Organoid frequently asked questions

Organoids are complex, multicellular 3-dimensional in vitro cell models that closely mimic in vivo organs.

In 2009 Hans Clevers, the pioneer of organoid biology, and fellow investigators, at the Hubrecht institute, Netherlands, first demonstrated that a single LGR5+ expressing intestinal stem cell could self-organize into crypt-villus structures typical of the adult intestine. Following this work organoids have been derived from numerous tissues including but not limited to brain, colon, kidney, liver, pancreas, lung, tumour biopsies and induced pluripotent stem cells (ipscs). 

Biobanks of human organoids have been generated from various tissues containing a wide range of cellular and molecular phenotypes. For example, establishing a collection of normal and diseased gastrointestinal organoids has aided in the research of colon cancer and other GI related diseases such as crohn’s, irritable bowel syndrome (IBS), and ulcerative colitis. Biobanks have also been developed for wider cellular research applications and for personalized medical research. 

An organoid is derived from a stem cell or isolated from primary tissue and contain multiple cell types with complex structures. Organoids are typically cultured in a defined serum-free medium with a supporting matrix or hydrogel, e.G. Matrigel. They have an unlimited lifespan in culture and are said to be phenotypically stable. Due to their complexity and stem cell-derived component cells they are considered to have a high physiological relevance for research studies and drug screening 

A spheroid is established in culture for single cells derived from an immortalized cell line. Spheroids usually contain a single cell type and develop simple structures within its three dimensional they are cultured as freely floating aggregates in low adhesion plates. Spheroids have a lower physiological relevance due tom the formation of nutrient and hypoxic gradients forming within the structure. Due to these gradients spheroids have a limited lifespan in culture as a physiologically-relevant models. 

Organoids can be derived from either adult tissue, termed patient-derived organoids (pdos), or developed from pluripotent stem cells (human ES/ipscs). Isolating and expanding the LGR5+ stem cell population is critical to maintain the self-renewing capability of the organoids. Other critical factors to maintain organoids in culture include using lot-qualified hydrogels, e.G. Growth factor reduced (GFR) matrigel, adherence to proper passaging techniques and sourcing high quality organoid-specific media and cell culture reagents. 

Patient-derived organoids can be established from both healthy and tumour tissue biopsies by many research groups. Tumour organoids (a.K.A. Tumoroids) have been shown to retain key genetic and phenotypic characteristics of their tissue of origin and tumour subtype. In doing so they represent a better in vitro model for maintaining intra-tumoral heterogeneity. Recently, tumour pdos have been used in the clinical setting as an in vitro predictor of chemotherapeutic treatment response. These observations demonstrate their importance as models in identifying new anti-cancer treatments through drug screening. 

• Once isolated, organoids are cultured in specialized media embedded in a rich extracellular matrix (ECM) basement membrane extract hydrogel such as growth factor reduced matrigel (corning).
• Media is usually replenished every other day and cells are passaged once per week (7-12 days). This protocol prevents the organoids becoming too large or necrotic.
• Organoids can be passaged/sub–cultured as either small clump fragments or as single cells using mechanical dissociation/enzyme-free passaging reagents.
• Depending on organoid type and confluency of the culture, split ratios of roughly 1:3-1:4 can be used when passaging organoids. For sensitive organoid types and subcultures involving single cell passaging,  the rho-kinase inhibitor rocki (Y27632) can be added during passaging to help in promoting cell viability. 

Historically organoids have been cultured matrigel to promote their proper growth. Matrigel would be recommended for the early stages of establishment of an organoid cultures. Culture of organoids in an alternative matrix/hydrogel systems would have to be determined by the investigator. The matrigel matrix provides the necessary chemical signalling cytokines and structural ECM proteins required to mimic the natural in vivo 3D environment. Matrigel is often used in an undiluted format (8 mg/ml or higher) to create the matrigel domes used to culture organoids. It has been reported recently that higher throughput suspension cultures of organoids can be generated by adding diluted matrigel to the media rather than utilizing the matrigel dome method. 

The dome-based method for growing organoids involves seeding organoid fragments within a single drop of matrigel ECM and allowing polymerization of the gel. Once the matrigel ECM polymerizes, media is added to the well on top of the dome. One matrigel dome is typically seeded per well. 

The growth media is removed and replace with cold PBS. The matrigel dome(s) is disrupted mechanically using a pipette tip and diluted further with cold PBS. Matrigel can be removed by centrifugation at 1100 rpm for 5 minutes. After centrifugation, a thin layer of matrigel visible above the organoid cell pellet can be carefully aspirated. Organoids should be washed with growth medium or PBS and centrifuged again to remove any additional matrigel. Alternatively proprietary formulations such as corning® cell recovery solution can be used to recover organoids from matrigel domes. 

Each organoid cell type requires its own unique serum-free media. Commonly used media and supplements would include advanced DMEM/F12, N-2 , N-27, niacinamide, n-acetyl-l-cysteine, gastrin wnts, r-spondin-1, noggin, fgfs, egfs or small molecule inhibitors such as CHIR99021, SB202190, and A-83-01. To reduce the cost of expensive recombinant proteins, L-WRN (WNT, r-spondin, noggin) conditioned media containing, r-spondin-1 and WNT conditioned media have been used as an inexpensive alternative to recombinant proteins to supplement organoid media. 

