Friday, October 22, 2010

Understanding the Process of compaction of DNA

Pure (“naked”) DNA has little chance to enter a cell. DNA is a huge, negatively charged and hence highly hydrophilic molecule. Cells are surrounded by a hydrophobic plasma membrane and, in addition, bear a negative surface charge. The plasma membrane contains several highly selective transporter units, which allow for the well-controlled introduction and excretion of certain molecules. Foreign DNA is normally not amongst the molecules allowed to enter the cell.

The first and best-understood step of transfection is therefore the necessity for “compaction” of the large, negatively charged DNA molecule. A suitable compacting agent is a positively charged molecule able to interact with the DNA and to neutralize or even overcompensate the negative charges. During compaction, the DNA forms stable complexes with the compaction agent, which either stay in solution or form a precipitate. In a typical transfection experiment, the complexes are formed in a reaction mixture containing the given amounts of purified DNA as well as the compaction agent under defined pH and salt conditions. The complex formation occurs spontaneously upon mixing. Within the next 30 minutes the complexes are added to the target cells. Usually, cells are exposed for several hours to the complexed DNA. Subsequently, the medium is exchanged in order to minimize possible toxic effects.

Two groups of molecules are currently investigated as compaction agents: cationic lipids and cationic polymers. Protonated amino groups provide the required positive charges in both cases. Amino groups are also found in some of the naturally occurring compaction agents such as spermine and spermidine. They are clearly the group of choice, since they allow the generation of a positive charge at physiological (neutral) pH. In addition, eukaryotic cells developed over eons of evolution special proteins (nucleosomes) with a high affinity to DNA, which also can complex DNA. The structure of these nucleosomes may in the future inspire the design of novel compaction agents. Prominent representatives are histones or protamines, naturally occurring ubiquitous DNA binding (compacting) proteins.

Cationic lipids are usually fairly small molecules, which mimic the structure of the cell’s plasma membrane and hence facilitate the passage of DNA into the cell by increasing the solubility of the DNA in the plasma membrane. These molecules consist of a hydrophobic (hydrocarbon) tail and a positively charged head-group. The hydrophobic tail promotes in aqueous solutions self-aggregation into larger structures (micelles, double layers) capable of interaction or even fusion with the cellular membrane.

The cationic polymers (such as polyethyleneimine, polyvinyl pyrrolidone) commonly used for transfection are fairly large molecules (up to 1,000,000 g/mol). They are soluble in water at neutral pH due to their positive charges. Linear as well as branched molecules are employed for transfection. In contrast to the cationic lipids, which usually were developed as dedicated transfection reagents, most cationic polymers have been developed for other applications and purposes. They are therefore available from several suppliers in a wide variety of purity and chemical homogeneity.

Tags: Bio Technology, Bio Genetics, Gene Compaction

Monday, August 16, 2010

What are the barriers to Efficient Gene Transfer?

DNA, the common carrier of the genetic information for all living entities on this planet, is omnipresent and we are daily exposed to large quantities of foreign DNA (e.g., by food or bacterial infections). Under these circumstances, nature had to provide powerful barriers against the spontaneous insertion of foreign DNA sequences into the genomic DNA of cells. Barriers are the plasma membrane of the cell, the envelope of the cell’s nucleus, but also the possibility for DNA degradation in lysosomes and the cytoplasm. These protective mechanisms work rather well and even under optimized conditions it is by no means easy to genetically modify an eukaryotic cell (the terminus usually employed for this modification is to “transfect” the cell). However, the necessity to transfect cells for research purposes, the discovery of new and efficient reporter systems to verify the success of a transfection experiment (luciferase, green fluorescent protein) as well as the availability of powerful transfection reagents have spurred research in the area for many years. Several methods to transfer genes into cells have been developed during the last 30 years. However, considerable efforts to develop new techniques or to improve the efficiency of old ones are still being made.

Transfection reagents help to overcome the natural barriers to gene transfer by various strategies.

The steps involved in the transfer of a “gene” from the outside into the genome of the cell comprise of the following:

1. Compaction of the DNA,

2. Attachment to the cell surface,

3. Transport into the cytoplasm,

4. Import into the nucleus and

5. Insertion into the chromosomal DNA.

The mechanism by which a certain barrier is overcome is an important feature of the respective transfection reagent. In order to elucidate the difficulties in optimizing the genetic engineering of mammalian cells, the major steps of transfection as well as putative agents for reaching this goal will be discussed in detail in the following sections. The mechanisms for many of the above-mentioned five steps of transfection are still under discussion. This is especially the case for the later steps taking place inside the cell, i.e., transport into the cell and most importantly into the nucleus. The earlier stages of compaction and interaction with the cell surface are better understood. This has important consequences for our current ability to engineer transfection agents and procedures. It should be noted that man-made transfection procedures are still orders of magnitude less efficient than nature’s transfection agents, the viruses are. One to five infectious particles, i.e., viruses, per cell are sufficient in that case, compared to the 105– 106 plasmid molecules needed in most nonviral transfection methods.

