Fabrication of biomimetic topological micropattern arrays to induce anisotropic/isotropic orientation of fibroblast

Abstract

During fibroblast activation, fibroblasts exhibit remarkable heterogeneity characterized by distinct subtypes with unique molecular markers and specialized functions. In cancer and fibrosis, fibroblasts drive extracellular matrix (ECM) remodeling and actin stress fiber contractility, thereby inducing a dysregulated ECM mechanical microenvironment, which in turn sustains their persistent activation. ECM topology is a key characteristic of the mechanical microenvironment. ECM topology refers to ECM fiber orientation, which interacts reciprocally with cellular orientation. In cancer, ECM and cellular orientations shift from random (isotropic) to aligned (anisotropic), whereas in fibrosis, these orientation transitions exhibit distinct patterns across different organs and tissues. Thus, understanding the relationship between fibroblast orientation and its activation and heterogeneity in cancer and fibrosis may facilitate identifying novel therapeutic targets. This protocol is established to fabricate biomimetic topological micropattern array chips with simple operation and cost effectiveness to induce anisotropic/isotropic fibroblast.

A method is established to fabricate rectangular (300 × 50 μm) and square (300 × 300 μm) micropattern array chips via microcontact printing, utilizing decellularized ECM as the bioink.

Micropattern array chips induce anisotropic/isotropic fibroblasts, with fibroblast orientation analyzed by F-actin staining and Fast Fourier transform analysis.

These chips enable in situ immunofluorescence staining and imaging.

Graphical abstract

Specifications table

Subject area	Engineering
More specific subject area	Biomedical engineering
Name of your method	Biomimetic topological micropattern arrays generated by Microcontact printing for regulating fibroblast anisotropy and isotropy
Name and reference of original method	Zhu X, Mao S, Yang Y, Liu X, Liu Q, Zhang N, Yang Y, Li Y, Gao M, Bao J, Li W, Li Y. Biomimetic Topological Micropattern Arrays Regulate the Heterogeneity of Cellular Fates in Lung Fibroblasts between Fibrosis and Invasion. ACS Nano. 19(1) (2025) 580–599.https://doi.org/10.1021/acsnano.4c11113
Resource availability	NA

Background

Fibroblasts are recognized as the key regulators in wound healing, cancer, and fibrosis, exerting diverse and dynamic functions via the fibroblast activation [1,2]. During the process of fibroblast-to-myofibroblast transition (FMT), fibroblasts exhibit remarkable heterogeneity and phenotypic plasticity, which is characterized by distinct subtypes with unique molecular markers and specialized functional properties [[3], [4], [5]]. In wound healing, myofibroblasts (MFs) rapidly revert to a quiescent state or undergo apoptosis upon completion of tissue repair [1]. However, in cancer and fibrosis, the persistent activation of MFs drives excessive extracellular matrix (ECM) deposition and remodeling and enhances the contractility of actin stress fibers, which contributes to the dysregulated ECM mechanical microenvironment [6,7]. Notably, a dynamic reciprocity exists whereby MFs generating this dysregulated mechanical microenvironment can in turn sustain their own persistent activation [7,8]. Generally, the dysregulated ECM mechanical microenvironment is primarily characterized by altered stiffness, viscoelasticity, and topological features [8]. While the effects of ECM stiffness and viscoelasticity on FMT and fibroblast heterogeneity have been extensively elucidated, the role of ECM topology remains poorly understood.

ECM topology refers to ECM fiber orientation, which interacts reciprocally with cellular orientation [9]. The aligned or random orientations of ECM or cells are commonly referred to as ECM or cellular anisotropy or isotropy, respectively [10]. These transitions of ECM or cellular orientation are easily observed by histological analysis of cancer and fibrosis tissues. Generally, during the process of tumor progression, cancer-associated fibroblasts deposit ECM and mediate actomyosin contraction, resulting in the transition from isotropic ECM to anisotropic ECM [7]. A study manifested that anisotropic fibroblasts could facilitate tumor cell migration and promote the expression of PDGFRα (a biomarker related to reactive tumor stroma) [11]. In fibrosis, transitions of ECM or cellular orientation display distinct patterns across different organs and tissues. In idiopathic pulmonary fibrosis, in vivo ECM and fibroblasts showed a more random orientation relative to normal lung tissue. Using rectangular and square micropatterns to induce anisotropic/isotropic fibroblasts, isotropic fibroblasts exhibited cytoskeletal remodeling and developed a highly invasive phenotypic profile [9]. In myocardial infarction, MFs were predominantly localized in anisotropic ECM fibers. In vitro experiments further showed anisotropic fibroblasts had a higher rate of MFs differentiation [12]. In a study of keloid scars, fibroblast and ECM orientation interacted reciprocally. Isotropic normal dermal fibroblasts induced ECM isotropy (few cell-cell adhesions and low cell/actin/focal adhesion alignment), while anisotropic keloid fibroblasts caused ECM anisotropy (more cell-cell adhesions, high cell/actin/focal adhesion alignment) [13].

