Author: Liu Fang
Editor: HS
Using a chip to instantly turn your skin tissue into a blood vessel or nerve cell sounds too sci-fi. However, researchers at Indiana University and Ohio University not only made it a reality, but also open-sourced the chip-making technology. In other words, if you have the medical knowledge you need, you can make your favorite VIP tissue conversion chip in five to six days according to the instructions!
In the paper, published in Nature Protocols titled Fabrication and Use of Silicon Hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo, Indiana scientists elaborate on this non-invasive and harmless black technology.
Using electroporation, you can channel plasmid DNA directly into a specific depth of mouse (or yourself) skin in milliseconds, transfecting the skin into blood vessels or nerve cells.
Schematic diagram of nanotransfection | Source: Indiana University
According to the authors, the equipment required for this "tissue nanotransfection" (TNT) can be standardized for mass production. The biggest advantage of "tissue nanotransfection technology" is that there is no need to use the virus as a carrier, thus minimizing the risk of inflammatory response and cell death.
Chanda Sen, director of the Indiana Center for Regenerative Medicine and Engineering, who invented the technology, said: "This paper will allow more people to participate in regenerative medicine. ”
Prepare hollow microneedle arrays
According to the "Nanotransfection" manual, changing tissue function is divided into three steps (25 specific procedures): preparation of hollow microneedle arrays, preparation of TNT devices, and transfection of cells. The first thing to consider is which microneedle array to use, there is always a different form of microneedle that is right for you. For the introduction of plasmid DNA into the local skin, the authors recommend using a microneedle with a flat tip (see Figure 1A). For the introduction of deep tissue, the authors recommend using microneedles with sharp tips to facilitate tissue penetration (see Figure 1B or 1C).
Various microneedle | Source: Papers
After choosing the style, we will have to make it by hand.
A layer of photoresist is first coated with a layer of photoresist on a double-sided polished 4-inch silicon (Si100) wafer, followed by a deep reactive ion etching (DRIE) of the wafer using the Bosch process. Three different silicon hollow microneedles can be prepared depending on the transfection needs, including a flattened type I at the apex (Figure 1E, Type I), a protruding Type II at the apex, or type III with eccentric pores (Figure 1F, g). The latter two sharp-tipped microneedle arrays are more helpful in penetrating into tissues.
If the diameter of the nanochannel of the needle is reduced, the speed at which the needle outputs biomolecules will increase accordingly; however, this may also limit the total number of molecules passing through the needle. In nanochannels the diameter is too small (
In addition, the researchers connected a Transwell on each silicon chip to serve as a repository for plasmid DNA.
The paper emphasizes that in order to make a high-quality microneedle array, special attention must be paid to two aspects. The first is to be precisely aligned when flipping the chip to make the nanochannels of the microneedles (step 1A (Xix)), and the second is to be gentle during the fabrication process to avoid clogging in the pinholes (steps 1A (xxii, xxvii and xxiv)).
Nanotransfection introduces the short film | Source: Indiana University
Preparation of TNT devices
After the chip is made, it needs to be integrated with the electroporation device to achieve the introduction of plasmid DNA. First, a reservoir is mounted on the TNT chip to store the solution with plasmid DNA. In order to achieve nano-perforation, a precisely controlled electrical pulse generator is also required. The researchers mixed PDMS with curing agents to form a PDMS film that could be embedded in the chip. After that, the front, bottom and bottom surfaces of the PDMS membrane are treated with an equal particle washer and then gently pressed together to form a complete bond between the layers. The thickness of the PDMS film should be adjusted to 2-4 mm.
The paper shows that PDMS membranes larger than 4 mm are not flexible enough to provide a complete leak-proof connection between the chips, and can also damage the chips during the connection process. PDMS membranes smaller than 2 mm are too soft, creating folds and air gaps between the chip and the film.
Mouse experiments | Source: Papers
In vivo transfection using TNT devices
When electroporation is used to introduce DNA into the body, the voltage applied determines how much migration is present on the DNA molecules, and thus how far it is introduced under a constant pulse. At the same time, the voltage also determines the pore density and average pore size on the cell membrane during electroporation. Medium voltage (100-150 V/mm) balances the introduction distance and porosity parameters. Excessively high voltages can damage cell and tissue structures. In the experiment, the researchers used male mice that were 8 to 12 weeks old. However, this does not mean that live mouse experiments are limited to any gender or age, and any mouse can participate.
In order to facilitate the TNT chip's access to deeper layers of cellular tissue in the skin, the mice in the experiment were dehaired. Electrical impulses then reach the skin through a reservoir filled with plasmid solution and microneedles. The electrical pulse is square wave and the parameters are adjustable. Starting at a voltage of 200 V, the perforation pulse voltage can be continuously increased to facilitate the formation of pores in the cell membrane, followed by a drive pulse of 200 V to introduce the plasmid into the tissue. Higher voltages (>250 V) increase the drive force and create larger pores in the cell membrane, which facilitates the delivery of plasmids. However, if the voltage is too large, it will cause toxicity to the cells.
The ultimate goal of the TNT chip with silicon-hollow microneedle arrays is to reprogram tissue, the authors said. With the help of the chip, the mice avoided tissue necrosis of the hind limbs, and even the nerve cells transplanted into the brain improved the nerve function of the stroke mice.
Currently, scientists have not conducted large-scale clinical trials in humans. However, after the method of preparing nanochips is open sourced, it is bound to attract the interest of a large number of biological hackers.
We will see what magical discoveries will be made at that time.
Source: Academic Headlines