Tags: Bio Technology, Bio Genetics, Gene Delivery

Thursday, July 8, 2010

Understanding of DNA Molecules

The structure of the large DNA molecule, which was known to be the main material of the chromosomes, remained a mystery until Watson and Crick proposed the double helix structure in 1953.6 Chromosomal (genomic) DNA consists of two complementary polyanionic chains made up of long sequences of four different nucleic bases. Since the four bases are complementary, the double stranded DNA molecule is capable of exact self-replication from either strand. The diameter of the double helix is about 2 nm, while the length of the DNA polymer can be enormous, i.e., several centimeters in a putatively “stretched out”-state.

In a typical human cell, DNA molecules with a total length of 1 meter have to be packed into a nucleus of about 5 m in diameter. The compaction is mediated by the so-called nucleosomes, which contribute about 50% of the total mass of the chromosomes. Nucleosomes are formed by 4 to 5 different types of histones; small, basic proteins with a high proportion of positively charged amino acids (25% lysine or arginine). Histones, which bind tightly to the sugar-phosphate backbone of DNA, also have important regulatory functions. Compacted DNA is not active, meaning it can be neither replicated nor transcribed into RNA and finally into proteins. The histones control the compaction and the compaction-reversal through a regulated process that is gene or sequence specific. The exact biochemical basis of this regulation strategy is still unknown, but the essential role of histones in life is supported by the fact that their amino acid sequence is among the best conserved throughout evolution. Apparently, even minor changes in the histone structure have dire consequences for the organism in question.

Plasmid DNA is an independent type of DNA, which occurs naturally in many microorganisms in addition to the genomic DNA of the respective organism. Plasmids are comparatively small (typically 5–10 kba), circular DNA molecules that can multiply independently from the genomic (chromosomal) DNA. They occur naturally in the supercoiled (major percentage) and the open circular form. Linearized fragments of plasmid DNA can be obtained by “digestion” of the plasmid with restriction endonucleases, i.e., enzymes that cut the DNA at specific base pair sequences. For various reasons, plasmid molecules are the preferred tools for genetic engineering. Plasmids can easily be amplified in bacteria. They are separated from the larger chromosomal bacterial DNA by a denaturation/renaturation process, where the chromosomal DNA forms an insoluble precipitate, because it renatures more slowly. Purified plasmids can be transferred into eukaryotic cells either in their natural, supercoiled form or as linearized molecules.

Tags: Bio Technology, Bio Genetics, Gene Delivery

Tuesday, June 15, 2010

Understanding the impact of molecular biology on everyday life

The impact of molecular biology on everyday life has increased enormously over the last two decades. Medical, pharmaceutical and lately even agricultural applications of “gene technology” have become standard, if sometimes controversially viewed procedures. The feasibility of this “revolution” is based on a few biological facts; most importantly the relationship between DNA, RNA and proteins. DNA carries the information for protein production.

Basic units of information are called genes, which typically are DNA sequences of about 1500 base pairs (bp). Usually, one gene carries the information for one protein. While the proteins are highly specific to a species, the genetic code is universal and shared among all living organisms. Therefore, if a human gene is transferred into a bacterium, the bacterium will be able to translate this DNA sequence into the “correct”, i.e., human, amino acid sequence (protein). The insertion of foreign genes into bacteria has become a routine laboratory procedure1 and genetically modified bacteria have been widely used to produce so-called “recombinant” proteins for the pharmaceutical industry. A well-known example is the production of human insulin in E. coli.

