3D Bioprinting of Brown Adipose Tissue

The vascularized tissue that I would like to 3D bioprint is brown adipose tissue. Brown adipose tissue is a fatty tissue that is used in the body to generate heat from energy stored in the form of lipids (5). Brown adipose tissue is primarily found in human babies, hibernating animals, and human adults that are in need of an extra source of heat. In most adults however, a majority of the brown adipose tissue that was prevalent as a child is lost and no longer used, this is shown to the left in Figure 1. This being said, brown adipose tissue is generally more important in children as it is used for several reasons. In babes, brown adipose tissue is used to create heat that is needed for the body. This is due to Figure 1 : Brown adipose tissue in babies and adults (6) the fact that babies have more

heat loss through the head due to the head to body ratio, they are unable to use other methods to keep warm like adults, and their bodies are not able to quickly respond to cold (5). As for adults, it has been found that there are many other benefits to having brown adipose tissue that do not only involve the creation of heat (5). One area that has been heavily researched is the usage of brown adipose tissue to aid in fat loss (6). This application of brown adipose tissue makes sense, because the energy that is used up by brown adipose tissue is stored in the form of lipids. This means that the brown adipose tissue can essentially use up excess fat and create heat from it. This is a very attractive feature for scientists and companies because there is a large fat loss market.

The purpose of creating brown adipose tissue by 3D bioprinting is to create a method of fat loss that is biological and safe for adult humans. This means that brown adipose tissue will have to be 3D bioprinted and then inserted into the body, near especially fatty areas (6). This will allow for that brown adipose tissue to consume the fat around it, heating up the body in the process. Not only would this be useful for people trying to lose fat, it could also be useful for people that live in or travel to especially cold climates.

The design of the 3D printed brown adipose tissue is especially important compared to other tissues because brown adipose tissue contains more capillaries than most tissues (3). This is due to the fact that a large amount of blood is needed in order to supply the tissue with the correct nutrients, bring waste out of the tissue, and supply the rest of the body with the heat that is generated. This means that one of the key design considerations is that the tissue has to be highly vascularized. In order to achieve this design, there are two possible ways to create vascularization. One method is to just create pores in the 3D design, and the other is to create blood vessels throughout the design by printing a sacrificial bioink where vessels should be. Either way this would allow for there to be optimal distances between the pores or vessels, which would allow there to be enough exchange of nutrients, waste, and heat. In previous 3D bioprint studies of brown adipose tissue, it was found that the cell viability increased as the 3D print was more solid (1). This means that it was found that 3D bioprints with many pores were actually less viable than 3D bioprints with fewer pores. This is surprising, however, since data suggests this, I would attempt this strategy. One way to limit the complexity of the design is to create a thin 3D bioprint, so that less vascularization is needed. This could be a good idea to think about because then the bioprint would not be as complicated and it would potentially make less of an impact when implanting into the body. Since creating a thin bioprint will not only make vascularization easier, but also make it easier to implant into the body, the brown adipose tissue that will be 3D bioprinted will have this feature. The shapes of the print in the other directions is not all that important however. The normal anatomical shapes of adult brown adipose tissue are circles or ovals, which can be seen in Figure 1. This being said, the best shape option for a 3D bioprinted brown adipose tissue is a thin oval or sphere, which is the most anatomically correct shape for most adults.

The actual design of the bioprint for brown adipose tissue would feature blood vessels, such as capillaries, that would allow for enough vascularization. The capillaries would be in a structure such that there is no brown adipose tissue that is further than 200 micrometers away from any capillary (7). This will allow there to be adequate vascularization for the entire structure. The design would also be flat, preferably less than 1 centimeter thick (7). The reasoning behind this is to make it easier to incorporate the vascularization because the thicker the design is, the more complex the vascularization would need to be. Another reason is so that the implant into the body would be easier. Since the 3D print would not be filling an area, but rather being placed subcutaneously, it needs to be thin in order for the print to fit into the body without creating a rather large bulge. This thin 3D structure would also be similar to actual adult brown adipose tissue, as it is usually very thin and there is not very much of is present.

