In this manuscript authors wants to discuss about in-vitro meat production systems and about its pros and cons. It is a concept in which edible animal tissues can be produced by the culturing through tissue engineering techniques. These techniques offer health and environmental advantages over existing meat production systems. Though, the production of highly-structured, unprocessed meats faces considerably greater technical challenges in respect to its safety and quality. Through the use of various techniques like Scaffold-based techniques and self-organizing techniques we can produce edible tissues. The draw back of this system is its structural integrity as the whole animal tissues and its safety for human consumption.
The term in vitro meat is generally applied for cultured meat. The technique of producing edible animal muscle (i.e., meat) in vitro is known as tissue engineering techniques. Most edible animal meat is made of skeletal muscle tissue. Thus the production of cultured meat in vitro must draw upon techniques developed for skeletal muscle tissue engineering (Kosnik et al., 2003). Invitro or cultured meat is an animal product produced that has been never part of a complete living animal. Now it is possible to grow many cells types of both animals and plant origin without the organisms of which it was derived is called invitro culturing. Probably the first research on invitro meat was performed by M.A. benzamine from Tauro-college. The first edible form was produced by the NSR/Tauro Applied Bioscience Research Consortium in year 2000. In this concern, Gold fish cell grown to resemble fish fillet. Modern research on invitro meat arouse of experiments conducted by NASA- attempting to find improved form of long term food for astronauts. The technique adopted for its production was approved by US Food and Drug Administration (FDA) in 1995 and NASA is conducting research on this topic since 2001.
Necessity of invitro meat production:
Techniques Adopted for in-vitro Meat Production
In vitro meat production is a process of using muscle cells and applying a protein on the cells to grow into large portions of meat. Once the initial cells have been achieved, additional animals would not be required – akin to the production of yogurt cultures. There are two techniques adopted for invitro-meat production i.e. scaffold-based and self-organizing techniques:
In scaffold-based techniques, embryonic myoblasts or mature skeletal muscle satellite cells are proliferated, attach to a scaffold or carrier, such as a collagen meshwork or microcarrier beads, and then perfuse with a culture medium in a stationary or rotating bioreactor. By introducing a variety of environmental cues, these cells fused into myotubes, which can then differentiate into myofibers. The resulting myofibers may then harvested, cooked, and consumed as meat. Van Eelen, Van Kooten, and Westerhof embrace a Dutch patent for this general approach to producing cultured meat (Van Eelen et al., 1999). However, Catts and Zurr appear to have been the first to have actually produced meat using the method (Catts and Zurr, 2002). A scaffold-based technique may be appropriate for producing processed meats, such as hamburger or sausage. But it is not suitable for producing highly structured meats, such as steaks. To produce these, one would need a more ambitious approach, creating structured muscle tissue as self-organizing constructs (Dennis and Kosnik, 2000) or proliferating existing muscle tissue in vitro. The detailed procedure for Invitro-meat production is given as under:
Culturing of Muscle Cell
It is possible to grow or culture muscle fibre invitro however, the problem of its proliferation may occur. As an alternate satellite cells can be cultured. Typically neonate individuals are selected for the isolation of Myo-satellite cells because these cells are much more abundant in the muscle of the young animals than the older animals. The capacity of differentiation into variety of cells is more in young than older ones. Most of the cells are taken from still born pig foetus. There are always some dead ones. The cells are taken from the semitendinosus muscle of the pig hamstring. After its freezing in liquid nitrogen it can be utilized for years together. Its isolation requires the mincing of complete muscle followed by enzymatic treatment or separation of the satellite cells by differential centrifuge. Preplating, precell gradients or combination of these on removal of the growth factor from the culture medium these myoblasts fuse from myofibers. Growth factors contain hormones, growth may have basic amino acids, glucose, minerals, serum harvested from animals usually calves and artificial serum has been produced from the limp muscle.
Co-culturing Muscle Cell
Myoblasts cell are specialized to produce contractile proteins but produce only little extracellular matrix and as such other cells likely need to be introduced to engineer muscle. Fibroblasts residing in the muscle are mainly responsible for the production of extracellular matrix which could be beneficial to add to the culture system (Brady et al., 2008). However, due to the difference in growth rate, co-culturing involves the risk of fibroblasts overgrowing the myoblasts. Meat also contains fat and a vasculature and possibly, co-culture with fat cells should also be considered (Edelman et al., 2005). The problem of vascularisation is a general issue in tissue engineering and currently we can only produce thin tissues because of passive diffusion limitations. To overcome the tissue thickness limit of 100 to 200 um, a vasculature needs to be created (Jain et al., 2005).
