Microorganospheres
Next-generation organoids
Maxim Chubik, PCR.news
Not so long ago, researchers had the opportunity to study not only two-dimensional cell cultures, but also three-dimensional tissue structures, or organoids. After the appearance of the first organoid, there was rapid progress in the artificial cultivation of organoids, including with the help of microfluidic technologies.
A group of scientists from the USA, Germany and the Netherlands proposed using drip microfluidics to obtain organoids — microorganospheres (MOS) from cell lines or biopsies of patients. MOS can be used to study the mechanisms of immune therapy, interactions in the pathogen-host system, as well as for high-performance screening of potential drugs.
MOS are miniature 3D models of tissues encapsulated in small volume droplets. For the production of MOS, the authors developed a microfluidic device in which the required number of cells are mixed with commercial reagents: Cultrex BME or Matrigel (a natural protein extract obtained from the secretions of mouse sarcoma cells). The device includes a cooling module that prevents the mixture from solidifying during the formation of MOS, and a heating module to accelerate solidification at the final stage.
In addition, scientists have developed a new method of demulsification using a membrane made of hydrophobic polyvinylidene fluoride, which allows you to quickly and efficiently isolate ready-made MOS from the emulsion. In a study using induced pluripotent stem cells (iPSC) as a cellular component, the authors demonstrated the high viability of MOS obtained by the new method.
The authors created a number of MOS cultures from both pathological tissues (colorectal cancer cell line) and samples of normal human organ tissues (colon and duodenum, human embryonic liver).
All the MOS obtained had morphology and key histopathological characteristics similar to traditional organoids obtained from similar organs. At the same time, the small size and large surface-to-volume ratio of MOS allow for more efficient and uniform diffusion of growth factors, nutrients and oxygen to cells and gives MOS an advantage both in growth rate and in the degree of viability. This is evidenced by the larger sizes of organoids and a smaller number of necrotized MOS compared to traditional organoids.
Scientists have demonstrated that MOS can be created from many different tissues. They retain histopathological morphology, the ability to differentiate and genetic expression, just like ordinary organoids. For example, when cultivating human duodenal MOS, organoids retained the ability to differentiate goblet-shaped and neuroendocrine cells. MOS can be frozen and cultured, just like traditional organoids.
MOS is a promising platform for studying the nuances of immune therapy. Thus, the researchers observed the effective penetration of activated T cells into the MOS and the destruction of tumor cells in them. Such a degree of infiltration could not be obtained using conventional organ models. The authors believe that the method of fast and inexpensive creation of an accurate tumor model from a limited amount of biopsy tissue makes it possible to study the sensitivity of tumors to therapy.
A team of scientists developed MOS from the tissues of the human respiratory and digestive systems and infected them with SARS-COV-2, after which they conducted drug screening. Unlike conventional organoids, MOS can be directly infected with viruses without removing and suspending cells from the surrounding framework of Cultrex BME or Matrigel.
Traditional methods of mass production of organoids require a significant amount of manual labor — they are difficult to scale and automate for high-performance screening. The microfluidic device designed by the researchers makes it possible to obtain about 35 thousand microorganospheres in one technological cycle from 500 µl of a mixture of cells with a commercial reagent. Using an automated dosing system, the researchers placed the resulting MOS in 1,750 wells with a density of 20 MOS per well in less than 30 minutes.
The authors proposed to integrate an image analysis system developed on the basis of deep machine learning technology into an automated pipeline for creating MOS. The algorithm of the analytical visualization system is able to distinguish cytotoxic and cytostatic effects of drugs, identify drug-resistant tumor clones, distinguish tumor MOS from microorganospheres containing normal tissues. According to the researchers, MOS technology can be used in a variety of areas of clinical medicine.
Article by Wang et al. Rapid tissue prototyping with micro-organospheres is published in the journal Stem Cell Reports.
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