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Capture and 3D culture of colonic crypts and colonoids in a microarray platform
- Source :
- Lab on a Chip. 13:4625
- Publication Year :
- 2013
- Publisher :
- Royal Society of Chemistry (RSC), 2013.
-
Abstract
- Crypts are the basic structural and functional units of colonic epithelium and can be isolated from the colon and cultured in vitro into multi-cell spheroids termed “colonoids”. Both crypts and colonoids are ideal building blocks for construction of an in vitro tissue model of the colon. Here we proposed and tested a microengineered platform for capture and in vitro 3D culture of colonic crypts and colonoids. An integrated platform was fabricated from polydimethylsiloxane which contained two fluidic layers separated by an array of cylindrical microwells (150 μm diameter, 150 μm depth) with perforated bottoms (30 μm opening, 10 μm depth) termed “microstrainers”. As fluid moved through the array, crypts or colonoids were retained in the microstrainers with a >90% array-filling efficiency. Matrigel as an extracellular matrix was then applied to the microstrainers to generate isolated Matrigel pockets encapsulating the crypts or colonoids. After supplying the essential growth factors, epidermal growth factor, Wnt-3A, R-spondin 2 and noggin, 63 ± 13% of the crypts and 77 ± 8% of the colonoids cultured in the microstrainers over a 48–72 h period formed viable 3D colonoids. Thus colonoid growth on the array was similar to that under standard culture conditions (78 ± 5%). Additionally the colonoids displayed the same morphology and similar numbers of stem and progenitor cells as those under standard culture conditions. Immunofluorescence staining confirmed that the differentiated cell-types of the colon, goblet cells, enteroendocrine cells and absorptive enterocytes, formed on the array. To demonstrating the utility of the array in tracking the colonoid fate, quantitative fluorescence analysis was performed on the arrayed colonoids exposed to reagents such as Wnt-3A and the γ-secretase inhibitor LY-411575. The successful formation of viable, multi-cell type colonic tissue on the microengineered platform represents a first step in the building of a “colon-on-a-chip” with the goal of producing the physiologic structure and organ-level function of the colon for controlled experiments.
- Subjects :
- Colon
Cell Culture Techniques
Biomedical Engineering
Mice, Transgenic
Bioengineering
Tissue Array Analysis
Biology
Time-Lapse Imaging
Biochemistry
Article
Extracellular matrix
Mice
Intestinal mucosa
Epidermal growth factor
Wnt3A Protein
Animals
Intestinal Mucosa
Progenitor cell
Cells, Cultured
Matrigel
Alanine
Spheroid
Azepines
General Chemistry
Anatomy
Cell biology
Drug Combinations
Cell culture
Proteoglycans
Collagen
Laminin
Amyloid Precursor Protein Secretases
Subjects
Details
- ISSN :
- 14730189 and 14730197
- Volume :
- 13
- Database :
- OpenAIRE
- Journal :
- Lab on a Chip
- Accession number :
- edsair.doi.dedup.....e320aabf86f44127e9cc9d1059df2eab