However, there are limitations to the use of bacteria for the production of proteins, especially of complex proteins from higher organisms. While the genetic code is universal, the machinery for protein processing is not and bacteria lack the enzymes and organelles, which, for example, in mammalian cells are responsible for further processing and modification of the proteins (e.g., glycosylation, disulfide bridge formation, cleavage). Especially in the case of larger proteins, bacteria are often not able to fold the amino acid chain into the correct three-dimensional structure required for “biological activity”. Last but not least, the tendency of bacteria to store produced proteins inside the cell in the form of denatured precipitates, so-called inclusion bodies, has been known to considerably reduce the yield of active protein. For this reason, mammalian cells, which have been adapted to propagation in single cell culture, are nowadays used to produce the more complex but also more valuable products of modern biotechnology. Well-known examples are the various CHO cell lines derived from Chinese hamster ovary cells. In order to enable such mammalian cells to produce a desired – human - protein, they too need to be genetically modified. The genetic manipulation of mammalian cells (“transfection”) is much more difficult than that of bacteria. Over the last years a number of transfection strategies have been developed, amongst the methods that utilize (semi-) synthetic polymers. A controllable and successful transfection strategy is not only the basis for the production of recombinant proteins, but even more so for gene therapy. Considerable attention has therefore been paid to the development of synthetic polymers as vehicles for gene delivery. This chapter will focus on the current state of knowledge in regard to the requirements for putative transfection vehicles, but also will summarize and compare the various applications of such systems.

Tags: Bio Technology, Bio Genetics, Organ Rejection

Saturday, May 22, 2010

What are the areas needing Efficient Gene Delivery

Today an enormous amount of genetic information is available from databases, which are continuously fed by worldwide genome sequencing programs. Every day, the human genome-sequencing program alone provides new information about human genes with potential therapeutic value. On a diagnostic level, this will allow detecting “genetic defects” and also a prediction as to which amino acid in a given protein is concerned. However, unless the change in amino acids is meaningless or the malformed protein can be replaced, this information has limited therapeutic impact since curing the DNA defect is at present not possible. Another aspect concerns the large number of genes with unknown function. Since it is not possible to predict the three dimensional structure of a protein, let alone its biological function (interactions with other biological substances), from its amino acid sequence, the only way to “mine” the genetic information consists in a laborious transfection of a mammalian cell with the gene in question to enable said cell to produce the protein. Subsequently this allows studying the activity of the protein either directly within the cell or in vitro once enough of the material has been produced for further characterization.

The problem of quickly producing a certain amount of protein for further characterization and study is a major bottleneck in several areas of the life sciences and the related bioindustry. The list of sequenced genes for which the function of the corresponding protein is poorly understood is long. In addition, it is fairly easy to mutate genes in vitro, so a variety of new proteins can be encoded, some of which might have considerable therapeutic value. In contrast to the quick generation of new genes, the establishing of a stable recombinant production cell line requires at least a year for transfection, screening/amplification and scale up due to the difficulties of inserting the gene stably into a transcriptionally active region of the cell's chromosomal DNA. Recently, a much faster method -transient transfection - has been discussed as a means to produce quickly (within days) milligrams of a given protein. In this case, the foreign DNA is not inserted into the genome of the cell. The method, which until recently was only used for the production of smaller amounts of proteins through-out, had been shown to be compatible with at least the 1 L scale. If transfections could be established at the 100 liter scale or more, gram amounts of any protein could be produced within days. Screening of putative biopharmaceuticals but also basic research would profit enormously. Such large-scale transfections have not yet been achieved.

Gene therapy is another domain where efficient transfer of genes is essential. Many severe human diseases are caused by a genetic defect leading to the mal- or over-/ under-expression of the corresponding protein. Patients could be permanently cured if the missing genes could be transferred in a functional form into the concerned organs. Delivery of genes to specific tissues could become the most efficient medical treatment in the future, but for obvious reasons, the establishment of a very safe and well-controlled method for gene delivery is imperative.

Tags: Bio Technology, Bio Genetics, Gene Delivery

Monday, May 3, 2010

Understanding of Bioartificial Organ Rejection

The process of rejection may begin with the diffusion of immunogens from the graft across the membrane barrier. There are several possible sources for these antigens, including molecules shed from the cell surface, protein secreted by live cells and cytoplasmic protein liberated from dead cells. Recognition and display of these antigens by antigen presenting cells initiates the cellular and humoral immune response. The former leads to activation of cytotoxic cells, macrophages and other cells of the immune system. These cells must be prevented from contacting grafted tissue, a requirement relatively easy to meet. More difficult is keeping out components of the humoral immune response. These include cytokines, for example, interleukin-1, which can have detrimental effects on beta cells, as well as the antibodies formed as a response to the antigens, which have leaked across the barrier. In addition, there may always be some antibodies already present in the antibody spectrum of the blood serum which correspond to cell surface antigens (e.g., major histocompatibility complexes) on allo- or xenografts. Antibodies produced during preexisting autoimmune disease, such as type I diabetes, might also bind to surface antigens on allogeneic cells. Finally, macrophages and certain other immune cells can secrete low-molecular weight reactive metabolites of oxygen and nitrogen including free radicals, hydrogen peroxide, and nitric oxide that are toxic to cells in a nonspecific manner. These agents can diffuse large distances if their lifetime exceeds 1s.