There are two subsequent stages involved in the dying of the old cell culture. First one is senescence-in which cell normally die and second crisis-which occurs when cells for some reason have survival senescence. Senescence can be overcome by start fresh cell culture when needed, immobilize cell culture and by the use of an embryonic stem cell.
Perfusion of Growing Muscle Tissue
Actual growth of muscle tissue in culture is problematic because of the absence of blood circulation. It has been approved that it is possible to grow small muscle like organs termed as myoids denovo from co-culture of myoblast and fibroblast. These organs are able to contract both spontaneously and by electrical stimulation. An electrode ensures an electrical current about 1 Hz passing through the cells to make these skeletal muscle cells develop into muscle. They need to be constantly exercised just like into body.
This technique was employed by Benjaminson, Gilchriest and Lorenz (Benjaminson et al.,2002). They placed skeletal muscle explants from goldfish (Carassius auratus) in diverse culture media for seven days and observed an increase in surface area between 5.2 and 13.8 percent. When the explants were placed in a culture containing dissociated Carassius skeletal muscle cells, explant surface area grew by 79 percent. Explants have the advantage of containing all the cells that make up muscle in their corresponding proportions, thus closely mimicking an in vivo structure. However, lack of blood circulation in these explants makes substantial growth impossible, as cells become necrotic if separated for long periods by more than 0.5 mm from a nutrient supply (Dennis and Kosnik, 2000). Thus without vascularization, the production of large, highly structured meats will not be possible. Future efforts in culturing meat will have to be the limitations of modern techniques through advances that make cultured cells, scaffolds, culture media, and growth factors both edible and affordable.
Requisites for in-vitro Meat Production
Cells are the basic necessity for invitro meat production. As a cell skeletal muscle is best suited tissue because it is consisting of several cell types. The fibers of skeletal muscles are formed by the proliferation, differentiation and fusion of embryonic myoblasts. In the postnatal animal, satellite cells are capable to form large multinucleated syncytia (McFarland et al., 1999). Attempts to force skeletal muscle fibers to proliferate are typically counterproductive, as most myonuclei remain postmitotic (Kosnik et al., 2003). Although embryonic stem cells are characterized by high proliferation and differentiation potential, considerable effort must be applied to force them to differentiate and cell yields from harvests are low. Thus the most practical cell source for cultured meat is probably embryonic myoblasts or postnatal/post hatch skeletal muscle cells called satellite cells. Satellite cells with high proliferative potential have been isolated and characterized from the skeletal muscle of chickens, turkeys, pigs, lambs and cattle (McFarland et al., 1991, 1997; Blanton et al., 1999 and Dodson et al., 1986; 1987).
To enable skeletal muscle tissue engineering, an appropriate cell source is necessary that is able to sustain proliferation to produce large numbers of cells, but retains the ability to differentiate into skeletal muscle when appropriately stimulated. As adult skeletal muscle is post-mitotic, we set out to find a precursor cell source in adult pig and mice muscle and induce myogenesis in these cells. To this end, several multipotent muscle progenitor cell populations were isolated from muscle biopsies using different methods. In order to select the most promising cell type, proliferation capacity was tested and differentiation towards myotubes was evaluated.
Muscle Derived Stem Cells
Muscle derived stem cells (MDSCs) were first described by the group of Huard (Qu-Petersen et al. 2002). These cells show avid proliferation and differentiation into multiple lineages, making them a promising candidate for muscle tissue engineering. MDSCs are isolated and selected by preplating. Thus, the specific adhesion avidity of MDSCs to collagen is used as a selection criterion. Using the described methods (Qu-Petersen et al. 2002), we were unable to reproduce their results. We performed several trials to isolate these cells from pig and mouse muscle. In our first trial, a pig muscle biopsy was coarsely cut and enzymatically digested (using collagenase type 1, proteinase K and trypsin). Afterwards, the slurry was shearedthrough needles in order to generate a suspension containing all cells available in muscle. This suspension was plated out (preplate (PP) 1) and floating cells were replated after 2 hours (PP2). Consecutively, every 24 hours, floating cells were removed and replated (PP3, PP4 and PP5). After 96 hours, PP6 was left for another 72 hours and should then contain MDSCs, albeit very few. Cells from each preplate (PP) were isolated (Figure 1.2) and readily proliferated for more than 20 passages, but we were unable to induce differentiation (fusion) by serum reduction and insulin addition (Figure 1.3). The medium switches did terminate proliferation.
