Organoid culture – Future opportunities on tumour organoid culture standardisation

by | Jan 24, 2024 | Cancer, CRISPR, NGS, Organoids, Single Cell, Stem Cell

REVIEW HIGHLIGHTS

TUMOUR ORGANOID CULTURE: FUTURE OPPORTUNITIES ON STANDARDISATION

The organoid has developed as an attractive in vitro platform for tumour biology research and high-throughput drug screening in cancer medicine [1]. However, there are no standardized methods to guide the culture of organoids, leading to confusion in organoid studies that may disturb accurate judgements of tumour biology[1].

In a recent journal article published by Cancer Medicine, Zhou et al. presented the latest research findings on organoid standardisation and shared future opportunities for tumour organoid culture standardisation, including organoid formation, culture medium and extracellular matrix (ECM).

Dolly from Lab-A-Porter extracted some of the highlights from the review article while we featured some of the organoid research reagents and tools that may support your next organoid research project.

Image 1: Cancer cells

TUMOUR ORGANOID CULTURE 

(1) LIMITATIONS

 

WHAT ARE THE LIMITATIONS OF THE CURRENT TUMOUR ORGANOID CULTURE SYSTEM?

 

The protocols of organoid construction vary widely[1]. Although each culture method demands to be appropriate for the research of tumour biology and therapeutic efficacy, the current culture techniques have not unified, which limits their preclinical application[1]. The limitations of the current organoid culture system include the limitations of tumour tissue sampling, the limitations of primary tissue processing methods and the limitations of the organoid culture environment[1].

TUMOUR ORGANOID CULTURE

(2) PROGRESS ON STANDARDISATION

 

WHAT PROGRESS HAS BEEN MADE ON TUMOUR ORGANOID CULTURE STANDARDISATION?

Currently, there is progress made in the culture standardisation of tumour organoids, like the standardisation of organoid source sample collection, organoid construction process, and culture environment including culture medium, organoid ECM and tumour microenvironment (TME) [1].

(A) ORGANOID SOURCE SAMPLE COLLECTION

  • Advances in the standardisation of organoid culture began with organoid-based studies aimed at identifying intra- and inter-tumour heterogeneity[1]. Tissue samples from multiple tumour sites or sub-regions will be able to establish more accurate tumour organoid models[1].
  • Zhou et al. from this review shared examples from different research groups regarding progress on tissue sampling from multiple tumour sites when establishing organoid model.
    • Kopper et al. established a multi-point organoid model by sampling the primary sites and multiple metastatic sites of an ovarian cancer patient to analyse genomics, transcriptomics, morphology, and pharmaceutical response of organoids[1].
    • Using the similar approach, Vlachogiannis et al. showed that colorectal and oesophagal cancer organoids mimic intra- and inter-tumoral heterogeneity in drug sensitivity[1].
  • The authors from this review also presented examples from Walsh et al. on how advanced procedures have broken technical bottlenecks regarding organoid source sample collection. Walsh et al. recovered organoids from cryopreserved breast cancer tissues[1]. Those organoids derived from quick-frozen tissues thawed after 6-12 months of storage had similar drug response profiles compared with organoids constructed from fresh samples of the same tissue origin[1].

(B) ORGANOID CONSTRUCTION PROCESS

Zhou et al. from this review shared examples from different research groups regarding progress on the organoid construction process:

  • Horowitz et al developed a microdissection procedure to generate millimetre-scale cuboid tissue sections which improves the uniformity of tissue cutting compared with traditional tissue section technique[1].
  • Brandenberg et al. invented a novel U-shaped microwell. In artificial microwells, automated imaging techniques showed a uniform increase in organoid size and morphology[1].
  • Li et al. showed that 3D air-liquid interface cultures could support primary organoid generation, carcinogenic transformation, and long-term in vitro culture of murine gastrointestinal tissue fragments[1].

(C) CULTURE ENVIRONMENT:

ORGANOID CULTURE MEDIUM

 

Tumour organoids should maintain reproducibility for clinical translation[1]. To meet this requirement, production and purification of growth factors should comply with to standardized platform[1]. The authors from this review presented examples from different research groups regarding progress on organoid culture medium standardisation.

  • Tüysüz et al. introduced phospholipid- and cholesterol-based liposomes to enhance the stability and activity of recombinant Wnt-3a[1].
  • Janda et al. developed water-soluble surrogate Wnt agonists by inviting Frizzled-LRP5/6f heterodimerization and activating downstream beta-catenin signalling pathways. They designed a ‘next-generation surrogate’ Wnt which could produce a similar level of downstream signalling at a concentration 50-fold lower than that of the previous generation[1].
  • Urbischek et al. developed a unique method to purify R-spondin 1 and Gremlin-1 in Escherichia coli [1].

(D) CULTURE ENVIRONMENT:

ORGANOID ECM

 

The present biomaterial platform provides favourable conditions for the three-dimensional culture of primary tumour tissue and extends the research of tumour biology and tumour therapy. Zhou et al. shared several examples from different research groups regarding progress on organoid ECM standardisation.