Any attempts to evaluate biocompatibility in vitro would show some lack of predictability for in vivo experiments. Therefore, implantation experiments are necessary to correlate these phenomena. The majority of experiments have been performed on rodents, and there are only a few reports on systematic experiments in large animal models. The choice of an animal model should reflect the human situation. In diabetes research, the diabetic BB-rat, NOD-mice and STZ-treated mice have generally been accepted to be a representative animal model of autoimmune diabetes.

Tags: Bio Technology, Bio Genetics, Organ Rejection

Tuesday, April 20, 2010

What is Tissue Sourcing

Organs and cells of animal origin are being considered as a source of tissue for xenotransplantation. If islet transplantation is to become a widespread treatment for type 1 diabetics, solutions must be found for increasing the availability of insulin-producing tissue and for overcoming the need for continuous immunosuppression. Insulin-producing cells being considered for clinical transplantation include porcine and bovine islets, fish-Brockman bodies, genetically engineered insulin-secreting cell lines and in vitro produced “human” beta-cells.

Both primary tissue and cultured cell lines have been employed in small animal xenotransplantation, including cells that have been genetically modified. Substantial efforts have also been made in the isolation of primary tissue, especially for pancreatic islets, though further improvements are necessary for practical, large-scale processing. The most urgent problem in transplantation is the shortage of donor organs and tissue.

Xenotransplantation could offer some advantages over the use of human organs. Xenotransplantation could be planned in advance, the organ would be transplanted while it was still fresh and undamaged. In addition, a planned transplantation allows the administration of therapeutic regimens that call for the pretreatment of the recipient. Another advantage is the possibility that animal sources could be genetically engineered in order to lower the risk of rejection by expressing specific genes for the benefit of the patient. However, the concern over retroviruses has led to political moratoriums on the clinical use of xenotransplantation. It has yet to be established in nonrodent models as a viable alternative.

Alternative Tissue Sources

The optimal source of xenogeneic islets remains controversial. Islets have been isolated from primates and xenografted into immunosuppressed, diabetic rodents, with short-term reversal of diabetes. However, there are ethical issues surrounding the use of primates for these studies. Other promising islet sources are porcine, bovine and rabbit islets, all of which function remarkably well in diabetic rodents. Long-term human, bovine and porcine islet xenograft survival has been documented in nude mice and rats, suggesting that, in the absence of an immune response, sufficient islet-specific growth factors are present in xenogeneic recipients.

Porcine islets are at present receiving the greatest attention since pigs produce an insulin which is structurally very similar to human insulin and pigs are, on the other hand, the only large animals slaughtered in sufficient quantities to supply the estimated demand from type 1 diabetics. In addition, porcine islets within microcapsules have been reported to correct diabetes in cynomologus monkeys. Elaborate studies are in progress to engineer a “perfect pig”, having adequate levels of complement-inhibiting factors. Thus, porcine sources are

perhaps most likely to provide islets for an inaugural human xeno-islet trial. However, porcine islets are fragile and have poor long-term stability. The in vitro glucose-stimulated insulin secretion rate per unit islet volume appears to be substantially smaller for porcine islets than for other species including human. Lastly, there is significant current concern regarding the potential for transmission of infectious agents from porcine organ sources to human xenograft recipients, and to the population at large. None of these characteristics bode well for their practical large-scale use, and serious consideration and investigation is being given to alternate animal sources. There is also speculation that neonatal porcine islets, which culture better and present minimal infrastructure problems, would be an ultimate substitute. Isolation of bovine islets is technically easier and calf islets are glucose-responsive. However, adult bovine islets are relatively insensitive to glucose. The rabbit pancreas is also an attractive source of islets since rabbit insulin differs from human insulin at only one amino acid and rabbit islets are glucose responsive.

