of a solid porogen like scpl. first, a synthetic polymer is dissolved into a suitable solvent (e.g. polylactic acid in dichloromethane) then water is added to the polymeric solution and the two liquids are mixed in order to obtain an emulsion. before the two phases can separate, the emulsion is cast into a mold and quickly frozen by means of immersion into liquid nitrogen. the frozen emulsion is subsequently freeze-dried to remove the dispersed water and the solvent, thus leaving a solidified, porous polymeric structure. while emulsification and freeze-drying allow for a faster preparation when compared to scpl (since it does not require a time-consuming leaching step), it still requires the use of solvents. moreover, pore size is relatively small and porosity is often irregular. freeze-drying by itself is also a commonly employed technique for the fabrication of scaffolds. in particular, it is used to prepare collagen sponges: collagen is dissolved into acidic solutions of acetic acid or hydrochloric acid that are cast into a mold, frozen with liquid nitrogen and then lyophilized. thermally induced phase separation similar to the previous technique, the tips phase separation procedure requires the use of a solvent with a low melting point that is easy to sublime. for example, dioxane could be used to dissolve polylactic acid, then phase separation is induced through the addition of a small quantity of water: a polymer-rich and a polymer-poor phase are formed. following cooling below the solvent melting point and some days of vacuum-drying to sublime the solvent, a porous scaffold is obtained. liquid-liquid phase separation presents the same drawbacks of emulsification/freeze-drying. electrospinning electrospinning is a highly versatile technique that can be used to produce continuous fibers ranging in diameter from a few microns to a few nanometers. in a typical electrospinning set-up, the desired scaffold material is dissolved within a solvent and placed within a syringe. this solution is fed through a needle and a high voltage is applied to the tip and to a conductive collection surface. the buildup of electrostatic forces within the solution causes it to eject a thin fibrous stream towards the oppositely charged or grounded collection surface. during this process the solvent evaporates, leaving solid fibers leaving a highly porous network. this technique is highly tunable, with variation to solvent, voltage, working distance (distance from the needle to collection surface), flow rate of solution, solute concentration, and collection surface. this allows for precise control of fiber morphology. on a commercial level however, due to scalability reasons, there are 40 or sometimes 96 needles involved operating at once. the bottle-necks in such set-ups are: 1) maintaining the aforementioned variables uniformly for all of the needles and 2) formation of "beads" in single fibers that we as engineers, want to be of a uniform diameter. by modifying variables such as the distance to collector, magnitude of applied voltage, or solution flow rate – researchers can dramatically change the overall scaffold architecture. historically, research on electrospun fibrous scaffolds dates back to at least the late 1980s when simon showed that electrospinning could be used to produced nano- and submicron-scale fibrous scaffolds from polymer solutions specifically intended for use as in vitro cell and tissue substrates. this early use of electrospun lattices for cell culture and tissue engineering showed that various cell types would adhere to and proliferate upon polycarbonate fibers. it was noted that as opposed to the flattened morphology typically seen in 2d culture, cells grown on the electrospun fibers exhibited a more rounded 3-dimensional morphology generally observed of tissues in vivo. cad/cam technologies because most of the above techniques are limited when it comes to the control of porosity and pore size, computer assisted design and manufacturing techniques have been introduced to tissue engineering. first, a three-dimensional structure is designed using cad software. the porosity can be tailored using algorithms within the software. the scaffold is then realized by using ink-jet printing of polymer powders or through fused deposition modeling of a polymer melt. a 2011 study by el-ayoubi et al. investigated "3d-plotting technique to produce (biocompatible and biodegradable) poly-l-lactide macroporous scaffolds with two different pore sizes" via solid free-form fabrication (ssf) with computer-aided-design (cad), to explore therapeutic articular cartilage replacement as an "alternative to conventional tissue repair". the study found the smaller the pore size paired with mechanical stress in a bioreactor (to induce in vivo-like conditions), the higher the cell viability in potential therapeutic functionality via decreasing recovery time and increasing transplant effectiveness. laser-assisted bioprinting in a 2012 study, koch et al. focused on whether laser-assisted bioprinting (labp) can be used to build multicellular 3d patterns in natural matrix, and whether the generated constructs are functioning and forming tissue. labp arranges small volumes of living cell suspensions in set high-resolution patterns. the investigation was successful, the researchers foresee that "generated tissue constructs might be used for in vivo testing by implanting them into animal models" (14). as of this study, only human skin tissue has been synthesized, though researchers project that "by integrating further cell types (e.g. melanocytes, schwann cells, hair follicle cells) into the printed cell construct, the behavior of these cells in a 3d in vitro microenvironment similar to their natural one can be analyzed", which is useful for drug discovery and toxicology studies. self-assembled recombinant spider silk nanomembranes gustafsson et al. demonstrated free‐standing, bioactive membranes of cm-sized area, but only 250 nm thin, that were formed by self‐assembly of spider silk at the interface of an aqueous solution. the membranes uniquely combine nanoscale thickness, biodegradability, ultrahigh strain and strength, permeability to proteins and promote rapid cell adherence and proliferation. they demonstrated growing a coherent layer of keratinocytes. these spider silk nanomembranes have also been used to create a static in-vitro model of a blood vessel. assembly methods a persistent problem within tissue engineering is mass transport limitations. engineered tissues generally lack an initial blood supply, thus making it difficult for any implanted cells to obtain sufficient oxygen and nutrients to survive, or function properly. self-assembly self-assembly methods have been shown to be promising methods for tissue engineering. self-assembly methods have the advantage of allowing tissues to develop their own extracellular matrix, resulting in tissue that better recapitulates biochemical and biomechanical properties of native