preparation of single cell suspensions from human intestinal organoids differentiated as 96 well monolayers

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

The small intestinal epithelium has two distinct zones: the crypt that houses the intestinal stem cell (ISC) and the villus, which is comprised of differentiated cells of the secretory and absorptive lineages. Adding to this complexity is the regional specificity of the epithelium that provides unique functional properties between the regions of the small intestine. Pioneering work established culture conditions in which both the human small intestinal crypt and villus zones can be generated ex vivo from surgical tissues or tissue biopsies1. These cultures are bridging the gap between animal studies and clinical trials and are revealing previously unrecognized cell biology, biochemistry, and physiology of the gastrointestinal tract. The HIOs are propagated as 3D spherical structures using a media with growth factors that promote stem cell viability and extracellular support matrices. These conditions result in a crypt-like HIO model that consists mostly of progenitors and stem cells. Removal of the growth factors promotes ISC differentiation (villus-like model) and production of mature intestinal epithelial cells (goblet, enteroendocrine, tuft, enterocyte) in appropriate ratios along with cell proliferation and differentiation, polarization, barrier integrity, regional specific features, and appropriate physiological responses2. HIO cultures are genetically stable and can be propagated indefinitely in their crypt-like state. Easy access to the apical surface of these cultures is provided by culture conditions that allow growth in a monolayer format3. HIOs also allow considerations of host individual variability such as genetics, age, sex, ethnicity, and disease status to be included in biological analyses. Analytic and functional assessment tools are identical to those used in approaches centered on transformed cell lines and include a variety of molecular techniques such as flow cytometry, microscopy, transcriptomics, proteomics, and metabolomics.
Single cell transcriptomics is revolutionizing our understanding of the biology and physiology of the small intestine by providing insight into the individual contributions of each cell type to a biological process. Pioneering work using this technology has provided a landscape of the cell types present in the native human intestine4,5,6. Single cell transcriptional profiling platforms allow exploration of the transcriptional landscape of individual cells, allowing cell heterogeneity to come to the forefront of scientific exploration. In some single cell transcriptional profiling platforms, microfluidic partitioning and barcoding are used to generate cDNA libraries from cellular polyadenylated mRNAs obtained from up to 10,000 cells per sample. On this platform, droplets containing single cells, barcoded oligonucleotides, reverse transcription reagents, and oil form a reaction vesicle that results in all cDNAs from a single cell having the same barcode. The barcoded cDNAs can then be efficiently sequenced using next generation sequencing. Data generated can be handled through software and visualization tools, which convert the barcoded sequences into visualization maps and single cell transcriptional profiles. Cell populations can be identified using publicly available databases of human small intestinal epithelium4,5,6. Although many studies have utilized this platform to interrogate murine intestinal organoids at the single cell level, the analysis of single cell transcriptional responses of HIOs has lagged behind7,8,9,10,11,12.
Here, we provide a step-by-step guide to isolating viable cells from small intestinal HIOs for processing on single cell transcriptional profiling platforms (Figure 1). We provide culturing and differentiation guidelines along with media components that have been optimized for epithelial differentiation. We outline cell recovery methods for three different culture formats: three dimensional (3D) and monolayer cultures either on plastic or on membrane cell culture inserts. We provide sample clustering data obtained using open-source software to derive differentially expressed genes in each cluster.

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

We are integrating single-cell transcriptomics and organoid technology to better understand the biology of the small intestine. These technologies are allowing us deeper insight into the cellular heterogeneity that exists within that tissue. The tools that improve the throughput, sensitivity, and the resolutions are the keys that advanced research in the field.Some examples are improved computational methods and the technological innovations. Cost reductions also a major focus. The process of preparing and sequencing the libraries of samples can cause transcription variations, which is one challenge.For organoids, specific challenges include limited spatial context or information about temporal dynamics. Single-cell analysis of the human intestinal organoids provides previously unattainable information into the cell types that make up the intestinal epithelium. This, thereby, opens the window to the transcriptional landscape of those cells.We are using these technologies to identify biomarkers and mechanisms of human intestinal diseases. These biomarkers and mechanisms will advance our ability to utilize personalized medicine-based approaches to treat human disease and improve human health overall.

