Antrobots

Micro-robots from human cells started regeneration of nervous tissue in a test tube

Oleg Lischuk, N+1

American researchers have created spheroid mobile microrobots from unmodified living human cells. In an in vitro experiment with their help, it was possible to start rapid healing of nervous tissue. The work is available as a preprint on the bioRxiv resource (Gumuskaya et al., Motile Living Biobots Self-Construct from Adult Human Somatic Progenitor Seed Cells).

The idea of using living cells to create biorobots is very attractive, since such structural components have a ready-made set of organelles, signaling pathways and receptors that allow them to respond to biochemical, biomechanical and bioelectric stimuli, as well as generate and exchange them. Often, when creating such structures, cells are combined with inanimate components, for example, polymer substrates, wire and plastic frames.

The staff of the laboratory of Michael Levin from Tufts University decided to choose the path of synthetic biology — cooperating with colleagues from other scientific centers in the USA, they have been creating organoid biobots for several years. In 2020, researchers presented an evolutionary algorithm for the development of such structures and created with its help millimeter biorobots from embryonic cells of a smooth spur frog (Xenopus laevis), called xenobots. During the year, they were improved and taught to move around the site, turn in mazes, remember the experience of meeting with blue light and clear the site of garbage, raking it into heaps, as well as successfully recover from injuries.

Now the same scientific group with the participation of Simon Garnier from the New Jersey Institute of Technology has moved on to experiments on human tissues. As a starting material, the researchers used the atrial fibrillation epithelium of the trachea obtained from sputum samples of patients. The reason for this choice was that his cells have movable cilia (in the body they serve to clean the lungs, promoting bronchial secretions outward).

Isolated progenitor epithelial cells were cultured in a gel extracellular matrix with differentiation factors. After 14 days, monoclonal multicellular spheroid organoids were formed from each cell with cilia of cells facing inward, as in the respiratory tract. They were extracted from the matrix and placed in a liquid low-adhesive nutrient medium. Perceiving it as a tracheobronchial secret, the cells turned outward with cilia within seven days (apico-basal translocation occurred), forming spheroidal organoids ranging in size from 30 to 500 micrometers, many of which were mobile. Their lifetime ranged from 45 to 60 days. The authors called them antrobots (from the ancient Greek ἄνθρωπος — "man").

Approximately 200 randomly selected mobile organoids were filmed in groups of four or five in a time lapse for five hours, fixing the coordinates of the movements. The recording was divided into 30-second intervals for greater detail and the clustering algorithm was launched without a teacher according to the indices of straightness and rounding of movement. He identified four separate clusters among the antrobots according to the type of movement: rectilinear, curved, circular and eclectic.

After that, the immunocytochemical method determined the shape of about 350 organoids and subjected it to a cloud PCA analysis according to eight parameters: proportions, maximum radius, smoothness of shape, homogeneity and polarity of the distribution of cilia, the proportion of the surface covered by them and the number of point cilia moving independently of the cluster ("noise points"). According to the results, three morphotypes were revealed among the antrobots: the first was the smallest, smoothly spherical, densely and evenly covered with cilia; the second was the largest and shapeless with a less dense and uniform distribution of cilia; the third was intermediate in shape with the most polarized ciliated cover.

Then the parameters of movement patterns were added to the PCA cloud to reveal their relationship with morphological characteristics. 62 percent of motionless (including oscillating in one place) organoids fully represented the first morphotype, the remaining 38 percent fell into the second. Accordingly, all the mobile ones were found among the second and third morphotypes, with about 85 percent of antrobots with rectilinear movement detected in the second, and 88 percent with circular movement in the third. Thus, a clear correlation was shown between morphology and type of movements.

To assess the ability of antrobots to move in living tissue, the researchers grew a tissue sample consisting of adjacent two-dimensional layers of neurons from human induced neural stem cells. A "microtrauma" was cut through it and organoids were placed at a distance of several diameters from it. Moving arbitrarily, the antrobots "examined" the damage after a while, and those who moved moderately curvilinearly at a speed of at least 10 micrometers per second achieved the greatest success.

After that, the most suitable organoids were placed in a tight enough space to achieve their self—aggregation into larger clusters - "superbots". They were placed on different edges of the damaged area of the grown nerve tissue so that they formed a bridge between them. After 72 hours without additional effects, a significant growth of the original tissue was observed under this bridge, "stitching" the edges of the damage. In other places, the wound remained intact. Quantitative analysis showed that the neural density of the "crosslinking" is indistinguishable from the rest of the tissue.

Thus, genetically unmodified, immune neutral, non-self-reproducing and biodegradable for a limited time organoids started regeneration of nervous tissue after damage. According to the authors of the work, promising areas of their application include the delivery of regenerative molecules to the site of damage, targeted destruction of malignant cells in the intestine, the creation of bioengineered tissues for transplantation and personalized drug testing.

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