Friday, April 2, 2010

What are the techniques of Interfacial Polymerization & Photo Polymerization

Interfacial polymerization is a method developed for encapsulation of mammalian cells. Cells are coextruded with a generally hydrophobic polymer solution through a coaxial needle assembly. Shear and mechanical forces due to a coaxial air/liquid stream flowing past the tip of the needle assembly causes the hydrogel to envelop the cells and fall off. The encapsulated cells fall subsequently through a series of oil phases, which cause precipitation of the hydrogel around the cell. This process, based on membrane phase inversion, is used primarily when encapsulating cells with hydrogels from the polyacrylate family. Polyacrylates are well tolerated by the host’s immune system and have exceptional hydrolytic stability. A potential disadvantage of this technique is that organic solvents, which may be harmful to living cells, are used to precipitate the hydrogel. To eliminate the use of organic solvents, complex coacervation was developed using acidic and basic water-soluble polymers. Briefly, a droplet containing one of these polymers and cells is added to the other polymer. A thin membrane encapsulates the droplet due to ionic interactions of the two polymers. The major disadvantage of this method is that the capsules may be unstable due to high water uptake in the capsule wall. Modifications have been made to better control permeability and stability of the hydrogel capsules.

Photopolymerization has also been used to conformally coat hydrogel capsules to:

1) Improve their biocompatibility and

2) Reduce the volume to a minimum in order to reduce implant size, a critical issue if an internal organ is the intended transplantation site.

Photopolymerization permits gelation of the polymer membrane in the presence of dissolved oxygen, which is helpful for cell survival during the encapsulation process. The advantage of this technique is that the membrane is directly in contact with the encapsulated cells. Minimizing diffusion distance for oxygen, nutrients, and cell products is important for eliminating necrosis at the center of the capsule12 and for improving therapeutic efficiency.

Tags: Bio Technology, Bio Genetics, Polymerization

Thursday, March 25, 2010

Methods adopted for Micro-capsule Formation

The most widely used procedure for micro-capsule formation involves the gelation of charged poly-electrolytes around the cell core. The popular alginate-L-polylysine micro-capsules, for example, are obtained in the following sequence:

1) The cells are embedded in alginate droplets with the aid of a droplet generator (air / liquid jet or an electrostatic generator);

2) The droplets are transformed into rigid beads by inducing cross-linking with calcium ions;

3) The beads are coated with polylysine and alginate, thereby forming the semi-permeable capsule; and

4) The alginate core is liquefied with a chelating agent.

Micro-capsules surrounding individual cells or clusters such as islets should be physically durable, smooth and spherical for optimal bio-compatibility. Smoothness is one factor, which, in addition to the interfacial composition, reduces tissue irritation, which decreases the probability of cell overgrowth on the capsule surface if aggregated tissue such as beta-cell clusters (beta cells transform blood glucose concentration stimuli into a regulated, pulsatile, insulin secretion) is employed. The capsules should be as small as possible in relation to the islet size to optimize nutrient ingress and hormone egress.

The poly-electrolyte complexation technique used to make alginate-polylysine capsules is advantageous since the capsules are formed under very mild conditions. A disadvantage, however, is the impurities and batch to batch irreproducibility of the alginate, a naturally derived polysaccharide.The high mannuronic acid content of alginate was shown to be responsible for fibrotic tissue response. Fibrosis was reduced and a more resistant micro-capsule was fabricated by decreasing the mannuronic acid level of the alginate at the expense of the guluronic acid content, although these conclusions have been questioned by some authors. Another disadvantage of alginate-polylysine micro-capsules is that the alginate-polylysine membrane, a weak polyelectrolyte complex, gives the micro-capsules relatively poor mechanical properties.

Local changes in pH or ionic concentration may have influence on the integrity of these microcapsules drastically.

Several different hydrogels have been investigated to determine the efficacy of encapsulation therapy as treatment for multiple diseases in a variety of animal models. For instance, alginate-polylysine-alginate micro-capsules have been employed to encapsulate islets and to reverse the effects of diabetes in rats and mice. The mild encapsulation procedure preserved the integrity of the islet’s secretory function with long term viability maintained. Modified alginate-polylysine micro-capsules, which are smaller and stronger than the previous versions, improved the survival of the xenographic tissue grafts. Coating alginate-polylysine capsules with a poly(ethylene glycol)hydrogel or incorporating monomethoxy poly(ethylene glycol) pendant chains to the polylysine polymer backbone has led to improved biocompatibility compared to unmodified capsules. In an attempt to simultaneously control biocompatibility and permeability, polymer blends have been selected that were optimal with respect to islet cytotoxicity (as measured by in vivo tests or) as well as thermodynamic (swelling / shrinking) and mechanical parameters.