  • Xiao et al. demonstrated that adjustable matrix platforms facilitate organoid phenotyping and drug sensitivity analysis[1].
  • Gjorevski et al. described a PEG-based synthetic matrix for culturing purified Lgr5+ intestinal stem cells and showed that different mechanical environments and ECM components are required at different stages of the process[1].
  • Cruz-Acuña et al. also revealed that synthetic matrix was beneficial to intestinal organoids derived from human pluripotent stem cells[1].
  • Hernandez Gordillo et al. designed a similar synthetic ECM with tuneable biomolecular and biophysical properties to identify the gel components that support the primary human intestine[1].
  • Broguiere et al. pointed out that fibrin gels were suitable for both mouse and human epithelial organoids when supplemented with purified laminin[1].

(E) CULTURE ENVIRONMENT:

ORGANOID TUMOUR MICROENVIRONMENT (TME)

 

Different organoid culture methods can mimic the immune microenvironment of tumours in vitro, and currently include two main models[1]:

  • The reconstituted TME model
  • The holistic native TME model

THE RECONSTITUTED TME MODEL

  • Tumour tissue is separated into single cells or tiny clusters of cells using physical splicing or enzymatic hydrolysis[1].
  • Organoids were cultured in ECM[1].
  • Exogenous immune cells were isolated and co-cultured with the organoids[1].
  • Factors such as Wnt-3a, R-spondin, epidermal growth factor (EGF), Noggin, and bone morphogenetic (BMP) inhibitors, are commonly used to promote stem cell self-renewal and differentiation[1].
  • Different growth factors and pathway inhibitors need to be added according to the tissue type subsequently[1].

 

Cons:

Organoids cultured in this way are highly consistent with the original tumour tissue at both the genetic and pathological levels, so they can be used for in vitro disease modelling and drug screening and can functionally mimic the response of tumour patients to clinical treatment[1].

Pros:

This culture method of completely immersing organoids does not preserve stromal cells, so the exogenous addition of immune cells is required to build the TME[1].

THE HOLISTIC NATIVE TME MODEL

 

The holistic native TME model is a unit in which tumour epithelial cells are cultured with stromal endogenous immune cells as a whole, mainly including air-liquid interface (ALI) culture and microfluidic 3D culture[1].

Air-liquid interface (ALI) culture method:

  • To physically cut tumour tissue containing immune cells into tissue fragments and culture them in a transwell coated with collagen gel[1].
  • The top of the gel is exposed to air to allow the cells to receive an adequate supply of oxygen[1].
  • The medium in the outer dish diffuses into the inner dish to form an ALI[1].

 

Pros:

  • The ALI method preserves the basic genetic characteristics of the original tumour and also preserves the complex cellular composition and structure of TME[1].
  • In ALI organoid cultures, most tumours can grow in their native state, thus preserving a variety of endogenous immune cells, including T-cells, B cells, NK cells and macrophages[1].
  • ALI patient-derived organoid (PDO) cultures can accurately reflect histologic and other features of the original tumour within a short period (at least 30 days) [1].
  • ALI culture method provides an integrated strategy for immune TME modelling in vitro, which can explore the interaction between multiple different cell populations[1].

 

Besides the ALI organoid culture methods, Zhou et al also shared examples of the use of an organoids-on-a-chip system for co-culture of organoids with cells in TME including immune cells  and printing of 3D bio-scaffolds regarding microfluidic 3D culture[1].

FUTURE OPPORTUNITIES

(1) ORGANOID FORMATION

 

  • The development of precision medicine, or personalised medicine, will accelerate the process of organoid standardisation[1].
  • Accumulating techniques including cellular barcoding and machine learning-based imaging may facilitate quantitation monitoring organoid expansion at the cellular level and its clinical application[1].

(2) ORGANOID CULTURE MEDIUM

 

  • Continued development of next-generation organoid culture medium for organoid culture requires understanding individual tumour niches and modelling them with standard methods[1].
  • A decent description of tumour features before organoid culture encourages us to develop a suitable medium to better mimic tumour signalling and predict drug response in tumour organoids[1].
  • Emerging micromachining technologies (e.g. two-photon mode) enable us to visualize four-dimensional models of active growth factors[1].

(3) ORGANOID ECM

 

  • An important goal of the biomaterial community is to develop precision-engineered material platforms to explore minimum requirements for matching the biological output and efficiency of Engelbreth-Holm-Swarm (EHS) matrices[1].
  • Future of polymer and material engineering disciplines should address the limitation on biodegradability, reconfigurability, and frequency in incorporating ECM components and cell-interacting ligands, as well as the level of spatiotemporal control over biochemical and mechanical properties to mimic dynamic TMEs of engineered matrices[1].

References:

1. Zhou, Changchun et al. “Standardization of organoid culture in cancer research.” Cancer medicine vol. 12,13 (2023): 14375-14386. doi:10.1002/cam4.5943

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