Human intestinal enteroids derived from adult stem cells offer a relevant ex vivo system to study biological processes of the human gut. They recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids) albeit limited by the lack of associated cell types that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium, support barrier function, and deploy effector functions. We have established a co‐culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Enteroids are generated from biopsies or resected tissue from any segment of the human gut and maintained in long‐term cultures as three‐dimensional structures through supplementation of stem cell growth factors. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC). Monocytes from PBMC are differentiated into macrophages by cytokine stimulation prior to co‐culture. The methods are divided into the two main components of the model: (1) generating enteroid/colonoid monolayers and isolating immune cells and (2) assembly of enteroid/colonoid‐immune cell co‐cultures with separate apical and basolateral compartments. Co‐cultures containing macrophages can be maintained for 48 hr while those involving neutrophils, due to their shorter life span, remain viable for 4 hr. Enteroid‐immune co‐cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions. © 2020 Wiley Periodicals LLC.
A Minireview of Microfluidic Scaffold Materials in Tissue Engineering
In 2020, nearly 107,000 people in the U.S needed a lifesaving organ transplant, but due to a limited number of donors, only ∼35% of them have actually received it. Thus, successful bio-manufacturing of artificial tissues and organs is central to satisfying the ever-growing demand for transplants. However, despite decades of tremendous investments in regenerative medicine research and development conventional scaffold technologies have failed to yield viable tissues and organs. Luckily, microfluidic scaffolds hold the promise of overcoming the major challenges associated with generating complex 3D cultures: 1) cell death due to poor metabolite distribution/clearing of waste in thick cultures; 2) sacrificial analysis due to inability to sample the culture non-invasively; 3) product variability due to lack of control over the cell action post-seeding, and 4) adoption barriers associated with having to learn a different culturing protocol for each new product. Namely, their active pore networks provide the ability to perform automated fluid and cell manipulations (e.g., seeding, feeding, probing, clearing waste, delivering drugs, etc.) at targeted locations in-situ . However, challenges remain in developing a biomaterial that would have the appropriate characteristics for such scaffolds. Specifically, it should ideally be: 1) biocompatible —to support cell attachment and growth, 2) biodegradable —to give way to newly formed tissue, 3) flexible —to create microfluidic valves, 4) photo-crosslinkable —to manufacture using light-based 3D printing and 5) transparent —for optical microscopy validation. To that end, this minireview summarizes the latest progress of the biomaterial design, and of the corresponding fabrication method development, for making the microfluidic scaffolds.
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- Award ID(s):
- 1953890
- NSF-PAR ID:
- 10316398
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 8
- ISSN:
- 2296-889X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Basic Protocol 1 : Seeding enteroid fragments onto Transwells for monolayer formationAlternate Protocol : Seeding enteroid fragments for monolayer formation using triturationBasic Protocol 2 : Isolation of monocytes and derivation of immune cells from human peripheral bloodBasic Protocol 3 : Isolation of neutrophils from human peripheral bloodBasic Protocol 4 : Assembly of enteroid/macrophage or enteroid/neutrophil co‐culture -
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Abstract Tissue engineered grafts show great potential as regenerative implants for diseased or injured tissues within the human body. However, these grafts suffer from poor nutrient perfusion and waste transport, thus decreasing their viability post‐transplantation. Graft vascularization is therefore a major area of focus within tissue engineering because biologically relevant conduits for nutrient and oxygen perfusion can improve viability post‐implantation. Many researchers used microphysiological systems as testing platforms for potential grafts owing to an ability to integrate vascular networks as well as biological characteristics such as fluid perfusion, 3D architecture, compartmentalization of tissue‐specific materials, and biophysical and biochemical cues. Although many methods of vascularizing these systems exist, microvascular self‐assembly has great potential for bench‐to‐clinic translation as it relies on naturally occurring physiological events. In this review, the past decade of literature is highlighted, and the most important and tunable components yielding a self‐assembled vascular network on chip are critically discussed: endothelial cell source, tissue‐specific supporting cells, biomaterial scaffolds, biochemical cues, and biophysical forces. This paper discusses the bioengineered systems of angiogenesis, vasculogenesis, and lymphangiogenesis and includes a brief overview of multicellular systems. It concludes with future avenues of research to guide the next generation of vascularized microfluidic models.
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Ellis, K ; Ferrell, W ; Knapp, J. (Ed.)Three-dimensional bio-printing is a rapidly growing field attempting to recreate functional tissues for medical and pharmaceutical purposes. Development of functional tissues and organs requires the ability to achieve large full-scale scaffolds that mimic human organs. It is difficult to achieve large scaffolds that can support themselves without damaging printed cells in the process. The high viscosity needed to support large prints requires high amounts of pressure that diminishes cell viability and proliferation. By working with the rheological, mechanical, and microstructural properties of different compositions, a set of biomaterial compositions was identified to have high structural integrity and shape fidelity without needing a harmful amount of pressure to extrude. Various large scale-scaffolds were fabricated (up to 3.0 cm, 74 layers) using those hybrid hydrogels ensuring geometric fidelity. This effort can ensure to fabricate large scaffolds using 3D bio-printing processes ensuring proper internal and external geometries.more » « less
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Atomic force microscopy (AFM) in conjunction with microfluidic delivery was utilized to produce three-dimensional (3D) lipid structures following a custom design. While AFM is well-known for its spatial precision in imaging and 2D nanolithography, the development of AFM-based nanotechnology into 3D nanoprinting requires overcoming the technical challenges of controlling material delivery and interlayer registry. This work demonstrates the concept of 3D nanoprinting of amphiphilic molecules such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Various formulations of POPC solutions were tested to achieve point, line, and layer-by-layer material delivery. The produced structures include nanometer-thick disks, long linear spherical caps, stacking grids, and organizational chiral architectures. The POPC molecules formed stacking bilayers in these constructions, as revealed by high-resolution structural characterizations. The 3D printing reached nanometer spatial precision over a range of 0.5 mm. The outcomes reveal the promising potential of our designed technology and methodology in the production of 3D structures from nanometer to continuum, opening opportunities in biomaterial sciences and engineering, such as in the production of 3D nanodevices, chiral nanosensors, and scaffolds for tissue engineering and regeneration.more » « less
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Regenerative medicine and tissue engineering strategies have made remarkable progress in remodeling, replacing, and regenerating damaged cardiovascular tissues. The design of three-dimensional (3D) scaffolds with appropriate biochemical and mechanical characteristics is critical for engineering tissue-engineered replacements. The extracellular matrix (ECM) is a dynamic scaffolding structure characterized by tissue-specific biochemical, biophysical, and mechanical properties that modulates cellular behavior and activates highly regulated signaling pathways. In light of technological advancements, biomaterial-based scaffolds have been developed that better mimic physiological ECM properties, provide signaling cues that modulate cellular behavior, and form functional tissues and organs. In this review, we summarize the in vitro, pre-clinical, and clinical research models that have been employed in the design of ECM-based biomaterials for cardiovascular regenerative medicine. We highlight the research advancements in the incorporation of ECM components into biomaterial-based scaffolds, the engineering of increasingly complex structures using biofabrication and spatial patterning techniques, the regulation of ECMs on vascular differentiation and function, and the translation of ECM-based scaffolds for vascular graft applications. Finally, we discuss the challenges, future perspectives, and directions in the design of next-generation ECM-based biomaterials for cardiovascular tissue engineering and clinical translation.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2021.783268