Thus, fibroblast orientation modulates their activation and heterogeneity in cancer and fibrosis, with insights into this process facilitating the identification of new therapeutic targets. This study established a protocol to fabricate biomimetic topological micropattern array chips with simple operation and cost effectiveness to induce anisotropic/isotropic fibroblasts (Fig. 1).

Fig. 1.

Schematic illustration of the stepwise process for fabricating biomimetic topological micropattern array chips to induce anisotropic/isotropic orientation of fibroblast.

Method details

Preparation of decellularized ECM (dECM) scaffolds

• •Male Bama miniature pigs, weighing 30–40 kg, were procured from Sainuo Biomedical (Chengdu, China) and housed in biosecure facilities. Prior to experimentation, the pigs were anesthetized with Zoletil 50 (10 mg/kg body weight, Virbac, France), and maintained with propofol (6 mg/kg/h, Qingyuan Jiabo, China). The lungs were excised, and the pulmonary artery was cannulated. To remove residual blood, the lungs were perfused with phosphate buffer saline (PBS) supplemented with heparin for 10 min, after which they were frozen at −20 °C for subsequent use. Before initiating the decellularization process, the lungs were frozen and thawed for three times to disrupt the cellular structure.
• •The thawed lungs were perfused continuously via the pulmonary artery with double-distilled water (ddH₂O) at a rate of 100 mL/min for 1 hour.
• •The decellularization process involved sequential perfusion with different solutions at 100 mL/min: 0.2 % (w/v) dextrose (Sigma-Aldrich) for 5 hours, 1 % (v/v) Triton X-100 (Biofroxx) for 3 hours, 1 % (v/v) sodium lauryl ether sulfate (SLES) (Biofroxx) for 6 hours, and a second round of 1 % (v/v) Triton X-100 for 3 hours. After completing these perfusion steps, ddH₂O was perfused at 100 mL/min for 3 hours to remove residual detergents.
• •The decellularized lung was cut into 1 cm × 1 cm × 1 cm cubes for lyophilization. The lyophilized cubes were stored at −80 °C for the subsequent preparation of dECM hydrogel.

Preparation of dECM hydrogel

• •The lyophilized lung cubes were ground into powders by a Wiley Mill (Retsch, MM400, Germany), and lung dECM powders were stored at −80 °C.
• •One gram of dECM powder was digested in 50 mL of 2 mg/mL pepsin (Sigma-Aldrich) dissolved in 0.01 M hydrochloric acid (HCl). The digestion mixture was stirred at a rate of 60 rpm for 48 hours at room temperature to ensure complete digestion.
• •The digested dECM solution was neutralized to a pH range of 7.2–7.4 by adding 0.1 M sodium hydroxide (NaOH). The final concentration of the dECM hydrogel was adjusted to 10 mg/mL using 1 × PBS.
• •The prepared dECM hydrogel was stored at −4 °C and used as the bioink for the subsequent fabrication of micropattern array chips.

