intestinal epithelial monolayers derived from established murine colonoids

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation3. However, all these systems have important shortcomings that limit their ability to recapitulate the epithelial changes during IBD4. Most cell lines used to study IBD are transformed, have the ability to form a monolayer, and can differentiate3 but intrinsically propagate differently than non-transformed intestinal epithelial cells in the host. Several different murine models of inflammation are used to study IBD, some of which include knockout models, infectious models, chemical inflammatory models, and T-cell transfer models5,6,7,8. While each can study certain etiological aspects of IBD, such as genetic predispositions, barrier dysfunction, immune deregulation, and the microbiome, they are limited in their ability to study the multifactorial nature of the disease.
Intestinal organoids, including enteroids and colonoids, have been established over the last decade as a useful in vitro model for studying not only the dynamics of intestinal stem cells but also their role the barrier integrity and function of the intestinal epithelium play in intestinal homeostasis and disease. These entities have significantly contributed to our understanding of the pathogenesis of IBD9 and have opened new opportunities for personalized medicine. Colonoids, or stem cell-derived, self-organizing tissue cultures from the colon, have been developed from both murine and human tissue in a process that allows stem cells located within intestinal crypts to propagate and be maintained indefinitely10. The stem cell niche in vivo relies on extracellular factors to support its growth, notably the canonical Wnt signaling and bone-morphogenic protein signaling pathways11. The addition of these factors promotes the health and longevity of colonoids but also drives the culture toward a stem cell-like state that is not reflective of the in vivo epithelial cellular architecture, which consists of both self-renewing and terminally differentiated cells12,13. While the functionality of the intestinal epithelium is dependent upon the continual crosstalk between the stem cell compartment and differentiated cells, the ability to have both in a colonoid culture system is fairly limited. Despite these limitations, the organoid culture system remains the gold standard to study the intrinsic properties of the epithelium ex vivo. Nonetheless, alternative culture strategies may need to be considered to answer the scientific question at hand.
It has been shown that mice on a continuous 7 day regimen of dextran sodium sulfate (DSS) develop both epithelial inflammation and barrier dysfunction14. Furthermore, mitochondrial biogenesis failure and metabolic reprogramming within the intestinal epithelium, which have been shown to be evident in human IBD, have also been captured in this DSS model of colitis15. However, our preliminary data demonstrate that the characteristics of mitochondrial biogenesis failure are not retained in colonoids derived from the crypts of DSS-treated animals (Supplementary Figure 1). Thus, alternative culture methods must be used when examining how inflammation drives epithelial changes during murine intestinal inflammation. Here, we outline a protocol we have developed that describes 1) how to isolate crypts from whole colonic tissue for the establishment of murine colonoids, 2) how to terminally differentiate this cell population to reflect the cell population as it stands in vivo, and 3) how to induce inflammation in this in vitro model. To study drug interactions within the inflamed epithelium, we have developed a protocol to establish 2-dimensonal (2D) monolayers from murine colonoids that can be basally treated with inflammatory mediators and apically treated with drug therapies.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

Through our research, we aim to explore how murine colonoids could be modified from a conventional colonoid culture system, to study certain aspects of intestinal physiology and their alteration in the presence of an inflamed disease state. Studying barrier function and intestinal physiology in disease states like inflammatory bowel disease is limited when using a traditional murine colonoid culture system that reflects only stem cell physiology. Our protocol is advantageous in that it provides the reader with multiple techniques to study how inflammatory mediators affect the intestinal epithelium, by either terminally differentiating them, or creating a 2D monolayer system.In the future, our laboratory will concentrate on comprehending how the human colonoid culture system can be altered to investigate different aspects of intestinal physiology and diseases, particularly inflammatory bowel disease.