The next important design consideration is the method of how the brown adipose tissue will be 3D printed. It is important to consider each of the types of 3D printers and the advantages or disadvantages of using each of them. The six main methods of 3D bioprinting are using droplet, extrusion, laser-based, bio-paper-based, 2-photon polymerization, and acoustophoretic printers. The most widely adopted printers are droplet, extrusion, and laser-based printers, so a good place to start is with these three options.

Droplet 3D printers are usually either thermal or piezoelectric. Laser printers actually shine a laser at the hydrogels to cause them to crosslink. Since brown adipose tissues can be rather sensitive, it is possible that using a droplet of laser printer could kill some of the cells that are being used. This is important to consider because the best print will have the best viability. Also, droplet printers do not make solid lines of material, but rather many individual droplets. This could make it difficult for vascularization to be incorporated, as the hydrogels and cells that are printed are usually not going to be solids.

Extrusion 3D printers are the most widely used 3D bioprinters. There are several different methods in which these printers extrude, including using pneumatics, pistons, and screws. All of these methods of extrusion are easier on the material that is being printed than droplet based printers because there is no need for heat and electricity to be used. This means that using an extrusion printer will not cause as much damage to cells. Extrusion printers are not perfect though, cells can become damaged if the size of the tip is not large enough. This is because cells need to squeeze through the tip, and if the cells are too large, shear forces will rupture the cells. Careful consideration must be made when deciding upon an extrusion tip, as there is a delicate balance between having a tip large enough for the cells being used and having a tip small enough for the desired resolution of the print. The best choice for printing brown adipose tissue is the extrusion printer.

Another important feature of the printer is that it needs to have multiple printing tips. This is key in order for the print to involve more than one type of bioink. For the brown adipose tissue, there will need to be two different printing tips. The first tip will contain the cells and the hydrogel that will make up the brown adipose tissue. The second tip will contain a sacrificial bioink that will be used to create vasculature throughout the entire structure. The print will have to be done layer by layer, incorporating vessels created by the sacrificial bioink throughout.

There are many types of materials and hydrogels that are important to research before using to 3D bioprint. Some materials and hydrogels that can be used sacrificially include glass fibers, polymer fibers, gelatin, and pluronic (8). For creating a 3D bioprint of brown adipose tissue, pluronic is a great choice to create the vasculature that is needed (4). Pluronic is great to use for extrusion printing, so this is a good choice to use with the extrusion printer. Pluronic is also a good choice for a sacrificial bioink because it undergoes reverse gelation, which means that it crosslinks with increasing temperatures. On the other hand, if the pluronic is cooled down, it will no longer be crosslinked. This is the method that will be used for printing the brown adipose tissue. The pluronic will be printed in the form of vasculature and after the entire print, the brown adipose tissue will be cooled down, which will allow for the pluronic to turn to liquid and evacuate the rest of the print.

It is also important to consider the type of hydrogel that the brown adipose cells will be in. For this print, it has been found that the best prints have a combination of hyaluronic acid and gelatin (1). Gelatin promotes cell adhesion, differentiation, migration, and proliferation, so it can be extremely beneficial to the cells that it contains. Hyaluronic acid has great biocompatibility and helps in embryogenesis. The combination of these two hydrogels allows for there to be maximal benefits to the cells that will be involved. Both gelatin and hyaluronic acid can be used in an extrusion 3D printer, so this will also work with the strategy that was planned earlier.

One important consideration involving both of these hydrogels, is that they need to be crosslinked after the print. Crosslinking allows for the bioprint to have a more solid 3D structure rather than the liquid that is printed. Crosslinking can be done in many methods, but the common one is photo-crosslinking. This method will be used in creating the brown adipose tissue, as it is quick and simple to shine light on the print either while it is printing or shortly after. The duration the light is shone on the print determines the extent of crosslinking of the hydrogels. In previous studies, it has been found that the more crosslinking there is, the stiffer the 3D print will be and the better the cell viability is (1). This means that the crosslinking should be done for a time around 90 seconds, which would allow for maximal crosslinking and therefore cell viability. Gelatin and hyaluronic acid are the best hydrogels to use for 3D bioprinting brown adipose tissue.

