基于PEGDA的微图案化底物构建及其在组织工程中表面形貌与细胞响应关系研究中的应用

Mohd Khairul Akma Darwis; Victoria Levario-Diaz; Sadaf Pashapour; Jonah Luka Voigt; Eloïse Lebaudy; Norhayati Sabani; Ahmad Shuhaimi Abu Bakar; Nihal Engin Vrana; Philippe Lavalle; Elisabetta Ada Cavalcanti-Adam; Siti Hawa Ngalim

doi:10.21769/BioProtoc.5323

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Development of Polyethylene Glycol Diacrylate-Based Micropattern Substrate to Study the Interplay Between Surface Topography and Cellular Response for Tissue Engineering Applications

基于PEGDA的微图案化底物构建及其在组织工程中表面形貌与细胞响应关系研究中的应用

Mohd Khairul Akma Darwis

VL Victoria Levario-Diaz

AB Ahmad Shuhaimi Abu Bakar

NV Nihal Engin Vrana

PL Philippe Lavalle

EC Elisabetta Ada Cavalcanti-Adam

SN Siti Hawa Ngalim email

发布: 2025年05月20日第15卷第10期 DOI: 10.21769/BioProtoc.5323 浏览次数: 1082

评审: Rajesh RanjanEVANGELOS THEODOROUAnonymous reviewer(s)

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Abstract

A key goal in the bioengineering field is the development of surface patterning of proteins that guide and control cellular organization. To this end, we developed a method to create a microstructured hydrogel based on soft-lithography techniques using polydimethylsiloxane (PDMS) and polyethylene glycol diacrylate (PEGDA). This approach involves the design of microfluidic geometries using graphical software, employing PDMS as a mold and leaving PEGDA as a substrate for the fabrication of microstructures and, thus, patterning extracellular matrix (ECM) proteins to promote cell adhesion. The combination of these techniques allows the fabrication of hydrogel microstructures without following conventional photolithography methods, such as the use of a photomask, the alignment required to produce the patterns, and the associated expenses. This study highlights the versatility and potential of PEGDA-based hydrogels as platforms to advance tissue engineering strategies.

Key features

• This protocol focuses on investigating the feasibility of patterning PEGDA as a substrate for protein surface patterning and further tissue engineering applications.

• Optimization of the fabrication of PEGDA hydrogels into simple shapes and angular patterns, ensuring a robust substrate capable of guiding cellular responses.

Keywords: PEGDA (PEGDA)

Micropatterns (微图案)

PDMS (PDMS)

Protein patterning (蛋白图案化)

Biomaterials (生物材料)

Graphical overview

Fabrication of biofunctionalized microtopography hydrogels using soft lithography and fibronectin-deposition approach. The protocol consists of designing micropatterns for the fabrication of the silicon (Si) wafer using QCAD graphical software (step 1), followed by mixing and casting the PDMS precursor on the Si wafer to obtain PDMS molds with the different micropatterns (step 2). Finally, developing PEGDA hydrogel on the PDMS mold to transfer micropatterns to the hydrogel and depositing fibronectin on the hydrogel’s surface to functionalize it (step 3).

