Science and medicine have become increasingly “human‐centric” over the years. A growing shift away from the use of animals in basic research has led to the development of sophisticated in vitro models of various tissues utilizing human‐derived cells to study physiology and disease. The human cornea has likewise been modeled in vitro using primary cells derived from corneas obtained from cadavers or post‐transplantation. By utilizing a cell's intrinsic ability to maintain its tissue phenotype in a pre‐designed microenvironment containing the required growth factors, physiological temperature, and humidity, tissue‐engineered corneas can be grown and maintained in culture for relatively long periods of time on the scale of weeks to months. Due to its transparency and avascularity, the cornea is an optimal tissue for studies of extracellular matrix and cell‐cell interactions, toxicology and permeability of drugs, and underlying mechanisms of scarring and tissue regeneration. This paper describes methods for the cultivation of corneal keratocytes, fibroblasts, epithelial, and endothelial cells for in vitro applications. We also provide detailed, step‐by‐step protocols for assembling and culturing 3D constructs of the corneal stroma, epithelial‐ and endothelial‐stromal co‐cultures and isolation of extracellular vesicles. © 2020 Wiley Periodicals LLC.
Engineered tissue models comprise a variety of multiplexed ensembles in which combinations of epithelial, stromal, and immune cells give rise to physiologic functions. Engineering spatiotemporal control of cell–cell and cell–matrix interactions within these 3D multicellular tissues would represent a significant advance for tissue engineering. In this work, a new method, entitled CAMEO (
- NSF-PAR ID:
- 10452886
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Functional Materials
- Volume:
- 30
- Issue:
- 48
- ISSN:
- 1616-301X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Basic Protocol 1 : Isolating and culturing human corneal keratocytes and fibroblastsBasic Protocol 2 : Isolating and culturing human corneal epithelial cellsBasic Protocol 3 : Isolating and culturing human corneal endothelial cellsBasic Protocol 4 : 3D corneal stromal construct assemblyBasic Protocol 5 : 3D corneal epithelial‐stromal construct assemblyBasic Protocol 6 : 3D corneal endothelial‐stromal construct assemblyBasic Protocol 7 : Isolating extracellular vesicles from corneal cell conditioned mediumSupport Protocol : Cryopreserving human corneal fibroblasts, corneal epithelial cells, and corneal endothelial cells -
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Abstract Many cell types require direct cell–cell interactions for differentiation and function; yet, this can be challenging to incorporate into 3‐dimensional (3D) structures for the engineering of tissues. Here, a new approach is introduced that combines aggregates of cells (spheroids) with similarly‐sized hydrogel particles (microgels) to form granular composites that are injectable, undergo interparticle crosslinking via light for initial stabilization, permit cell–cell contacts for cell signaling, and allow spheroid fusion and growth. One area where this is important is in cartilage tissue engineering, as cell–cell contacts are crucial to chondrogenesis and are missing in many tissue engineering approaches. To address this, granular composites are developed from adult porcine mesenchymal stromal cell (MSC) spheroids and hyaluronic acid microgels and simulations and experimental analyses are used to establish the importance of initial MSC spheroid to microgel volume ratios to balance mechanical support with tissue growth. Long‐term chondrogenic cultures of granular composites produce engineered cartilage tissue with extensive matrix deposition and mechanical properties within the range of cartilage, as well as integration with native tissue. Altogether, a new strategy of injectable granular composites is developed that leverages the benefits of cell–cell interactions through spheroids with the mechanical stabilization afforded with engineered hydrogels.
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Abstract Capillary scale vascularization is critical to the survival of engineered 3D tissues and remains an outstanding challenge for the field of tissue engineering. Current methods to generate micro‐scale vasculatures such as 3D printing, two photon hydrogel ablation, angiogenesis, and vasculogenic assembly face challenges in rapidly creating organized, highly vascularized tissues at capillary length‐scales. Within metabolically demanding tissues, native capillary beds are highly organized and densely packed to achieve adequate delivery of nutrients and oxygen and efficient waste removal. Here, two existing techniques are adopted to fabricate lattices composed of sacrificial microfibers that can be efficiently and uniformly seeded with endothelial cells (ECs) by magnetizing both lattices and ECs. Ferromagnetic microparticles are incorporated into microfibers produced by solution electrowriting and fiber electropulling. By loading ECs with superparamagnetic iron oxide nanoparticles, the cells could be seeded onto magnetized microfiber lattices. Following encapsulation in a hydrogel, the capillary templating lattice is selectively degraded by a bacterial lipase that does not impact mammalian cell viability or function. This study introduces a novel approach to rapidly producing organized capillary networks within metabolically demanding engineered tissue constructs which should have broad utility in the fields of tissue engineering and regenerative medicine.
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Abstract Stem cell−based therapies for various ailments have attracted significant attention for over a decade. However, low retention of transplanted cells at the damaged site has hindered their potential for use in therapy. Tissue engineered grafts with fibrillar structures mimicking the extracellular matrix (ECM) can be potentially used to increase the retention and engraftment of stem cells at the damaged site. Moreover, these grafts may also provide mechanical stability at the damaged site to enhance function and regeneration. Among all the methods to produce fibrillar structures developed in recent years, electrospinning is a simple and versatile method to produce fibrous structures ranging from a few nanometers to micrometers. Coaxial electrospinning enables production of a mechanically stable core with a cell‐binding sheath for enhanced cell adhesion and proliferation. Furthermore, this process provides an alternative to functionalized engineered scaffolds with specific compositions. The present article describes the protocol for developing a polycaprolactone (PCL) core and gelatin/gelatin methacrylate (GelMA) sheath laden with stem cells for various regenerative engineering applications. © 2021 Wiley Periodicals LLC.
This article was corrected on 18 July 2022. See the end of the full text for details.
Basic Protocol 1 : Uniaxial PCL electrospinningBasic Protocol 2 : Coaxial electrospinningSupport Protocol 1 : Scaffold characterization for Basic Protocols 1 and 2Basic Protocol 3 : Cell seeding on uniaxial and coaxial electrospun scaffolds and MTS assaySupport Protocol 2 : Preparation of scaffold with cells for scanning electron microscopy
