tissue. these cells provide an ex-vivo model of cell behavior without any genetic, epigenetic, or developmental changes; making them a closer replication of in-vivo conditions than cells derived from other methods. this constraint however, can also make studying them difficult. these are mature cells, often terminally differentiated, meaning that for many cell types proliferation is difficult or impossible. additionally, the microenvironments these cells exist in are highly specialized, often making replication of these conditions difficult. secondary cells a portion of cells from a primary culture is moved to a new repository/vessel to continue being cultured. medium from the primary culture is removed, the cells that are desired to be transferred are obtained, and then cultured in a new vessel with fresh growth medium. a secondary cell culture is useful in order to ensure that cells have both the room and nutrients that they require to grow. secondary cultures are most notably used in any scenario in which a larger quantity of cells than can be found in the primary culture is desired. secondary cells share the constraints of primary cells (see above) but have an added risk of contamination when transferring to a new vessel. genetic classifications of cells autologous: the donor and the recipient of the cells are the same individual. cells are harvested, cultured or stored, and then reintroduced to the host. as a result of the host's own cells being reintroduced, an antigenic response is not elicited. the body's immune system recognizes these re-implanted cells as its own, and does not target them for attack. autologous cell dependence on host cell health and donor site morbidity may be deterrents to their use. adipose-derived and bone marrow-derived mesenchymal stem cells are commonly autologous in nature, and can be used in a myriad of ways, from helping repair skeletal tissue to replenishing beta cells in diabetic patients. allogenic: cells are obtained from the body of a donor of the same species as the recipient. while there are some ethical constraints to the use of human cells for in vitro studies (i.e. human brain tissue chimera development), the employment of dermal fibroblasts from human foreskin demonstrates an immunologically safe and thus a viable choice for allogenic tissue engineering of the skin. xenogenic: these cells are derived isolated cells from alternate species from the recipient. a notable example of xenogeneic tissue utilization is cardiovascular implant construction via animal cells. chimeric human-animal farming raises ethical concerns around the potential for improved consciousness from implanting human organs in animals. syngeneic or isogenic: these cells describe those borne from identical genetic code. this imparts an immunologic benefit similar to autologous cell lines (see above). autologous cells can be considered syngenic, but the classification also extends to non-autologously derived cells such as those from an identical twin, from genetically identical (cloned) research models, or induced stem cells (isc) as related to the donor. stem cells stem cells are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. stem cells are divided into "adult" and "embryonic" stem cells according to their source. while there is still a large ethical debate related to the use of embryonic stem cells, it is thought that another alternative source – induced pluripotent stem cells – may be useful for the repair of diseased or damaged tissues, or may be used to grow new organs. totipotent cells are stem cells which can divide into further stem cells or differentiate into any cell type in the body, including extra-embryonic tissue. pluripotent cells are stem cells which can differentiate into any cell type in the body except extra-embryonic tissue. induced pluripotent stem cells (ipscs) are subclass of pluripotent stem cells resembling embryonic stem cells (escs) that have been derived from adult differentiated cells. ipscs are created by altering the expression of transcriptional factors in adult cells until they become like embryonic stem cells. multipotent stem cells can be differentiated into any cell within the same class, such as blood or bone. a common example of multipotent cells is mesenchymal stem cells (mscs). scaffolds scaffolds are materials that have been engineered to cause desirable cellular interactions to contribute to the formation of new functional tissues for medical purposes. cells are often 'seeded' into these structures capable of supporting three-dimensional tissue formation. scaffolds mimic the extracellular matrix of the native tissue, recapitulating the in vivo milieu and allowing cells to influence their own microenvironments. they usually serve at least one of the following purposes: allowing cell attachment and migration, delivering and retaining cells and biochemical factors, enabling diffusion of vital cell nutrients and expressed products, and exerting certain mechanical and biological influences to modify the behaviour of the cell phase. in 2009, an interdisciplinary team led by the thoracic surgeon thorsten walles implanted the first bioartificial transplant that provides an innate vascular network for post-transplant graft supply successfully into a patient awaiting tracheal reconstruction. this animation of a rotating carbon nanotube shows its 3d structure. carbon nanotubes are among the numerous candidates for tissue engineering scaffolds since they are biocompatible, resistant to biodegradation and can be functionalized with biomolecules. however, the possibility of toxicity with non-biodegradable nano-materials is not fully understood. to achieve the goal of tissue reconstruction, scaffolds must meet some specific requirements. high porosity and adequate pore size are necessary to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients. biodegradability is often an essential factor since scaffolds should preferably be absorbed by the surrounding tissues without the necessity of surgical removal. the rate at which degradation occurs has to coincide as much as possible with the rate of tissue formation: this means that while cells are fabricating their own natural matrix structure around themselves, the scaffold is able to provide structural integrity within the body and eventually it will break down leaving the newly formed tissue which will take over the mechanical load. injectability is also important for clinical uses. recent research on organ printing is showing how crucial a good control of the 3d environment is to ensure reproducibility of experiments and offer better results. materials material selection is an essential aspect of producing a scaffold. the materials utilized can be natural or