In addition to embryonic stem cells, adult stem cells could be a potential source for muscle tissue engineering. Different types of adult muscle stem cells have been isolated from skeletal muscle: muscle derived stem cells (MDSCs) (Peng and Huard 2004), side population (SP) cells (Asakura et al. 2002; Tamaki et al. 2003) and satellite cells (SCs) (Asakura et al. 2001; Zammit et al. 2004). Satellite cells are resident muscle stem cells responsible for regeneration and repair in the adult and are already programmed to differentiate into skeletal muscle. Therefore, these cells are an appealing source for muscle tissue engineering. Activated satellite cells differentiate to MPCs, which then proliferate and migrate in order to repair defects. The function of the other types of muscle derived adult stem cells as well as bone marrow derived stem cells (Gussoni etal. 1999; Gang et al. 2004) in physiological circumstances remains unclear. However, adult stem cells derived from either the muscle or the bone marrow, including hematopoietic and mesenchymal stem cells, appear to have conserved the capacity to differentiate into skeletal muscle and therefore remain potential candidates for muscle regeneration (Gussoni et al. 1999; Gang et al. 2004).
Unfortunately, at present, the proliferative ability of adult stem cells does not equal that of embryonic stem cells, mostly because they tend to differentiate spontaneously in vitro. It is anticipated that this issue will be tackled by optimizing the culture circumstances, for example by mimicking the in vivo environment (niche) of the cells (Boonen and Post 2008). An advantage of using mature stem cells over embryonic stem cells is that pure adult stem cell populations, when stimulated to differentiate into skeletal muscle, will give rise to a homogeneous tissue.
For the growth and proliferation of cells there is a need of optimum field. Mechanical, electromagnetic, gravitational, and fluid flow fields have been found to influence the proliferation and differentiation of myoblasts (Kosnik et al., 2003 and De Deyne, 2000). Powell and others found that repetitive stretch and relaxation equal to 10 percent of length, 6 times per hour, increased differentiation into myotubes (Powell et al., 2002). Yuge and Kataoka seeded myoblasts with magnetic micro particles and induced differentiation by placing them in a magnetic field, without adding special growth factors or any conditioned medium (Yuge and Kataoka,2000). Electrical stimulation also contributes to differentiation, as well as sarcomere formation within established myotubes (Kosnik et al., 2003 and De Deyne, 2000).
Culture Media and Growth Factors
For the growth of any substance affordable medium is required. Such medium must contain the necessary nutritional components and be presented in a form freely available to myoblasts and complementary cells, as no digestive system are involved. In this concern, McFarland and others developed a serum-free medium that supported the proliferation of turkey satellite cells in culture (McFarland et al., 1991). Kosnik, Dennis, and Vandenburgh refer to serum-free media developed by Allen et al., Dollenmeier et al., and Ham et al (Kosnik et al., 2003). Benjaminson and others succeeded in using a serum-free medium made from maitake mushroom extract that achieved higher rates of growth than fetal bovine serum (Benjaminson et al., 2002). And it has recently been shown that lipids such as sphingosine-1-phosphate can replace serum in supporting the growth and differentiation of embryonic tissue explants (W. Scott Argraves, Medical University of South Carolina, personal communication, 22 May 2004). The successful system must be capable of altering the growth factor composition of the media.
Cultured meat production is probable to require the development of new bioreactors that sustain low shear and uniform perfusion at large volumes. Much recent skeletal muscle tissue engineering research has employed NASA rotating bioreactors. Their major advantages are that cells are in near-continuous suspension, fluid shear is minimal, and suspension is probable for tissue assemblies up to 1 centimetre. These bioreactors can sustain biomass concentrations up to 108 cells per mL. Research size revolving bioreactors (10 to 250ml) have been scaled up to three litres and, theoretically, scale up to industrial sizes should not affect the physics of the system. Industrial scales are already available for low-shear particle-based biofilm reactors, allowing biomass concentrations as high as 30 kg per m3 (Nicolella et al., 2000).
A laminar flow of the medium is created in revolving wall vessel bioreactors by revolving the cylindrical wall at a speed that balances centrifugal force, draw force and gravitational force, leaving the 3-dimensional culture submerged in the medium in a perpetual free fall state (Carrier et al., 1999) which improves diffusion with high mass transfer rates at minimal levels of shear stress, producing three dimensional tissues with structures very similar to those (Martin et al., 2004). Direct perfusion bioreactors appear more appropriate for scaffold based myocyte cultivation and flow medium through a porous scaffold with gas exchange taking place in an external fluid loop (Carrier et al., 2002). Besides offering high mass transfer they also offer significant shear stress, so determining an appropriate flow rate is essential (Martin et al., 2004). Direct perfusion bioreactors are also used for high density, uniform myocyte cell seeding (Radisic et al., 2003). Another method of increasing medium perfusion is by vascularizing the tissue being grown. Levenberg et al., (2005) had induced endothelial vessel networks in skeletal muscle tissue builds by using a co-culture of myoblasts, embryonic fibroblasts and endothelial cells coseeded onto a highly porous biodegradable scaffold.
