Illness and old age can reduce blood flow in the body so much that tissue is damaged by the lack of oxygen and nutrients, causing wounds and severe pain. An entirely new technology enables microscopic tissue spheroids made from a mixture of blood vessel cells and stem cells to be injected into the body, and then the cells organize themselves into new branched blood vessels that connect to the existing ones. The new technology has been tested on mice, and the next step is to carry out trials involving people. The researchers think that this technology can help people with poor blood circulation and ischemia and may also provide the opportunity to create other artificial organs that can be injected into the body – avoiding the need for surgery.
The dream of being able to create artificial organs and tissue is far from new. Researchers hope that tiny, self-organized three-dimensional tissue cultures derived from stem cells (organoids) will be used both to replace damaged organs inside the body and to test drugs outside the body.
However, the challenge has often been to get stem cells to develop into the right organ or tissue. So far, attempts have been made to force the stem cells to progress in the right direction by using growth factors and other expensive biochemical signals.
Now researchers have developed a new and surprising solution to this problem, giving the cells a natural three-dimensional cluster with neighbouring cells and structures and then letting the cells do the work themselves.
“Instead of bombarding the cells with growth factors to force them to develop in a certain direction, we allow the cells to aggregate into small spheroids and even organize themselves in relation to each other. The result is tiny microtissues 0.1 millimetres across with an outer cell layer that protects a core with a primitive blood vessel. Then we can inject these organoids into the body, where the blood vessels in the cores connect to each other in a new network of blood vessels,” explains Ninna Struck Rossen, Assistant Professor, Biotech Research & Innovation Centre (BRIC), University of Copenhagen, first author of the study that was carried out at Columbia University in New York and published in Cell’s iScience.
Using this approach, the researchers succeeded in creating new blood vessels and linking them to the existing ones in both healthy and ischemic mice, the latter having decreased blood supply and ischemia in one hindlimb.
"The technology saves the ischemic hindlimbs of the mice and enables the muscles to regenerate. This technology may also be used to create and inject other types of organs and tissue, avoiding the need for surgery."
Left the cells alone
Attempts to model healthy tissue have been going on for decades. Stem cells are usually differentiated into mature tissue using various types of growth factors, but the problem has been to produce tissue from stem cells under sufficient control to avoid the risk of the stem cells developing into the wrong types of tissue or, worse, becoming uncontrollable.
The idea for the new method is based on observations from and the further development of other methods of culturing tissue and cells.
“We were actually in the process of making skin tissue by placing cells in layers and applying growth factors. One major challenge of this method was that the cells contracted the tissue into a cluster. Although the clusters could not be used as skin tissue, we were interested in how the cells were organizing and developing, so we continued to cultivate some of the failed clusters, both with and without growth factors. We examined the cell clusters that had not received growth factors and found that the cells inside the clusters had organized themselves to form blood vessels in the centre of the clusters,” says Ninna Struck Rossen.
Another major problem with creating artificial organs and tissue has been producing enough cells or tissues. Previous techniques such as spinner culture or hanging drop culture are difficult to scale up or harsh to cells. The solution here was an innovative modification of an otherwise simple and fairly traditional method – microwell inserts in well plates for cell culture.
“We grow these cellular spheroids in culture plates with 1000 microwells in each, so we know exactly what and how much we have in each well. However, instead of the microwells being made of the usual plastic, they are made of alginate – a polymer extracted from algae that provides surfaces to which cells cannot adhere. This material can be easily dissolved, leaving us with 1000 well-defined uniform cellular spheroids, each with a tiny blood vessel at the core that is ready to be injected,” explains Ninna Struck Rossen.
Restoring blood flow
Robots can seed the culture plates, which can be stacked in layers in cell incubators and filled with growth medium, so that the researchers can mass-produce uniform microtissue clusters. Once they are fully cultivated, the plates are dissolved and the microtissues are ready for use.
“To test whether the blood vessel cores could form a new vascular network, we injected our microtissues into mice that had an ischemic hindlimb. Ischemia occurs in diseases with impaired blood supply, for example as a result of calcification or blood clots in veins. Ischemia can be mimicked in a disease model in mice, in which we ligate and excise the main artery in a mouse’s hindlimb. We then treated the mouse by injecting our microtissues with the endothelial cores into the muscle of the hindlimb. The most amazing thing is that the cells self-organize so that the new blood vessels form automatically. Cells seem to know best what they are and how to organize themselves to form functional tissue,” says Ninna Struck Rossen.
The new self-organizing system appears to enable small building blocks of relatively complex tissue to be delivered. In any case, in mice, new blood vessels were established that could assemble into vascular networks and connect with the existing blood vessels and restore blood flow – not just to replace blood flow of the excised main artery but also to enable ischemic tissue to be rapidly regenerated and recreate new muscle tissue in the hindlimb.
“Our next step is to have people test the system. If we can make it work, this may prove to be a very important tool for treating people with peripheral artery disease and critical ischemia, in which the blood vessels in the limbs of people with diabetes, for example, are slowly being destroyed. We also hope to be able to use the technology in completely different tissues, by making injectable organoids such as lung, liver and pancreatic tissues. Last but not least, we hope that others can benefit from the method by making large amounts of microtissues of different types that, for example, could be used to test drugs without laboratory animals and to treat people with diseases in which tissues or organs need to be restored,” says Ninna Struck Rossen.
“Injectable therapeutic organoids using sacrificial hydrogels” has been published in Cell’s iScience. In 2015, the Novo Nordisk Foundation awarded a Visiting Scholar Fellowship at Stanford Bio-X to Ninna Struck Rossen, Biotech Research & Innovation Centre (BRIC), University of Copenhagen.