Background

Central to the new era of tissue engineering is the development of biofunctionalized scaffolds where engineered structures are designed to mimic the intricate extracellular matrix (ECM) and support cellular growth [1–4]. Collaborative efforts by material scientists, electronic engineers, and cell biologists have led to cutting-edge techniques, such as microfabrication and nanotechnology, to create scaffolds that can closely imitate the native ECM to guide cell behaviour [1–6]. Although the interconnected roles of biochemical and biophysical cues are widely recognized [7–9], researchers often focus on biochemical aspects, overlooking the importance of biophysical cues in guiding cell behaviour. Hydrogel substrates have been created through pattern transfer using deep UV lithography and/or soft lithography, with a focus on fabricating polyethylene glycol diacrylate (PEGDA)-based hydrogels. Polyethylene glycol (PEG), being inherently inert to protein adsorption and cellular attachment, is considered ideal for anti-fouling coatings in biomedical applications and medical devices [17,18]. Linear PEG can be chemically modified into a reactive polymeric mesh network with a diacrylate (DA) crosslinker [10], making PEGDA suitable for various applications, such as drug delivery systems, tissue engineering, and wound healing [11–15]. The utility of PEGDA lies in its tunability of its mechanical and physico-chemical properties [10,16,17]. This versatility allows for precise control over the PEGDA’s characteristics, such as shape, stiffness, swelling behaviour, and degradation rate, making it an excellent choice for creating customized materials, including specific patterns and topographies [10,16,17]. However, since PEGDA remains inert to cell attachment, the coating of fibronectin, homogeneously or in selective PEGDA regions, enhances its bio-functionality and promotes cell adhesion on the PEGDA surface. Overall, the feasibility of creating and using patterned PEGDA allows for the optimization of surface properties to guide cellular organization, advancing its applications in tissue engineering.

Materials and reagents

Biological materials

1. C2C12 cell line (ATCC, catalog number: CRL-1772)

2. Fetal bovine serum (FBS) (Sigma Life Science, catalog number: F7524)

3. Fibronectin (Sigma-Aldrich, catalog number: F1141)

4. Fibrinogen (Thermo Fisher Scientific, catalog number: F35200)

Reagents

1. Dulbecco’s modified Eagle medium (DMEM) supplemented with 4.5 g/L D-glucose, L-glutamine, and pyruvate (Gibco, catalog number: 41966-029)

2. Penicillin/Streptomycin (Thermo Fisher Scientific, catalog number: 15140122)

3. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, catalog number: 25200072)

4. 1× phosphate saline buffer (PBS) (Thermo Fisher Scientific, catalog number: 14190169)

5. Trypan blue (Invitrogen, catalog number: T10282)

6. Alamar blue (Thermo Fisher Scientific, catalog number: DAL1025)

7. Absolute ethanol (Carl Roth, catalog number: 9065.5)

8. Acetic acid (Honeywell, catalog number: 607-002-00-6)

9. 3-(Trimethoxysily) propyl methacrylate (Silane A174) (Sigma-Aldrich, catalog number: 440159)

10. Polyethylene glycol diacrylate (PEGDA) (MW = 700) (Sigma-Aldrich, catalog number: 455008)

11. Poly(L-lysine) (PLL) (Biosynth, catalog number: FP14985)

12. PLPP gel photoinitiator (Alvéole)

13. 70% ethanol

14. Isopropanol

15. Acetone

16. Sylgard 184 elastomer base and curing agent (VWR, catalog number: 634165S)

17. 4% paraformaldehyde (PFA)

18. Phalloidin-TRITC (Sigma-Aldrich, catalog number: P1951)

19. Hoechst (Thermo Fisher Scientific, catalog number: H1398)

20. Mowiol (Carl Roth, catalog number: 0731.2)

21. Sodium hydroxide solution (NaOH) (lab-made)

22. Ultrapure water (lab-made)

23. Bovine serum albumin (BSA) powder (Sigma-Aldrich, catalog number: A2153)

24. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: C-40020)

Solutions

1. Complete cell culture medium (see Recipes)

2. Sodium hydroxide solution (see Recipes)

3. PLL mix solution (see Recipes)

4. PEGDA mix solution (150 mg/mL) (see Recipes)

5. PEGDA mix solution (250 mg/mL) (see Recipes)

6. PEGDA mix solution (350 mg/mL) (see Recipes)

7. Hydrogel precursor solution (see Recipes)

8. Alamar blue solution (see Recipes)

9. Silanization immersion solution (see Recipes)

10. HEPES solution (see Recipes)

11. Micropattern labeling solution (see Recipes)

12. 5% BSA solution (see Recipes)

13. 1% BSA solution (see Recipes)

14. PDMS solution (see Recipes)

15. Immunostaining solution (see Recipes)

Recipes

1. Complete cell culture medium (50 mL)

Storage: 4 °C, up to 3 months.