Fabrication of micropattern array chips by microcontact printing

• •Custom made polydimethylsiloxane (PDMS) seals (1 cm × 1 cm) were fabricated by laser etching of the designed micropatterns on a silicon wafer. The surface of each PDMS seal featured an array of cube shaped protrusions, available in two specifications.
• •Specification A: Cubes with dimensions 300 μm (length) × 50 μm (width) × 20 μm (height) and a 50 μm spacing between adjacent cubes. This specification was used to fabricate rectangular micropattern (300 μm × 50 μm) chips.
• •Specification B: Cubes with dimensions 300 μm (length) × 300 μm (width) × 20 μm (height) and a 50 μm spacing between adjacent cubes. This specification was used to fabricate square micropattern (300 μm × 300 μm) chips.
• •The surface of each PDMS seal was coated with 0.2 mL dECM hydrogel (0.1 mg/mL) containing 20 μg/mL fluorescein isothiocyanate isomer (FITC) (Sigma-Aldrich) for 20 min at room temperature.
• •The dECM hydrogel was removed from the surface of the PDMS seals, and PDMS seals were dried at 37 °C for 10 min.
• •The coated PDMS seal was placed on a 35 mm diameter nontreated cell culture dish (BIOFIL) and pressed with a force of 0.2 N for 10 min.
• •The micropattern array chips were imaged by the confocal microscope (Nikon).

Culture of fibroblasts on micropattern array chips

• •A commercial human lung fibroblast cell line (MRC-5) was procured from Wuhan Pricella Biotechnology Co., Ltd. MRC-5 cells were cultured in modified Eagle’s medium (MEM) supplemented with 10 % fetal bovine serum (FBS; Gibco, NY, USA), 1 % nonessential amino acids (NEAA; Gibco), 1 % sodium pyruvate (Gibco), 1 % GlutaMAX (Gibco), and 1 % penicillin−streptomycin solution (HyClone). The cells were cultured in an incubator at 37 °C under 5 % CO₂ with saturated humidity.
• •To prevent nonspecific cell adhesion, the micropattern array chips were incubated with 10 mg/mL pluronic F-127 (Sigma-Aldrich) at room temperature for 1 hour.
• •The micropattern array chips were sterilized by ultraviolet irradiation for 1 hour.
• •A total of 1 × 10⁵ MRC-5 cells were suspended in 3 mL culture medium, and then seeded into each micropattern array chip.
• •After 6 hours of incubation, the culture medium was removed, and the chips were washed three times with PBS to remove the unattached cells. Subsequently, 1.5 mL culture medium was added to each chip, and the cells were cultured for an additional 3 days before further analysis.

Analysis of fibroblast orientations

• •Following 3 days of culture, MRC-5 cells were washed three times with PBS and subsequently fixed with the 4 % neutral paraformaldehyde solution for 20 min. After fixation, the paraformaldehyde solution was removed, and the cells were washed three times with PBS.
• •The cell culture region was demarcated by the immunohistochemical pen. 100 μL 100 nM rhodamine labeled phalloidin working solution (Cytoskeleton) was added to fully cover the cells on the micropattern array chips, and the samples were stained at room temperature for 45 min. After staining, the phalloidin working solution was removed, followed by washing three times with PBS.
• •100 μL 10 μg/mL DAPI staining working solution (Biosharp) was then added, and the samples were stained at room temperature for 5 min. The DAPI staining solution was removed, followed by washing three times with PBS. Finally, 2 mL PBS was added to each chip for sample preservation.
• •The stained samples were imaged the confocal microscope . To quantify the orientation of MRC-5 cells, the F-actin staining images were further analyzed via fast Fourier transform (FFT) analysis.

Fast Fourier transform (FFT) analysis

• •The F-actin staining images of MRC-5 cells were processed using ImageJ software to generate the frequency spectra.
• •For each frequency spectrum, the pixel intensity values were calculated across the angular range of −180° to 0° and 0° to 180° by ImageJ.
• •FFT plots were finally plotted by either ImageJ or Origin. The degree of alignment of MRC-5 cells was reflected by the height and shape of the peak in the FFT plots. The higher peak indicated a more uniform alignment of cells along a single axis of orientation.

