3D culture of human organoids - A new tool for human disease research


3D culture of human organoids - A new tool for human disease research
Currently, 2D culture systems have been widely used in cancer research, but 2D culture systems have many limitations and can't be a good model for clinical trials. Because 3D culture can simulate the microenvironments of tissues or organs in the body, direct cell-cell interaction, gene/protein expression mimic to the in vivo expression, observe the drug response of clinical drugs, and explore the cellular oxygen, nutrient and cytokine messaging. Therefore, 3D cell culture may allow a better understanding of cancer biology and facilitate research on biomarkers and targeted therapies.

3D Culture Patterns
Depending on the cell source, the cells can be distinguish into different kinds of 3D structured cells, either from organ-specific adult stem cells or multifunctional stem cells (e.g., iPSCs, ESCs) and developed into organoids by adding different growth factors (e.g., BMP, EGF, FGF, etc.). For general cancer disease research, cells can be formed into 3D structures (spheroids) using Ultra-Low Attachment (ULA), hydrogel, scaffold, etc.

ABclonal provides cytokine proteins for organoid research

Establish 3D culture structure
How should we prepare 3D structures? There are three main types, scaffold-free, scaffold-base and hybrid.
◆Scaffold-free: The cells can be formed into spheroids by using special culture plates (Ultra-Low Adherent Plate), hanging drop culture, and spinner flask bioreactor.
◆Scaffold-base: Natural materials (collagen, gelatin) or synthetic materials (polymer) are used to form a hydrogel, which allows cells to be inoculated into gel, thus forming a spherical structure.
◆Hybrid: 3D printing, organotypic raft culture, and organ-on-a-chip are used to create 3D culture models.

TOOLS provides Scaffold-base 3D culture reagent: TOOLS 3D Culture Plus Kit (#TTL-3D48)

Product Features:
  • Scaffold-base and realistic simulation of in vivo growth pattern
  • Stable structure without crushing cytoskeleton and organelles
  • Rapid gelation of the substrates with stable quality and convenient operation
  • Use special lysis reagent which does not affect cell activity and east collects complete spheroids

HCT116 cells were cultured in TOOLS 3D Culture Plus Kit. After different time observations, the cells were able to form a complete spheroid.
Cancer Research
To investigate the severe combined immune deficiency (SCID) caused by RAG1 and RAG2 gene defects, Cameron L. Gardner's team used iPSCs knocking-in a promoterless RAG2 cDNA and built an artificial thymic organoid (ATO) to maintain RAG2 gene expression. CRISPR technology has been shown to restore RAG2-deficient cells to normal levels (1). In addition, a transfection reagent can be used to transfect specific genes into 3D culture and observe the protein expression by fluorescence microscopy.
U87MG spheroid observed microscope showed that spheroid has high transfection efficiency by using  using TOOLSFect transfection reagent (#TTM-TF01).

  • No need to change cell culture medium before or after transfection
  • With specially formulated working solution no need to prepare extra serum-free medium for preparation
  • High cell survival rate after transfection
  • High successful transfection rate proven by various cell lines

Proteomics research
The lumen structure of many epithelial tissues is disrupted by the Apical-basal cell polarity in cancer diseases, Li-Ting Wang's team used Caco2 cells to build a 3D lumen formation and screened PARD3B, RALB and HRNR, which can act as new regulators of lumen formation (2). In addition to the common cancer research, Thayna Mendonca Avelino's team, in order to understand the metabolic imbalance caused by obesity and related mechanisms, could also use adipocytes (3T3-L1) cultured in 3D and analyze spheres and mouse adipose tissue by LC-MS/MS, the 3D culture shared 83% of proteins with animal tissues and discovered the pathways involved in obesity The 3D culture was found to share 83% of the proteins with the animal tissues and found that the pathways involved in obesity are adipogenesis and fatty acid metabolism, so the 3D culture can serve as a good model (3).



BIOTOOLS provides sophisticated Mass Spectrometry services and offers complete proteomics analysis services according to customer's experimental needs. (A) From sample extraction, protein identification and bioinformatics analysis. We also provide Customized drafting service.

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  1. Cameron L Gardner et al. Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System. J Clin Immunol. 2021 Jul;41(5):852-862.
  2. Li-Ting Wang et al. A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation. Science Report. 2021; 11, 22807.
  3. Thayna Mendonca Avelino et al. Mass spectrometry-based proteomics of 3D cell culture: A useful tool to validate culture of spheroids and organoids. SLAS Discovery. 2021; Volume 27, Issue 3, 167 - 174
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