Organoids

Abstract

Organoids have attracted increasing attention because they are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration, and repair in human tissues. Organoids can also be used in diagnostics, disease modeling, drug discovery, and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer serves to highlight the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community is also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and key priorities in organoid engineering for the coming years.

Fig. 1. Components to engineer organoids

The set up of organoid-based culture requires considerations about four major components that make up organoid cultures – cells, soluble factors, matrix, and physical cues and how to integrate these components

Fig. 2. Flowchart of the procedures

Organoids can be generated from TDC or iPSC.

Fig. 3. Representative results of pancreatic islet organoids validation analysis

1. Representative view of pancreatic islet organoids.

2. Cell types and hormones secretion level validation by immunofluorescence or immunohistochemical staining.

3. Real-time qPCR analysis for some key transcription factors and differentiation markers.

4. The maturation of the organoids can be induced through prolonged culture for a total of 30 days at any passage.

5. Schematic of the pancreatic islet organoids function validation in vitro

6. Measurement of the secreted C-peptide by ELISA

7. Intensity of calcium signalling traces imaging indicating the capability of responding acutely to glucose

8. Schematic of the islet organoids function validation in vivo

Figure 4.. typical characterization of cancer organoids: liver cancer subtype

1. Isolation of cells from patient samples and organoid culture; schematic of tissue isolation and processing;

   HCC, hepatocellular carcinoma; CC, cholangiocarcinoma; CHC, combined HCC/CC tumors.

2. Histological analysis of liver cancer samples: top, tissue; middle, organoid brightfield images; bottom, histological H&E staining of organoids; scale bar, middle row, 100 μm; top and bottom rows, 50 μm.

3. Analysis of specific marker gene expression: immunofluorescene staining for AFP (hepatocyte/HCC marker; red) and EpCAM (ductal/CC marker; green); blue - DAPI, scale bar, 30 μm.

4. Organoid formation efficiency: growth and splitting curves; dot, splitting time point, arrow, continuous expansion.

5. Transplantation into immunodeficient mice: xenograft and histopathology analysis, matching to the patient origianal tissue sample; scale bars, left, 125 μm; right, 62.5 μm

6. Analysis of genetic changes in the cancer organoids and their concordance to the mutations in the original tumor sample.

7. Organoid sensitivity to drugs: IC50 curves for gemcitabine treatment.

Fig. 5. Reducing the heterogeneity with complexity reduction

Simpler models of reduced dimensions to recapitulate the essential tissue structures and functions of interest are gaining momentum. Micropatterned 2D mono- or co-culture allow the formation of reproducible initial 2D condition which can further form the initial 3D structure^,. Then a high degree of spatiotemporal control, such as stretching and osmotic forces can be applied to direct certain tissue morphogenesis.

Fig. 6. Side-by-side comparison of the current limitations for organoid culture and approaches to overcome them

Top panel: Accummulation of dead cells and cell debris inside of cystic organoid lumina (left) has been overcome by (right) designing a perfusable open-end structures that use inducible flow to wash out cell debris, which are compatible with long-term experiments. Middle panel: Organoids grown in Matrigel domes display high-variability of cell heterogeneity and morphology (left) which can be overcome by utilising (right) grids with patterned synthetic ECM which provide cues for cell differentiation. These platforms are additionally compatible with high-throughput screenings. Bottom panel: Single-cell type derived organoids do not recapitulate the cellular and physiological complexity of native tissue (left), but (right) combining organoids with organ-on-chip (OoC) as novel technology would enable creating controlled micro-envirorment, suitable for multiple cell types