Characterization of anisotropic/isotropic fibroblasts

• •After 3 days of culture, MRC-5 cells were fixed.. 100 μL goat serum containing 0.15 % Triton X-100 was added to permeabilize cell membranes and block nonspecific binding sites, and the samples were incubated at room temperature for 30 min. After incubation, the goat serum was removed, followed by washing three times with PBS.
• •The MRC-5 cells were stained with 100 μL primary antibody working solutions against α-smooth muscle actin (α-SMA, ab7817, 1:200, Abcam) and Ki-67 (ab16667, 1:100, Abcam). The samples were incubated overnight at 4 °C. The primary antibody solutions were removed, followed by washing three times with PBS.
• •100 μL secondary antibody (1:500, Invitrogen) against the primary antibody was added and incubated at room temperature for 2 hours. Following incubation, the fluorescent secondary antibody solution was removed, followed by washing three times with PBS.
• •100 μL 10 μg/mL DAPI staining working solution was added, and the samples were stained at room temperature for 5 min. The DAPI solution was removed, followed by washing three times with PBS. Finally, 2 mL PBS was added to each chip for sample preservation.
• •The stained samples were imaged by the confocal microscope, and the images were analyzed by ImageJ.

Method validation

The fabrication of micropattern array chips

Fig. 2A showed the stepwise process for fabricating biomimetic topological micropattern array chips. A 1 cm × 1 cm PDMS seal was coated with the dECM hydrogel and incubated for 20 min, followed by drying at 37 °C for 10 min. Subsequently, the coated PDMS seal was placed on a cell culture dish and pressed with a 0.2 N force for 10 min. Compared with conventional micropattern fabrication methods (photoresist method, stencil method, and mold method), microcontact printing, with cost effectiveness and convenience, is suitable for laboratory fabrication [14,15]. Furthermore, various microcontact printing devices have been developed for mechanization and automation [16,17]. This advancement holds great promise for realizing good manufacturing practice (GMP) compliant production and industrial scale implementation.

Fig. 2.

The fabrication of micropattern array chips. (A) A schematic diagram illustrating the stepwise fabrication process of biomimetic topological micropattern array chips. (B) Bright field images of the PDMS seals and 2D and 3D views of dECM micropattern array. Scale bar = 100 μm.

Fig. 2B showed that the surface of PDMS seals contained rectangular micropatterns (300 μm × 50 μm) and square micropatterns (300 μm × 300 μm), with a 50 μm spacing between adjacent micropatterns. Using the dECM hydrogel as the bioink, the rectangular and square micropattern arrays were fabricated by microcontact printing (Fig. 2B). The bioink acts not only as the cell adhesion substrate to confine the area of cellular spreading but also as the matrix for sustaining cell culture [14]. Currently, bioinks can be primarily categorized into biomacromolecules (proteins, carbohydrates, and peptides) and small molecules (amino acids and chemical substances). Among these, specific ECM proteins (fibronectin, collagen type Ⅰ, collagen type Ⅳ, and laminin) are the most extensively used to fabricate micropatterns [14,15]. Nevertheless, recent researches have demonstrated that a single type of ECM protein struggles to support and modulate cell survival, differentiation, and function [18,19]. Compound ECM protein mixtures, such as Matrigel and dECM, have been proven to offer more favorable matrices to support cells due to the abundant composition of ECM proteins, cytokines, chemokines, and growth factors [9,20]. Notably, dECM is capable of supplying the tissue specific matrix [21]. Consequently, the selection of tissue specific dECM as the bioink confers distinctive advantages for relevant applications.

Rectangular and square micropatterns to induce anisotropic/isotropic orientation of fibroblasts

F-127 was used to prevent nonspecific cell adhesion on the rectangular (300 μm × 50 μm) and square (300 μm × 300 μm) micropattern array chips. A total of 1 × 10⁵ MRC-5 cells were seeded into each micropattern array chip. Following 6 hours of incubation, the chips were washed three times with PBS. MRC-5 cells attached uniformly to the rectangular and square micropattern arrays, forming a cell monolayer (Fig. 3A). Cell growth was confined to the area of the micropatterns. After 3 days of culture, MRC-5 cells remained restricted to the micropattern arrays and maintained the monolayer structure (Fig. 3A).

Fig. 3.

Micropattern array chips to induce anisotropic/isotropic orientation of fibroblast. (A) Bright field images of MRC-5 cells cultured on rectangular (300 μm × 50 μm) and square (300 μm × 300 μm) micropattern array chips at 6 hours and on 3 days. (B) MRC-5 cells were stained with F-actin. FFT analysis of MRC-5 cells cultured on (C) rectangular and (D) square micropattern array chips. Scale bars: 100 μm.