Research size revolving bioreactors have been scaled up to three litters and, theoretically, scale up to industrial sizes should not affect the physics of the system. As cell feasibility and density positively correlate with the oxygen gradient in statically grown tissue cultures, it is necessary to have adequate oxygen perfusion throughout cell seeding and cultivation on the scaffold (Radisic et al., 2008). Adequate oxygen perfusion is mediated by bioreactors which increase mass transport between culture medium and cells and by the use of oxygen carriers to mimic hemoglobin provided oxygen supply to maintain high oxygen concentrations in solution, similar to that of blood. Modified versions of hemoglobin or artificially produced perfluorochemicals (PFCs) that are chemically inert are used as oxygen carriers (Lowe, 2006) but their bovine or human source makes them an unfit candidate and alternatively, human hemoglobin has been produced by genetically modified plants (Dieryck et al., 1997) and microorganisms (Zuckerman et al., 1998).
Biophotonics refers in general to the process of using light to bind together particles of matter. A new field, and one in which the mechanisms are still poorly understood, biophotonics relies on the effects of lasers to move particles of matter into definite organizational structures, such as three-dimensional chessboard, or hexagonal arrays. A amazing property of interacting light, this phenomenon produces so called ‘‘optical matter’’ in which the crystalline form of materials (such as polystyrene beads) can be held together by nets of infrared light that will fall apart when the light is removed. This is a phenomenon a step-up from ‘‘optical tweezers’’ that have been used for years to rotate or otherwise move tiny particles in laboratories. This has a binding effect among a group of particles that can lead them not only to be moved one by one to specific locations but that can coax them to form structures. Although primarily sparking interest in medical technologies such as separating cells, or delivering medicine or other microencapsulated substances to individual cells, there is an intriguing possibility that such a technology could be used for the production of tissues, including meat. A main researcher in bio photonics, Kishan Dholakia, reports in an interview that he and colleagues are already using the technology to create arrays of red blood cells and hamster ovaries (Mullins 2006). Given the success of making two-dimensional arrays, there is the chance of producing tissue formations that use only light to hold the cells together, thus eliminating the require for scaffoldings such as those mentioned above in other techniques.
One of the general problems with engineering suitable cultured meat is the consistency of the product. Current culturing techniques cannot provide the vascularization or the fat marbling or other basics of workable and suitably-tasting meat that are not simply versions of ground soft meat. A possible solution to such problems comes from research on producing organs for transplantation procedures. Not surprisingly, given the confluence of technologies, some of the same people who are working on culturing meat are also working on research in ‘‘organ printing’’ (Mironov et al. 2003). Organ printing is a simple yet astounding idea. Using the principles of ordinary printing technology—the kind of technology that inkjet printers use to produce documents like this one—researchers have essentially been able to use solutions containing single cells or balls of cells rather than ink and spray these cell mixtures onto gels that act as printing paper. The ‘‘paper’’ can actually be removed through a simple heating technique or could potentially be automatically degradable. What happens is essentially that live cells are sprayed in layers to create any shape or structure desired. After spraying these three-dimensional structures, the cells fuse into larger structures, such as rings and tubes or sheets. As a result, researchers argue that the feasibility of producing entire organs through printing has been proved. The organs would have not only the basic cellular structure of the organ but would also include, built layer-by-layer, appropriate vascularization providing a blood supply to the entire product. For applications focused on producing meat, fat marbling could be added as well, providing taste and structure. Essentially, sheets and tubes of appropriate cellular components could create any sort of organ or tissue you would like— whether for transplantation or for consumption (Mironov 2003; Aldhous 2006).
Benefits of in vitro Meat over Traditional Meat
In the development of in-vitro meat or laboratory meat there are several obstacles to overcome. Some of these may be as fallows:
Several research projects are going on experimentally growing in -vitro meat, but no meat has been produced for public consumption till date. The aim of in-vitro meat production is to grow fully developed muscle organs, but the first generation will most likely be minced meat products. The hindrances in this field are the structural integrity as the whole animal tissues and its safety for human consumption.
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