Selective Biofunctionalization of 3D Cell Imprinted PDMS with Collagen Immobilization for Targeted Cell Attachment

Cell culture

The ethics committee approved all experiments at the Institut Pasteur in Iran. Human umbilical vein endothelial cells (HUVEC, cell bank from the Pasteur Institute of Iran) were used to prepare the aligned cell pattern. After obtaining informed consent, adipose-derived stem cells (ADSC) were isolated from adipose tissue taken from 20-year-old healthy human bodies and used for culture on modified PDMS. First, the 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, USA) at 37°C for 2 h. The solution was passed through a 75 μm filter to remove undigested tissue, followed by enzyme neutralization 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 cell pellets. The solution obtained composed of ADSC was transferred into the culture medium composed of DMEM/Ham’s F12 supplemented with 100 μg/ml of streptomycin, 100 U/ml of penicillin and 10% of fetal bovine serum (FBS) and was incubated at 37°C in 5% CO22 incubator18. After 24 h, non-adherent cells and debris were discarded and new culture medium was added. ADSCs from the third passage were used for cell seeding.

Preparation of Aligned Cell-Imprinted PDMS Substrate Using a Microfluidic Chip

The aligned cell-printed substrate was better prepared to display protein immobilization in desired regions. The microfluidic chip-aligned cell-printed substrate was created based on the previous study19. Briefly, a set of 128 microchannels with a length of 20 mm and a depth of 50 μm was considered. The micro-channels are 40 μm wide and have the capacity to accommodate approximately 3 × 106 HUVEC cells in aligned rows. The microfluidic chip after washing with ethanol and sterilizing in the autoclave was placed on a cell culture plate with the channel side down. Solution of HUVEC cells with the concentration of 6 × 106 in 150 μl of DMEM/Ham’s F12 culture medium was injected into the chip at a flow rate of 50 μl/min by a syringe pump. After filling the microchannel with 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 10:1 PDMS was cast on it. After peeling off the hardened PDMS from the fixed cells, a wash with 1 M NaOH solution was used to remove any remaining cells or residues (Fig. 1A-i,ii).

Figure 1

Schematic illustration of (A-i,ii) create a micro-channel aligned cell imprinted PDMS substrate, (A-iii, iv) modification of the aligned cell imprint PDMS surface by immobilization of collagen, (B) functionalization of the glass substrate by GPTMS as a buffer layer, (VS-i) prepare the selective immobilization of collagen in the desired regions of the PDMS substrate using the epoxy resin silane buffer method, (VS-ii) Coomassie brilliant blue staining of the aligned cell imprinted PDMS surface with random and (VS-iii) selective immobilization of collagen, (D-i) cell seeding on selective PDMS immobilized with collagen, (D-ii) crystal violet staining of ADSC grown on the random samples and (D-iii) Collagen-immobilized cell-printed selective PDMS after 24 h.

Selective biofunctionalization of the PDMS substrate by immobilization of collagen

The PDMS substrate consisting of an 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 the APTES solution and washing twice with nuclease-free water, the samples were incubated with a 20 μg/ml bovine collagen solution (Nanozistaraye, Institut Pasteur, Iran) on a rocker for 2 h, then stored at 4°C overnight. followed by removal of collagen solution and washing with nuclease-free water. The collagen-coated PDMS substrate was stamped onto an epoxy silane-modified blade to remove collagen from the unwanted regions of the unwanted regions. Finally, the resulting substrate was sterilized in 70% ethanol and placed under UV light for 45 min. The substrate immobilized with non-selective collagen whose entire surface was covered with collagen was used as a negative control. The schematic presentation of the surface modification of PDMS is shown in Fig. 1A-iii, iv, and the method of selective detachment of immobilized collagen is shown in Fig. 1C-i.

Surface preparation activated by epoxy silane

Untreated slides were washed in 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 with water, washed twice with 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 sonication step. Then they were carefully washed with ethanol and dried20. Finally, they were baked in a vacuum oven at 50°C for 1 h and were ready to be used as buffer surfaces (Fig. 1B).

Assessment of selective protein adhesion

We performed a Coomassie brilliant blue staining assay to identify the selective adhesion of collagen in the pits on the PDMS substrate. Collagen-immobilized random and selective PDMS, after three washes with PBS, were treated with a solution of Coomassie brilliant blue (1% w/v Coomassie brilliant blue in 50% methanol and 10% acetic acid icy) for 1 h with stirring at 25°C After the mentioned time, the Coomassie Brilliant Blue solution was aspirated, the samples were washed three times with deionized water21. Then, the samples were imaged under an optical microscope (BEL, INV2, Italy). The images were analyzed with the ImageJ program and the area of ​​the stained region was measured. Then, the position of the immobilized collagen and the percentage of the area covered were quantified.

Stability of collagen immobilized on the PDMS substrate

To check the stability of collagen immobilized on the PDMS substrate, a micro-BCA protein assay kit (Thermo Scientific, USA) was used. Collagen retention on random and selective collagen immobilized PDMS was measured on days 0, 7 and 14 after immobilization and application of the epoxy silane buffer method. For this purpose, the samples (4 cm2) were stored in sterilized 1× PBS buffer, pH 7.2 at 37°C and 5% CO2. In order to get rid of any floating proteins, the samples were treated with 0.5% Tween 20 (Sigma-Aldrich, USA) for 30 min, then washed twice with nuclease-free water at the indicated times. Collagen retention on the surfaces was assessed using the kit’s specified procedure. Sample absorbance was measured at 562 nm with the Multiskan Spectrum microplate reader (Thermo Scientific, Singapore)22. Therefore, the final amount of collagen remaining on the substrates was calculated based on the initial collagen concentration, which was 20 µg/ml.

Assessment of Cell Viability on Random and Selective Collagen-Immobilized PDMS Substrate

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

Assessing the Desire of ADSCs to Attach to Collagen-Immobilized PDMS Substrate

Crystal violet staining was performed to indicate the affinity of cell attachment to the selective collagen-immobilized region. 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 seeding ADSCs. We sowed 1 × 104 ADSC per cm2 in DMEM/Ham’s F12 culture medium (3:1 ratio) and 10% (v/v) FBS on the samples and they were incubated at 37°C with 5% CO2 overnight. A schematic presentation of cell seeding on the selective collagen-immobilized PDMS substrate has been shown in Fig. 1D-i. After 24 h of culture, the cells were fixed with a 4% glutaraldehyde solution for 24 h. Thus, the 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. The images were analyzed with the ImageJ program. The location of attached cells and the total area covered by the cells were quantified and statistically analyzed.

statistical analyzes

Statistical analysis was performed using OriginLab software. Data were statistically analyzed using one-way ANOVA analysis, followed by Tukey’s multiple comparisons test to determine statistical significance. A p

Ethical standard

In vitro and in vivo experiments have been recorded at the Institut Pasteur in Iran and confirmed by its ethics committee. This project was supported by grant n° 1582 from the Pasteur Institute of Iran.

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