F-actin was stained with phalloidin to visualize the cytoskeletal distribution and morphological arrangement of MRC-5 cells. As shown in Fig. 3B, after 3 days of culture, MRC-5 cells on the rectangular micropatterns displayed the aligned orientation (anisotropy). In contrast, those on the square micropatterns showed the random orientation (isotropy). FFT analysis revealed higher and concentrated peaks for MRC-5 cells on rectangular micropatterns, indicating a more uniform alignment of MRC-5 cells along a single axis of orientation . However, the FFT results also confirmed the random cellular orientation for MRC-5 cells on the square micropatterns (Fig. 3C-D). In cell culture, the shapes of micropatterns are mainly circles, squares, or lines, and the sizes of micropatterns range from microns to millimeters [15]. Notably, the shape and size of micropatterns influence the cell morphology. For instance, circular and square micropatterns (diameter: 100 μm) are widely used to culture cell spheroid [18,19,21]. In this study, rectangular (300 μm × 50 μm) and square (300 μm × 300 μm) micropatterns were utilized to culture MRC-5 cells, and cell spheroid was not observed. However, these micropatterns could effectively induce the anisotropic/isotropic orientation of fibroblasts. This result is presumably associated with the shape and size of micropattern, and the cell inherent characteristics.

Characterization of anisotropic/isotropic fibroblasts

During FMT, fibroblasts show increased ECM synthesis and stress fiber contraction. α-SMA, a classic marker for MFs, was chosen to characterize fibroblast phenotypic and functional traits. Immunofluorescence staining and fluorescence quantitative analysis demonstrated that isotropic MRC-5 cells exhibited lower α-SMA expression levels compared to anisotropic MRC-5 cells (Fig. 4A-B). Moreover, cell proliferation reflects the quiescent and activated states of fibroblasts. Immunofluorescence staining showed significantly more Ki-67-positive cells in isotropic than in anisotropic MRC-5 cells (Fig. 4C and D).

Fig. 4.

Characterization of anisotropic/isotropic fibroblasts. Immunofluorescence staining of (A) α-SMA and (B) Ki-67 in anisotropic/isotropic MRC-5 cells. (C) Fluorescence quantitative analysis of α-SMA and (D) the count of Ki-67-positive cells in anisotropic/isotropic MRC-5 cells, according to immunofluorescence staining. Scale bars: 100 μm.

In conclusion, the biomimetic topological micropattern array chips can serve as a novel platform to induce the anisotropic/isotropic orientation of fibroblasts. Understanding the relationship between fibroblast orientation and its activation and heterogeneity in cancer and fibrosis may facilitate identifying novel therapeutic targets.

Limitations

On the one hand, micropattern array chips are currently fabricated via microcontact printing, a process that remains labor intensive. Thus, the development of automated microcontact printing devices is imperative for advancing this technology. Furthermore, micropattern fabrication lacks established standards. GMP grade micropatterns has not been reported. Developing GMP compliant micropatterns is critical for commercialization. Additionally, since dECM is used as the bioink for micropattern generation, maintaining the bioactivity of dECM is a key consideration. The storage temperature and the storage time of micropatterns are very significant for the bioactivity of dECM. These storage conditions are similar to those for commercial cell culture dishes that are coated with collagen or Matrigel and should be stored at −20 °C.

On the other hand, in this study, only the lung fibroblast cell line was cultured. It remains unclear whether the optimized micropattern parameters (shape and size) are also applicable for inducing anisotropic or isotropic orientation in fibroblasts derived from other organs and tissues.

Ethics statements

All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and Animal Experiment Center of Sichuan University (Approval No. 20,231,008,011) and met the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). All animals were cared for in accordance with the requirements of the Laboratory Animal Welfare Act and amendments thereof. Male Bama miniature pigs weighing 30–40 kg were used to carry out experiments. There was no observed difference of sex on the results of preparation of the dECM.

Supplementary material and/or additional information [OPTIONAL]

None

CRediT author statement

Ping Yang and Xinglong Zhu: Methodology, Investigation and Writing- Original draft preparation; Yi Li, Hehan Feng, Yang Deng, and Yanyan Zhou: Validity tests; Ji Bao: Conceptualization, Supervision and Writing- Reviewing and Editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.