If the LGR5+ stem cell population is maintained using proper media and passaging techniques most organoids can be expanded indefinitely. However, it is recommended to perform marker expression and karyotype analysis every 5-10 passages to ensure organoid quality and identity. 

Organoids can be passaged in either small clump fragments or single cells using mechanical dissociation or enzyme-free passaging reagents. 

Yes. Depending on the organoid cell type, optimized organoid cell freezing media can be used to cryopreserve organoids. There are several cryopreservation formulations available from commercial suppliers.  Most patient-derived organoid types can be cryopreserved. However, some ipsc-derived mature organoid cell types such as lung organoids can’t be frozen successfully. Organoids should be pre–treated with rocki (Y27632) before freezing to help promote cell viability. It is recommended that freezing of 5-10 matrigel domes into 1 cryovial using a controlled mr. Frosty freezing container be followed. The average organoid density of each dome should be ~90% at the time of freezing to ensure the highest cell viability. Try to use organoids that are still in growing state and have the least amount of dead cells or cellular debris. 

It is critical to use antibodies prequalified for organoid analysis. Organoids can be stained with antibodies for immunocytochemical (ICC)/immunohistochemical (IHC) analysis using either whole-mount or paraffin embedding/sectioning techniques.  

Due to the variable nature of organoids and their original tissue types, most traditional assays require further experimental optimization. For example, the common live dead cell viability assay (thermo fisher) has been optimized for 3D cultures including organoids. The assay relies upon calcein-am (live cells), propidium iodide (dead cells) and hoechst 33342 (all cells). Another common organoid assay is the forskolin-induced organoid swelling assay that measures CFTR function. 

Traditional DNA/RNA isolation kits can be used to prepare genomic material for sequencing (rna-seq, scrna-seq, WGS, chip-seq etc). For some applications with large organoids, tissue specific DNA/RNA extraction kits are recommended. TRI reagent (thermo fisher) can be added to the dissociated organoids before RNA extraction and can be stored at -80 °C until future use. 

Cells grown in 3D hydrogels have proven valuable for numerous applications. Traditional immortalized cell lines can be cultured embedded within hydrogels to form multicellular complexes called spheroids. Spheroids have been demonstrated to provide a wealth of information not apparent from the same cells cultured under 2D conditions. Stem cells grown in 3D hydrogels can differentiate into multiple cell types that closely resemble native tissues. They can be programmed to form 3D organoids, differentiated mini-organ structures that have been used as highly predictive, reproducible, and scalable in vitro models of complex physiology. Other applications of culturing cells within hydrogels have focused on interrogating the physical properties of the hydrogel itsel to assess its mechano-sensitive behaviours for cells including the formation of morphological features and cell motility. 

Cells in their physiological setting typically interact with extracellular factors in three dimensions (3D). The extracellular matrix (ECM) is a complex formulation of biological polymers composed of peptides, polysaccharides, proteins, enzymes and signalling molecules that reside outside of cells. Interactions between cells and their surrounding ECM provide cues that can influence cellular polarization, shape, motility, differentiation and many other phenotypes. Traditional cell culture is done on a two-dimensional (2D) substrate, usually polystyrene or glass, and cells grown in this manner often do not fully recapitulate many phenotypes observed by those same cell types in vivo. One approach to making cells cultured in vitro more closely resemble their corresponding physiological tissue type is to embed and culture cells within a 3D hydrogel that mimics the physiological ECM. This strategy is a subset to the larger approach of culturing cells in three-dimensions, typically referred to as 3D cell culture. 

Hydrogels can be defined as water-swollen networks of polymers. Most are liquids at 4 degrees or room temperature but will form a gel when incubated at 37 °C. Because of their properties, cells can be embedded inside hydrogels by mixing cell solution with hydrogel before gel formation: the mix is then dispensed in a cell culture vessel and during the gelation process cells will be encapsulated inside the gel. Compared to classical, 2D cell culture, cells cultured in 3D, embedded in gels, recover different characteristics they have when placed in their natural environment. 

In order to analyze the cytotoxic effects of drug compounds, luciferase-based assays that measure ATP such as the cell viability luciferase assay or celltiter-glo® 3D cell viability assays are often used on organoid cultures. These assays are available from promega. 

There are several research institutes, biorepositories and universities that have collections of ethically sourced high quality patient-derived PDOs, as well as IPSC derived organoids. 

Some organoid types, such human colon organoids, can be single cell passaged using tryple express dissociation reagents. Seeding equivalent numbers of organoids per well produces more uniform organoid cultures than traditional mechanical or enzymatic clump passaging techniques. When single cell passaging, it is critical to add rocki  (Y27632) at a final concentration of 10 μm to the media to maintain cell viability. Another technique to reduce assay variability is to manually remove organoids with abnormal morphologies while maintain organoids of similar sizes. 

Pdos: A shorter time is required to generate organoids from patient material. They are more representative of mature tissues with greater complex cell heterogeneity. They are more genetically diverse (biobanks possible) and representative of patient specific disease state.

In contrast, using IPSC–derived organoids it is possible to generate multiple cell types from same genetic background, it is easier to source originating material and perform gene editing.