Tags: Bio Technology, Bio Genetics, Bio Artificial organs

Wednesday, March 10, 2010

Understanding of Bioartificial Organs

Tissue engineering involves the in vitro or in vivo generation of organoids such as cartilage, skin or nerves. More ambitious projects seek to ameliorate the quality of life of diseased or injured patients and reduce the economic burden of treatment. Bioartificial organs involve an in vitro prepared tissue-material interface fabricated into a durable device. A typical example is the bioartificial pancreas, which will be discussed in the following section as a case study. The extra-corporeal bioartificial liver and more recently the bioartificial kidney14 are examples of the transient replacement of organ functions, the former intended as a bridge to stabilize comatose patients until a whole organ can be procured. As the bioartificial pancreas is often microcapsule based, a specific section will be dedicated to review encapsulation technology prior to the application of this bioartificial organ for in situ insulin production.

Bioartificial organs require the combination of several research areas. The understanding of cellular differentiation and growth and how extracellular matrix components affect cell function comes under the umbrella of cell biology. Immunology and molecular genetics will also be needed to contribute to the design of cells or cell transplant systems that are not rejected by the immune system. Cell source and cell preservation are other important issues. The transplanted cells may come from cell lines or primary tissues—from the patients themselves, other human donors, animal sources or fetal tissue. In choosing the cell source, a balance must be struck between ethical issues, safety issues and efficacy. The sterilization and depyrogenation of the polymers involved in transplants is also critical. The materials used in tissue engineering and polymer processing are other key issues. The development of controlled release systems, which deliver molecules over long time periods, will be important in administering numerous tissue controlling factors, growth factors and angiogenesis stimulators. Finally, it will be useful to develop methods of surface analysis for studying interfaces between cell and materials and mathematical models and in vitro systems that can predict in vivo cellular events.

Tags: Bio Technology, Bio Genetics, Bio Artificial organs

Thursday, February 25, 2010

Microencapsulation for Cell Delivery

Microencapsulation is currently the most widely used form of cell delivery with preparation methods including:

1. Gelation and polyelectrolyte complexation,
2. Interfacial polymerization/phase inversion and
3. Conformal coating.

Microencapsulation involves surrounding a collection of cells with a thin generally micrometer sized, semipermeable membrane. Its primary purpose is to protect the encapsulated cells from the host’s immune system, while allowing the exchange of small molecules and thereby ensuring cell survival and function. There are several requirements for polymer capsules or hydrogels used as components of microcapsules:

# Noncytotoxicity to the encapsulated cells

# Biocompatibility with the surrounding environment where capsules are to be implanted (e.g., minimal fibrotic response)

# Adequate permeability for diffusion of essential nutrients (e.g., oxygen and glucose for islets of Langerhans) and cell secretory products (such as insulin, metabolic waste)

# Impermeability to secreted antibodies of the host’s immune system (e.g., immunoglobulins and glycoproteins after complement activation

# Chemical and mechanical stability

From the technological point of view, the requirements for microencapsulation include:

# Small capsule diameters to ensure sufficient diffusion and internal organ transplantability (depending on application, < 400 μm for bioartificial pancreas), with the cell centering within the microcapsule

# Minimum shrinking/swelling due to changes in osmotic conditions upon transplantation

# Uniform wall thickness for optimum transport of molecules across the membrane and effective immunoprotection.

In addition, the technology used for encapsulation must be nontraumatic to the encapsulated cells. This includes minimizing the mechanical stress during encapsulation and solvent toxicity (if any), as well as optimizing temperature, viscosity, pH and ionic strength. This, in turn, limits the concentration and molecular mass which can be employed. In addition, the ionic content of the polymer backbone (density distribution of charges in the polymer chain), the chemistry and location of functional group attachment, the chain rigidity, aromaticity, conformation and extent of branching were identified as important variables in the type of complex produced. The presence of secondary hydrogen bonding interactions was also found to be significant.

Several problems may prevent wide scale application of microcapsules in the clinic. The capsules can clump together, in which case the cells towards the center may suffer severely from limited diffusion of oxygen and nutrients. A substantial fraction of the capsules may also adhere to tissue. If the capsules degrade, the liberated islet cells, even if nonviable, would greatly increase the antigenic burden on the patient. Semipermeable polymeric membranes have been developed with the aim of permitting the transplantation of xenogenic cells thus removing the need for immunosuppression therapy. However, early clinical implementations is not likely to involve xenografts or genetically modified cells but rather auto- and allografts supplemented by immunosuppression when necessary.

