optimizing human intestinal enteroid cultivation for bioluminescence assays

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

Circadian clock 
All organisms, from bacteria to mammals, have a complex and dynamic relationship with their environment. Within this relationship, adaptation to environmental changes is critical for the survival of organisms. Most organisms possess circadian rhythms that enable them to adapt and optimize their functions to diurnal cycles of approximately 24 h. The circadian clock is a hierarchical network of central and peripheral clocks that work in cooperation to maintain physiological homeostasis and keep organisms synchronized with daily changes1,2. In mammals, the central or master clock located in the suprachiasmatic nucleus (SCN) receives external cues, such as light, and transmits the information to the peripheral clocks via an advanced interplay of neural and humoral signaling pathways3. In addition to the central clock, peripheral tissues possess their own cell-autonomous circadian clock mechanism, maintained by a transcriptional-translational negative feedback loop (TTFL) regulating tissue-specific clock-controlled genes (CCGs)4,5. This molecular machinery produces approximately 24 h rhythmicity in cellular and physiological events, such as gene expressions, signaling pathways, immune responses, and digestion. The circadian clock is present in almost all mammalian cells, and it has been demonstrated that up to 50% of the genes&#39; expression patterns exhibit circadian rhythmicity6. Considering the abundance of CCGs, disruption of this clock mechanism may result in critical physiological problems. Hence, investigations into circadian rhythms are necessary to elucidate essential biological mechanisms and develop novel therapeutic strategies.
Luciferase reporter system 
In circadian studies, real-time monitoring is critical for a better understanding of cellular behaviors and responses because it allows tracking of temporal changes in gene expression and/or protein levels, providing insights into the molecular mechanisms regulated by the circadian clock. Furthermore, real-time monitoring enables researchers to study the effects of environmental changes on molecular mechanisms7,8. There are numerous techniques for real-time monitoring studies, including bioluminescence assay, which is widely used to track gene expression or protein levels over time. Bioluminescence assay is a method to detect biological processes using light production as a readout. In this assay, an oxidative enzyme that produces bioluminescence (e.g., luciferase) is either transiently or stably transfected into cells of interest, and the bioluminescence readout is measured in the presence of a substrate (e.g., luciferin) over time. For example, the luciferase enzyme produces bioluminescence by oxidizing the substrate luciferin in the presence of ATP9. Due to its short half-life, 3-4 h10, firefly luciferase is a powerful tool for circadian studies in terms of providing real-time dynamic monitoring with minimal background noise11,12,13. For the insertion of DNA with a luciferase-tagged promoter or open reading frame (ORF), the lentiviral gene delivery system is a reliable method that provides high transduction efficacy, stable integration, and low immunogenicity. Stable transduction of a bioluminescent reporter provides robust expression in dividing and non-dividing cells, generating consistent data for circadian studies14.
Organoid as a model 
Traditional immortalized two-dimensional cell lines have been instrumental in biological studies ranging from uncovering fundamental molecular mechanisms of circadian rhythms to drug screening. Despite the convenience of utilizing homogenized cell lines, they lack multicellular structures and intercellular interactions. In contrast, organoids are in vitro 3D multicellular &#34;organ-like&#34; structures that mimic organ structure in a dish by displaying similarity to in vivo tissue architecture and multicellularity, including stem, progenitor, and differentiated cell types15,16. Possessing self-organization, multicellularity, and functionality features makes organoids a remarkable in vitro model representing the cellular and physiological processes occurring in real tissues17. Different types of organoids can be derived from pluripotent stem cells via directed differentiation or adult stem cells harvested from various organs, including the small intestine, brain, liver, lung, and kidney18,19. Since organoid structures possess a real tissue-like architecture and function with multicellularity and dynamic cell-to-cell interaction, they are superior to homogenized cell lines for understanding the cellular events occurring in in vivo tissues. Organoids are also easily manipulated and can be grown under controlled conditions, making them useful for circadian studies20.
The main purpose of this work is to introduce a real-time monitoring method utilizing a bioluminescence assay specifically tailored for studying circadian rhythms in multicellular 3D organoids. Real-time monitoring of cellular events using a bioluminescence assay technique has been widely performed for cell cultures lacking the multicellular complexity and intercellular communications that exist in real tissues. 3D organoids present unique opportunities to investigate the functions of circadian rhythms in multicellular systems in vitro. For example, one could investigate circadian rhythms in the organoids with altered cell compositions or organoids derived from patients&#39; diseased tissues. This protocol enables the utilization of a bioluminescence assay to investigate different aspects of circadian rhythms in a more physiologically relevant in vitro model, organoids, which will help us better understand the roles of circadian rhythms in peripheral organs.

Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Оптимизация культивирования кишечных энтероидов человека для биолюминесцентных анализов

Для начала разложите все материалы, необходимые для переноса кишечных энтероидов человека, или ГИЭ, на рабочую платформу. Смешайте HIE с 3D-матрицей культуры со сниженным фактором роста. Высадите смесь на 24-луночный планшет с тремя каплями по 10 микролитров 3D-матрицы культуры на лунку и инкубируйте в течение 20 минут при температуре 37 градусов Цельсия.Затем добавьте 350 микролитров питательной среды HIE. Через семь дней соберите HIE из четырех лунок в пробирки объемом 1,5 миллилитров. Центрифугируйте HIE при 2000 г в течение 40 секунд, чтобы удалить мусор.С помощью пипетки удалите излишки матрицы 3D-культуры из пробирки. Добавьте в пробирку 0,5 миллилитра холодного PBS. Используя инсулиновый шприц объемом в один миллилитр, наложите на нее физическую турбулентность путем спринцевания до 10 раз, чтобы диссоциировать ГИЭ.Центрифугируйте диссоциированные HIE при 2000 г в течение 40 секунд и аккуратно удалите надосадочную жидкость. Затем поместите трубку на лед примерно на три минуты. Добавьте в пробирку уменьшенную 3D матрицу культуры с фактором роста и смешайте ее с гранулой.Засейте смесь HIE в 3D-матрице культуры в центре 35-миллиметровой круглой чашки. Инкубируйте чашку при температуре 37 градусов Цельсия в течение 20 минут, чтобы 3D-матрица культуры застыла. Затем добавьте в блюдо два миллилитра предварительно подогретой кишечной органоидной среды для роста и выдерживайте при температуре 37 градусов Цельсия с 5% углекислым газом в течение 48 часов.На третий день замените питательную среду двумя миллилитрами предварительно подогретой дифференцировочной среды и инкубируйте при температуре 37 градусов Цельсия с 5% углекислым газом в течение 48 часов. На пятый день замените энтероидную среду двумя миллилитрами предварительно подогретой дифференцировочной среды и продолжайте инкубацию в течение 24 часов.

Research

JoVE Journal

Biology

Optimizing Human Intestinal Enteroid Cultivation for Bioluminescence Assays

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

Synchronizing Circadian Rhythms in Human Intestinal Enteroids for Bioluminescence Monitoring

Синхронизация циркадных ритмов в кишечных энтероидах человека для мониторинга биолюминесценции