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Long-Term Culture of Engineered Skeletal Muscle on Micromolded Gelatin Hydrogels Gio C. Suh1, Archana Bettadapur1, Holly Huber1, Clara Hua1, Evelyn R. Wang1,2, Alyssa A. Viscio1, Joon Young Kim1, Julie Strickland1, Megan L. McCain1,3

1Laboratory for Living Systems Engineering, Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California

2Keck School of Medicine, University of Southern California3Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California

Introduction• Skeletal muscles are

vital for movement and survival in our environment

• Progressive skeletal muscle degeneration limits mobility and decreases quality of life

• Skeletal muscle is derived from satellite cells, which differentiate into precursor cells, called myoblasts

This work was supported by the Viterbi School of Engineering, Women in Science and Engineering, the

Provost Fellowship, and the USC Undergraduate Research Associates Program

Methods Conclusion

Future Directions

Acknowledgements

Micropatterning PDMS Coverslips• Stiff PDMS (Sylgard 184) and soft PDMS (Sylgard 527 and Sylgard

184 in a 20:1 mass ratio) coated glass coverslips were prepared using a spincoater and plasma oxidized in the UVO cleaner

• Patterned 15x2 PDMS coverslips were microcontact printed with fibronectin solution

• Isotropic PDMS coverslips were placed PDMS side down onto a drop of fibronectin solution for 5 minutes

Micromolding Gelatin Coverslips• Taped coverslips were activated with APTES and glutaraldehyde• A solution of 10% gelatin was dropped onto the coverslips,

micromolded with a 10x10 (patterned) or flat stamp (isotropic), and incubated overnight

• Coverslips were soaked in 10% microbial transglutaminase solution for 4 hours

Culture Conditions• C2C12 mouse myoblast cells were then seeded onto the

coverslips with 10% fetal bovine serum media, which promotes myoblast growth and proliferation

• 2% horse serum media was added when the cells were confluent, which promotes myoblast differentiation (Day 1)

• Differentiation media was changed every other day

Immunostaining• Coverslips with each scaffold (stiff PDMS, soft PDMS, and gelatin)

were fixed after one week, two weeks, and three weeks• After fixing, coverslips were stained with DAPI (stains for nuclei),

488 Phalloidin (stains for actin filaments) and 547 α-actinin (stains for sarcomeres)

• We optimized protocols for long term culturing and differentiating skeletal muscle fibers

• On micropatterned stiff PDMS, engineered skeletal muscle:○ Were initially highly aligned○ Had very low cell numbers after one week in culture

due to delamination○ Did not express differentiation marker ɑ-actinin

• On micropatterned soft PDMS, engineered skeletal muscle:○ Stayed aligned throughout○ Showed decreased signs of delamination○ Fused into thin myofibers with relatively few nuclei

• On micromolded gelatin hydrogels, engineered skeletal muscle: ○ Stayed aligned throughout○ Maintained high cell numbers○ Formed thick multinucleated fibers○ Fused into thick myofibers with many nuclei

Our results suggest that gelatin hydrogels are the optimal substrate for maintaining engineered skeletal muscle tissue due to the additive effects of chemical

and mechanical properties.

• Improve the gelatin fabrication protocol by:○ Making coverslip activation more efficient○ Using a plasma treater to activate coverslips○ Determining a new way to micromold without the use of

tape• Use human cells for applications in human-relevant drug

testing and disease modeling• Stain for other muscle markers, such as Myo-D• Measure function of engineered muscle, using muscular

thin films, calcium trackers, and mitochondrial stains• Use qPCR to measure the α-actinin levels in mRNA

Our objective is to determine if the structural, mechanical, or chemical properties of ECM substrates

can be tuned to extend the culture lifetime of engineered skeletal muscle tissues.

McCain, Megan L., et al. "Micromolded gelatin hydrogels for extended culture of engineered cardiac tissues." Biomaterials 35.21 (2014): 5462-5471.

Microcontact Printing Protocol (Courtesy of ML McCain)

• Nikon inverted fluorescent microscope was used to image stained tissues

• Four types of analyses were implemented in Fiji: cell count, sarcomeric α-actinin thresholding, fiber thickness, and nuclei in fiber count

Data Analysis

ResultsMicropatterned Soft PDMS (25-30 kPa)

Micromolding Protocol (Courtesy of Christian Trujillo)

Cell Count Threshold Fiber Thickness Nuclei in Fiber

• Myoblasts fuse and differentiate into multi-nucleated, striated muscle fibers that generate force in response to neural or electrical stimulation

• Sarcomeres are the force-generating units in the cytoskeleton and indicate muscle maturation and differentiation

• Skeletal myoblasts typically delaminate shortly after fusing into fibers, limiting extended studies of skeletal muscle development and disease in vitro

Week 1 Week 2 Week 3

Micropatterned Stiff PDMS (2.5 MPa) Micromolded Gelatin Hydrogels (50 kPa)

Week 1 Week 2 Week 3 Week 1 Week 2 Week 3• Green: actin; Red: alpha actinin; Blue: nuclei

• Cell adhesion was higher on micromolded gelatin hydrogels compared to both stiff and soft PDMS

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• Myotubes were thicker on gelatin hydrogels • Myoblast fusion into myotubes was higher on gelatin hydrogels

• Sarcomeric alpha-actinin expression was higher on gelatin hydrogels than soft PDMS

1. Mask 25mm coverslip with tape

2. Make gelatin solution

Day 1 Day 2

3. Micromold gelatin on coverslip

4. Dry overnight

5. Remove stamp6. Remove tape

7. Soak coverslip in MTG for 4 hr

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Micropatterned stiff PDMSMicropatterned soft PDMSMicromolded gelatinp < 0.05 Stiff PDMS vs. soft PDMSp < 0.05 Stiff PDMS vs. gelatinp < 0.05 Soft PDMS vs. gelatin

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