substrate by GPTMS as stamp layer, (C-i) preparing the selective collagen-immobilization into the desired regions of PDMS substrate by using epoxy silane resin stamp method, (C-ii) Coomassie brilliant blue staining of the aligned cell-imprinted PDMS surface with random and (C-iii) selective collagen immobilization, (D-i) cell seeding on selective collagen-immobilized PDMS, (D-ii) crystal violet staining of cultured ADSCs on the random and (D-iii) selective collagen-immobilized cell-imprinted PDMS after 24 h.

Selective biofunctionalization of PDMS substrate by collagen immobilization

PDMS substrate consists of aligned cell pattern was treated with argon plasma (Harrick Plasma-PDC 32G) for 3 min at the pressure of 0.3 mbar followed by their immersion in 3-Aminopropyl triethoxysilane (APTES) (Sigma-Aldrich, USA) 10% in ethanol at 50 °C for 2 h. After removing APTES solution and washing twice with nuclease-free water, the samples were incubated with 20 μg/ml of bovine collagen (Nanozistaraye, Pasteur Institute, Iran) solution on a rocker shaker for 2 h and then were stored at 4 °C overnight followed by removing the collagen solution and washing with nuclease-free water. The collagen-coated PDMS substrate was stamped on an epoxy silane-modified slide to remove the collagen from the undesired regions from the undesired regions. Eventually, the resulting substrate was sterilized at ethanol 70% and was placed under UV light for 45 min. The non-selective collagen-immobilized substrate whose entire surface was coated with collagen was used as the negative control. Schematic presentation of the surface modification of PDMS is depicted in Fig. 1A-iii,iv, and the method for selective detachment of immobilized collagen showed in Fig. 1C-i.

Epoxy silane activated surface preparation

Untreated slides were washed with ethanol and then etched by immersion in 10% NaOH at 25 °C for 1 h, followed by sonication for 15 min in 10% NaOH. Later, it was rinsed four times in water, washed twice in ethanol, and derivatized in the coating solution: 2.5% (3-glycidoxypropyl) trimethoxysilane (GPTMS) and 10 mM acetic acid in ethanol at 25 °C for 1 h, again followed by a sonification step. Subsequently, they were washed thoroughly with ethanol and driedtwenty. Finally, they baked in a vacuum oven at 50 °C for 1 h and were ready to use as stamp surfaces (Fig. 1B).

Evaluation of the selective protein adhesion

We performed Coomassie brilliant blue staining assay to identify the selective collagen adhesion into the pits on the PDMS substrate. The random and selective collagen-immobilized PDMS, after washing three times with PBS were treated with Coomassie brilliant blue solution (1% w/v Coomassie brilliant blue in 50% methanol and 10% glacial acetic acid) for 1 h with shaking at 25 ° c. After the mentioned time, the Coomassie brilliant blue solution aspirated off, the samples were washed three times with deionized watertwenty-one. Afterwards, the samples were imaged under an optical microscope (BEL, INV2, Italy). Images were analyzed with the ImageJ program, and the area of ​​the stained region was measured. Afterward, the position of immobilized collagen and the percentage of the covered area was quantified.

Stability of immobilized collagen on the PDMS substrate

To check the stability of immobilized collagen on the PDMS substrate, a micro-BCA protein assay kit (Thermo Scientific, USA) was used. Collagen retention on the random and selective collagen-immobilized PDMS was measured on days 0, 7, and 14 after immobilization and applying the epoxy silane stamp method. For this purpose, the samples (4 cmtwo) were stored in sterilized 1× PBS buffer, pH 7.2 at 37 °C and 5% COtwo. In order to get rid of any free-floating proteins, samples were treated with 0.5% Tween 20 (Sigma-Aldrich, USA) for 30 min and then washed twice with nuclease-free water at the indicated times. Collagen retention on the surfaces was evaluated using the kit’s specified procedure. The absorbance of samples was measured at 562 nm with the Multiskan Spectrum microplate reader (Thermo Scientific, Singapore)22. Hence, the final amount of remaining collagen on the substrates was calculated based on the initial concentration of collagen, which was 20 µg/ml.

Evaluation of the cell viability on the random and selective collagen-immobilized PDMS substrate

To evaluate the effect of the epoxy silane stamp method and the process for preparing the collagen-immobilized substrate on cell viability, the MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma) assay was performed. Random and selective collagen-immobilized polydimethylsiloxane (PDMS) substrates were submerged in 70% ethanol for 30 min, dried under a laminar hood, and then irradiated with UV light for 45 min before being seeded with cells. ADSCs were sown on the sterile samples at a density of 104 cells/well. A cell-imprinted PDMS without collagen immobilization was used as a control. The samples were incubated for 1, 3, and 7 days at 37 °C with 5% CO2. At mentioned time points, the MTT solution at a concentration of 0.5 mg/ml was added to each well, then cells were stored in the incubator for 4 h at 37 °C. After the formation of formazan crystal, the medium was removed, and the crystals were dissolved in isopropanol. The plate was placed in the orbital shaker for 15 min to enhance the dissolution process. Optical density was measured by the ELISA reader (ELX800 Universal Microplate Reader, BIO-TEK Instruments, USA) at 570 nm23.

Evaluation of ADSCs’ desire to attach to the collagen-immobilized PDMS substrate

Crystal violet staining was performed to indicate the affinity of cell attachment to the selective collagen-immobilized region. The random and selective collagen-immobilized PDMS substrates were immersed in 70% ethanol for 30 min, and dried under a laminar hood followed by UV light irradiation for 40 min before ADSCs seeding. We seeded 1 × 104 ADSCs per cmtwo in culture medium DMEM/Ham’s F12 (3:1 ratio) and 10% (v/v) FBS on the samples and they were incubated at 37 °C with 5% COtwo overnight. A schematic presentation of cell seeding on the selective collagen-immobilized PDMS substrate was shown in Fig. 1D-i. After 24 h of culture, the cells were fixed with 4% glutaraldehyde solution for 24 h. Thus, fixed cells were stained with 1% crystal violet in 50% methanol for 10 min at 25 °C, then washed with distilled water. Stained viable cells cultured on the substrates were observed under an optical microscope (BEL, INV2, Italy)24.25. Images were analyzed with the ImageJ program. The location of attached cells and the total area covered by cells were quantified and statistically analyzed.

Statistical analyzes

Statistical analysis was performed using OriginLab software. Data statistically were analyzed using one-way analysis of ANOVA, followed by Tukey multiple comparison tests to determine the statistical significance. A p<0.05 was considered statistically significant.

Ethical standard

In vitro and in vivo experiments were registered at the Pasteur Institute of Iran and confirmed by its ethics committee. This project was supported by grant No. 1582 in Pasteur Institute of Iran.

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