Tags: Bio Technology, Bio Genetics, Cell Encapsulation

Monday, February 15, 2010

Adhesion based Immobilization Techniques

Each immobilization method has specific properties and advantages. Therefore, the selection of a cell delivery technique depends heavily on the intended application.

Adhesion: Adhesion to a three-dimensional structure is used to immobilize cells for culture or analytical procedures as well as to provide a structural template directing cell growth and differentiation. Adhesion alone does not offer immunoisolation. For in vivo investigations, adhesion-based immobilization must be used in conjunction with either a polymeric membrane or matrix entrapment methods. This method is effective for surface binding, either on top of gel films or within hydrogel foams. Several hydrogels can be engineered with bioadhesive properties by methods which include interfacial polymerization, phase separation, interfacial precipitation and polyelectrolyte complexation. Factors affecting cell affinity and behavior on hydrogels include the general chemistry of the monomers and the crosslinks, hydrophilic and hydrophobic properties, and the surface charge and functionality. One method to enhance cell adhesion is by adding immobilized cell-adhesive proteins or oligopeptides, such as the arginine-glycine-aspartic acid sequence, in the hydrogel. The physical characteristics of the hydrogel also govern the adhesion affinity. Therefore, altering the pore size and network structure can modify cell adhesion as well as morphology and function. For some adhesion applications the mechanical strength is also important with a lower fractional porositygenerally creating stronger networks. Furthermore, closed pore systems make stronger hydrogels than open pore ones. With the adhesion approach, cells are generally plated onto the hydrogel and allowed to attach and migrate. Supplemented culture media provide the cells with essential nutrients for growth and development as well as a means of oxygen and metaboli product transport while in vitro.

Macroporous hydrogel membranes are manufactured by several techniques. One method of constructing pores large enough for cell growth is by phase separation in the polymer and solvent mixture. The “freeze thaw” and the porosigen techniques are two other approaches. The hydrogel is polymerized around a crystalline matrix made from freezing the aqueous solvent (freeze-thaw technique) or around a porosigen of desired size (porosigen technique). With the “freeze-thaw” method, the ice-based crystalline matrix is then thawed after UV polymerization, leaving a macroporous foam. The porosigen technique also requires removal of the crystalline porosigens, in this case usually by leaching or dispersion after polymerizing of the hydrogel with free-radical initiators has taken place. Another method for constructing hydrogel foams uses gas bubbles from sodium bicarbonate to create the macroporous network. Bubbles are trapped during the gelation stage. Thus, the foam morphology is dependent on the polymerization kineics and varies for different hydrogel compositions.

Tags: Bio Technology, Bio Genetics, Cell Encapsulation

Thursday, January 28, 2010

Understanding of Immunoisolation

A variety of systems can be employed for cell or enzyme immobilization. These include, for example, microcarriers, gel entrapment, hollow fibers, encapsulation and conformal coatings. The latter three have been extensively tested in small animal models over the last 20 years, particularly in the area of diabetes therapy. The polymeric materials used in bioartificial endocrine devices (the terms bioartificial and endocrine device are often distinguished from ‘artificial organs’ due to the presence of tissue in the former two) serve two major purposes:

1. As a scaffold and an extracellular matrix they favor the attachment and differentiation of functional cells or cell clusters and keep them separate from one another;

2. As permselective envelopes which provide immunoisolation of the transplant from the host.

The central concept of immunoisolation is the placement of a semipermeable barrier between the host and the transplanted tissue. The properties required for the semipermeable membranes used in cell transplantation depend strongly on the source of cells. An allograft is a transplant between individuals within one species, while a xenograft is a graft between individuals from different species.

Immunoisolation of transplanted cells by artificial barriers that permit crossover of low molecular weight substances, such as nutrients, electrolytes, oxygen, and bioactive secretory products, though not of immune cells and high molecular weight proteins such as antibodies (IgG, IgM), provides great promise for developing new technologies to overcome these problems in a reasonable time frame.

Device Geometry Considerations

The immunoisolation of allogeneic or xenogeneic islets can be achieved via two main classes of technology: macroencapsulation and microencapsulation. Macroencapsulation refers to the reliance on larger, prefabricated “envelopes” in which a slurry of islets or cell clusters is slowly introduced and sealed prior to implantation. An intravascular device usually consists of a tube through which blood flows, on the outside of which is the implanted tissue contained within a housing. The device is then implanted as a shunt in the cardiovascular system. Extravascular devices are implanted directly into tissue in a body space such as the peritoneal cavity, though some have also been vascularized into a major artery such as in Calafiore’s clinical trial. Geometrical alternatives include cylindrical tubular membranes containing tissue within the lumen and planar diffusion chambers comprised of parallel flat sheet membranes between which the implanted tissue is placed.