Preparation of Single Cell Suspensions from Human Intestinal Organoids Differentiated as 96 Well Monolayers

To begin, prepare enzymatic cell digestion reagent and PBS/EDTA for human intestinal organoids digestion. Under a brightfield microscope, confirm the growth of differentiated three-dimensional human intestinal organoids as monolayers. Discard the cell culture media from monolayer culture and replace it with 100 microliters of PBS/EDTA.After removing PBS/EDTA, add 100 microliters of warm enzymatic cell digestion reagent to each well. Place the plate at 37 degrees Celsius in 5%carbon dioxide incubator and pipette the entire volume five times every 10 minutes for 20 to 30 minutes. Transfer one to two microliters of cell suspension to a glass slide every 10 minutes to assess dissociation by observing the percentage of single cells.After dissociation, combine three replicate wells per condition in a single 1.5 milliliter microcentrifuge tube. Add 700 microliters of cell culture differentiation medium containing 10 micromolar ROCK inhibitor, and mixed gently by pipetting. Filter the entire volume through a 70 micron tip strainer, collecting the flow-through in a fresh 1.5 milliliter tube.Filter the flow-through obtained through a 70 micron strainer on a 40 micron tip strainer. Add five microliters of 0.4%Trypan Blue to five microliters of cell suspension, and count viable single cells. Dilute the sample with cell culture media containing ROCK inhibitor to obtain 1, 000 cells per microliter.A total of 9, 952 single cells were isolated from differentiated intestinal human intestinal organoids grown in 96 well plates.

preparation-single-cell-suspensions-from-human-intestinal-organoids

Research

JoVE Journal

Biology

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...

Preparation of Single Cell Suspensions from Differentiated 3D Human Intestinal Organoids for Single Cell Transcriptomics

To begin, thaw the enzymatic cell digestion reagent at 37 degrees Celsius for 30 minutes. Under a brightfield microscope at 10x magnification, observe the differentiated three-dimensional human intestinal organoids to ensure cell health. Replace the media from the wells of a 24-well plate containing differentiated organoids with 500 microliters of cold cell recovery solution.Pipette up and down to release the cells from the basement membrane. Transfer the cell suspension to a 1.5-milliliter microcentrifuge tube. Incubate the tube on ice for 10 minutes, pipetting up and and down every five minutes.Centrifuge the cell suspension at 400 g for three minutes at four degrees Celsius. Remove the supernatant from the tube without disturbing the pellet. Resuspend the pellet in 500 microliters of prewarmed enzymatic cell digestion reagent, pipetting up and down 10 times.Place the organoids for 30 minutes at 37 degrees Celsius in a carbon dioxide incubator. Every 10 minutes, pipette the entire volume 10 times using a P1000 pipette to help dissociate the cells. Centrifuge the cell suspension at 400 g for three minutes at four degrees Celsius, and resuspend the pellet in one milliliter of differentiation media.Rapidly pipette the cells up and down 50 times on ice to dissociate organoids into single cells. Filter the entire volume through a 70 micron tip strainer and collect the eluate in a fresh 1.5-milliliter tube. Then filter the eluate obtained through a 70 micron strainer on a 40 micron tip strainer.Add five microliters of 0.4%Trypan Blue to five microliters of cell suspension, and count viable single cells using Trypan Blue Exclusion. Dilute the sample with cell culture media to obtain 1000 cells per microliter. A total of 4, 402 single cells were isolated from differentiated ileal three-dimensional human intestinal organoids, representing expected cell types, including absorptive enterocytes and secretory cells.

Preparation of Single Cell Suspensions from Human Intestinal Organoids Differentiated on Membrane Cell Culture Insert

To begin, place the 15 milliliter tube containing prewarmed enzymatic cell digestion reagent in a 37 degree Celsius incubator until use. Aliquot 500 microliters of freshly prepared PBS/EDTA into the wells of a 24 well plate. Under a brightfield microscope, observe the growth of differentiated three dimensional human intestinal organoids and transwells.Transfer the cell culture inserts from the growth media to the PBS/EDTA solution. Remove the culture media from the apical side and replace it with 100 microliters of PBS/EDTA without disturbing the cell layer. After discarding PBS/EDTA, remove the insert from the well, blot it gently onto the edge of a paper towel and inverted on the bench top.Using a sharp scalpel blade, cut around the inner circumference of the membrane to remove the membrane from the insert. Transfer the membrane with forceps to the 1.5 milliliter tube containing 200 microliters of prewarmed enzymatic cell digestion reagent. Place the membrane for 30 minutes at 37 degrees Celsius in a carbon dioxide incubator.Every 10 minutes, pipette the entire volume five times using a P 200 pipette. Transfer, one to two microliters of cell suspension to a glass slide every 10 minutes to assess dissociation by qualitatively assessing the percentage of single cells. After dissociation, combine three replicate wells per condition in a single 1.5 milliliter micro centrifuge tube.Add 400 microliters of cell culture differentiation medium containing 10 micromolar ROCK inhibitor and mix gently by pipetting. Filter the entire volume through a 70 micron tip strainer, collecting the flowthrough in a fresh 1.5 milliliter tube. Then filter the flowthrough obtained through a 70 micron strainer on a 40 micron tip strainer.Add five microliters of 0.4%trypan blue to five microliters of cell suspension and count viable single cells. Dilute the sample with WRNE growth medium to obtain 1, 000 cells per microliter. A total of 5, 623 single cells were isolated from differentiated jejunal human intestinal organoids grown on membrane cell culture inserts.