As an emerging technology, bio-3D printing is increasingly favored by biotechnology experts and so on. Its printer theory is usually to build complex solid biological tissues by stacking layer by layer. This technology is widely used in biomedical engineering and regenerative medicine due to its advantages such as flexible design and personalized customization, and has a very broad application prospect.
When it comes to bioprinting, similar to everyday printing, it is also necessary to print "ink", but the ink here is derived from active cells and biomaterials. We know that the survival of cells requires a certain microenvironment, so the "bioink" stored here needs to be designed to facilitate the efficient preservation of cells during bioprinting and storage.
With the development of new materials and micro-nano technologies, the design of such components has become more flexible and diverse, so that cells can survive in more extreme and vicious environments, one of the most striking technologies is low-temperature bioprinting technology.
Professor Y. Shrike Zhang of Harvard Medical School focuses on innovative biomedical engineering techniques, including bio-3D printing, organ chips, microfluidics, bioanalysis, and more to reconstruct functionalized tissues and their biomimetic models. His research group has published more than 260 papers in science, Nat Rev Mater, Nat Commun, PNAS, Matter, Adv Mater and other journals.
Recently, Professor Y. Shrike Zhang's team reported on Mater an unconventional bioprinting technique called cryo-bioprinting, which utilizes bioprinting and cryopreservation methods in synergy, and by using a new printing device and bioink, printed biological tissue can be stored for a longer period of time and thawed/crosslinked as needed and cultured and used after recovery. The development of the technology promises to expand bioprinting applications, such as storable biological tissue and tissue models, so that they can be shipped to the desired location at any time. The research is titled "Freeform cell-laden cryobioprinting for shelf-ready tissue fabrication and storage".
Figure | Related Papers (Source: Matter)
Biology presents challenges in the production and storage of 3D printed organizational structures. On the one hand, most bioprinting technologies, such as the widely used extrusion bioprinting, have the disadvantage of a more complex printing process, so this manufacturing technology is difficult to achieve in many environments. On the other hand, due to the lack of functional design for long-term storage of cells, pre-printed biological tissue products cannot achieve high spot rates.
Combining advanced biomanufacturing techniques and storage methods such as bioprinting and cryopreservation is a potential solution to these barriers. As a result, bioprinted tissue can be prepared off-site in advance, allowing direct storage at low temperatures for later laboratory or clinical use. But one of the challenges of this approach is to address the fact that the formation and recrystallization of ice crystals can negatively affect the cellular activity of the prepared tissue.
To address this challenge, bioink design is important to efficiently preserve cells during bioprinting and cryo-storage/resuscitation. Studies have shown that cryoprotectants (CPAs) play a key role in eliminating or reducing ice crystal formation. In the absence of CPA, ice crystals grow in large numbers and rapidly, destroying cell membranes and jeopardizing the viability of cells.
Dimethyl sulfoxide (DMSO) is a traditional CPA that is widely used for cryopreservation to reduce the formation of ice crystals. In addition, CPA with a large molecular weight protects cells from osmotic damage caused by extracellular hypertonicity. Such as a variety of disaccharides such as sucrose, trehalose, lactose, and maltose, as well as trisaccharides such as maltose and raffinose, etc., have shown good low temperature protection.
In addition, soft materials, especially the hydrogel materials used in this article, constitute a 3D network structure that can further reduce the formation of ice crystals, thereby providing cells with an inherent physical cryogenic protection microenvironment, that is, cell-loaded hydrogel scaffolds containing CPA provide a good cryopreservation environment, resulting in higher cell viability and cell function after resurrection.
The design emphasized in the article is a method of simultaneous biological 3D printing and cryopreservation cell-bearing tissue construction. In simple terms, by designing a cryolate that allows precise temperature control during the printing process, bio tissues can be printed using bioink containing CPA and living cells under low temperature controllable conditions. Specifically, in the following figure, in the device, the cell-carrying bioink is printed on a frozen plate with a strictly controlled temperature, and stored for a long time under low temperature conditions. Prior to subsequent cultures, cryogenic bioprinted structures can be transported to where needed for resuscitation and crosslinking, and subsequent use.
Figure | Schematic diagram of frozen bioprinting for simultaneous cryopreservation of tissue structures (Source: Matter)
Figure | Simulation and actual structure of sample frozen bioprinting construction (Source: Matter)
Subsequently, to further validate the technique, the team used the functionality of mesenchymal stem cells after osteogenesis, lipidization, and cartilage differentiation to investigate the survival, proliferation, and differentiation capabilities of post-print biological tissues. Interestingly, this design proves that frozen bioprinted cell vectors can effectively guarantee cell viability and functionality. In addition, the study also verified that cryogenic bioprinted tissues maintain their ability to induce angiogenesis, suggesting that such designs do not cause damage to cells or growth factors.
An important highlight of the study is the design of a cryo-plate for the construction of low-temperature conditions, which is a key component to ensure the cell activity microenvironment, and at the same time, the equipment will also preserve cells, ensure the function of bioprinting tissue and provide effectiveness for surface cells, opening up a new innovative path for bone tissue engineering or cell tissue modeling.
Based on this research, one can imagine that cells can survive in this microenvironment and can print specific biological tissues and organs through this design, and ensure the proliferation and differentiation of tissue cells, this innovative design can be said to be a blessing for any hospital, any patient, because it can be tailored for patients with an active cell carrier of tissues and organs, and can be prepared and stored in advance for emergency needs, which is of great help to improve patient survival and tissue repair Of course, there is also a great improvement in the transshipability and spot rate of the in vitro tissue model.
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reference:
Ravanbakhsh et al., Freeform cell-laden cryobioprinting for shelf-ready tissue fabrication and storage,Matter(2021), https://doi.org/10.1016/j.matt.2021.11.020