Establishment and Optimisation of Small and Large Intestinal Organoid Cultures for in vitro Studies on Murine Rotavirus

Abstract

Despite available vaccines, rotavirus (RV) gastroenteritis remains to be an alarming cause of under-five mortality, especially in developing countries. Research on RV has been limited by the lack of faithful in vitro models that mimic virus-host interaction and RV pathogenesis in the intestine. Most recently, adult stem cell-derived organoids have advanced the understanding of the cellular mechanisms in homeostasis and disease. Intestinal organoids, derived from isolated pluripotent stem cells, exhibit a similar architecture, cellular composition and many functions to the region-specific aspects of the intestinal epithelium.
Here we established and optimised a long-term small and large intestinal organoid culture system by applying previously defined insights to intestinal epithelial growth requirements. By in situ production of Wnt3a, R-spondin1 and Noggin we demonstrated a cost-effective alternative of adding these growth factors in the culture medium. Further, we showed that intestinal epithelial organoids were successfully expanded from murine small and large intestinal tissue through culture settings that mimic the extracellular matrix and the intricate system of signalling growth factors in vivo. Through microscopy, we observed the progress of differentiation, from initially hollow spherical structures, to more complex budding structures. The organoids were stably maintained for four months, with the possibility of a longer lifespan. Additionally, we found insignificant loss of organoids and their proliferative capacity following cryopreservation. Varying the ratio of growth factors in the culture medium revealed the possibility to manipulate the differentiation state of the organoids.
Although we did not achieve true infection in this study, the established small and large
intestinal organoids cultures and quantitative-PCR (q-PCR) specific for RV detection indicate that we are on the verge of this, and can consequently initiate on studies on the susceptibility of the intestinal epithelial cells as first responders to RV infection.

Popular Abstract

No Guts, No Glory: How to Make a Mini-Gut in a Culture Dish

People often say that if you have no guts, you have no courage. However, it would be more accurate to say that if you have no guts, you have no protection against outside intruders in your body, that is, if you are still alive. The intestine and the immune system are intricately connected and the walls of the intestine act as the first line of defence against enteric pathogens such as the rotavirus (RV). RV is a virus that attacks and damages the cells lining the intestinal wall, causing gastroenteritis. Infection is usually asymptomatic in adults but can severely affect infants and children under the age of five, and in extreme cases even cause fatality, especially in poverty-stricken countries.

The positive thing is that there are vaccines to help combat this virus, but the negative thing is, the efficacy of these vaccines is not always optimal due to varying factors. One of the main hindrances is that the gut is so complex and intertwined with other biological entities and processes. Another is that quite little is known about RV pathogenesis, undoubtedly due to the limitations of studying RV. Scientists often have two choices of simplified models when studying any biological question (RV included) and these are either in vivo or in vitro studies. In vitro studies make use of cell lines that have been manipulated to behave and multiply indefinitely. However, many cell lines are handpicked from tumour cells, making them corrupt with genetic mutations that when used, may not act the way normal cells in a body would. Furthermore, some cell lines commonly used are from a range of different animals or tissues completely different to the organ under investigation. This makes the results unreliable or give too small of an insight to truly be an effective method. The counterpart to this, in vivo studies, involves lab animals. These studies can be used to answer questions that the cell lines might have difficulty with, such as an immune response towards RV. The main obstacle with in vivo studies remains to be the mismatch that arise with the use of different host-specific viruses. Also, the problem of complexity and interfering variables is still present in this smaller body.

Therefore, we opted to establish a system that can combine the best of the two models, in vitro organoid cultures. We collected stem cells from the gut and nurtured them into mini-guts, by mimicking the signalling cues these cells are subjected to in a real intestine. These signalling factors were manufactured inhouse and a satisfactory amount was obtained. To nudge the stem cells in the right direction, these, together with the signalling factors, were cultured in a setting that resembles the physical environment of the cells lining the gut. Et voilà, mini-guts! The mini-guts look like hollow balls with protruding buds, where the main body contains the self-renewing stem cells and the differentiated cells sit along the buds. The hollow space is alike the hollow space, or lumen, in the gut which is where RV attacks.

These mini-guts showed not only to fully contain all the cells that are normally in the real thing, but also the ability to be long-lived, multiplied and stored for later use. We also found that we could manipulate their maturation by adjusting the signalling cues. This meant that we could produce mini-guts to suit our needs, without buds i.e. less differentiated structures if desired. These mini-guts would be beneficial in our overall aim to propagate RV in our lab and to unravel how the cells of the intestinal walls act when combating RV. This is still ongoing and hopefully, the answer is right around the corner.

Master’s Degree Project in Molecular Biology, Medical Biology, 45 credits, 2017-2018
Department of Biology, Lund University

Advisor: Katharina Lahl
Co-advisor: Konjit Getachew
Department of Experimental Medical Science, Lund University

• author: Ulmert, Isabel
• supervisor: Katharina Lahl; Konjit Getachew Muleta
• organization: Degree Projects in Molecular Biology
• course: MOBN02 20172
• year: 2018
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8958883
• date added to LUP: 2018-09-17 12:33:22
• date last changed: 2018-09-17 12:33:22