25 March 2008

The bioprinter created a functioning piece of heart muscle

Membrane, illustrations by Forgacs et al.

There has been a breakthrough in the technology of printing human organs. Biologists are already studying the first samples of printed living tissues and spatial structures constructed from them, which are still quite simple, in laboratories. There is still a lot of work ahead. But, according to scientists, the first "marketable" fabrics from the printer will appear on the market in the very next few years.

A few years ago it was shown that printing biological tissues is not a fantasy. However, more than one year will pass from early experiments to the mass application of such technology in medicine. It seems to be a simple principle: the build-up of cellular tissue layer by layer using a printer that resembles an ordinary device. But the main thing here is to think through all the subtleties of technology, to identify its pitfalls.

This is what Professor Gabor Forgacs and his Forgacslab laboratory are doing as part of the Organ Printing project.

Forgach and his colleagues from the University of Missouri (University of Missouri-Columbia) created functional blood vessels and pieces of heart tissue using their promising method of organ printing, which they wrote an article about in the journal Tissue Engineering.

By the way, we briefly talked about the first successful experiments of Forgach and the method of printing living tissues developed by him. To put it simply, the University of Missouri experiments use a three-dimensional bioprinter (built by order of scientists by nScrypt), fueled with live "ink". He follows the commands of the computer and builds the necessary "structure" layer by layer.

I must say that there are different ways of cultivating tissues for the same purposes (as an implant), in particular, heart tissues. However, in all of them, in order to grow even a simple heart patch, it is first necessary to create a "mounting frame" that would set the shape of the future organ or transplant.

The advantage of the new method is that such a basis is not required at all – the shape of the vessel, a piece of liver or heart muscle is set by the printer itself. But any "framework" for cells that got into the body as part of an implant is a potential initiator of inflammation, Gabor notes.

We have already said that the experimenter does not print individual cells (as other scientists working in the same field try), but conglomerates numbering tens of thousands of cells. How does it turn out that they form the necessary tissue in composition and structure?

Here are the details of the technology.

Схема печати органа 

General scheme of printing of the organ "by Forgach"
First, a special device cuts a pre-cultured tissue (which is not, however, an organ) or, more precisely, a dense cell suspension into microscopic cylinders with a diameter-length ratio of 1:1 (a).
Then these cylinders are rounded in the nutrient medium, forming microspheres – "biochernila". One drop of them is shown in the photo. Its diameter is 500 micrometers. Orange color is given to it by a special dye injected into the cell membranes (b).
The printer cartridge (c) contains micropipettes filled with such microspheres one after another.
A three-dimensional printer (d) can take turns producing these balls (scientists also call them "spheroids") with micron precision.
Micropipettes and the area of operation of the print head can be observed by researchers in real time using cameras built into the printer (e).
The device prints three "colors" at once. Two of them are spheroids with target cells (in Forgach's recent experiments, these were heart muscle cells and epithelial cells), and the third is a bonding gel containing collagen, growth factor and a number of other substances. The future organ needs it to maintain its shape until the moment when the target cells fuse together. It is important that the gel is printed together with the "spare part", in the form of sequentially applied two-millimeter layers, into which microspheres with cells of different types (f) are immersed.

Нанесённые вперемешку с гелем сфероиды постепенно объединяются в нужную ткань

Spheroids mixed with gel (with thousands of cells each) are gradually combined into the desired tissue, and the gel is removed

"We will never be able to fully print the liver, with all its details," says Gabor, "but this is not required. If you can initiate the process, nature will do everything for you." In other words, the Forgach method does not involve printing completely ready-made organs that are no different from those that work in the human body, but creating live blanks that are very close to organs. Blanks, the refinement of which will be taken over by the laws of developmental biology.

The authors of the experiments say that what happens in the printed piece of tissue is identical to the processes going on in the embryo at the early stages of organ development. Specialized cells, following internal "instructions", combine into exactly the system that is expected of them.

According to Nature (How to print out a blood vessel, 03.20.2008), when printing with endothelial cells mixed with heart cells, the Forgach group received a piece of workable muscle in which all the cells united into a single system 70 hours after printing and began to contract synchronously after 90 hours. At the same time, the endothelial cells were collected into some tubes resembling capillaries.

Примеры напечатанных на принтере тканей

Examples of Gabor-printed fabrics and biological "spare parts":
(a) ringed "biochernil" particles (fluorescent due to the dye in two colors) immediately after printing and after 60 hours;
(b) evolution of the tube made up of the rings shown in the picture (a);
(c, above) a 12-layer tube made up of cells of smooth muscle fibers of the umbilical cord;
(c, bottom) branched tube (prototype of vessels for transplantation);
(d) construction of contracting cardiac tissue. On the left is a grid (6x6) of spheroids with heart muscle cells (without endothelium) printed on collagen "bio-paper".
If endothelial cells are added to the same "ink" (the second picture is red, cardiomyocytes are shown here in green), they first fill the space between the spheroids, and after 70 hours (d, right) the entire tissue becomes a single whole. Below: a graph of the reduction of cells of the resulting tissue. As you can see, the amplitude (measured vertically) of the contractions is about 2 microns, and the period is about two seconds (the time is marked horizontally).

Similarly, scientists printed just individual small vessels. In the process of "recruiting" their walls, collagen gel (otherwise – "bio-paper") was fed not only to the edges, but also to the middle of the vessel. After the cells were joined into the tissue, the core was easily removed, leaving a passage for blood flow. In this way, the Forgach team can already create branching vessels to order, of any desired shape.

Now researchers are working on a way to build muscle on such tubes to make them (printed vessels) strong enough to be stitched with real vessels during surgery.

At the same time, the group is working on vessels that are particularly difficult to manufacture, with a diameter of less than 6 millimeters. The fact is that for larger vessels there have long been successful synthetic substitutes used as transplants. But it is not yet possible to create good small vessels from bare synthetics, and even more so – capillaries. Therefore, their cultivation would be the real way out.

Структура напечатанных на принтере тканей сердца

The structure of the heart tissues obtained by scientists Forgach and his colleagues created the company Organovo, also based in Missouri, which will develop the technology and bring it to market.

And now they say: within a few years, the first product of the company should be on sale. These will be simple tissue fragments intended for toxicological tests (for example, printed pieces of human liver). Such samples could replace laboratory animals.

Printed grafts should also appear a little later. First it will be blood vessels.

Well, then it will be possible to get a little closer to printing more complex organs to order. For example, the developers of this technology say that they will be one of the first such "spare parts" to start printing human kidneys. Interestingly, these organs probably won't look like kidneys outwardly, the experimenters report, but they should work no worse in the body.

And let the turn of organs, arranged much more complicated and working not so simply, come later, the first harbingers of these man-made, but at the same time living transplants have already been created. So the efficiency of the method, at least at its core, can be considered proven.

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