tissue. self-assembling engineered articular cartilage was introduced by jerry hu and kyriacos a. athanasiou in 2006 and applications of the process have resulted in engineered cartilage approaching the strength of native tissue. self-assembly is a prime technology to get cells grown in a lab to assemble into three-dimensional shapes. to break down tissues into cells, researchers first have to dissolve the extracellular matrix that normally binds them together. once cells are isolated, they must form the complex structures that make up our natural tissues. liquid-based template assembly the air-liquid surface established by faraday waves is explored as a template to assemble biological entities for bottom-up tissue engineering. this liquid-based template can be dynamically reconfigured in a few seconds, and the assembly on the template can be achieved in a scalable and parallel manner. assembly of microscale hydrogels, cells, neuron-seeded micro-carrier beads, cell spheroids into various symmetrical and periodic structures was demonstrated with good cell viability. formation of 3-d neural network was achieved after 14-day tissue culture. additive manufacturing main article: organ printing it might be possible to print organs, or possibly entire organisms using additive manufacturing techniques. a recent innovative method of construction uses an ink-jet mechanism to print precise layers of cells in a matrix of thermo-reversible gel. endothelial cells, the cells that line blood vessels, have been printed in a set of stacked rings. when incubated, these fused into a tube. this technique has been referred to as "bioprinting" within the field as it involves the printing of biological components in a structure resembling the organ of focus. the field of three-dimensional and highly accurate models of biological systems is pioneered by multiple projects and technologies including a rapid method for creating tissues and even whole organs involve a 3-d printer that can bio-print the scaffolding and cells layer by layer into a working tissue sample or organ. the device is presented in a ted talk by dr. anthony atala, m.d. the director of the wake forest institute for regenerative medicine, and the w.h. boyce professor and chair of the department of urology at wake forest university, in which a kidney is printed on stage during the seminar and then presented to the crowd. it is anticipated that this technology will enable the production of livers in the future for transplantation and theoretically for toxicology and other biological studies as well. recently multi-photon processing (mpp) was employed for in vivo experiments by engineering artificial cartilage constructs. an ex vivo histological examination showed that certain pore geometry and the pre-growing of chondrocytes (cho) prior to implantation significantly improves the performance of the created 3-d scaffolds. the achieved biocompatibility was comparable to the commercially available collagen membranes. the successful outcome of this study supports the idea that hexagonal-pore-shaped hybrid organic-inorganic micro-structured scaffolds in combination with cho seeding may be successfully implemented for cartilage tissue engineering. scaffolding in 2013, using a 3-d scaffolding of matrigel in various configurations, substantial pancreatic organoids was produced in vitro. clusters of small numbers of cells proliferated into 40,000 cells within one week. the clusters transform into cells that make either digestive enzymes or hormones like insulin, self-organizing into branched pancreatic organoids that resemble the pancreas. the cells are sensitive to the environment, such as gel stiffness and contact with other cells. individual cells do not thrive; a minimum of four proximate cells was required for subsequent organoid development. modifications to the medium composition produced either hollow spheres mainly composed of pancreatic progenitors, or complex organoids that spontaneously undergo pancreatic morphogenesis and differentiation. maintenance and expansion of pancreatic progenitors require active notch and fgf signaling, recapitulating in vivo niche signaling interactions. the organoids were seen as potentially offering mini-organs for drug testing and for spare insulin-producing cells. aside from matrigel 3-d scaffolds, other collagen gel systems have been developed. collagen/hyaluronic acid scaffolds have been used for modeling the mammary gland in vitro while co-coculturing epithelial and adipocyte cells. the hystem kit is another 3-d platform containing ecm components and hyaluronic acid that has been used for cancer research. additionally, hydrogel constituents can be chemically modified to assist in crosslinking and enhance their mechanical properties. tissue culture in many cases, creation of functional tissues and biological structures in vitro requires extensive culturing to promote survival, growth and inducement of functionality. in general, the basic requirements of cells must be maintained in culture, which include oxygen, ph, humidity, temperature, nutrients and osmotic pressure maintenance. tissue engineered cultures also present additional problems in maintaining culture conditions. in standard cell culture, diffusion is often the sole means of nutrient and metabolite transport. however, as a culture becomes larger and more complex, such as the case with engineered organs and whole tissues, other mechanisms must be employed to maintain the culture, such as the creation of capillary networks within the tissue. bioreactor for cultivation of vascular grafts another issue with tissue culture is introducing the proper factors or stimuli required to induce functionality. in many cases, simple maintenance culture is not sufficient. growth factors, hormones, specific metabolites or nutrients, chemical and physical stimuli are sometimes required. for example, certain cells respond to changes in oxygen tension as part of their normal development, such as chondrocytes, which must adapt to low oxygen conditions or hypoxia during skeletal development. others, such as endothelial cells, respond to shear stress from fluid flow, which is encountered in blood vessels. mechanical stimuli, such as pressure pulses seem to be beneficial to all kind of cardiovascular tissue such as heart valves, blood vessels or pericardium. bioreactors in tissue engineering, a bioreactor is a device that attempts to simulate a physiological environment in order to promote cell or tissue growth in vitro. a physiological environment can consist of many different parameters such as temperature, pressure, oxygen or carbon dioxide concentration, or osmolality of fluid environment, and it can extend to all kinds of biological, chemical or mechanical stimuli. therefore, there are systems that may include the application of forces such as electromagnetic forces, mechanical pressures, or fluid pressures to the tissue. these systems can be two- or three-dimensional setups. bioreactors