Angela M. Taylor

M. Hammersley, J. Hendricksen, R. Shah, A. Domenighetti

Developing a 3-D Printed Cell Culture Assay

to Study Muscle Contractures in Children

with Cerebral Palsy

DISCLAIMER

No conflicts of interest to disclose.

CEREBRAL PALSY (CP)

Etiology: Impaired CNS

development due to ischemia or

malformation of the developing

brain

Onset: Pregnancy to ~3 y.o

Prevalence:

• 2-3 in 1,000 births in

USA/Europe.

• Most common cause of childhood

disability

[Graham et al. Nat Rev Dis Primers. 2016]

CLINICAL MANIFESTATIONS

Musculoskeletal Defects:

• Muscle contractures

• Decreased range of motion

Our Main Goals:

• Treat or prevent contractures

• Use new stem cell or drug therapies

• To maintain or grow muscle mass

[Graham et al. Nat Rev Dis Primers. 2016]

MUSCLE STEM CELLS

Muscle Satellite cells (MuSCs):

• Muscle stem cells that reside between the basal lamina (a layer of ECM) and the

sarcolemma (the plasma membrane) of muscle fibers.

Function:

• Pre- and post- natal muscle growth

• Muscle repair when injury occurs

MYOGENESIS: From Stem Cell to Muscle Fiber

SATELLITE CELLS (MuSC)

QUIESCENT

DIFFERENTIATIONPROLIFERATION

MUSCLE FIBER

MYOTUBES

ACTIVATED

MYOBLASTS

Fusion

(UNDER CONTROL OF PRO-MYOGENIC GENETIC PROGRAMS)

MYOGENESIS: From Stem Cell to Muscle Fiber

SATELLITE CELLS (MuSC)

QUIESCENT

DIFFERENTIATIONPROLIFERATION

MUSCLE FIBER

MYOTUBES

ACTIVATED

MYOBLASTS

Fusion

(UNDER CONTROL OF PRO-MYOGENIC GENETIC PROGRAMS)

MYOGENESIS: From Stem Cell to Muscle Fiber

SATELLITE CELLS (MuSC)

QUIESCENT

DIFFERENTIATIONPROLIFERATION

MUSCLE FIBER

MYOTUBES

ACTIVATED

MYOBLASTS

Fusion

(UNDER CONTROL OF PRO-MYOGENIC GENETIC PROGRAMS)

↓ number of MuSCs in CP ↓ fusion capacity in CP

OBJECTIVES: In the Context of Contractures

Long-term goals:

To create an in vitro 3D-bioprinted skeletal muscle tissue

1. To investigate cell-cell and cell-matrix interactions

2. To test new drug or cell therapies to support muscle rehabilitation and surgery in CP

Project Aims:

1. Demonstrate feasibility of growing myotubes on 3D bio-printed gelatin-made hydrogels

2. Improve or rescue myogenic potential of CP myoblasts in vitro

Hypotheses:

1. In a 2D stiff environment, myoblasts from children with CP will show decreased myotubeformation and myogenic potential vs. TD

2. 3D culturing on softer substrate +/- matrix-producing fibroblasts (co-culture) would improve myotube formation in CP conditions.

DESIGN OF 3-D PRINTED HYDROGELS

MUSCLE BIOPSIES

FROM SURGICAL

PROCEDURES

(SEMITENDINOSUS,

GRACILIS)

SAT. CELLS SORTING (FACS)

(CD31−/CD45−/CD56+/ITGA7+)

MuSC CULTURE

PAX7

MYOD

N = 4 patients/group

Age = CP 9.0±6.4 vs. TD 15.3±2.6 y.o.; 5 males, 3 females

ENZYMATIC DIGESTION OF MUSCLE TISSUE

(COLLAGENASE + DISPASE)

MuSC ISOLATION & CULTURE

CO-CULTURE 1:1

(TD MUSCLE FIBROBLASTS)

MYOTUBES

DIFFERENTIATION

HIGH SERUM

MEDIA:

20% FBS + bFGF

in Ham’s F10

LOW SERUM

MEDIA:

5% HS in DMEM

2 weeks

ACTA2

WGA

DAPI

DIFFERENTIATION & ANALYSIS

MuSCs

2-4 days

MYOBLASTS

FIXATION (4% PFA)

IMMUNOSTAINING

PROLIFERATION

W/T or W/OUT FIBROBLASTS

ACTA2

WGA

DAPI

TD CP

50 mm 50 mm

ACTA2

RESULTS – Differentiation over 2D plastic support

ACTA2

WGA

DAPI Rid

ge

Gro

ove

Rid

ge

Gro

ove

RESULTS – Differentiation over 3D soft gelatin support

TD CP

TD CP

# Condition (CP/TD) * Substrate (2D/3D) P=0.001 (n=4/group, 2-way ANOVA)

RESULTS – Fusion Index

Substrates: 2D = hard, plastic thin-coated with 0.5% gelatin 3D = soft, 15% gelatin scaffoldCo-culture: added TD “Fibroblasts” at 1:1 ratio.

#

CONCLUSIONS

1. Feasibility: Culturing myoblasts and producing myotubes

on 3D gelatin-based scaffolds is possible.

2. Substrate effect: Myogenic potential of CP myoblasts is

improved in 3D versus 2D cell cultures.

3. Cellular effect: Co-culturing CP myoblasts with TD

“fibroblasts” may improve myogenic potential of CP and TD

myoblasts on 3D-printed gelatin scaffolds, but not rescue

on gelatin-coated hard plastic 2D substrate.

ONGOING RESEARCH AND CLINICAL APPLICATIONS

Ongoing Research:• Optimize scaffold stiffness (5-40 kPa)

• Optimize scaffold geometry

• Optimize cell composition (co-cultures)

Clinical Applications: To build an in vitro muscle tissue model for use as …

• As a tool for drug discovery

• As a therapeutic intervention for contractures: a transplantable stem cell-laden “patch” or scaffold, to improve outcomes of muscle-tendon lengthening and other orthopedic surgical procedures

ACKNOWLEDGEMENTS