Reagent	Final concentration	Quantity or Volume
DMEM	n/a	44.5 mL
FBS	10% (v/v)	5 mL
Penicillin/streptomycin	1% (v/v)	0.5 mL
Total	n/a	50 mL

2. Sodium hydroxide solution (100 mL, 0.15 M, pH 7.8)

Storage: room temperature, up to 6 months.

Reagent	Final concentration	Quantity or Volume
NaOH solution (1 M)	0.15 M	15 mL
Ultrapure water	n/a	85 mL
Total	n/a	100 mL

3. PLL mix solution (100 mg/mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
PLL powder	100 mg/mL (w/v)	100 mg
NaOH solution (0.15 M)	n/a	1 mL
Total	n/a	1 mL

4. PEGDA mix solution (150 mg/mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
PEGDA solution (1 M)	150 mg/mL (v/v)	26.8 μL
Ultrapure water	n/a	173.2 μL
Total	n/a	200 μL

5. PEGDA mix solution (250 mg/mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
PEGDA solution (1 M)	250 mg/mL (v/v)	44.6 μL
Ultrapure water	n/a	155.4 μL
Total	n/a	200 μL

6. PEGDA solution (350 mg/mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
PEGDA solution (1 M)	350 mg/mL (v/v)	62.5 μL
Ultrapure water	n/a	137.5 μL
Total	n/a	200 μL

7. Hydrogel precursor solution (0.42 mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
PEGDA mix solution	150/250/350 mg/mL (v/v)	200 μL
PLL mix solution	100 mg/mL (w/v)	200 μL
PLPP gel photoinitiator	5%	20 μL
Total	n/a	420 μL

8. Alamar blue solution (100 μL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
Alamar blue	10%	10 μL
Complete culture medium	n/a	90 μL
Total	n/a	100 μL

9. Silanization immersion solution (300 mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
Ultrapure water	n/a	10 mL
Acetic acid	1 (ratio)	18.75 mL
Silane A174	1 (ratio)	18.75 mL
Absolute ethanol	14 (ratio)	252.5
Total	n/a	300 mL

10. HEPES solution (500 mL)

Storage: 4 °C, up to 3 months.

Reagent	Final concentration	Quantity or Volume
HEPES (1 M)	10 mM	5 mL
Ultrapure water	n/a	495 mL
Total	n/a	500 mL

11. Micropattern labeling solution (0.7 mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
Fibronectin	50 μg/mL	17.5 μL
Fibrinogen	10 μg/mL	4.7 μL
HEPES solution (10 mM)	n/a	677.8 μL
Total	n/a	700 μL

12. 5% BSA solution (50 mL)

Storage: 4 °C, one week.

Reagent	Final concentration	Quantity or Volume
BSA powder	5% (w/v)	2.5 g
1× PBS	n/a	50 mL
Total	n/a	50 mL

13. 1% BSA solution (50 mL)

Storage: 4 ˚C, one week.

Reagent	Final concentration	Quantity or Volume
BSA powder	1% (w/v)	0.5 g
1× PBS	n/a	50 mL
Total	n/a	50 mL

14. PDMS solution (50 mL)

Storage: -20 ˚C, up to 5 months.

Reagent	Final concentration	Quantity or Volume
Sylgard 184 elastomer base	10 (ratio)	50 g
Curing agent	1 (ratio)	5 g
Total	n/a	55 g

15. Immunostaining solution (0.7 mL)

Immediate use.