synthetic and can be biodegradable or non-biodegradable. additionally, they must be biocompatible, meaning that they don't cause any adverse effects to cells. silicone, for example, is a synthetic, non-biodegradable material commonly used as a drug delivery material, while gelatin is a biodegradable, natural material commonly used in cell-culture scaffolds the material needed for each application is different, and dependent on the desired mechanical properties of the material. tissue engineering of long bone defects for example, will require a rigid scaffold with a compressive strength similar to that of cortical bone (100-150 mpa), which is much higher compared to a scaffold for skin regeneration. there are a few versatile synthetic materials used for many different scaffold applications. one of these commonly used materials is polylactic acid (pla), a synthetic polymer. pla – polylactic acid. this is a polyester which degrades within the human body to form lactic acid, a naturally occurring chemical which is easily removed from the body. similar materials are polyglycolic acid (pga) and polycaprolactone (pcl): their degradation mechanism is similar to that of pla, but pcl degrades slower and pga degrades faster. pla is commonly combined with pga to create poly-lactic-co-glycolic acid (plga). this is especially useful because the degradation of plga can be tailored by altering the weight percentages of pla and pga: more pla – slower degradation, more pga – faster degradation. this tunability, along with its biocompatibility, makes it an extremely useful material for scaffold creation. scaffolds may also be constructed from natural materials: in particular different derivatives of the extracellular matrix have been studied to evaluate their ability to support cell growth. protein based materials – such as collagen, or fibrin, and polysaccharidic materials- like chitosan or glycosaminoglycans (gags), have all proved suitable in terms of cell compatibility. among gags, hyaluronic acid, possibly in combination with cross linking agents (e.g. glutaraldehyde, water-soluble carbodiimide, etc.), is one of the possible choices as scaffold material. due to the covalent attachment of thiol groups to these polymers, they can crosslink via disulfide bond formation. the use of thiolated polymers (thiomers) as scaffold material for tissue engineering was initially introduced at the 4th central european symposium on pharmaceutical technology in vienna 2001. as thiomers are biocompatible, exhibit cellular mimicking properties and efficiently support proliferation and differentiation of various cell types, they are extensively used as scaffolds for tissue engineering. furthermore thiomers such as thiolated hyaluronic acid and thiolated chitosan were shown to exhibit wound healing properties and are subject of numerous clinical trials. additionally, a fragment of an extracellular matrix protein, such as the rgd peptide, can be coupled to a non-bioactive material to promote cell attachment. another form of scaffold is decellularized tissue. this is a process where chemicals are used to extracts cells from tissues, leaving just the extracellular matrix. this has the benefit of a fully formed matrix specific to the desired tissue type. however, the decellurised scaffold may present immune problems with future introduced cells. synthesis tissue engineered vascular graft tissue engineered heart valve a number of different methods have been described in the literature for preparing porous structures to be employed as tissue engineering scaffolds. each of these techniques presents its own advantages, but none are free of drawbacks. nanofiber self-assembly molecular self-assembly is one of the few methods for creating biomaterials with properties similar in scale and chemistry to that of the natural in vivo extracellular matrix (ecm), a crucial step toward tissue engineering of complex tissues. moreover, these hydrogel scaffolds have shown superiority in in vivo toxicology and biocompatibility compared to traditional macro-scaffolds and animal-derived materials. textile technologies these techniques include all the approaches that have been successfully employed for the preparation of non-woven meshes of different polymers. in particular, non-woven polyglycolide structures have been tested for tissue engineering applications: such fibrous structures have been found useful to grow different types of cells. the principal drawbacks are related to the difficulties in obtaining high porosity and regular pore size. solvent casting and particulate leaching solvent casting and particulate leaching (scpl) allows for the preparation of structures with regular porosity, but with limited thickness. first, the polymer is dissolved into a suitable organic solvent (e.g. polylactic acid could be dissolved into dichloromethane), then the solution is cast into a mold filled with porogen particles. such porogen can be an inorganic salt like sodium chloride, crystals of saccharose, gelatin spheres or paraffin spheres. the size of the porogen particles will affect the size of the scaffold pores, while the polymer to porogen ratio is directly correlated to the amount of porosity of the final structure. after the polymer solution has been cast the solvent is allowed to fully evaporate, then the composite structure in the mold is immersed in a bath of a liquid suitable for dissolving the porogen: water in the case of sodium chloride, saccharose and gelatin or an aliphatic solvent like hexane for use with paraffin. once the porogen has been fully dissolved, a porous structure is obtained. other than the small thickness range that can be obtained, another drawback of scpl lies in its use of organic solvents which must be fully removed to avoid any possible damage to the cells seeded on the scaffold. gas foaming to overcome the need to use organic solvents and solid porogens, a technique using gas as a porogen has been developed. first, disc-shaped structures made of the desired polymer are prepared by means of compression molding using a heated mold. the discs are then placed in a chamber where they are exposed to high pressure co2 for several days. the pressure inside the chamber is gradually restored to atmospheric levels. during this procedure the pores are formed by the carbon dioxide molecules that abandon the polymer, resulting in a sponge-like structure. the main problems resulting from such a technique are caused by the excessive heat used during compression molding (which prohibits the incorporation of any temperature labile material into the polymer matrix) and by the fact that the pores do not form an interconnected structure. emulsification freeze-drying this technique does not require the use