Are there any recommendations for working with organoid tissue for 10x single cell assays?

Question: Are there any recommendations for working with organoid tissue for 10x single-cell assays?

Answer: Organoids are 3-dimensional (3D) cell cultures that represent fundamental characteristics and cellular organization of an organ. These 3D cultures provide useful information regarding the biology of healthy or diseased organ models. Organoids are generated using relevant cell culture strategies based on the cells of origin. For example, organoid cultures could be generated using iPSCs, hESCs, or adult stem cells. Below, you will find a schematic diagram of an ideal workflow along with some of our recommendations for working with organoid tissue as input to 10x Genomics Single-Cell Assays.

Note that we have not specifically tested these approaches, they are meant to be general guidelines. A pilot study is recommended before starting a large-scale experiment.

If organoids are prepared from adult stem cells it is important to take proper care in preparing a single-cell suspension from the tissue of origin. Experimental procedures should be optimized to minimize unnecessary prolonged tissue handling and enzymatic digestion during tissue association.
It is important to consider the seeding density of cells at the start of the organoid culture. Each organoid culture is limited by the surface area of the culture dish. Avoid overcrowding of the organoid cultures. High seeding density could result in undesirable outcomes such as smaller organoids and potential cell death.
Typically, organoids are placed in 3D cultures using Matrigel® or collagen. Prior to processing organoids for single-cell analysis, the 3D culture should be dissociated using reagents such as Dispase or Collagenase. In order to obtain healthy cells for the analysis, it is important to minimize incubation time during 3D matrix dissociation.
After matrix dissociation, preparation of single cells from organoids could be achieved by using different enzymes such as Accutase®, Papain, Trypsin, TrypLE™ or Dispase. Refer to the table below for the use of appropriate enzymes based on the organoid type. Optimization of enzyme digestion time should be considered in order to obtain healthy single cells during dissociation steps.
Cell viability should be higher than 90% post-organoid dissociation step in order to obtain the best results for single-cell analysis. Wash steps should be performed to remove any unwanted debris.
It is important to note that the cells from organoid cultures could potentially show different gene expression profiles than those observed from primary tissues.

Table: Examples of various organoid cultures, culture conditions, and commonly used enzymes for organoid dissociation.

Organoid Type	Organoid Culture	Cell Density	Dissociation Enzyme	References
Prostate	Dissociated prostate cells in Matrigel®	500-5000 cells	Dispase, TrypLE	Tara McCray 2019
Mammary	Dissociated mammary gland to obtain organoids. Mammospheres from single cells in Matrigel®.	5e3- 1e4 cells	Collagenase, Trypsin, TrypLE	Nicholas Pervolarakis 2020 Sergey Agnezov 2020
Kidney	iPSC cultured on Matrigel®.	5e5 cells	Trypsin, Accutase	Minoru Takasato 2016 Belinda Phipson 2019 Ayshwarya Subramanian 2019
Lung	Dissociated Lung cells in Matrigel®.	5e3 lung epithelial cells + 5e4 lung mesenchymal cells	Dispase, Trypsin	Jarod Zepp 2017 David Frank 2016
Brain	iPSC or hESC in low-adhesion plates.	2e3 – 9e3 Cells	Papain, ACCUMAX	Laura Pellegrini 2021 Zhisong He 2020 Silvia Velasco 2019
Gut	Dissociated crypt cells or hESC in Matrigel®.	500 – 1000 cells	TrypLE	Rosa Elmentaite 2020 Yu-Hwai Tsai 2017 Toshiro Sato 2011
Skin	hESC in Matrigel®.	3.5e3 cells	TrypLE	Jiyoon Lee 2020