Reagent	Final concentration	Quantity or Volume
Phalloidin	1:200	3.5 μL
Hoechst	1:1,000	0.7 μL
5% BSA	n/a	695.8 μL
Total	n/a	700 μL

Laboratory supplies

1. T-75 cell culture flask (Fisher Scientific, catalog number: 50-809-260)

2. Glass bottom 6-well plate (lab custom-made)

3. 96-well plate (Fisher Scientific, catalog number: 92096)

4. 6-well plate (Fisher Scientific, catalog number: 92006)

5. Hemocytometer (Sigma-Aldrich, catalog number: Z359629-1EA)

6. 20 × 20 mm² glass coverslip (Carl Roth, catalog number: H873.2)

7. 18 × 18 mm² glass coverslip (Carl Roth, catalog number: 0657.2)

8. Teflon rack (custom-made)

9. Glass box (custom-made)

10. 20 mL syringe (B. Braun Inkjet, catalog number: 4606736V)

11. 50 mm length needle (B. Braun Sterican, catalog number: 466 5503)

12. 1.5 mL microcentrifuge tubes (Axygen, catalog number: 10620934)

13. 0.6 mL microcentrifuge tubes (Nest Biotechnology, catalog number: 605001)

14. 15 mL conical tube (Fisher Scientific, catalog number: 20160618-058)

15. 50 mL conical tube (Cellstar, catalog number: 227261)

16. 5 mL serological pipette (Eppendorf, catalog number: 0030127714)

17. 10 mL serological pipette (Eppendorf, catalog number: 0030127722)

18. 25 mL serological pipette (Eppendorf, catalog number: 0030127730)

19. Aluminum foil (Reynolds Consumer Products LLC, catalog number: 1090000084)

20. Chromium mask (Toppan Photomasks, Inc.)

21. Micropattern master (IMSEAM, Heidelberg)

22. 100 mm diameter Petri dish (Corning, catalog number: 430167)

23. 60 mm diameter Petri dish (Falcon, catalog number: 353004)

24. 35 mm diameter Petri dish (Falcon, catalog number: 353001)

25. Spatula

26. Scalpel No. 11 (Fisher Scientific, catalog number: 11708353)

27. 76 mm × 26 mm microscope slides (Marienfeld, catalog number: 1000200)

Equipment

1. Incubator (Thermo Scientific, model: BBD 6220)

2. Automation cell Countess II FL (Invitrogen, model: AMQAF1000)

3. Multi-mode microplate reader (Tecan, model: Spark)

4. Nitrogen gun (MPI)

5. UV crosslinker chamber (Vilber Lourmat, model: BLX312)

6. Ultraviolet-ozone cleaner machine (Jetlight Company Inc., model: 342-220)

7. Vortex machine (StarLab, model: N2400-6110)

8. Fume hood (Thermo Fisher Scientific, model: EN 12469)

9. Weighing balance (Kern, model: AEJ 200-5CM)

10. Centrifuge machine (VWR, model: Mega Star 600R)

11. Desiccator (Duran, model: DN150)

12. -20 ˚C blast freezer (Liebherr, model: MediLine)

13. Oven (VWR, model: INCU Line)

14. Optical microscope (Zeiss, model: Primovert)

15. Optics11 Chiaro Nanoindeter (Optics11 Life)

16. Delta Vision (Olympus)

Software and datasets

1. QCAD software (3.31.1, 19.04.2024)

2. ImageJ software (1.8.0, 07.06.2024)

3. GraphPad Prism (8.4.3, 02.09.2024)

4. Optics11 Life DataViewer (V2, 31.07.2024)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Feb 24, 2025

接收日期: Apr 18, 2025

在线发布日期: May 8, 2025

出版日期: May 20, 2025

版权信息

如何引用

Darwis, M. K. A., Levario-Diaz, V., Pashapour, S., Voigt, J. L., Lebaudy, E., Sabani, N., Bakar, A. S. A., Vrana, N. E., Lavalle, P., Cavalcanti-Adam, E. A. and Ngalim, S. H. (2025). Development of Polyethylene Glycol Diacrylate-Based Micropattern Substrate to Study the Interplay Between Surface Topography and Cellular Response for Tissue Engineering Applications. Bio-protocol 15(10): e5323. DOI: 10.21769/BioProtoc.5323.