Brown adipose tissue is made up of both white adipose tissue and brown adipose tissue. White adipose tissue is fat deposits that have large lipid droplets within the cell. This tissue makes up the majority of all fat within the human body. Brown adipose tissue is brown due to the large amount of mitochondria in the cells and the smaller lipid droplets. This tissue makes up a much smaller amount of fat in the human body. These tissues are what make up brown adipose tissue, meaning that brown adipose tissue is not only brown adipose tissue, but also white adipose tissue. This means that the cells that make up both of these tissues are necessary to create this 3D bioprint. Both brown and white adipose tissue cells come from adipocytes (2). In order to create many

Figure 2 : Process of 3D Bioprinting Brown Adipose Tissue (2)

healthy cells, it is important to grow these adipocytes in a cell culture. After this, the adipocyte cells can then be differentiated in order that they can become brown and white adipose cells (2). Once the brown and white adipose cells are obtained, they can be added to the hydrogels that have already been prepared. This series of events is shown in Figure 2 above. First the stem cells or adipocytes are obtained, but in this case only adipocytes are needed. Next for this 3D print, the cells go into the mixture of gelatin and hyaluronic acid. There is no need for the cells to be mixed into the pluronic because it is being used as a sacrificial bioink and it will all be removed shortly after the print to create the vasculature. The next step, as shown by Figure 2 is to do the 3D bioprint. After completion, the transplantation can be completed and the 3D bioprint should begin to function within the body.

The placement of the 3D bioprint of brown adipose tissue in the body is important once the print is completed. Since brown adipose tissue essentially uses up fat that is stored within the body, it should be placed in areas of high fat content in the body. The specific area where the brown adipose tissue would be placed could depend person to person. For example, if someone has more fat near their hips, then the brown adipose tissue should be placed within that area. The goal of the brown adipose tissue is to help with fat loss, so if the brown adipose tissue is successful, there should be fat loss in and around areas where the tissue was implanted.

It is possible in this case to measure the function of the brown adipose tissue by simply looking for fat loss in the areas where the tissue was implanted. This may be true, however it is important to have a more exact method of measuring the function of the brown adipose tissue. One of the most common ways to assess the function of tissues or cells is to do a live dead cell assay. This method uses two different types of dyes, one that dyes the live cells green and one that dyes the dead cells red. The green live cell dye is membrane permeable and non-fluorescent until ester groups are removed and make the molecule fluorescent. The red dead cell dye is not membrane permeable and binds to DNA with high affinity (9). This means that it is able to mark cells that have a compromised cell membrane, which are dead. In order to check the viability of the brown adipose tissue cells that were printed, the print can be soaked in a mixture of the green and the red dyes for 10 minutes (9). After the cells are done soaking, they can be observed using fluorescence microscopy. This allows for both the live cells and the dead cells to be easily seen. If the cells are mostly green, the print was likely successful and the viability and function of the cells is good.

Many options have been considered for the 3D bioprinting of brown adipose tissue. The method and details of one possible way to print brown adipose tissue have been discussed above. In summary, the goal of this paper was to identify a possible method of 3D bioprinting brown adipose tissue, with the purpose of using the tissue as a possible fat and weight loss solution. The bioprinting method that will be used is an extrusion printer with two different printing tips, which will allow for the printer to print more than one bioink at a time. Extrusion was the chosen printer type due to the safety pertaining to the cells and the ease of use. The two cell types that are needed to make brown adipose tissue are brown and white adipose tissue. Both of these types of cells are found in biological brown adipose tissue, so it makes sense to use both in this situation (1). These cells can be printed within hydrogels in order to print them. A mixture of hyaluronic acid and gelatin creates a great environment for the cells to be in, and it allows for the 3D bioprint to be printed and then cross linked afterwords using light. A sacrificial bioink, pluronic, is also key in the design of the brown adipose tissue, as it functions to create the vasculature needed for the cells to survive and thrive. With all of these factors that have been considered, it is likely that this method will create a viable brown adipose tissue.

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