Watch this Scientific Journal Video about Intestinal Epithelial Monolayers Derived from Established Murine Colonoids at JoVE.com

Intestinal Epithelial Monolayers Derived from Established Murine Colonoids  

Begin by preparing all the reagents prior to the experiment and thaw the basement membrane matrix on ice. Then prepare the murine monolayer medium. Dilute the thawed basement membrane one to 20 with the cold murine monolayer medium, and keep it on ice.Place a 0.4 micrometer transparent cell culture insert, using sterile forceps, into a single well of a 24 well tissue culture plate. Grab a corner prong of the insert and transfer it into the well. Repeat until the appropriate number of cell culture inserts are available for culture.To cover the apical surface of each cell culture insert with diluted basement membrane matrix, place the tip of the P 200 pipette in the center of the insert and expel 150 microliters matrix. Then, transfer the plate to a sterile 37 degrees incubator with 5%carbon dioxide, for at least one hour. At the end of the incubation, rotate the 24 well plate at a 45 degree angle.Place a single finger on the corner of the prong to stabilize the insert. Gently place the P 200 pipette tip along the bottom side of the insert and remove the basement matrix. Remove any excess residue by washing each cell culture insert with 150 microliters of sterile DPBS without calcium or magnesium ions and allow them to dry in a biological safety cabinet for a minimum of 10 minutes.Once the inserts are dried, add 400 microliters of the murine monolayer medium to the bottom, and 75 microliters on top of the cell culture insert. Repeat until all the cell culture inserts are immersed. Leave the plate in the biological safety cabinet or the incubator until needed.Then remove the 24 well plate of mature intestinal colonoids from the incubator and place it under the biological safety cabinet. Aspirate the medium using sterile tips and wash each well with one milliliter of sterile DPBS at room temperature. Using sterile tips, remove the DPBS without calcium or magnesium ions.Digest each plug of the basement membrane matrix by adding 500 microliters of enzymatic dissociation reagent in each well to be passaged. To break up the plug, rotate the 24 well plate at a 45 degree angle. Place the tip of the pipette at the edge of the plug and gently dislodge it by pipetting each plug approximately six to 10 times.Place the 24 well plate back in a sterile incubator at 37 degrees with 5%carbon dioxide, for three to four minutes. After incubation, pipette the trypsin up and down five to seven times for each well under a biological safety cabinet. Transfer the dissociated colonoids from each well into a sterile 15 milliliter conical tube and bring up to a volume of 10 milliliters with an ice cold mouse wash medium.Next, centrifuge the partially digested colonoids at 300 G for five minutes at four degrees Celsius in a swinging bucket centrifuge. Under the biological safety cabinet, remove the supernatant from the pellet using a 10 milliliter pipette. Also, remove any remaining medium using a P 200 pipette.Then, re-suspend the colonoid pellet by adding 75 microliters of murine monolayer medium. Dispense 75 microliters of the colonoid suspension to the center of each cell culture insert. Gently pipette the suspension once or twice before adding it to the well to ensure uniform platting.Place the 24 well plate with cell culture inserts on a rotating platform for 10 minutes to allow for uniform dispersion across the cell culture insert, before placing the plate in a sterile 37 degree Celsius in 5%carbon dioxide incubator. At the end of the incubation, dislodged the unattached colonoids fragments by placing the tip alongside the inside of the cell culture and pipetting the medium three to five times with a P 200 pipette. Avoid disrupting the attached intestinal cells.Remove the medium and colonoid mixture immediately. Repeat until all the cell culture inserts have been cleaned. Now add 150 microliters of pre-warmed murine monolayer medium to the apical side of each cell culture insert.Add 400 microliters of warmed murine monolayer medium to each well of a new 24 well plate. Transfer the cell culture insert to the new plate by grabbing its prong using sterile forceps. Change the medium every other day until the desired confluency is attained.The enzymatically disrupted colonoids plated on the cell culture inserts initially appeared in cellular clusters ranging from 15 to 150 cells. On day three, the cells flattened out and slowly covered the cell culture insert, generally reaching confluency. Over the next couple of days, monolayers continued to grow and formed a continuous barrier that can be quantitatively measured.

intestinal-epithelial-monolayers-derived-from-established-murine

Research

JoVE Journal

Biology

143 visualizações.  UPMC Children’s Hospital of Pittsburgh. Comece preparando todos os reagentes antes do experimento e descongele a matriz da membrana basal no gelo. Em seguida, prepare o meio monocamada murino. Diluir a membrana basal descongelada de um a 20 com o meio monocamada murino frio e mantê-lo no gelo.Coloque uma pastilha de cultura de células transparente de 0,4 micrômetros, usando pinças estéreis, em um único poço de uma placa de cultura de tecidos de 24 poços. Pegue um pino de canto da pastilha e transfira-a para o poço. ...