Home › Cell Biology › Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration
Cell Biology JoVE (Open Access) Citable · DOI

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

DOI: 10.3791/51826-v
What you'll learn
  • ✓ Design and fabricate microstereolithography templates for corneal biomaterial devices
  • ✓ Produce electrospun PLGA membranes with integrated microfeatures using hybrid techniques
  • ✓ Culture limbal explants on microstructured scaffolds to assess corneal regeneration
Protocol

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Difficulty
advanced
Total time
~5–7 days (template fabrication 1 day, membrane production 1 day, cell isolation and culture 3–5 days)
Model organism
Rabbit (limbal explants for corneal regeneration)
Biosafety
BSL-1

Steps

1
Fabricate PEGDA templates by microstereolithography

Design and optically cure poly(ethylene glycol) diacrylate (PEGDA) templates using two-photon microstereolithography to create precise micropatterns. These templates serve as molds for subsequent membrane fabrication.

▶ 02:15
2
Produce biodegradable PLGA membranes using electrospinning

Electrospin poly(lactide-co-glycolide) solution onto the PEGDA templates to create nanofibrous membranes with integrated micropockets. Cross-link or cure as needed to stabilize the composite structure.

▶ 03:58
3
Store PLGA microfabricated membranes long-term

Preserve fabricated PLGA membranes under appropriate conditions (e.g., desiccation, controlled temperature) to maintain structural integrity and bioactivity for extended periods before cell seeding.

▶ 05:48
4
Isolate limbal explants from rabbit cornea

Harvest and isolate limbal tissue from rabbit cornea following established protocols. Prepare tissue for culture on microstructured scaffolds.

▶ 06:34
5
Integrate ring scaffolds into 3D corneal models

Seed limbal explants onto ring-shaped PLGA scaffolds equipped with micropockets and culture in 3D configuration to support corneal cell behavior and regeneration.

▶ 07:53
6
Evaluate corneal regeneration on microstructured membranes

Assess cell proliferation, differentiation, and tissue regeneration on synthetic microstructured membranes using histology, immunofluorescence, or live imaging to confirm biocompatibility and regenerative capacity.

▶ 08:46
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