Microencapsulation refers to the formation of a spherical gel around each group of islets, cell cluster or tissue fragment. Microcapsules based on natural or synthetic polymers have been used for the encapsulation of both mammalian and microbial cells as well as various bioactive substances such as enzymes, proteins and drugs. A review of alternative semipermeable microcapsules prepared from oppositely charged water soluble polyelectrolyte pairs has been presented in recent papers. The main advantage of this approach is that cells, or bioactive agents, are isolated from the body by a microporous semipermeable membrane and the encapsulated material is thus protected against the attack of the immune system. In the case of microencapsulated pancreas islets, a suspension of microcapsules is typically introduced in the peritoneal cavity to deliver insulin to the portal circulation.

Tags: Bio Technology, Bio Genetics, Cell Encapsulation

Thursday, January 21, 2010

Future of Plant-cell Tissue Culture

The commercial potential of plant-cell tissue culture has not yet been fully recognized and is underexploited. Plant-cell tissue culture has two primary products: plant tissue for efficient micropropagation of plants and the use of plant-tissue culture to produce specialty chemicals.

Plant-cell, tissue, and organ cultures can be used in processes analogous to traditional fermentation processes for producing chemicals. Although less than 5% of the world's plants have even been identified taxonomically, from among the known plants over 20,000 chemicals are produced - about 4 times as many as from all microorganisms. Very few of the chemicals in pure or semipure form have been tested for their pharmacological activity for other uses. The enzymatic systems in plants can be used to generate completely new compounds when supplied with analogues of natural substrates; thus, plants contain an underused biochemical diversity. Even the limited use of this vast biochemical potential has had important impacts on mankind; in western countries, about one-fourth of all medicines are derived from compounds extracted from plants. Other plant products are used as flavors, fragrances, or pesticides.

Plant-cell tissue culture to produce chemicals commercially has been exploited in Japan, although regulatory approval for medicinal uses has proved difficult and commercial production is restricted to food uses and pigment production. In Japan, a government-sponsored consortium of universities and corporations was recently developed to establish a foundation for plant-cell culture exploitation (i.e., a precompetitive research thrust). In the US plant-cell tissue is not being exploited for chemical production, although some companies are developing processes for the production of the chemotherapeutic agent taxol.

The major technical barriers to the commercial exploitation of plant-cell tissue culture are low growth rates and relatively low product yields. To mitigate those problems, research is needed in subjects as diverse as bioreactor strategies to maintain high-density cultures and enable large-scale production of chemicals through organ cultures and a mechanistic understanding of the role of elicitors in activating pathways for secondary metabolites that could lead to higher productivities of compounds with therapeutic value.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering

Tuesday, January 5, 2010

Role of Bio-Process engineering in agriculture and food

Bioprocess engineering in agriculture and the food industry involves the application of biocatalysts (living cells or their components) to produce useful and value-added products, and it offers opportunities to design and produce new or improved agricultural and food products and their manufacturing processes. This will likely have a great impact on the food-processing industry. In the increasingly health-conscious society, genetically engineered microorganisms and specialty enzymes will find increased use in improving the nutritional, flavoring, and storage characteristics and safety of food products. Products under development range from genetically improved strains of freeze-resistant yeast used in frozen bakery products to phage-resistant dairy (yogurt) starter cultures. Chymosin, a product of recombinant E. coli, is already used in the milk-clotting step of cheese manufacture, and a recombinant maltogenic amylase is being used as an antistaling agent. Enzyme-based immunoassays could develop into a widely used method for detecting pesticides in foods at parts-per-billion concentrations. Challenges that must be addressed include the economics of production and regulatory issues.

The most important applications of bioprocess-engineering research and development related to agriculture and food involve production of agricultural chemicals for control of animal and plant diseases, growth-stimulating agents for improved yield, and biological insecticides and herbicides; increasing bioprocess efficiencies for fermented foods, natural food additives, food enzymes as processing aids, and separation and purification of the products; use of plant-cell culture systems to produce secondary metabolites or chemical substances of economic importance; and efficient use of renewable biomass resources for production of liquid fuel and chemical feedstocks and efficient treatment and management of agricultural wastes and wastes from food-processing industries.

Tags: Bio Technology, Bio Genetics, Bio Process Engineering